parts of essay analysis

How To Write an Analytical Essay

If you enjoy exploring topics deeply and thinking creatively, analytical essays could be perfect for you. They involve thorough analysis and clever writing techniques to gain fresh perspectives and deepen your understanding of the subject. In this article, our expert research paper writer will explain what an analytical essay is, how to structure it effectively and provide practical examples. This guide covers all the essentials for your writing success!

What Is an Analytical Essay

An analytical essay involves analyzing something, such as a book, movie, or idea. It relies on evidence from the text to logically support arguments, avoiding emotional appeals or personal stories. Unlike persuasive essays, which argue for a specific viewpoint, a good analytical essay explores all aspects of the topic, considering different perspectives, dissecting arguments, and evaluating evidence carefully. Ultimately, you'll need to present your own stance based on your analysis, synthesize findings, and decide whether you agree with the conclusions or have your own interpretation.

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How to Structure an Analytical Essay

Crafting an excellent paper starts with clear organization and structuring of arguments. An analytical essay structure follows a simple outline: introduction, body, and conclusion.

Introduction: Begin by grabbing the reader's attention and stating the topic clearly. Provide background information, state the purpose of the paper, and hint at the arguments you'll make. The opening sentence should be engaging, such as a surprising fact or a thought-provoking question. Then, present your thesis, summarizing your stance in the essay.

Body Paragraphs: Each paragraph starts with a clear topic sentence guiding the reader and presents evidence supporting the thesis. Focus on one issue per paragraph and briefly restate the main point at the end to transition smoothly to the next one. This ensures clarity and coherence in your argument.

Conclusion: Restate the thesis, summarize key points from the body paragraphs, and offer insights on the significance of the analysis. Provide your thoughts on the topic's importance and how your analysis contributes to it, leaving a lasting impression on the reader.

Meanwhile, you might also be interested in how to write a reflection paper , so check out the article for more information!

How to Write an Analytical Essay in 6 Simple Steps

Once you've got a handle on the structure, you can make writing easier by following some steps. Preparing ahead of time can make the process smoother and improve your essay's flow. Here are some helpful tips from our experts. And if you need it, you can always request our experts to write my essay for me , and we'll handle it promptly.

Step 1: Decide on Your Stance

Before diving into writing, it's crucial to establish your stance on the topic. Let's say you're going to write an analytical essay example about the benefits and drawbacks of remote work. Before you start writing, you need to decide what your opinion or viewpoint is on this topic.

• Do you think remote work offers flexibility and improved work-life balance for employees?
• Or maybe you believe it can lead to feelings of isolation and decreased productivity?

Once you've determined your stance on remote work, it's essential to consider the evidence and arguments supporting your position. Are there statistics or studies that back up your viewpoint? For example, if you believe remote work improves productivity, you might cite research showing increased output among remote workers. On the other hand, if you think it leads to isolation, you could reference surveys or testimonials highlighting the challenges of remote collaboration. Your opinion will shape how you write your essay, so take some time to think about what you believe about remote work before you start writing.

Step 2: Write Your Thesis Statement

Once you've figured out what you think about the topic, it's time to write your thesis statement. This statement is like the main idea or argument of your essay.

If you believe that remote work offers significant benefits, your thesis statement might be: 'Remote work presents an opportunity for increased flexibility and work-life balance, benefiting employees and employers alike in today's interconnected world.'

Alternatively, if you believe that remote work has notable drawbacks, your thesis statement might be: 'While remote work offers flexibility, it can also lead to feelings of isolation and challenges in collaboration, necessitating a balanced approach to its implementation.'

Your thesis statement guides the rest of your analytical essay, so make sure it clearly expresses your viewpoint on the benefits and drawbacks of remote work.

Step 3: Write Topic Sentences

After you have your thesis statement about the benefits and drawbacks of remote work, you need to come up with topic sentences for each paragraph while writing an analytical essay. These sentences introduce the main point of each paragraph and help to structure your essay.

Let's say your first paragraph is about the benefits of remote work. Your topic sentence might be: 'Remote work offers employees increased flexibility and autonomy, enabling them to better manage their work-life balance.'

For the next paragraph discussing the drawbacks of remote work, your topic sentence could be: 'However, remote work can also lead to feelings of isolation and difficulties in communication and collaboration with colleagues.'

And for the paragraph about potential solutions to the challenges of remote work, your topic sentence might be: 'To mitigate the drawbacks of remote work, companies can implement strategies such as regular check-ins, virtual team-building activities, and flexible work arrangements.'

Each topic sentence should relate back to your thesis statement about the benefits and drawbacks of remote work and provide a clear focus for the paragraph that follows.

Step 4: Create an Outline

Now that you have your thesis statement and topic sentences, it's time to create an analytical essay outline to ensure your essay flows logically. Here's an outline prepared by our analytical essay writer based on the example of discussing the benefits and drawbacks of remote work:

Step 5: Write Your First Draft

Now that you have your outline, it's time to start writing your first draft. Begin by expanding upon each point in your outline, making sure to connect your ideas smoothly and logically. Don't worry too much about perfection at this stage; the goal is to get your ideas down on paper. You can always revise and polish your draft later.

As you write, keep referring back to your thesis statement to ensure that your arguments align with your main argument. Additionally, make sure each paragraph flows naturally into the next, maintaining coherence throughout your essay.

Once you've completed your first draft, take a break and then come back to review and revise it. Look for areas where you can strengthen your arguments, clarify your points, and improve the overall structure and flow of your essay.

Remember, writing is a process, and it's okay to go through multiple drafts before you're satisfied with the final result. Take your time and be patient with yourself as you work towards creating a well-crafted essay on the benefits and drawbacks of remote work.

Step 6: Revise and Proofread

Once you've completed your first draft, it's essential to revise and proofread your essay to ensure clarity, coherence, and correctness. Here's how to approach this step:

• Check if your ideas make sense and if they support your main point.
• Make sure your writing style stays the same and your format follows the rules.
• Double-check your facts and make sure you've covered everything important.
• Cut out any extra words and make your sentences clear and short.
• Look for mistakes in spelling and grammar.
• Ask someone to read your essay and give you feedback.

What is the Purpose of an Analytical Essay?

Analytical essays aim to analyze texts or topics, presenting a clear argument. They deepen understanding by evaluating evidence and uncovering underlying meanings. These essays promote critical thinking, challenging readers to consider different viewpoints.

They're also great for improving critical thinking skills. By breaking down complex ideas and presenting them clearly, they encourage readers to think for themselves and reach their own conclusions.

This type of essay also adds to academic discussions by offering fresh insights. By analyzing existing research and literature, they bring new perspectives or shine a light on overlooked parts of a topic. This keeps academic conversations lively and encourages more exploration in the field.

Analytical Essay Examples

Check out our essay samples to see theory in action. Crafted by our dissertation services , they show how analytical thinking applies to real situations, helping you understand concepts better.

With our tips on how to write an analytical essay, you're ready to boost your writing skills and craft essays that captivate your audience. With practice, you'll become a pro at analytical writing, ready to tackle any topic with confidence. And, if you need help to buy essay online , just drop us a line saying ' do my homework for me ' and we'll jump right in!

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How to Write an Analytical Essay?

What is an analytical essay.

Daniel Parker

is a seasoned educational writer focusing on scholarship guidance, research papers, and various forms of academic essays including reflective and narrative essays. His expertise also extends to detailed case studies. A scholar with a background in English Literature and Education, Daniel’s work on EssayPro blog aims to support students in achieving academic excellence and securing scholarships. His hobbies include reading classic literature and participating in academic forums.

is an expert in nursing and healthcare, with a strong background in history, law, and literature. Holding advanced degrees in nursing and public health, his analytical approach and comprehensive knowledge help students navigate complex topics. On EssayPro blog, Adam provides insightful articles on everything from historical analysis to the intricacies of healthcare policies. In his downtime, he enjoys historical documentaries and volunteering at local clinics.

Guide to Writing an Analytical Essay

An analytical essay is a type of academic writing in which a complicated topic or idea is broken down into smaller parts, analyzed and looked at, and a well-structured argument or evaluation is given. The main purpose of an analytical essay is to show that the writer has a deep understanding of the topic and can also think critically about it. Analytical essays are important in many fields, such as literature, history, science, and sociology. They require a deep knowledge of the topic and the ability to think critically and objectively about the information given. Analytical essays include things like a literary analysis, a research paper , or an analysis of a piece of rhetoric. Writing an analytical essay is useful because it helps the writer improve their analytical and critical thinking skills. In analytical essays, students must look at and evaluate different sources, find patterns and relationships, and come to meaningful conclusions. When students write analytical essays, they also improve their research skills because they have to find relevant information from multiple sources and put it all together in a logical argument.

Also, analytical essays are important in academic writing because they help students understand ideas, theories, and concepts that are hard to understand. By breaking a topic down into smaller parts, students can better understand it and figure out what the main ideas and themes are. Analytical essays help students get better at writing by making them present their arguments in a way that is clear, concise, and well-organized. Writing an analytical essay is a skill that students need to learn if they want to do well in school and in their careers. For analytical essays, you need to be able to think critically , do research, and write well, all of which are important for success in many fields. By getting better at these skills, students can become better writers and thinkers, which will help them reach their academic and career goals.

What You'll Learn

Understanding the Basics of Analytical Essays

There are a few main things that set an analytical essay apart from other types of essays. One of the most important things about it is that it requires a thorough look at the subject. An analytical essay isn’t just a description of a topic or a point of view. Instead, it calls for a thorough look at the subject, breaking it down into its different parts and evaluating each one carefully.

Argumentative essays try to convince the reader to agree with a certain point of view. Descriptive essays, on the other hand, try to give a detailed description of a topic. Analytical essays, on the other hand, require the writer to look at the topic objectively and judge it, as well as use evidence from different sources to back up their claims. In an analytical essay, you can’t say enough about how important analysis is. Analysis is the process of breaking down big ideas or thoughts into smaller, more manageable pieces. By analyzing the topic, the writer can find the main ideas, patterns, and connections, which can then be used to back up their arguments.

Also, analysis lets the author draw conclusions that make sense based on the evidence given. If there wasn’t any analysis in an analytical essay, it would just be a list of facts and opinions. Analysis is what gives depth and substance to an analytical essay and lets the writer make a well-reasoned, evidence-based argument.

Analytical essays are different from other types of essays because they focus on analysis and evaluation. They require a thorough look at a subject, breaking it down into its different parts and judging each one objectively. Students can get the skills they need to do well in school and in the workplace by learning what makes an analytical essay unique and what role analysis plays in this type of writing.

Choosing a Topic for Your Analytical Essay

It can be hard to decide what to write about in an analytical essay, but there are several ways to come up with ideas. One way to do this is to make a list of possible topics based on your interests, your schoolwork, or what’s going on in the world right now. You can also find possible topics by reading articles, books, or other materials in your field of study. When choosing a topic for your analytical essay, you should think about a few things. First and foremost, the topic must fit with the needs of the course or assignment. It should also be narrow enough to allow for a detailed analysis while still having enough information for research . Also, the topic should be something you’re interested in as a writer. This will make the writing process more interesting and fun.

To help you get started, here are some examples of potential analytical essay topics:

1. The impact of social media on mental health

2. Analyzing the themes of race and identity in Toni Morrison’s “Beloved”

3. The role of technology in modern education

4. An analysis of the effectiveness of the Affordable Care Act

5. Examining the causes and consequences of income inequality in the United States

6. The portrayal of gender roles in Shakespeare’s plays

7. Analyzing the impact of climate change on global food production

8. A critical analysis of the role of the media in shaping public opinion

9. A comparison of different political ideologies and their impact on society

10. An analysis of the ethical implications of gene editing technology.

A topic for an analytical essay must be chosen with care, taking into account several factors such as relevance, scope, and personal interest. By brainstorming ideas, researching different sources, and applying these criteria, you can choose a topic that is both interesting and informative, allowing you to write a well-researched and well-argued analytical essay.

Conducting Research for Your Analytical Essay

In order to write an analytical essay, you need to do research. It lets the writer gather relevant information, find patterns and relationships, and come to conclusions that make sense. If you didn’t do research for your analytical essay, it wouldn’t have much substance or credibility, and the arguments you made would be weak and not backed up. You can gather information for your analytical essay from a number of different places. There are many examples, such as books, academic journals, online databases, government reports, and reputable news sources. When choosing sources, think about how relevant, reliable, and trustworthy they are. Academic sources like peer-reviewed journals and scholarly books are more reliable and credible than popular sources like blogs and social media posts.

To conduct effective research for your analytical essay, here are some tips to keep in mind:

1. Start early: Give yourself plenty of time to conduct research, as it can be a time-consuming process.

2. Use multiple sources: Gather information from a variety of sources to ensure you have a well-rounded understanding of the topic.

3. Take notes: Keep detailed notes on the information you gather, including the source and page number, to make it easier to cite your sources later.

4. Evaluate your sources: Assess the reliability and credibility of your sources, looking for biases or conflicts of interest that may affect the information presented.

5. Organize your research: Create a system for organizing your research, such as using annotated bibliographies or note-taking apps, to keep track of your sources and ideas.

You can conduct effective research for your analytical essay by following these tips, gathering reliable and credible information that supports your arguments and improves the overall quality of your writing.

Developing a Thesis Statement for Your Analytical Essay

A thesis statement is a short sentence that sums up the main argument or point of an essay. It gives the reader a clear idea of where the author stands on the subject and acts as a road map. In an analytical essay, the thesis statement is very important because it sets the tone for the whole essay and shows the writer how to analyze and evaluate the subject . In an analytical essay, you can’t say enough about how important a strong thesis statement is. A well-written thesis statement states the writer’s main point in a clear and concise way, making it easier for the reader to follow the writer’s thought process and understand the purpose of the essay. A strong thesis statement also helps the writer focus their analysis and evaluation, making sure that each paragraph supports the main point and builds on it.

To develop a strong thesis statement for your analytical essay, here are some tips to consider:

1. Start with a question: Ask yourself a question related to your topic and use the answer to develop your thesis statement.

2. Be specific: Your thesis statement should be specific and focused on the main argument of your essay .

3. Use evidence: Support your thesis statement with evidence from your research, such as quotes or statistics, to give it more credibility and strength.

4. Be original: Your thesis statement should be unique and original, providing a fresh perspective on the topic.

5. Revise as needed: As you write your essay , revisit your thesis statement and revise it if necessary to ensure it remains relevant and accurate.

By following these tips, you can develop a strong thesis statement for your analytical essay, providing a clear and concise statement of your main argument and guiding the reader through your analysis and evaluation of the topic.

Analytical Essay Structure

An analytical essay is made up of an introduction, body paragraphs, and a conclusion, just like a regular essay. The purpose of the introduction is to give background on the topic, introduce the thesis statement, and get the reader interested. Through analysis and evaluation of the topic, the body paragraphs should support the thesis statement with evidence and examples . The conclusion should sum up the main points of the essay and restate the thesis statement in a new way that makes sense.

Here is a more detailed breakdown of the structure of an analytical essay:

1. Introduction: The introduction should set the tone for the essay by providing background information on the subject as well as a clear thesis statement. It should also engage the reader and convey the writer’s point of view on the subject.

2. Body paragraphs: The body of the essay should be divided into several paragraphs, each focusing on a different aspect of the topic. Each paragraph should start with a clear topic sentence that supports the thesis statement and then proceed to an analysis and evaluation of the subject matter, using evidence and examples to support the writer’s argument.

3. Conclusion: The conclusion should summarize the essay’s main points and restate the thesis statement in a new and meaningful way. It should also provide the reader with a sense of closure, leaving them with a clear understanding of the writer’s point of view on the subject.

To organize your ideas in an analytical essay, here are some tips to consider:

1. Create an outline: Before you start writing your essay , create an outline that organizes your ideas and supports your thesis statement. This will help you to organize your thoughts and ensure that each paragraph supports the main argument.

2. Use topic sentences: Each paragraph should begin with a clear topic sentence that supports the thesis statement and provides a roadmap for the reader.

3. Use evidence: Use evidence and examples to support each point you make in your essay , ensuring that each paragraph reinforces the main argument.

4. Use transitions: Use transitional phrases and sentences to connect your ideas and ensure that your essay flows smoothly from one paragraph to the next.

By following these tips, you can organize your ideas effectively in an analytical essay, producing a well-structured and well-supported piece of writing.

Writing the Introduction Paragraph

An analytical essay’s introduction paragraph serves as a road map for the reader, providing background information on the topic, introducing the thesis statement, and engaging the reader. The introduction’s purpose is to set the tone for the essay , grab the reader’s attention, and provide a clear understanding of the writer’s viewpoint on the topic.

To write an engaging introduction paragraph for your analytical essay, here are some tips to consider:

1. Start with a hook: Begin your introduction with a hook that captures the reader’s attention and makes them want to keep reading. This could be a surprising statistic, a provocative question, or a striking image.

2. Provide background information: Provide some context for the topic by providing background information and explaining why it is important.

3. Introduce the thesis statement: Clearly state your thesis statement in the introduction, providing a roadmap for the reader and guiding the rest of your essay.

4. Be concise: Keep your introduction concise and to the point, avoiding unnecessary information or tangents.

5. Revise as needed: As you write your essay, revisit your introduction and revise it as needed to ensure that it remains relevant and engaging.

Here are some examples of effective introduction paragraphs for an analytical essay:

1. “Throughout history, the concept of justice has been a subject of debate and controversy. From the ancient Greeks to modern-day philosophers, the definition and application of justice have been explored in depth. In this essay, I will examine the concept of justice in the context of criminal justice reform and argue that a more restorative approach is needed to address the root causes of crime and reduce recidivism.

2. The rise of social media has had a profound impact on our society, transforming the way we communicate, share information, and interact with the world around us. However, this transformation has not been without its challenges. In this essay, I will explore the impact of social media on mental health and argue that we need to take a more proactive approach to addressing the negative effects of social media on our well-being.

3. “Climate change is one of the most pressing issues of our time, affecting everything from the environment to the economy. Despite the overwhelming scientific evidence, there are still those who deny the reality of climate change or refuse to take action. In this essay, I will analyze the reasons for this denial and argue that we need to take urgent action to address the threat of climate change before it’s too late.”

Writing the Body Paragraphs

An analytical essay’s body paragraphs are where the writer presents their analysis and evaluation of the topic. The purpose of the body paragraphs is to provide evidence and examples to support the thesis statement, while using clear and concise language to make the argument as persuasive as possible.

To write clear and concise body paragraphs for your analytical essay, here are some tips to consider:

1. Use topic sentences: Each body paragraph should begin with a clear topic sentence that supports the thesis statement and provides a roadmap for the reader.

2. Provide evidence: Use evidence and examples to support each point you make in your essay , ensuring that each paragraph reinforces the main argument. The evidence should be relevant, reliable, and credible.

3. Use analysis and evaluation: Analyze and evaluate the evidence you present to demonstrate how it supports your argument. This shows the reader that you have thought deeply about the topic and considered multiple perspectives.

4. Be clear and concise: Use clear and concise language to make your argument as persuasive as possible. Avoid using jargon, complex sentences, or overly technical language that may confuse the reader.

5. Use transitions: Use transitional phrases and sentences to connect your ideas smoothly and ensure that your essay flows from one paragraph to the next.

Here are some examples of effective body paragraphs for an analytical essay:

1. The first reason why a restorative approach to criminal justice reform is necessary is that it addresses the root causes of crime. Rather than simply punishing offenders, restorative justice encourages dialogue between the offender and the victim, allowing both parties to understand the harm that has been caused and work together to find a solution. This approach has been shown to reduce recidivism rates and promote healing within communities.”

2. One of the most significant negative effects of social media on mental health is the increase in anxiety and depression. Studies have shown that social media use is associated with feelings of isolation, comparison, and low self-esteem, all of which contribute to poor mental health . In order to address this issue, we need to take a more proactive approach to promoting mental health and well-being, such as limiting social media use, encouraging face-to-face interactions, and providing mental health resources for those in need.

3. One of the main reasons why climate change denial persists is due to the influence of special interest groups and political ideology . These groups use their resources to spread misinformation and discredit the overwhelming scientific consensus on climate change. In order to combat this, we need to prioritize education and awareness, promote scientific literacy, and hold those who spread misinformation accountable for their actions.”

Writing the Conclusion Paragraph

The conclusion paragraph of an analytical essay summarizes the author’s main points and emphasizes the significance of the thesis statement. The conclusion’s goal is to leave a lasting impression on the reader by bringing the essay to a close and reinforcing the main argument .

To write a strong conclusion for your analytical essay, here are some tips to consider:

1. Restate the thesis statement: The conclusion should restate the thesis statement in a new and meaningful way, emphasizing its importance and relevance to the topic.

2. Summarize the main points: Summarize the main points of the essay, highlighting the evidence and examples that support the thesis statement .

3. Provide a final thought: End the essay with a final thought or reflection on the topic, leaving the reader with something to think about or consider.

4. Be concise: Keep the conclusion concise and to the point, avoiding any new information or arguments.

5. Make it memorable: Use language and phrasing that is memorable and impactful, leaving a lasting impression on the reader.

Here are some examples of effective conclusion paragraphs for an analytical essay:

1. “In conclusion, a restorative approach to criminal justice reform is not only necessary but essential. By addressing the root causes of crime, promoting dialogue and understanding, and fostering healing within communities, we can create a more just and equitable society for all.”

2. In order to address the negative effects of social media on mental health , we need to take a more proactive approach to promoting well-being. By limiting social media use, encouraging face-to-face interactions, and providing mental health resources, we can create a healthier and more connected society.

3. “Climate change is one of the most pressing issues of our time, and we must take urgent action to address it. By promoting education and awareness, prioritizing scientific literacy, and holding those who spread misinformation accountable, we can create a more sustainable and equitable future for generations to come.”

Editing and Proofreading Your Analytical Essay

The editing and proofreading stages of the essay writing process are critical. It ensures that the essay is clear, concise, and error-free, improving overall writing quality and the credibility of the arguments presented.

To edit and proofread your analytical essay, here are some tips to consider:

1. Take a break: After completing your essay, take a break before editing and proofreading it. This will allow you to approach the essay with fresh eyes and a clear mind.

2. Read it out loud: Reading the essay out loud can help you to identify awkward phrasing, grammatical errors, and other issues.

3. Be consistent: Ensure that you are consistent in your use of language, formatting, and citation styles throughout the essay.

4. Use a checklist: Use a checklist to ensure that you have addressed all the necessary components of the essay, such as the thesis statement, evidence, and analysis.

5. Get feedback: Ask a friend or colleague to read your essay and provide feedback on areas that need improvement.

Common mistakes to avoid in analytical essays include:

1. Using overly complicated language or jargon that may confuse the reader.

2. Failing to provide evidence or examples to support the thesis statement.

3. Neglecting to address counterarguments or alternative perspectives on the topic.

4. Making unsupported claims or presenting opinions as facts.

5. Failing to proofread and edit the essay thoroughly, leading to grammatical errors and typos.

By following these tips and avoiding common mistakes, you can edit and proofread your analytical essay effectively, producing a well-written and error-free piece of writing.

Analytical Essay Examples

Here are some examples of successful analytical essays:

1. “The Symbolism of the Green Light in The Great Gatsby” by F. Scott Fitzgerald: This essay analyzes the symbolism of the green light in the novel, arguing that it represents Gatsby’s hopes and dreams and his ultimate failure to achieve them.

2. “The Rhetoric of Malcolm X” by Malcolm X: In this essay, Malcolm X analyzes his own rhetorical strategies, explaining how he uses language and persuasion to achieve his goals.

3. “The Role of Women in Shakespeare’s Macbeth” by William Shakespeare: This essay analyzes the role of women in the play, arguing that they are often marginalized and oppressed by the male characters.

Each of these essays follows a clear and well-structured format, with a strong thesis statement, supporting evidence, and a clear conclusion. The writers use analysis and evaluation to present their arguments, using evidence and examples to support their claims.

When using analytical essay examples to improve your writing, here are some tips to consider:

1. Choose examples that are relevant to your topic or subject matter.

2. Read the example essays carefully, paying attention to the structure, language, and evidence used.

3. Identify the thesis statement and main arguments of the essay.

4. Analyze the evidence used to support the arguments, evaluating its relevance and credibility.

5. Consider how the writer uses language and rhetoric to persuade the reader.

6. Use The examples as a guide for structuring your own essay, but be sure to use your own unique ideas and perspective.

7. Practice writing your own analytical essays and seek feedback from others to improve your writing skills.

8. Avoid copying or plagiarizing the example essays, as this can lead to serious academic consequences.

Frequently Asked Questions

1. what is an analytical essay.

An analytical essay is a type of academic writing that requires the writer to analyze and evaluate a specific topic or subject matter. The writer presents an argument or thesis statement and supports it with evidence and examples, using analysis and evaluation to persuade the reader.

2. What are the main characteristics of an analytical essay?

The main characteristics of an analytical essay include:

– A clear and concise thesis statement that presents the writer’s argument

– Use of evidence and examples to support the argument

– Analysis and evaluation of the evidence presented

– Clear and logical structure, with well-developed paragraphs and transitions between them

– Use of formal and academic language

– Objective and impartial tone

3. What are some tips for writing an analytical essay?

Here are some tips for writing an analytical essay:

– Choose a topic that interests you and that you can analyze in depth

– Develop a clear and concise thesis statement that presents your argument

– Use evidence and examples to support your argument, ensuring that they are relevant, reliable, and credible

– Analyze and evaluate the evidence presented, demonstrating your critical thinking skills

– Use a clear and logical structure, with well-developed paragraphs and transitions between them

– Use formal and academic language, avoiding slang and colloquialisms

– Edit and proofread your essay thoroughly, ensuring that it is error-free and well-written.

To summarize, writing an effective analytical essay necessitates a thorough understanding of the subject matter, a well-developed thesis statement, strong evidence and analysis, and a clear and logical structure. To summarize the main points covered in this guide:

– The writer of an analytical essay must analyze and evaluate a specific topic or subject matter.

– A clear thesis statement, evidence and examples to support the argument, analysis and evaluation of the evidence, a clear and logical structure, formal and academic language, and an objective tone are the main characteristics of an analytical essay.

– Choosing a relevant topic, developing a clear thesis statement, using credible evidence and examples, analyzing and evaluating the evidence, using a clear and logical structure, using formal and academic language, and thoroughly editing and proofreading the essay are all tips for writing a successful analytical essay.

An effective analytical essay demonstrates critical thinking skills as well as the ability to analyze and evaluate complex issues. It is an important skill for both academic and professional success.

Finally, practicing writing on a regular basis, seeking feedback from others, and reading and analyzing examples of successful analytical essays can all help you improve your own writing skills.

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The Power of Analysis: Tips and Tricks for Writing Analysis Essays: Home

Crystal sosa.

Helpful Links

• Super Search Webpage Where to start your research.
• Scribbr Textual analysis guide.
• Analyzing Texts Prezi Presentation A prezi presentation on analyzing texts.
• Writing Essays Guide A guide to writing essays/
• Why is it important?
• Explanation & Example
• Different Types of Analysis Essays

Text analysis and writing analysis texts are important skills to develop as they allow individuals to critically engage with written material, understand underlying themes and arguments, and communicate their own ideas in a clear and effective manner. These skills are essential in academic and professional settings, as well as in everyday life, as they enable individuals to evaluate information and make informed decisions.

What is Text Analysis?

Text analysis is the process of examining and interpreting a written or spoken text to understand its meaning, structure, and context. It involves breaking down the text into its constituent parts, such as words, phrases, and sentences, and analyzing how they work together to convey a particular message or idea.

Text analysis can be used to explore a wide range of textual material, including literature, poetry, speeches, and news articles, and it is often employed in academic research, literary criticism, and media analysis. By analyzing texts, we can gain deeper insights into their meanings, uncover hidden messages and themes, and better understand the social and cultural contexts in which they were produced.

What is an Analysis Essay?

An analysis essay is a type of essay that requires the writer to analyze and interpret a particular text or topic. The goal of an analysis essay is to break down the text or topic into smaller parts and examine each part carefully. This allows the writer to make connections between different parts of the text or topic and develop a more comprehensive understanding of it.

In “The Yellow Wallpaper,” Charlotte Perkins Gilman uses the first-person point of view and vivid descriptions of the protagonist’s surroundings to convey the protagonist’s psychological deterioration. By limiting the reader’s understanding of the story’s events to the protagonist’s perspective, Gilman creates a sense of claustrophobia and paranoia, mirroring the protagonist’s own feelings. Additionally, the use of sensory language, such as the “smooch of rain,” and descriptions of the “yellow wallpaper” and its “sprawling flamboyant patterns,” further emphasize the protagonist’s sensory and emotional experience. Through these techniques, Gilman effectively communicates the protagonist’s descent into madness and the effects of societal oppression on women’s mental health.

There are several different types of analysis essays, including:

Literary Analysis Essays: These essays examine a work of literature and analyze various literary devices such as character development, plot, theme, and symbolism.

Rhetorical Analysis Essays: These essays examine how authors use language and rhetoric to persuade their audience, focusing on the author's tone, word choice, and use of rhetorical devices.

Film Analysis Essays: These essays analyze a film's themes, characters, and visual elements, such as cinematography and sound.

Visual Analysis Essays: These essays analyze visual art, such as paintings or sculptures, and explore how the artwork's elements work together to create meaning.

Historical Analysis Essays: These essays analyze historical events or documents and examine their causes, effects, and implications.

Comparative Analysis Essays: These essays compare and contrast two or more works, focusing on similarities and differences between them.

Process Analysis Essays: These essays explain how to do something or how something works, providing a step-by-step analysis of a process.

Analyzing Texts

• General Tips
• How to Analyze
• What to Analyze

When writing an essay, it's essential to analyze your topic thoroughly. Here are some suggestions for analyzing your topic:

Read carefully: Start by reading your text or prompt carefully. Make sure you understand the key points and what the text or prompt is asking you to do.

Analyze the text or topic thoroughly: Analyze the text or topic thoroughly by breaking it down into smaller parts and examining each part carefully. This will help you make connections between different parts of the text or topic and develop a more comprehensive understanding of it.

Identify key concepts: Identify the key concepts, themes, and ideas in the text or prompt. This will help you focus your analysis.

Take notes: Take notes on important details and concepts as you read. This will help you remember what you've read and organize your thoughts.

Consider different perspectives: Consider different perspectives and interpretations of the text or prompt. This can help you create a more well-rounded analysis.

Use evidence: Use evidence from the text or outside sources to support your analysis. This can help you make your argument stronger and more convincing.

Formulate your thesis statement: Based on your analysis of the essay, formulate your thesis statement. This should be a clear and concise statement that summarizes your main argument.

Use clear and concise language: Use clear and concise language to communicate your ideas effectively. Avoid using overly complicated language that may confuse your reader.

Revise and edit: Revise and edit your essay carefully to ensure that it is clear, concise, and free of errors.

• Understanding the assignment: Make sure you fully understand the assignment and the purpose of the analysis. This will help you focus your analysis and ensure that you are meeting the requirements of the assignment.

Read the essay multiple times: Reading the essay multiple times will help you to identify the author's main argument, key points, and supporting evidence.

Take notes: As you read the essay, take notes on key points, quotes, and examples. This will help you to organize your thoughts and identify patterns in the author's argument.

Take breaks: It's important to take breaks while reading academic essays to avoid burnout. Take a break every 20-30 minutes and do something completely different, like going for a walk or listening to music. This can help you to stay refreshed and engaged.

Highlight or underline key points: As you read, highlight or underline key points, arguments, and evidence that stand out to you. This will help you to remember and analyze important information later.

Ask questions: Ask yourself questions as you read to help you engage critically with the text. What is the author's argument? What evidence do they use to support their claims? What are the strengths and weaknesses of their argument?

Engage in active reading: Instead of passively reading, engage in active reading by asking questions, making connections to other readings or personal experiences, and reflecting on what you've read.

Find a discussion partner: Find someone to discuss the essay with, whether it's a classmate, a friend, or a teacher. Discussing the essay can help you to process and analyze the information more deeply, and can also help you to stay engaged.

• Identify the author's purpose and audience: Consider why the author wrote the essay and who their intended audience is. This will help you to better understand the author's perspective and the purpose of their argument.

Analyze the structure of the essay: Consider how the essay is structured and how this supports the author's argument. Look for patterns in the organization of ideas and the use of transitions.

Evaluate the author's use of evidence: Evaluate the author's use of evidence and how it supports their argument. Consider whether the evidence is credible, relevant, and sufficient to support the author's claims.

Consider the author's tone and style: Consider the author's tone and style and how it contributes to their argument. Look for patterns in the use of language, imagery, and rhetorical devices.

Consider the context : Consider the context in which the essay was written, such as the author's background, the time period, and any societal or cultural factors that may have influenced their perspective.

Evaluate the evidence: Evaluate the evidence presented in the essay and consider whether it is sufficient to support the author's argument. Look for any biases or assumptions that may be present in the evidence.

Consider alternative viewpoints: Consider alternative viewpoints and arguments that may challenge the author's perspective. This can help you to engage critically with the text and develop a more well-rounded understanding of the topic.

• Last Updated: Jun 21, 2023 1:01 PM
• URL: https://odessa.libguides.com/analysis

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5 Steps to Write a Great Analytical Essay

General Education

Do you need to write an analytical essay for school? What sets this kind of essay apart from other types, and what must you include when you write your own analytical essay? In this guide, we break down the process of writing an analytical essay by explaining the key factors your essay needs to have, providing you with an outline to help you structure your essay, and analyzing a complete analytical essay example so you can see what a finished essay looks like.

What Is an Analytical Essay?

Before you begin writing an analytical essay, you must know what this type of essay is and what it includes. Analytical essays analyze something, often (but not always) a piece of writing or a film.

An analytical essay is more than just a synopsis of the issue though; in this type of essay you need to go beyond surface-level analysis and look at what the key arguments/points of this issue are and why. If you’re writing an analytical essay about a piece of writing, you’ll look into how the text was written and why the author chose to write it that way. Instead of summarizing, an analytical essay typically takes a narrower focus and looks at areas such as major themes in the work, how the author constructed and supported their argument, how the essay used literary devices to enhance its messages, etc.

While you certainly want people to agree with what you’ve written, unlike with persuasive and argumentative essays, your main purpose when writing an analytical essay isn’t to try to convert readers to your side of the issue. Therefore, you won’t be using strong persuasive language like you would in those essay types. Rather, your goal is to have enough analysis and examples that the strength of your argument is clear to readers.

Besides typical essay components like an introduction and conclusion, a good analytical essay will include:

• A thesis that states your main argument
• Analysis that relates back to your thesis and supports it
• Examples to support your analysis and allow a more in-depth look at the issue

In the rest of this article, we’ll explain how to include each of these in your analytical essay.

How to Structure Your Analytical Essay

Analytical essays are structured similarly to many other essays you’ve written, with an introduction (including a thesis), several body paragraphs, and a conclusion. Below is an outline you can follow when structuring your essay, and in the next section we go into more detail on how to write an analytical essay.

Introduction

Your introduction will begin with some sort of attention-grabbing sentence to get your audience interested, then you’ll give a few sentences setting up the topic so that readers have some context, and you’ll end with your thesis statement. Your introduction will include:

• Brief background information explaining the issue/text
• Your thesis

Body Paragraphs

Your analytical essay will typically have three or four body paragraphs, each covering a different point of analysis. Begin each body paragraph with a sentence that sets up the main point you’ll be discussing. Then you’ll give some analysis on that point, backing it up with evidence to support your claim. Continue analyzing and giving evidence for your analysis until you’re out of strong points for the topic. At the end of each body paragraph, you may choose to have a transition sentence that sets up what the next paragraph will be about, but this isn’t required. Body paragraphs will include:

• Introductory sentence explaining what you’ll cover in the paragraph (sort of like a mini-thesis)
• Analysis point
• Evidence (either passages from the text or data/facts) that supports the analysis
• (Repeat analysis and evidence until you run out of examples)

You won’t be making any new points in your conclusion; at this point you’re just reiterating key points you’ve already made and wrapping things up. Begin by rephrasing your thesis and summarizing the main points you made in the essay. Someone who reads just your conclusion should be able to come away with a basic idea of what your essay was about and how it was structured. After this, you may choose to make some final concluding thoughts, potentially by connecting your essay topic to larger issues to show why it’s important. A conclusion will include:

• Paraphrase of thesis
• Summary of key points of analysis
• Final concluding thought(s)

5 Steps for Writing an Analytical Essay

Follow these five tips to break down writing an analytical essay into manageable steps. By the end, you’ll have a fully-crafted analytical essay with both in-depth analysis and enough evidence to support your argument. All of these steps use the completed analytical essay in the next section as an example.

#1: Pick a Topic

You may have already had a topic assigned to you, and if that’s the case, you can skip this step. However, if you haven’t, or if the topic you’ve been assigned is broad enough that you still need to narrow it down, then you’ll need to decide on a topic for yourself. Choosing the right topic can mean the difference between an analytical essay that’s easy to research (and gets you a good grade) and one that takes hours just to find a few decent points to analyze

Before you decide on an analytical essay topic, do a bit of research to make sure you have enough examples to support your analysis. If you choose a topic that’s too narrow, you’ll struggle to find enough to write about.

For example, say your teacher assigns you to write an analytical essay about the theme in John Steinbeck’s The Grapes of Wrath of exposing injustices against migrants. For it to be an analytical essay, you can’t just recount the injustices characters in the book faced; that’s only a summary and doesn’t include analysis. You need to choose a topic that allows you to analyze the theme. One of the best ways to explore a theme is to analyze how the author made his/her argument. One example here is that Steinbeck used literary devices in the intercalary chapters (short chapters that didn’t relate to the plot or contain the main characters of the book) to show what life was like for migrants as a whole during the Dust Bowl.

You could write about how Steinbeck used literary devices throughout the whole book, but, in the essay below, I chose to just focus on the intercalary chapters since they gave me enough examples. Having a narrower focus will nearly always result in a tighter and more convincing essay (and can make compiling examples less overwhelming).

#2: Write a Thesis Statement

Your thesis statement is the most important sentence of your essay; a reader should be able to read just your thesis and understand what the entire essay is about and what you’ll be analyzing. When you begin writing, remember that each sentence in your analytical essay should relate back to your thesis

In the analytical essay example below, the thesis is the final sentence of the first paragraph (the traditional spot for it). The thesis is: “In The Grapes of Wrath’s intercalary chapters, John Steinbeck employs a variety of literary devices and stylistic choices to better expose the injustices committed against migrants in the 1930s.” So what will this essay analyze? How Steinbeck used literary devices in the intercalary chapters to show how rough migrants could have it. Crystal clear.

#3: Do Research to Find Your Main Points

This is where you determine the bulk of your analysis--the information that makes your essay an analytical essay. My preferred method is to list every idea that I can think of, then research each of those and use the three or four strongest ones for your essay. Weaker points may be those that don’t relate back to the thesis, that you don’t have much analysis to discuss, or that you can’t find good examples for. A good rule of thumb is to have one body paragraph per main point

This essay has four main points, each of which analyzes a different literary device Steinbeck uses to better illustrate how difficult life was for migrants during the Dust Bowl. The four literary devices and their impact on the book are:

• Lack of individual names in intercalary chapters to illustrate the scope of the problem
• Parallels to the Bible to induce sympathy for the migrants
• Non-showy, often grammatically-incorrect language so the migrants are more realistic and relatable to readers
• Nature-related metaphors to affect the mood of the writing and reflect the plight of the migrants

#4: Find Excerpts or Evidence to Support Your Analysis

Now that you have your main points, you need to back them up. If you’re writing a paper about a text or film, use passages/clips from it as your main source of evidence. If you’re writing about something else, your evidence can come from a variety of sources, such as surveys, experiments, quotes from knowledgeable sources etc. Any evidence that would work for a regular research paper works here.

In this example, I quoted multiple passages from The Grapes of Wrath  in each paragraph to support my argument. You should be able to back up every claim you make with evidence in order to have a strong essay.

#5: Put It All Together

Now it's time to begin writing your essay, if you haven’t already. Create an introductory paragraph that ends with the thesis, make a body paragraph for each of your main points, including both analysis and evidence to back up your claims, and wrap it all up with a conclusion that recaps your thesis and main points and potentially explains the big picture importance of the topic.

Analytical Essay Example + Analysis

So that you can see for yourself what a completed analytical essay looks like, here’s an essay I wrote back in my high school days. It’s followed by analysis of how I structured my essay, what its strengths are, and how it could be improved.

One way Steinbeck illustrates the connections all migrant people possessed and the struggles they faced is by refraining from using specific titles and names in his intercalary chapters. While The Grapes of Wrath focuses on the Joad family, the intercalary chapters show that all migrants share the same struggles and triumphs as the Joads. No individual names are used in these chapters; instead the people are referred to as part of a group. Steinbeck writes, “Frantic men pounded on the doors of the doctors; and the doctors were busy.  And sad men left word at country stores for the coroner to send a car,” (555). By using generic terms, Steinbeck shows how the migrants are all linked because they have gone through the same experiences. The grievances committed against one family were committed against thousands of other families; the abuse extends far beyond what the Joads experienced. The Grapes of Wrath frequently refers to the importance of coming together; how, when people connect with others their power and influence multiplies immensely. Throughout the novel, the goal of the migrants, the key to their triumph, has been to unite. While their plans are repeatedly frustrated by the government and police, Steinbeck’s intercalary chapters provide a way for the migrants to relate to one another because they have encountered the same experiences. Hundreds of thousands of migrants fled to the promised land of California, but Steinbeck was aware that numbers alone were impersonal and lacked the passion he desired to spread. Steinbeck created the intercalary chapters to show the massive numbers of people suffering, and he created the Joad family to evoke compassion from readers.  Because readers come to sympathize with the Joads, they become more sensitive to the struggles of migrants in general. However, John Steinbeck frequently made clear that the Joads were not an isolated incident; they were not unique. Their struggles and triumphs were part of something greater. Refraining from specific names in his intercalary chapters allows Steinbeck to show the vastness of the atrocities committed against migrants.

Steinbeck also creates significant parallels to the Bible in his intercalary chapters in order to enhance his writing and characters. By using simple sentences and stylized writing, Steinbeck evokes Biblical passages. The migrants despair, “No work till spring. No work,” (556).  Short, direct sentences help to better convey the desperateness of the migrants’ situation. Throughout his novel, John Steinbeck makes connections to the Bible through his characters and storyline. Jim Casy’s allusions to Christ and the cycle of drought and flooding are clear biblical references.  By choosing to relate The Grapes of Wrath to the Bible, Steinbeck’s characters become greater than themselves. Starving migrants become more than destitute vagrants; they are now the chosen people escaping to the promised land. When a forgotten man dies alone and unnoticed, it becomes a tragedy. Steinbeck writes, “If [the migrants] were shot at, they did not run, but splashed sullenly away; and if they were hit, they sank tiredly in the mud,” (556). Injustices committed against the migrants become greater because they are seen as children of God through Steinbeck’s choice of language. Referencing the Bible strengthens Steinbeck’s novel and purpose: to create understanding for the dispossessed.  It is easy for people to feel disdain for shabby vagabonds, but connecting them to such a fundamental aspect of Christianity induces sympathy from readers who might have otherwise disregarded the migrants as so many other people did.

The simple, uneducated dialogue Steinbeck employs also helps to create a more honest and meaningful representation of the migrants, and it makes the migrants more relatable to readers. Steinbeck chooses to accurately represent the language of the migrants in order to more clearly illustrate their lives and make them seem more like real paper than just characters in a book. The migrants lament, “They ain’t gonna be no kinda work for three months,” (555). There are multiple grammatical errors in that single sentence, but it vividly conveys the despair the migrants felt better than a technically perfect sentence would. The Grapes of Wrath is intended to show the severe difficulties facing the migrants so Steinbeck employs a clear, pragmatic style of writing.  Steinbeck shows the harsh, truthful realities of the migrants’ lives and he would be hypocritical if he chose to give the migrants a more refined voice and not portray them with all their shortcomings. The depiction of the migrants as imperfect through their language also makes them easier to relate to. Steinbeck’s primary audience was the middle class, the less affluent of society. Repeatedly in The Grapes of Wrath , the wealthy make it obvious that they scorn the plight of the migrants. The wealthy, not bad luck or natural disasters, were the prominent cause of the suffering of migrant families such as the Joads. Thus, Steinbeck turns to the less prosperous for support in his novel. When referring to the superior living conditions barnyard animals have, the migrants remark, “Them’s horses-we’re men,” (556).  The perfect simplicity of this quote expresses the absurdness of the migrants’ situation better than any flowery expression could.

In The Grapes of Wrath , John Steinbeck uses metaphors, particularly about nature, in order to illustrate the mood and the overall plight of migrants. Throughout most of the book, the land is described as dusty, barren, and dead. Towards the end, however; floods come and the landscape begins to change. At the end of chapter twenty-nine, Steinbeck describes a hill after the floods saying, “Tiny points of grass came through the earth, and in a few days the hills were pale green with the beginning year,” (556). This description offers a stark contrast from the earlier passages which were filled with despair and destruction. Steinbeck’s tone from the beginning of the chapter changes drastically. Early in the chapter, Steinbeck had used heavy imagery in order to convey the destruction caused by the rain, “The streams and the little rivers edged up to the bank sides and worked at willows and tree roots, bent the willows deep in the current, cut out the roots of cottonwoods and brought down the trees,” (553). However, at the end of the chapter the rain has caused new life to grow in California. The new grass becomes a metaphor representing hope. When the migrants are at a loss over how they will survive the winter, the grass offers reassurance. The story of the migrants in the intercalary chapters parallels that of the Joads. At the end of the novel, the family is breaking apart and has been forced to flee their home. However, both the book and final intercalary chapter end on a hopeful note after so much suffering has occurred. The grass metaphor strengthens Steinbeck’s message because it offers a tangible example of hope. Through his language Steinbeck’s themes become apparent at the end of the novel. Steinbeck affirms that persistence, even when problems appear insurmountable, leads to success. These metaphors help to strengthen Steinbeck’s themes in The Grapes of Wrath because they provide a more memorable way to recall important messages.

John Steinbeck’s language choices help to intensify his writing in his intercalary chapters and allow him to more clearly show how difficult life for migrants could be. Refraining from using specific names and terms allows Steinbeck to show that many thousands of migrants suffered through the same wrongs. Imitating the style of the Bible strengthens Steinbeck’s characters and connects them to the Bible, perhaps the most famous book in history. When Steinbeck writes in the imperfect dialogue of the migrants, he creates a more accurate portrayal and makes the migrants easier to relate to for a less affluent audience. Metaphors, particularly relating to nature, strengthen the themes in The Grapes of Wrath by enhancing the mood Steinbeck wants readers to feel at different points in the book. Overall, the intercalary chapters that Steinbeck includes improve his novel by making it more memorable and reinforcing the themes Steinbeck embraces throughout the novel. Exemplary stylistic devices further persuade readers of John Steinbeck’s personal beliefs. Steinbeck wrote The Grapes of Wrath to bring to light cruelties against migrants, and by using literary devices effectively, he continuously reminds readers of his purpose. Steinbeck’s impressive language choices in his intercalary chapters advance the entire novel and help to create a classic work of literature that people still are able to relate to today. 

This essay sticks pretty closely to the standard analytical essay outline. It starts with an introduction, where I chose to use a quote to start off the essay. (This became my favorite way to start essays in high school because, if I wasn’t sure what to say, I could outsource the work and find a quote that related to what I’d be writing about.) The quote in this essay doesn’t relate to the themes I’m discussing quite as much as it could, but it’s still a slightly different way to start an essay and can intrigue readers. I then give a bit of background on The Grapes of Wrath and its themes before ending the intro paragraph with my thesis: that Steinbeck used literary devices in intercalary chapters to show how rough migrants had it.

Each of my four body paragraphs is formatted in roughly the same way: an intro sentence that explains what I’ll be discussing, analysis of that main point, and at least two quotes from the book as evidence.

My conclusion restates my thesis, summarizes each of four points I discussed in my body paragraphs, and ends the essay by briefly discussing how Steinbeck’s writing helped introduce a world of readers to the injustices migrants experienced during the dust bowl.

What does this analytical essay example do well? For starters, it contains everything that a strong analytical essay should, and it makes that easy to find. The thesis clearly lays out what the essay will be about, the first sentence of each of the body paragraph introduces the topic it’ll cover, and the conclusion neatly recaps all the main points. Within each of the body paragraphs, there’s analysis along with multiple excerpts from the book in order to add legitimacy to my points.

Additionally, the essay does a good job of taking an in-depth look at the issue introduced in the thesis. Four ways Steinbeck used literary devices are discussed, and for each of the examples are given and analysis is provided so readers can understand why Steinbeck included those devices and how they helped shaped how readers viewed migrants and their plight.

Where could this essay be improved? I believe the weakest body paragraph is the third one, the one that discusses how Steinbeck used plain, grammatically incorrect language to both accurately depict the migrants and make them more relatable to readers. The paragraph tries to touch on both of those reasons and ends up being somewhat unfocused as a result. It would have been better for it to focus on just one of those reasons (likely how it made the migrants more relatable) in order to be clearer and more effective. It’s a good example of how adding more ideas to an essay often doesn’t make it better if they don’t work with the rest of what you’re writing. This essay also could explain the excerpts that are included more and how they relate to the points being made. Sometimes they’re just dropped in the essay with the expectation that the readers will make the connection between the example and the analysis. This is perhaps especially true in the second body paragraph, the one that discusses similarities to Biblical passages. Additional analysis of the quotes would have strengthened it.

Summary: How to Write an Analytical Essay

What is an analytical essay? A critical analytical essay analyzes a topic, often a text or film. The analysis paper uses evidence to support the argument, such as excerpts from the piece of writing. All analytical papers include a thesis, analysis of the topic, and evidence to support that analysis.

When developing an analytical essay outline and writing your essay, follow these five steps:

Reading analytical essay examples can also give you a better sense of how to structure your essay and what to include in it.

What's Next?

Learning about different writing styles in school? There are four main writing styles, and it's important to understand each of them. Learn about them in our guide to writing styles , complete with examples.

Writing a research paper for school but not sure what to write about? Our guide to research paper topics has over 100 topics in ten categories so you can be sure to find the perfect topic for you.

Literary devices can both be used to enhance your writing and communication. Check out this list of 31 literary devices to learn more !

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Christine graduated from Michigan State University with degrees in Environmental Biology and Geography and received her Master's from Duke University. In high school she scored in the 99th percentile on the SAT and was named a National Merit Finalist. She has taught English and biology in several countries.

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How to write a good analytical essay

Published September 27, 2020. Updated June 7, 2022.

Analytical Essay Definition

An analytical essay is an essay that provides an analysis of a work or issue.

Overview of Analytical Essay Writing

An analytical essay is used to analyze just about anything. Mostly, students will be asked to analyze a piece of writing, a film, or a specific issue. Topics for analytical essays should be neither too broad nor too narrow and should have enough sources to support the analysis. An analytical essay should be structured with an outline that consists of an introduction, body, and conclusion. Enough time should be spent writing, rewriting, and revising thoroughly. Line editing, spell-checking, and proofreading should be done carefully, and the sentence flow should be checked to create the final analytical essay.

Worried about your writing? Submit your paper for a Chegg Writing essay check , or for an Expert Check proofreading . Both can help you find and fix potential writing issues.

What is an analytical essay?

So, first things first: what is an analytical essay? Analytical essays are just what they sound like — they are, simply put, an analysis. To analyze is to “study or determine the nature and relationship of the parts of something” (Merriam-Webster). An analytical essay can analyze just about anything, but most often, students are asked to analyze a piece of writing, a film, or a specific issue.

It’s important to note the difference between argumentative and analytical essays. Though they are similar, there is a distinction: argumentative papers aim to prove a point through a well-researched, persuasive argument, while analytical papers posit a question and explore possible answers.

Some analytical essays will set out to prove a point, which can make them easily confused with argumentative essays. Remember, the main goal of argumentative essays is to argue a point. The main objective of analytical essays is to analyze a work or idea. Often, a firm stance will be used as a vehicle to create a more structured analysis. But, it’s not the point of the essay.

How to prepare to write an analytical essay

Before you dive into brainstorming topics for your analytical essay, be sure to read and reread the rubric for the assignment. Depending on your field of study, the guidelines will vary. For instance, psychology, education, and the sciences tend to use APA format, while the humanities, languages, and the fine arts tend to use MLA or Chicago style.

Once you know which format to use, take heed of any specific expectations your instructor has for this assignment. For example:

• When is it due?
• What is the expected page count?
• Will your instructor expect to see an outline before the draft?
• Is there a set topic list, or can you choose your own?
• Is there someplace to look at sample analytical essays that got A’s?

If anything is unclear, don’t hesitate to ask your instructor.

How to brainstorm the perfect topic for your analytical essay

Some instructors will offer their students a set of essay topics to choose from. That makes it easy for you — just pick the topic that intrigues you the most! Since your instructor has approved all the topics, you shouldn’t have to worry about any of them being too “broad” or “narrow.”

On the other hand, many instructors expect students to brainstorm their own topics. In this case, you will need to ensure your topic is relevant and not too broad or narrow.

After you think of a topic that interests you and is neither too broad nor too narrow, make sure you can find an adequate number of reputable sources to substantiate your analysis. You’ll need to evaluate all your sources’ credibility and probably include a few peer-reviewed journal articles (tip: use a database).

Many good sources can be found online or at your school’s library (in-person and online). If you’re having trouble finding useful sources, it may be a warning sign that your idea is too broad or narrow. If you’re stuck finding sources at all, ask your librarian for help.

How to structure an analytical essay

Now that you’ve found a good topic, it’s time to get organized! Even if you prefer to write spontaneously, creating an outline (even a loose one) can help you stay on track while you draft. The traditional outline for an analytical essay looks like the following:

Introduction

• main point #1
• main point #2
• main point #3

Works cited

Let’s examine each section.

No good analytical essay is complete without a super-strong introductory paragraph. It’s like the title screen at the beginning of a movie. Without it, you’d have no idea what the movie’s about!

A good introduction should state:

• the topic of your essay
• your thesis statement (the one- or two-line gist of your paper)
• the question or idea you’ll analyze
• your research methodology

The body of your essay is not limited to three points, as shown above, but three is typically considered the minimum for a good analysis. To make your analysis more compelling, present your points and arguments in a “strong, stronger, strongest” format.

• strong supporting evidence #1
• stronger supporting evidence #2
• strongest supporting evidence #3

Many students struggle with writing conclusions for their essays. It can feel unnecessary to restate what’s already been said, right? But really, a strong conclusion does much more than repeat what’s already been said. Your conclusion should:

• restate your thesis statement
• hit on all the main points of the essay
• explore the implications of the main points

Works cited 

A works cited or bibliography page should be the final section of your paper. A works-cited page includes a list of the resources you quoted or cited within the body of your work. A bibliography includes these, plus any resources you consulted and didn’t refer to in the paper, or any resources that influenced your ideas on the topic. Check your assignment to see which of these two pages you will need to have.

How to write an analytical essay

It might not seem like it when you’re staring at that blank document and flashing cursor, but this is the easy part! If you’ve adequately researched and planned your analysis, the writing process will flow much more quickly.

Remember, it’s usually not possible to write an essay in one sitting. Don’t wait until the last minute to get started! You’ll need to factor in time for breaks, meetings with your writing tutor, and the dreaded writer’s block.

Don’t expect your first draft to be perfect. It is normal (and smart) to write multiple drafts. You may even need to change your main argument halfway through your draft. That’s okay! Be ready to re-brainstorm, re-outline, and rewrite.

How to revise an analytical essay

Revision may just be the most crucial step of the essay-writing process. Even if you brainstorm the perfect topic, create a brilliant outline, and write a strong first draft, none of that brilliance will shine through if your paper is full of typos, grammar errors, and rambling tangents.

You’ll want to complete these steps of editing, in this order:

• line editing
• spell-checking
• proofreading

Revision deals with broad issues, such as an argument that doesn’t make sense or a source that doesn’t support your thesis. Line editing, spell-checking, and proofreading have more to do with your writing, the flow of your sentences, and any spelling or grammatical errors.

After you finish, it also doesn’t hurt to check your paper for plagiarism !

Example analytical essay on folklore and current events

Before you turn in that paper, don’t forget to cite your sources in APA format , MLA format , or a style of your choice.

Key takeaways

• An analytical essay is, simply put, an analysis of a work or issue.
• Be sure to understand your instructor’s expectations before you dive into writing an analytical essay.
• Topics for analytical essays need to be neither too broad nor too narrow and should have enough sources to support your analysis.
• The basic outline for an analytical essay consists of an introduction, body, conclusion, and works cited (or bibliography).
• Leaving yourself enough time to write, rewrite, and revise thoroughly is a vital part of writing an analytical essay that earns an A.

“Analyze.” Merriam-Webster.com Dictionary , Merriam-Webster, www.merriam-webster.com/dictionary/analyze.

By Jolee McManus. Jolee earned a BA in English from the University of Georgia. She has several years of experience as a writing tutor and freelance copywriter and editor.

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The Ultimate Guide to Analytical Essay Writing: How to Craft an A-Grade Paper?

25 January, 2021

17 minutes read

Author:  Kate Smith

An analytical essay is often considered the most challenging piece of writing. However, those who have dealt with it at least once are a step closer to calling themselves masters of essay writing. This type of paper requires plenty of analytical skills to carry out an in-depth analysis of the assigned topic. Yet, the main goal of an analytical essay is not only to demonstrate your ability to learn the basics of the theme.

You also need to think critically, analyze facts, express your standpoint, and clearly show a deep understanding of key concepts. In short, your main task as an author is to prove the validity of your views by coming up with strong arguments that do not beg any questions.

The given guide provides a full analytical essay definition, as well as specifies its features and structural aspects. The following information will help you properly start your paper, choose a relevant topic, and come up with compelling conclusions. 

What is an Analytical Essay?

An analytical essay is a piece of writing aimed to provide a thorough analysis of a definite phenomenon using persuasive arguments and supporting assertions. Analysis in the analytical essay writing process stands for a method of research that allows one to study specific features of an object. Analytical papers also have to do with analysis of a specific problem; that is consideration of the problem itself and identification of its key patterns. The subject matter of analysis can be a well-known or little-studied scientific phenomenon, artistic work, historical event, social problem, etc.

The content of an analytical essay will totally depend on the object that has been chosen for analysis. Thus, when shedding light on any kind of scientific work, an analytical essay can be devoted to the analysis of research credibility, its relevance, or the adequacy of conclusions. When considering a work of art, an essay writer can focus on the analysis of the author’s artistic techniques or issues raised in the book. For this reason, it is essential to accurately determine the topic and subject matter of your future analytical essay.

Steps to Take Before Writing

The preparational stage of analytical essay writing cannot be omitted. It lays the basis for the A-grade paper and should be carefully completed. If you don’t know how to start an analytical essay, read a few handy tips that will ensure a solid foundation for your paper.  

Define a subject matter

You first need to clearly understand the issue you will base your essay on. Since analytical essays imply an in-depth analysis of a specific problem, you need to define its core. Try to split the analysis into several components and provide arguments taken either from a book, a research, a scientific work, or a movie (depending on the subject matter of your analysis), and support your views comprehensively.

Decide on the content of your analytical essay

If you are a student who was given an analytical essay topic, read the task several times before you are 100% sure that you clearly understand the requirements as to the analytical essay format. In case you were lucky to choose the topic of the analytical paper by yourself, make sure the theme you will be dealing with is familiar or at least seems interesting to you. 

Remember that different subject matters require a different approach to their analysis. If you examine some literature work, you can prove your opinion based on the deeds of a certain or several characters. But if you have been assigned the task to elaborate on some historic events, analyze their main causes, driving forces that have affected their course, and their global consequences.  

Take care of the proper start

Don’t forget to start your analytical essay with a thesis statement. It is a sentence or a couple of sentences that aim to summarize the key statements of your paper. A thesis statement should provide readers with a preliminary idea of what your essay is all about.  

Find extra reasoning

Make sure your thesis is supported by compelling arguments. To find enough evidence, you should carry out a thorough analysis of the assigned topic. List the crucial points of your research and ponder over the ways they can be used to prove your final opinion. 

Elaborate the outline

A sound outline elaborated at the preparation stage will help you ensure a proper analytical essay structure and make the overall writing process easier. As a rule, an analytical essay consists of an introduction, three body paragraphs, and a conclusion. Your outline plan should include the key arguments you want to discuss in each paragraph. 

Analytical Essay Thesis

A thesis statement represents the central idea of your paper and must serve as strong proof of your standpoint. While elaborating your thesis statement, it is crucial to include it at the end of the first paragraph and thus set a direction for the overall paper. 

Analytical Essay Outline

An outline is not a required element of analytical essays writing and should not be included in the text, but it can greatly facilitate the whole process of paper writing.

The analytical essay structure looks as follows:

Introduction

In the introduction of an analytical essay, you will need to identify your paper’s subject matter. Mention the purpose of your work and specify its scope of research. Don’t forget to include a thesis to let readers know what your work is about.

Body Section

As has already been mentioned, the body section covers three or more main paragraphs, each being supported with arguments and details. Besides, you need to provide a small conclusion to each statement to make your essay sound professional and persuasive. 

At this stage, you need to summarize the points elucidated in your paper and make sure there is a smooth and logical transition from the body section to the concluding part of the text. If you don’t know how to conclude an analytical essay, try to restate the thesis statement without copying it word for word.  

Analytical Essay Examples

Writing an analytical essay may seem to be a thorny way. If you are still not sure how to properly craft one, try to find some examples that will help you go in the right direction. Below, there are some great examples of analytical essays. Take a look at their structure and try to write something similar based on your views and ideas:

https://drive.google.com/file/d/1JeR4i4RIZIj448W3KVFyHP-eS3QPN7gW/view

https://stlcc.edu/docs/student-support/academic-support/college-writing-center/rhetorical-analysis-sample-essay.pdf

https://www.germanna.eduhttp://handmadewriting.com/wp-content/uploads/tutoring/handouts/Literary-Analysis-Sample-Paper.pdf

30 Analytical Essay Topics

If you were allowed to choose the theme for your paper by yourself, check on the following analytical essay topics. Each of them can bring you the highest score:

General topics

• The influence of social networks on the life of teens
• Are salaries of football players too high?
• Wearing uniforms in schools should be banned
• A person in society: the problems of loneliness and privacy
• Sociology of corporate relationships
• Does the observation of space need more investments?
• Should the voting age in the UK be decreased?
• Reasons why capital punishment should be brought back in the UK
• A world with no rules: a new human era or a road to the global collapse?
• Life without technologies: will modern people survive?
• Should scientists test drugs on animals to fight cancer?
• The problem of keeping the balance between career and family life
• The importance of listening to your body 
• Problems caused by the lack of communication
• Food addiction and the problems it causes
• Problems of vaccination in the XXI century
• Does evil really rule the world?
• How does body size affect life quality?
• Pros and cons of video games 
• The role of a family model in the life and career of a person

Analytical Essay Topics on Literature

• “Robinson Crusoe”: fantasy vs reality
• Observation of the artistic uniqueness in the comedy by W. Shakespeare “A Midsummer Night’s Dream” 
• Observe the social problems in the novel by John Steinbeck “The Grapes of Wrath”
• Convulsions and death of the “little man” in the networks of impersonal, alienated forces in the novel “The Metamorphosis”
• Observation of the problems of a man on a plagued land in the novel “The Plague”
• Revolt of the protagonist in the novel by J. Salinger “The Catcher in the Rye”
• Observation of friendship and love in the fate of humanity in the XX century
• The triumph of immorality in the novel by F. Sagan “Hello Sadness”
• Observation of the personality of an American student in the novel by J. Salinger “The Catcher in the Rye”
• Eternal tragedies of humanity in the tragedy by W. Shakespeare “The Tragedy of Hamlet, Prince of Denmark”

How to Write a Well-Structured Analytical Essay With a Solid Argument

Writing an analytical essay with a clear structure might be challenging unless you are thoroughly prepared. We decided to help you out and create a detailed guide listing the main things to consider when creating an analytical essay outline. You need to explain your main idea in a concise way to bring your point across. As analytical writing has high requirements, it pays off to find an analytical essay example and analyze how this text was written. It will allow you to understand the analytical essay format better and learn how to provide substantive analysis on various topics. Read on to learn how to write a top-level analytical paper and submit it on time.

Main Tips for Writing an Analytical Essay

An analytical essay should provide a comprehensive analysis of a chosen topic. What makes an analysis essay different from other assignments is that it includes a personal opinion of an author. This is why analytical writing should be persuasive.

Below, we have rounded up the key tips you need to follow when producing an analytical essay outline and the main body of your text. Read on to learn more about the analytical essay format and create a text that will fully meet the requirements.

Select an Analytical Essay Topic

Before creating an analytical essay outline, make sure to pick a topic that you are interested in. It should be provocative enough to engage your readers. A widely-debated topic will help you write an analytical essay that grabs the attention of a wide audience.

Consider your goals and conduct thorough research to see if you have enough sources to support the main thesis of your analysis essay.

Come Up With a Strong Analytical Thesis Statement

When writing an analytical essay, start by formulating a thesis statement that includes the topic and the main goal of your text. It will help you create an analytical essay outline and show your readers what you will discuss in your analysis essay.

Add it to the last paragraph of your analytical essay introduction. Due to this, your analytical essay outline will look better structured. Look at any analytical essay example to see how you can introduce your subject. In most cases, one sentence will suffice to state your analysis essay’s goal. However, a complex analytical essay outline might require you to use two sentences for a thesis statement.

Write an Analytical Essay Body with a Clear Structure

Your analytical essay outline should include 3-4 paragraphs. However, a literary analysis essay usually consists of 5 paragraphs. When it comes to analytical writing, it is important to cover a different point in each section of the main body of an analysis paper.

After writing an analytical essay, check whether each paragraph contains an introduction and the main point. Besides, it should contain evidence. An expertly written analytical essay outline will help you reach out to your target audience more effectively.

Conduct Research Before Writing an Analytical Essay Outline

While this step is preparatory, it is a must for those who want to write a well-grounded analytical paper.

• First, select the best ideas for your essay
• Then, emphasize the problems with works written by other researchers
• Finally, write your analytical essay outline to demonstrate what approach you want to take

Examine the context and find examples to illustrate the scope of the issue. You may draw parallels to emphasize your point and make your topic more relatable.

Analyze the Implications of the Evidence

After listing your pieces of evidence and demonstrating how it is related to your thesis, show why it is important. You need to explore it deeply and use it to support your argument. It will make your analytical essay outline well-grounded facts.

Write an Analytical Essay Conclusion

Whether you write a literary analysis essay or other types of assignments, there is no need to add any new data at the end of your analysis paper. Instead, summarize the arguments you mentioned in your analytical essay outline. The conclusion of your analysis essay should be short and clear. Here, you need to demonstrate that you have achieved your goals.

Analytical Essay Writing Tips

If you want to get the highest grade for your analytical essay, you need to know a little bit more than just the basics of paper writing. Read these handy tips to write a perfect essay you will be proud of:

• Double-check your paper for spelling and grammar mistakes. In case your essay contains too many errors, neither an in-depth analysis nor the elaborate writing style will make it look any better. Situations when essays of great value in terms of research and a message they convey are poorly assessed because of the abundance of mistakes are not rare. Make sure you have enough time to proofread your paper before submission. Also, you may consider asking somebody to take a fresh look at your essay and check it for you.
• Reading your analytical essay out loud helps you discover all types of errors or weak phrases. This method might seem a bit uncomfortable, but it has proved to be very effective for many students. Note that silent reading of your paper isn’t even half as helpful as reading it aloud. 
• Another great idea to check on the rhythm and flow of your paper is to ask someone to read it for you. While listening to the text, you could perceive it from another perspective and discover even more inconsistencies and mistakes.  
• Double-check the facts you use in your analytical essay. The names of people, books, research, publications, as well as dates of historical events are too important to be misspelled. Things like these show your professionalism and the way you treat your readers.

Write an Analytical Essay with HandmadeWriting

Writing an analytical essay requires time, strong writing skills, great attention to detail, and a huge interest in the assigned topic. However, life can be unpredictable sometimes, and students might find themselves at risk of failing their creative assignments. Stress, family issues, poor health, and even unwillingness to work on a certain topic may become significant obstacles on their way to the A-grade work.

If you have similar problems, there is no need to compromise your reputation and grades. You can always refer to HandmadeWriting professionals who are ready to help you with a paper of any type and complexity. They will understand your individual style and totally devote themselv

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Analytical Essay

Definition of analytical essay, difference between an analytical essay and a critical essay, types of analytical essay, examples of analytical essay in literature, example #1: liposuction: the key to energy independence (by barbara ehrenreich).

“I say to my fellow humans: It’s time to stop feeding off the dead and grow up! I don’t know about food, but I have a plan for achieving fuel self-sufficiency in less time than it takes to say ‘Arctic National Wildlife Refuge.’ The idea came to me from reports of the growing crime of French fry oil theft: Certain desperate individuals are stealing restaurants’ discarded cooking oil, which can then be used to fuel cars. So the idea is: why not skip the French fry phase and harvest high-energy hydrocarbons right from ourselves?”

Example #2: Freedom (by Joyce M. Jarett)

“On the first day of school, I was escorted by hordes of national guardsmen. Like a funeral procession, the steady stream of official-looking cars followed me to the campus. Some patrolmen were parked near campus gates, while others, with guns strapped to their sides, stood near building entrances. Though many of my escorts had given me smiles of support, still I was not prepared for what I encountered upon entering my new school.”

Example #3: The Ways of Meeting Oppression (by Martin Luther King, Jr.)

“The third way open to oppressed people in their quest for freedom is the way of nonviolent resistance. Like the synthesis in Hegelian philosophy, the principle of nonviolent resistance seeks to reconcile the truths of two opposites—the acquiescence and violence—while avoiding the extremes and immoralities of both. The nonviolent resister agrees with the person who acquiesces that one should not be physically aggressive toward his opponent; but he balances the equation by agreeing with the person of violence that evil must be resisted. He avoids the nonresistance of the former and the violent resistance of the latter. With nonviolent resistance, no individual or group need submit to any wrong, nor need anyone resort to violence in order to right a wrong.”

Function of Analytical Essay

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• How to write a rhetorical analysis | Key concepts & examples

How to Write a Rhetorical Analysis | Key Concepts & Examples

Published on August 28, 2020 by Jack Caulfield . Revised on July 23, 2023.

A rhetorical analysis is a type of essay  that looks at a text in terms of rhetoric. This means it is less concerned with what the author is saying than with how they say it: their goals, techniques, and appeals to the audience.

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Table of contents

Key concepts in rhetoric, analyzing the text, introducing your rhetorical analysis, the body: doing the analysis, concluding a rhetorical analysis, other interesting articles, frequently asked questions about rhetorical analysis.

Rhetoric, the art of effective speaking and writing, is a subject that trains you to look at texts, arguments and speeches in terms of how they are designed to persuade the audience. This section introduces a few of the key concepts of this field.

Appeals: Logos, ethos, pathos

Appeals are how the author convinces their audience. Three central appeals are discussed in rhetoric, established by the philosopher Aristotle and sometimes called the rhetorical triangle: logos, ethos, and pathos.

Logos , or the logical appeal, refers to the use of reasoned argument to persuade. This is the dominant approach in academic writing , where arguments are built up using reasoning and evidence.

Ethos , or the ethical appeal, involves the author presenting themselves as an authority on their subject. For example, someone making a moral argument might highlight their own morally admirable behavior; someone speaking about a technical subject might present themselves as an expert by mentioning their qualifications.

Pathos , or the pathetic appeal, evokes the audience’s emotions. This might involve speaking in a passionate way, employing vivid imagery, or trying to provoke anger, sympathy, or any other emotional response in the audience.

These three appeals are all treated as integral parts of rhetoric, and a given author may combine all three of them to convince their audience.

Text and context

In rhetoric, a text is not necessarily a piece of writing (though it may be this). A text is whatever piece of communication you are analyzing. This could be, for example, a speech, an advertisement, or a satirical image.

In these cases, your analysis would focus on more than just language—you might look at visual or sonic elements of the text too.

The context is everything surrounding the text: Who is the author (or speaker, designer, etc.)? Who is their (intended or actual) audience? When and where was the text produced, and for what purpose?

Looking at the context can help to inform your rhetorical analysis. For example, Martin Luther King, Jr.’s “I Have a Dream” speech has universal power, but the context of the civil rights movement is an important part of understanding why.

Claims, supports, and warrants

A piece of rhetoric is always making some sort of argument, whether it’s a very clearly defined and logical one (e.g. in a philosophy essay) or one that the reader has to infer (e.g. in a satirical article). These arguments are built up with claims, supports, and warrants.

A claim is the fact or idea the author wants to convince the reader of. An argument might center on a single claim, or be built up out of many. Claims are usually explicitly stated, but they may also just be implied in some kinds of text.

The author uses supports to back up each claim they make. These might range from hard evidence to emotional appeals—anything that is used to convince the reader to accept a claim.

The warrant is the logic or assumption that connects a support with a claim. Outside of quite formal argumentation, the warrant is often unstated—the author assumes their audience will understand the connection without it. But that doesn’t mean you can’t still explore the implicit warrant in these cases.

For example, look at the following statement:

We can see a claim and a support here, but the warrant is implicit. Here, the warrant is the assumption that more likeable candidates would have inspired greater turnout. We might be more or less convinced by the argument depending on whether we think this is a fair assumption.

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See an example

Rhetorical analysis isn’t a matter of choosing concepts in advance and applying them to a text. Instead, it starts with looking at the text in detail and asking the appropriate questions about how it works:

• What is the author’s purpose?
• Do they focus closely on their key claims, or do they discuss various topics?
• What tone do they take—angry or sympathetic? Personal or authoritative? Formal or informal?
• Who seems to be the intended audience? Is this audience likely to be successfully reached and convinced?
• What kinds of evidence are presented?

By asking these questions, you’ll discover the various rhetorical devices the text uses. Don’t feel that you have to cram in every rhetorical term you know—focus on those that are most important to the text.

The following sections show how to write the different parts of a rhetorical analysis.

Like all essays, a rhetorical analysis begins with an introduction . The introduction tells readers what text you’ll be discussing, provides relevant background information, and presents your thesis statement .

Hover over different parts of the example below to see how an introduction works.

Martin Luther King, Jr.’s “I Have a Dream” speech is widely regarded as one of the most important pieces of oratory in American history. Delivered in 1963 to thousands of civil rights activists outside the Lincoln Memorial in Washington, D.C., the speech has come to symbolize the spirit of the civil rights movement and even to function as a major part of the American national myth. This rhetorical analysis argues that King’s assumption of the prophetic voice, amplified by the historic size of his audience, creates a powerful sense of ethos that has retained its inspirational power over the years.

The body of your rhetorical analysis is where you’ll tackle the text directly. It’s often divided into three paragraphs, although it may be more in a longer essay.

Each paragraph should focus on a different element of the text, and they should all contribute to your overall argument for your thesis statement.

Hover over the example to explore how a typical body paragraph is constructed.

King’s speech is infused with prophetic language throughout. Even before the famous “dream” part of the speech, King’s language consistently strikes a prophetic tone. He refers to the Lincoln Memorial as a “hallowed spot” and speaks of rising “from the dark and desolate valley of segregation” to “make justice a reality for all of God’s children.” The assumption of this prophetic voice constitutes the text’s strongest ethical appeal; after linking himself with political figures like Lincoln and the Founding Fathers, King’s ethos adopts a distinctly religious tone, recalling Biblical prophets and preachers of change from across history. This adds significant force to his words; standing before an audience of hundreds of thousands, he states not just what the future should be, but what it will be: “The whirlwinds of revolt will continue to shake the foundations of our nation until the bright day of justice emerges.” This warning is almost apocalyptic in tone, though it concludes with the positive image of the “bright day of justice.” The power of King’s rhetoric thus stems not only from the pathos of his vision of a brighter future, but from the ethos of the prophetic voice he adopts in expressing this vision.

The conclusion of a rhetorical analysis wraps up the essay by restating the main argument and showing how it has been developed by your analysis. It may also try to link the text, and your analysis of it, with broader concerns.

Explore the example below to get a sense of the conclusion.

It is clear from this analysis that the effectiveness of King’s rhetoric stems less from the pathetic appeal of his utopian “dream” than it does from the ethos he carefully constructs to give force to his statements. By framing contemporary upheavals as part of a prophecy whose fulfillment will result in the better future he imagines, King ensures not only the effectiveness of his words in the moment but their continuing resonance today. Even if we have not yet achieved King’s dream, we cannot deny the role his words played in setting us on the path toward it.

If you want to know more about AI tools , college essays , or fallacies make sure to check out some of our other articles with explanations and examples or go directly to our tools!

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The goal of a rhetorical analysis is to explain the effect a piece of writing or oratory has on its audience, how successful it is, and the devices and appeals it uses to achieve its goals.

Unlike a standard argumentative essay , it’s less about taking a position on the arguments presented, and more about exploring how they are constructed.

The term “text” in a rhetorical analysis essay refers to whatever object you’re analyzing. It’s frequently a piece of writing or a speech, but it doesn’t have to be. For example, you could also treat an advertisement or political cartoon as a text.

Logos appeals to the audience’s reason, building up logical arguments . Ethos appeals to the speaker’s status or authority, making the audience more likely to trust them. Pathos appeals to the emotions, trying to make the audience feel angry or sympathetic, for example.

Collectively, these three appeals are sometimes called the rhetorical triangle . They are central to rhetorical analysis , though a piece of rhetoric might not necessarily use all of them.

In rhetorical analysis , a claim is something the author wants the audience to believe. A support is the evidence or appeal they use to convince the reader to believe the claim. A warrant is the (often implicit) assumption that links the support with the claim.

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Formal Analysis Paper Writing Guide

A formal analysis essay, aka a visual analysis essay , is an essay that is written to analyze a painting or a work of art. A properly written formal analysis paper will give the reader both objective and subjective information about an art piece.

This post will teach you the A to Z of how to write a formal analysis essay.

What is a formal analysis essay?

A formal analysis essay is an academic writing that comprehensively describes a work of art (usually a painting, image, or sculpture). An excellent formal analysis essay is one that not only describes a work of art but also analyzes it.

If you are enrolled in an art program in college or university, you will most likely have to complete a good number of formal or visual analysis essays before you graduate. They are common in art programs to help art students to understand and judge art better.

The 4 Main Steps for a Formal Analysis Essay

Every proper formal analysis essay must include four key elements – description, analysis, interpretation, and evaluation. In other words, when writing a formal analysis essay, you must describe, analyze, interpret, and evaluate the work of art.

1. Description

The first thing you need to do when writing a formal analysis essay is to describe the elements of the work of art you see at first glance.

So what can you quickly notice about the work of art you want to analyze? What is it that has been depicted? What medium has the artist used? What painting element? Your answers to all these questions will help you to fully describe the work of art to the reader.

In your description of the work of art, there are several things you must strongly consider and describe.

• Texture : How does the artist use texture? Is it implied or actual?
• Color : How does the artist use colors? What color scheme? Is the image dark, medium, or light?
• Organizing space : How does the artist utilize perspective? Does the artist use linear perspective?
• Mass and volume : How does the artist represent mass and volume? Is the work of art one-dimensional, two-dimensional, or three-dimensional?
• Shape : How does the artist use shapes? Are the shapes soft or hard-edged? Are they small or large? How are the shapes related?
• Line : How does the artist use lines? Are they soft, jagged, straight, mechanical, or expressive? Are they used to describe space?

2. Analysis

The second thing you need to do when writing a formal analysis essay is to analyze the artistic elements of the work of art. This is where the rubber meets the road. It is where your inner artist must come out, and you must discuss the true art aspects of the work of art.

Once you have described the physical or first-glance elements of the artwork, you have to go straight into the analysis aspect. The analysis aspect is all about discussing how the artist employs design aspects in the artwork.

In your analysis of the work of art, there are several things you must strongly consider and analyze.

• Design : What principles of design (art techniques) does the artist use?
• Variety: What elements does the artist use, and how does he use them to provide variety?
• Scale : Is the work to scale? Are all the elements on the same scale?
• Symmetry : Is the work symmetrical or asymmetrical?
• Emphasis : What does the artist use to create emphasis? What movement does the work convey?
• Patterns : What patterns are clear in the artwork?

3. Interpretation

The third thing you need to do when writing a formal analysis essay is to reveal the hidden meanings or aspects of the work of art you are analyzing.

The interpretation part of your formal analysis essay should capture two things – the use of symbolism in the artwork and what it means to the viewer. It is all about revealing the hidden meaning of what may mean one thing at first glance and something deeper upon further inspection.

Interpretations of artworks usually reveal themselves somewhat slowly. This is because one has to look at an art object repeatedly to identify symbolism and get the deeper meaning.

When providing an interpretation for symbolism, you must provide supporting evidence.

4. Evaluation

The last thing you need to do when writing a formal analysis essay is to evaluate the work of art you have described, analyzed, and interpreted.

In the evaluation part of your visual analysis essay, you need to discuss your overall opinion of the artwork. Specifically, you need to discuss what you discovered about it and about yourself when examining it.

A proper evaluation of an art object will detail your opinion and feelings about it. It will also discuss whether you find it pleasing or engaging.

When evaluating an art piece, it is crucial to remember that it is best to evaluate art pieces per the period they were created. This allows for better contextualization and proper evaluation.

Format of Formal Analysis Paper

A typical formal analysis paper will have three key sections – an introduction, a body, and a conclusion. Find out what each section entails in the visual analysis paper outline below.

1. Introduction

The first part of a formal analysis paper is the introduction. The introduction must provide the reader with all the essential information about the artwork to be analyzed. It must also be interesting enough to hook the reader.

The best way to provide the reader with all the essential information about the artwork to be analyzed is to describe it vividly. The vivid description should be followed by information about how the artwork was created and information about the creator.

An introduction with all the above information is sufficient to hook the reader's attention. Nevertheless, if there is something controversial about the artwork, this information must also be provided in the introduction. The purpose of giving this type of information is to ensure the reader is fully aware of all the crucial aspects of the art piece to be analyzed.

2. Thesis statement

A well-written visual analysis paper will have a thesis statement at the end of the introduction. The thesis statement must be clear, concise, and to the point. Moreover, it must quickly tell the reader the paper's main argument.

Think of the thesis statement of your formal analysis paper as your paper's central argument; the argument/claim you will be supporting during your analysis.

The body of a formal analysis essay is where the actual analysis happens. It must have four key elements for it to be considered complete. The elements are – description, analysis, interpretation, and evaluation.

Each element is typically covered in a separate paragraph for good organization and flow. And it supports the thesis statement in the introduction section.

By the time the reader is done reading the body section of the essay, they should have all the art analysis they expected after reading the introduction.

4. Conclusion

The conclusion is the last part of every formal essay analysis. It is where the writer must nicely wrap up the art analysis. This is usually done by first restating the thesis and the main supporting statements. The restatement is frequently followed by information comparing the artwork with similar pieces or information about whether the artwork fits in with the creator's other artworks.                                                                                                                                       

Steps for Writing a Formal Analysis Essay

As an art history student, it is essential to learn how to write a visual analysis paper. This is because you will most likely be asked to write such a paper several times before graduating. Moreover, learning how to write such a paper will also help you become a good art critic, curator, or appraiser.

Follow the steps below to write a brilliant visual analysis essay.

1. Gather general information about the artwork and the creator

The first thing to do to write a formal analysis essay is to gather general information about the artwork and the creator. The most crucial bits of information to collect include:

• The subject – what artwork will you be analyzing? What is it called, and when was it created?
• The creator – who is behind the art? Identify them by their full name.
• The date of creation – when was the artwork created? Is it typical art for the period and the artist?
• The location – where is the art being displayed? Has it been displayed somewhere else before?
• The medium and techniques – what medium was used to create the artwork? What techniques did the artist use?

2. Write the introduction

A good introduction paragraph to a formal analysis essay provides sufficient background information about the subject piece and grabs the reader's attention. Use the information you have gathered in the step above to write a good introduction.

Make your introduction brilliant by ensuring you provide information about the art piece in a logical and flowing manner. After writing your introduction, read it to ensure it can grab the reader's attention. If it can, you are good to go. If it can't, you should rewrite it until it can do so.

The last statement of your introduction paragraph should be your thesis statement (your central argument for the paper). It should quickly tell the reader your overall argument or opinion about the subject artwork. You can only develop a good thesis statement after researching a painting and finding out more about it and the period it was created. Researching an art piece will also help you describe and analyze it more objectively.

If, after writing your paper, you feel your thesis statement doesn't closely reflect its overall argument, you should rewrite the statement to make sure it does. It is totally okay to do so.

3. Describe the painting

After writing your introduction paragraph, you should write the body section of your paper. The body section of a typical formal analysis paper must have at least two distinct parts – the first part describing the painting and the second analyzing it. It can also have an interpretation part to discuss any symbolism or hidden meaning discovered in the art.

The best way to describe the subject painting is to provide all the visible information about it. The visible aspects of the artwork that you must capture in your description include:

• The figures – the characters displayed in the painting and their identities or what they represent.
• The theme – the story or theme depicted in the painting
• The setting – the background of the painting.
• The shapes and colors – the shapes and colors that stand out in the painting.
• The mood – the lighting and overall mood of the subject artwork.

Make sure your painting description is well-organized and has a good flow. If need be, rewrite it to enhance its flow.

Your description can be one paragraph long or two paragraphs long.

4. Analyze the painting

The second part of your paper's body section should be a detailed analysis of the subject painting. Your analysis should focus on the painting's art elements and design principles.

Your professor will most likely focus on the analysis part of your paper, so make sure you do a brilliant analysis. To ensure your professor is impressed with your painting analysis, you should analyze/discuss the art elements and the design principles separately.

The art elements of a painting are the painting techniques used. They are also known as the basics of composition. The art elements that shouldn't miss in your analysis include:

• The lines – what lines does the artist use? Are they implied, thick, curved, or straight? Talk about them in your paper.
• The shapes – what shapes does the artist use? Are they plain or hidden? Are they geometrical or out of proportion?
• The use of light – what is the light source in the painting? Is it obvious or hidden?
• The colors – what colors does the painter use? Are they primary or secondary colors? Is the tone cool or warm?
• The patterns – does the painting have distinct, repeated, textural, or hidden patterns? How do they make it look?
• The use of space – is the painting one-dimensional or two-dimensional? How does the artist show depth?
• The passage of time – how does the painting show the passage of time?

Remember, simply describing the art elements above is not enough. You must analyze them because this step is all about analysis; it is all about going deeper.

The design principles of a painting are the aspects of the painting that provide a thematic or broader perspective. Therefore, your inner artist should come out when analyzing the design principles of the painting.

The following things should feature in your analysis of the design principles of the painting:

• Symmetry and asymmetry – talk about the points of balance in the shapes, colors, patterns, and overall painting.
• Variety – talk about the varied techniques used by the artist and whether the overall feel is of chaos or unity.
• Emphasis – talk about the thematic and artistic points of focus. Mention the colors that the artist emphasizes.
• Proportions – discuss how the figures work together to depict the painting's volume, mass, and scale.
• Rhythm – discuss how the artist displays rhythm using figures and techniques.

Carefully and methodically detailing your analysis of the painting's art elements and design principles will help you to get a good grade in your visual analysis essay.

5. Write the conclusion

After describing and analyzing the painting, you should write the conclusion. A good visual or formal analysis essay conclusion provides an overall evaluation of the subject painting. The best way to write an overall evaluation of the subject painting is to think of the evaluation as your general assessment.

So what do you think of the painting based on your description and analysis? Is it standard, brilliant, or exceptional? Does it meet your expectations? Why yes or why not? Talk about these things in your conclusion to make it perfect.

6. Proofread and edit your essay

After writing your essay, you should proofread and edit it. Doing this will help you to transform it from ordinary to extraordinary.

The correct way to proofread your essay is to proofread it twice – first using a grammar checker like Grammarly and then using your own eyes.

Proofreading your paper using a grammar checker like Grammarly will help you quickly identify and edit basic grammar and typing errors. Proofreading your document one more time using your own eyes will help you to catch any mistakes the checker might have missed. It will also help you to identify and rewrite parts of your paper that are difficult to understand.

Your paper should be ready to submit after you proofread it as described above.

10 tips to help you write a brilliant formal analysis essay

Writing a visual analysis essay is not easy. This is because there are many things to include and consider to ensure the paper is complete. In this section, you will discover what you need to consider to ensure the final draft of your formal analysis essay is perfect.

• Consider your feelings. When analyzing a painting, it is crucial to be objective in your analysis. But at the same time, it is also vital to consider your feelings. All your feelings toward the painting are legitimate. Therefore, do not be afraid to say what you think about an artwork, especially in your analysis and conclusion. Your professor should be able to clearly see your judgment on the subject piece and not just your objective analysis.
• Focus on analysis. When most art students write a formal analysis essay, they spend a lot of time on the description rather than the analysis. This is wrong. It is okay for the first part of your analysis essay to include a description of the painting. However, at least 50 to 60 percent of your essay should be your analysis of the subject painting. Your professor expects this. So if you want to get a good grade, you should focus on analysis.
• Start with the obvious and go deeper. While it is true that a good formal analysis paper must focus on the analysis, a thorough description of the artwork is also a must. The first part of your paper's body section must be a detailed description of the subject painting. It is only after describing the artwork that you should proceed to analyze it. If you write your paper this way, your reader will know what exactly you are talking about in your analysis.
• Use the correct terms. You must use the correct terms to get a top grade when writing your visual analysis essay. This is because professors hate when students use non-official or inaccurate words to describe art elements. If you do not know the correct way to refer to an art element or design principle, you should search for it online in art term glossaries such as MOMA .
• Less is more. There are usually many art elements and design principles to consider when analyzing an art piece. However, discussing or describing all of them in your paper is not a must. Your analysis should focus on discussing the most significant art elements and design principles. You can, and you should, ignore all insignificant art elements and design principles.
• Proofread your work thoroughly. Many students ignore proofreading their papers before submission and end up submitting low-quality essays. You should not do the same. Instead, you should carefully proofread your work twice or thrice to remove all errors and mistakes. This will help you polish your essay to a level that is extremely easy to understand.
• See live artwork regularly. To write great visual analysis essays, you must visit art galleries, museums, and exhibitions frequently. Visiting such art establishments regularly will allow you to see, hear, and familiarize yourself with art critique. Of course, by familiarizing yourself with art critique, the exercise will become like second nature to you. Therefore, it will make it much easier for you to write great visual analysis essays.
• Consider audience and historical context. When analyzing art, you must strongly consider the audience and the historical context. Failure to do so will make your analysis shallow. Think of who the artist was trying to appeal to. Was he trying to appeal to the church? Art people? Or the masses? The target audience obviously influenced the way artists developed their paintings. Think also of the historical context. Judging a Renaissance period painter with Rococo era standards is not appropriate. You can only judge an art piece by the standards of the art period it was created.

Example of a formal or Visual or Formal Analysis Essay

The essay below is a short example of a formal analysis essay. It includes the three key sections of a formal analysis essay – introduction, body, and conclusion. Its body section consists of a brief description paragraph and a brief analysis paragraph.

Analysis of Snow Storm – Steam-Boat off a Harbour's Mouth

Snow Storm – Steam-Boat off a Harbour's Mouth is a chaotic oil on canvas painting by Englishman Joseph William Turner (1775- 1851, England). It is one of Turner's most famous paintings, and it is on display at Tate, one of the most important art organizations in the world. By looking at this painting, it is clear that Turner was a master at drawing abstract, chaotic paintings.

At first glance, this painting reveals a steamboat at the center of what looks like a storm. However, the painting is chaotic, so this is something one cannot tell for sure. Nevertheless, based on the title of the painting, one can see it is shaped like a steam-boat belching heavy smoke. The vessel is surrounded by swirling snow and pitching waves. The sky is somewhat depicted in blue despite the raging storm.

A closer inspection of the painting reveals a well-drawn chaotic artwork featuring long and heavy strokes. The long strokes create dramatic movement and enhance the artist's idea of a steamboat in a raging storm. It also reveals asymmetrical shapes, as is the case with most chaotic oil on canvas paintings.

In terms of design principles, the scale of the boat stands out. It looks somewhat tiny in the middle of a big storm. The tiny size of the vessel, plus the small indication of space above it created by a sliver of blue sky, indicates a scene of danger. The scene is reminiscent of humanity's struggle against powerful forces of nature.

This artwork by Turner is a beautiful work of art. Turner is considered one of the foremost painters of his time, and this work indeed shows that he was a master oil on canvas painter. The work depicts a boat in a big storm. It is a powerful reminder of humanity's struggle for survival in a cold and unforgiving world.

Final words

Professors and instructors know that formal analysis essay assignments help students to understand and judge art better. Because of this, they give such assignments from time to time. Therefore, if you are an art program student, you should learn how to do such assignments perfectly. Doing so will help you get an excellent grade whenever you are given such an assignment.

This post revealed everything crucial that you need to know to write a formal essay. The information includes the four steps for a formal analysis essay, the structure of a formal essay, and the steps to follow to write one. You can use the information to write a brilliant visual analysis essay on any work of art.

If the information is insufficient for you to write an excellent visual analysis essay or you are too busy to write one, you should work with us. If you do so, you will get 100% confidentiality, 100% quality, and 100% on-time delivery. Try Essay Maniacs today for quality assignment help !    

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Musk's Fellow Investors in X Are Part of a $24 Billion Loss Since 2022

A new analysis estimates musk's management of the social media platform produced an unprecedented "vaporization of wealth" for him and his backers, less than two years after the $44 billion buout of twitter..

Much like his political ally and  endorsed candidate  for the 2024 presidential election Donald Trump, serial founder  Elon Musk  seems to hate nothing more than being perceived that he's not the peerless business genius he claims to be. The increasingly prickly Musk must absolutely despise the  Washington Post  right now. The paper crunched the numbers on his 2022 $44 billion acquisition and subsequent management of social media platform Twitter, which he renamed X, and concluded it resulted in "a  vaporization of wealth  that has little parallel" in entrepreneurial history.

Just as bad, the paper said, a bevy of similarly wealthy  financial backers got soaked by the USS X taking on water under Musk's captaincy, losing over 50 percent of their initial stakes in the process.

How can the  Post --owned by Musk's fellow tech billionaire and  purported foe , Jeff Bezos--confidently estimate the loss of $24 billion in value of a company that has been privately held since 2022? 

It calculations were built on a line of inquiry first reported by Axios last December. It established investment giant Fidelity has continually readjusted the value of its initial $316 million stake in Twitter,   now X, to what early this year was just $88 million. The reason for the  72 percent write-down ? The flight of advertisers--the platform's main source of income--since Musk took it over, as well as his decision to fire the majority of its employees, transforming it into   libertarian loudspeaker for unfiltered speech whose endless controversies have sent countless users packing, too. 

More than a few of those scandals were provoked by Musk himself. Among the biggest of those was his notorious and  financially self-destructive  invitation last November to big name advertisers wary of his social media site to, ah ,  "eff-off." That reinforced the logic of bolting X in the first place, and his antics   over time appear to confirm warnings that Musk's billionaire status and perceived entitlement has blinded him to the serious income and business damage he's inflicted on the company.

This week, the  Post  report detailed just how much that destruction has also cost people who financed about 20 percent of Musk's $44 billion takeover of Twitter.

Dozens of backers were identified in documents a court ordered release last month, revealing names like disgraced rap mogul Sean "Diddy" Combs, hedge fund gadfly Bill Ackman, Saudi prince billionaire Alwaleed bin Talal, former Cisco co-founder Larry Ellison, and Twitter's co-creator Jack Dorsey. Institutional investors included Andreesen Horowitz, Binance, and Qatar's investment fund. The unsealing of those documents was ordered by a federal judge  in California hearing a case lodged by fired Twitter employees and an investigating journalist.

Based on those and other records, the Post calculated the eight biggest backers of Musk's acquisition have now seen lost   around $3.4 billion, or over half of their initial investments.

"(Musk's) and his partners' overall stake has shed $24 billion in value," the paper reported, calling that "a vaporization of wealth that has little parallel outside the realm of economic or industry-specific crashes, or devastating corporate scandals."

Virtually none of those savvy and experienced investors answered the  Post 's requests to sing a little about taking that deep financial bath. The exception was bin Talal, who claims to still be pleased with his holdings. He also believes failure to appreciate the current and future worth of xAI--the platform's artificial intelligence unit--has resulted in an under-valuation of the wider platform. Not everyone agrees.

The father of Twitter, Dorsey, initially tweeted (naturally) his backing of Musk's "mission to extend the light of consciousness" in taking over the platform. He later lamented "It all went south" as Musk fired staff and allowed content criticized as anti-Semitic, racist, and otherwise intolerant to flourish. For Dorsey, that meant an initial $1 billion stake alongside Musk losing $720 million of its value, the  Post  said--while Ellison's received an identical haircut.

Meanwhile, banks that loaned Musk about $13 billion for the acquisition have not only seen the worth of those sums evaporate by over 50 percent. They've also wound being stuck with the paperwork on  loans that they usually sell off to third-party buyers. This time, nobody wants them. That led the  Wall Street Journal  to recently declare their backing of Musk's Twitter takeover "the  worst merger-finance  deal for banks since the 2008-09 financial crisis."

So who comes out looking smart? Possibly people who refused to back Musk's bid in the first place, or who managed to cash out early when they realized he was intent on driving the platform into the wall. 

Still, some people who lost big on Twitter are refusing to second guess Musk's wider business successes, despite his costly social media debacle. Ross Gerber, CEO of Gerber Kawasaki Wealth and Investment Management and a longtime Tesla investor, seems to think losses like his nearly worthless $1 million stake in X are as painful for Musk to deal with as backers watching their holdings evaporate.

"Elon's done a tremendous amount of wealth destruction since he's purchased Twitter," Gerber told the  Post . "For the people who put capital into him for any amount... trying to explain to people how he lost (that) is not a fun conversation."

It should be humbling to boot, yet of late Musk has Xed that he "can't wait" to take his social media performance to Washington D.C. and assist a Trump White House in any capacity he can--including performing a hostile audit of federal agencies and programs. 

That partnership would involve the CEO who the  Post  says vaporized $24 billion of the $44 billion Musk mobilized to take over Twitter repurposing those same talents to ensure taxpayer money " is spent in  a good way." And he'd be doing so under orders of the so-called "king of the deal" president who has at least  six bankruptcies  to his record.

Taking into account how that could work out, voters may decide to deliver their own "eff-off" version of unfettered free speech in November. 

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IC 814 hijack, indecision, and 'goof-ups': Former RAW chief on what went wrong

Speaking to india today tv, as dulat, the former chief of research and analysis wing (raw), admitted that there were "goof-ups" when it came to decision-making and the hijacked ic 814 plane shouldn't have been allowed to leave amritsar..

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• Netflix series on IC 814 hijacking stirs controversy
• Ex-RAW chief AS Dulat admits decision-making 'goof-ups'
• Dulat says ISI definitely had a role in hijacking

The 1999 hijacking of an Indian Airlines flight, IC 814, has become a centre of controversy lately after the release of a mini-series on Netflix. The series has reignited debate on a number of issues, including the handling of the situation by the government and different agencies involved.

Speaking to India Today TV, AS Dulat, who was the chief of Research and Analysis Wing (RAW) in 1999, admitted that there were "goof-ups" when it came to decision-making.

"Once the plane landed in Amritsar, we had an opportunity to make sure that it didn't leave Indian territory. But when it left Amritsar, there was no other option but to make a deal. We did the best possible deal with the best possible negotiators," Dulat said.

Flight IC 814 of Indian Airlines, bound from Kathmandu to Delhi, was hijacked by five terrorists as soon as it entered Indian airspace on December 24, 1999. The plane landed in Amritsar for refuelling and was stationed for 50 minutes. Despite this, the authorities, including Punjab Police and central intel forces, could not capture the advantage.

"We were all there and should have taken a call. I don't want to name-blame; after so many years, it is not fair. I am also to blame as much as anybody else," said Dulat.

The former RAW chief talked about his long chat with then Director General of Police (DGP) for Punjab Sarabjit Singh over the hijack situation.

"I had a long chat with the Punjab DGP who told me that he was not KPS Gill, and he was not going to put his job on the line. He said the Chief Minister (Parkash Singh Badal) told him that he didn't want bloodshed in Amritsar. Even Delhi was indicating the same thing. The DGP said they could storm the plane, but they didn't know how many casualties could happen. So in the name of bloodshed, nobody wanted to take a call," he said.

Dulat said that the Punjab Police needed to be told that the aircraft should not leave Amritsar, which did not happen.

Interestingly, DGP Sarabjit Singh had said, on record, that he would have taken a decision if he had got clear instructions from Delhi.

ISI's ROLE IN 'IC 814' HIJACKING

When asked about the Inter-Services Intelligence's (ISI) role in the hijack, Dulat said Pakistan's spy agency was definitely involved.

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• Open access
• Published: 04 September 2024

Mechanisms that clear mutations drive field cancerization in mammary tissue

• Marta Ciwinska 1   na1 ,
• Hendrik A. Messal   ORCID: orcid.org/0000-0003-2259-0286 2   na1 ,
• Hristina R. Hristova 2   na1 ,
• Catrin Lutz 2 ,
• Laura Bornes   ORCID: orcid.org/0000-0001-9721-1223 2 ,
• Theofilos Chalkiadakis 3 ,
• Rolf Harkes 4 ,
• Nathalia S. M. Langedijk   ORCID: orcid.org/0000-0002-1069-2704 2 ,
• Stefan J. Hutten   ORCID: orcid.org/0000-0001-5395-8392 2 ,
• Renée X. Menezes 5 ,
• Jos Jonkers   ORCID: orcid.org/0000-0002-9264-9792 2 ,
• Stefan Prekovic 3 ,
• Grand Challenge PRECISION consortium ,
• Benjamin D. Simons   ORCID: orcid.org/0000-0002-3875-7071 6 , 7 , 8   na2 ,
• Colinda L. G. J. Scheele   ORCID: orcid.org/0000-0001-8999-5451 1   na2 &
• Jacco van Rheenen   ORCID: orcid.org/0000-0001-8175-1647 2   na2  

Nature volume  633 ,  pages 198–206 ( 2024 ) Cite this article

Metrics details

• Breast cancer
• Cancer imaging
• Self-renewal

Oncogenic mutations are abundant in the tissues of healthy individuals, but rarely form tumours 1 , 2 , 3 . Yet, the underlying protection mechanisms are largely unknown. To resolve these mechanisms in mouse mammary tissue, we use lineage tracing to map the fate of wild-type and Brca1 −/− ;Trp53 −/− cells, and find that both follow a similar pattern of loss and spread within ducts. Clonal analysis reveals that ducts consist of small repetitive units of self-renewing cells that give rise to short-lived descendants. This offers a first layer of protection as any descendants, including oncogenic mutant cells, are constantly lost, thereby limiting the spread of mutations to a single stem cell-descendant unit. Local tissue remodelling during consecutive oestrous cycles leads to the cooperative and stochastic loss and replacement of self-renewing cells. This process provides a second layer of protection, leading to the elimination of most mutant clones while enabling the minority that by chance survive to expand beyond the stem cell-descendant unit. This leads to fields of mutant cells spanning large parts of the epithelial network, predisposing it for transformation. Eventually, clone expansion becomes restrained by the geometry of the ducts, providing a third layer of protection. Together, these mechanisms act to eliminate most cells that acquire somatic mutations at the expense of driving the accelerated expansion of a minority of cells, which can colonize large areas, leading to field cancerization.

The acquisition of genetic aberrations in oncogenes and tumour suppressor genes is considered fundamental to tumorigenesis. Seventy per cent of women with germline mutations in BRCA1 or BRCA2 , two well-studied tumour suppressor genes, develop breast cancer by the age of 80 (refs. 4 , 5 ). However, sequencing studies show that mutant cells with alterations in key driver genes, such as P53 , are abundant in a wide variety of tissues in healthy individuals, including the breast 1 , 2 , 3 , 6 . This high abundance of mutant cells could be associated with a high frequency of independent mutagenic events, or could arise from a minority of mutant cells that spread over large fields of tissue. With the latter, such behaviour has the potential to create areas predisposed to transformation, a process referred to as field cancerization, and may play a crucial role in the initiation and recurrence of human breast cancer—the ‘sick lobe theory’ 7 , 8 . Yet, despite its importance, the underlying cellular mechanisms that protect breast tissue from the accumulation or spread of mutant cells remain largely unknown.

The mammary epithelium is a branched network of tubes with an outer layer of basal cells and an inner layer of hormone receptor-positive (HR + ) and -negative (HR − ) luminal cells. During the 4–7 days of the oestrous cycle, the mouse variant of the menstrual cycle, hormones act on HR + luminal cells, triggering the secretion of mitogenic paracrine signalling factors 9 , 10 , 11 . These factors initiate coordinated rounds of proliferation, also in basal and luminal cells negative for HR, thereby driving the growth of side branches, referred to as alveolar buds. In pregnancy, these side branches progress into lobuloalveolar structures, capable of milk production and secretion 12 , 13 . However, outside pregnancy, these side branches regress through coordinated cell death at the end of the cycle 14 , 15 . Thus, throughout life, each oestrous cycle drives coordinated rounds of localized proliferation and cell death 9 , 16 , 17 , 18 , 19 , 20 , 21 .

Long-term maintenance under conditions of continuous remodelling requires self-renewal activity, and is therefore thought to be driven by adult stem cells 22 . On the basis of transplantation assays and lineage tracing studies, the mouse mammary gland is known to consist of self-renewing unipotent mammary stem cells (MaSCs), also termed enduring progenitors 23 , and their short-lived descendants 14 , 24 , 25 , 26 , 27 , 28 , 29 . In line with the turnover of side branches, the self-renewing capacity of MaSCs is found to fluctuate during the oestrous cycle 9 , 19 , 20 , 21 , 30 . However, the size of MaSC-descendant units and their spatial distribution is as present unknown. As a result, it is unclear whether field cancerization in breast tissue involves the expansion of mutant clones within a single unit or across several units. Moreover, it remains unknown whether and how the normal cellular organization of the mammary gland epithelium protects against the retention of mutant cells and field cancerization. Yet, because the initiation and recurrence of breast cancer may depend on the spread of mutations over larger fields, such protection mechanisms are crucial to resolve. Here, to gain insight into the cellular mechanisms that inhibit the mutant clone expansion, and how they may be overcome by mutant cells to drive field cancerization, we map the fate of cells that acquire mutations in the mouse mammary epithelium.

Extensive spread of mutant cells precedes tumorigenesis

To map the fate of mutant clones, we studied confetti mice carrying homozygous floxed alleles of the tumour suppressor genes Brca1 and Trp53 (Fig. 1a ). At the beginning of adulthood (between 10 and 15 weeks) 31 , luminal and basal cells were recombined at a low induction frequency by intraductal TAT-Cre injection (Fig. 1a,b ), with a bias towards luminal cells (Extended Data Fig. 1a ). By quantitative PCR (qPCR) we confirmed that, in more than 87% of confetti-labelled cells, the Brca1 or Trp53 gene was recombined (Extended Data Fig. 1b–d ). By contrast, most confetti-negative cells were wild-type (WT), although a fraction of these cells also harboured recombined Brca1 or Trp53 genes (Extended Data Fig. 1b–d ), indicating that, in some cases, mutant confetti clones neighbour an unlabelled mutant clone. By imaging the whole mammary glands at cellular resolution, we observed outgrowth of clones throughout the ductal tree, without preferential localization (Extended Data Fig. 1e ). No stromal recombination was detected (Extended Data Fig. 1e ). While multipotent clones have been reported in the adult mammary gland 27 , 29 , especially under mutant conditions or following widespread damage 32 , 33 , 34 , 35 , we found only lineage-restricted clones based on E-cadherin (ECAD) staining for luminal cells and α-smooth muscle actin (SMA) staining for basal cells (Extended Data Fig. 2a–f ). However, a potential minority of the multipotent population may be missed by the low induction frequency. Moreover, as Brca1;Trp53 confetti clones are genomically unstable, we cannot exclude the possibility that some of the luminal clones originate from basal cells that acquired multipotency as a result of accumulating genetic alterations.

a , Schematic of the Brca fl/fl ;Trp53 fl/fl ;R26R-Confetti mouse model used in this study. Recombination was induced by an intraductal injection method with TAT-Cre recombinant protein, leading to sporadic deletion of the Brca1;Trp53 alleles and at the same time stochastic recombination of the Confetti construct resulting in the expression of one of the four fluorophores. b , Timeline of lineage tracing experiments performed in the adult mammary gland. c , d , Brca1;Trp53 confetti lesions with a transformed ductal morphology ( c ) and partially transformed ductal morphology and local invasion ( d ). e , Transformed luminal (L, orange) and basal (B, red) clones as a percentage of the total number of luminal and basal clones, respectively. Each dot indicates an individual mouse; boxplots mark the 25th and 75th percentile, the line indicates the median and the whiskers mark the minimum and maximum values. f , Representative whole-mount confocal images of non-transformed Brca1 −/− ;Trp53 −/− confetti clones showing extensive field cancerization within the existing ductal structure. c , d , f , Images show 3D rendering of Z -stacks, with the confetti-labelled cells in their respective colours and the mammary ducts labelled with an antibody against SMA (white). Representative examples of n  = 6 mice. g , Charts representing the fraction of non-transformed luminal and basal clones (grey), the transformed luminal clones (orange) and the transformed basal clones (red) at different time points after recombination for all analysed glands combined. The total number of quantified Brca1 −/− ;Trp53 −/− confetti clones is indicated below the charts and the number of transformed clones is indicated within the charts. See Supplementary Information  1 for sample sizes and descriptive statistics for e and g . Scale bars, 100 μm.

Source Data

As reported previously 31 , within 200–250 days of induction, mice developed palpable mammary tumours (Extended Data Fig. 2g ). At the microscopic level, transformation was determined on the basis of ductal deformation (Fig. 1c ), aberrant branch formation (Fig. 1d ) and (local) invasion (Fig. 1d and Extended Data Fig. 2h ). Most transformed clones were of luminal origin (Fig. 1e ), in line with previous reports 36 , 37 , 38 , 39 . We also observed many large non-transformed Brca1;Trp53 confetti clones (Fig. 1f,g and Extended Data Figs. 1e and 2e,f ). Quantification showed that only a minority of mutant clones transitioned to a transformed phenotype at 225 days post-induction (Fig. 1g ). In contrast to transformed Brca1 ; Trp53 confetti clones, non-transformed clones did not alter the ductal morphology, but extended over large areas of normal-looking ducts, as previously suggested by the sick lobe theory 7 , 8 .

Next, we isolated both normal-looking and transformed Brca1;Trp53 clones and performed low-coverage DNA sequencing. As anticipated for genetically unstable Brca1 ; Trp53 clones, we identified a heterogeneous but substantial number of chromosomal aberrations in both normal-looking and transformed clones (Supplementary Information 3 , copy number aberrations (CNA)). When examining the average CNA of all clones, common patterns of chromosomal loss emerged (Extended Data Fig. 3a ). To assess whether these patterns were also present in fully developed late-stage tumours, we compared our sequencing data with published CNA data from late-stage Brca1;Trp53 tumours 40 . Many genomic regions that are lost in the late-stage tumours were already lost in normal-looking and transformed clones (Extended Data Fig. 3a,b ). At the same time, late-stage tumours also featured chromosomal gains, which were not yet present in our early clones (Extended Data Fig. 3c ). This suggests that the loss of genomic regions is an early event in Brca1;Trp53 -driven tumorigenesis, occurring even before transformation, whereas genomic amplifications represent late-stage events in this tumour model. Moreover, these data illustrate that we are studying the earliest phases of tumorigenesis, in which clones have already accumulated large genomic alterations, yet still present a normal phenotype.

Spread of mutant cells is intrinsic to ductal turnover

To understand what drives clonal expansion, we compared the spread of mutant confetti clones with WT confetti clones (that is, recombined cells in R26R-Confetti mice). Sporadic recombination of WT cells was induced throughout the ductal tree by either intraductal injection of TAT-Cre recombinant protein or activation of CreERT2 with a low dose of tamoxifen (Extended Data Fig. 4a–e ). Similar to the Brca1;Trp53 confetti clones, we found that WT clones also showed the capacity to spread extensively, occupying substantial areas of the ductal network (Fig. 2a,b and Extended Data Fig. 5a–d ). Hereafter, we refer to this process as ‘field clonalization’. To study their spread, we quantified the size of 2,250 WT confetti clones in 75 glands of 17 mice using whole-gland three-dimensional (3D) imaging (Fig. 2b,c and Extended Data Fig. 5b,d–f ). Despite differences in the relative labelling efficiency between the luminal and basal lineage (Extended Data Figs. 1a and 4c,e ), WT and mutant clones showed a similar size distribution, suggesting a common mechanism of expansion. Moreover, the sparsity of labelling and the cohesive nature of clones provided confidence in the integrity of clonal assignments (Extended Data Fig. 4c,e ). Together, these data suggest that, similar to Brca1;Trp53 confetti-labelled cells, WT clones can spread over large areas of the ductal network, leading to field clonalization.

a , b , Representative confocal whole-mount images showing clonal expansion of luminal Brca1;Trp53 confetti clones ( a ) and luminal WT confetti clones ( b ) in the adult mammary gland over a lineage tracing time period of 225 days. Persisting clones form cohesive clusters of cells spanning many ducts and branch points. Images show 3D rendering of Z -stacks, with the confetti-labelled cells in their respective colours and the mammary ducts labelled with SMA or Keratin 14 (KRT14), both depicted in white. c , Clone size quantification of luminal (cyan dots) and basal (blue dots) Brca1;Trp53 confetti clones (left) and WT confetti clones represented on a logarithmic scale. For each time point at least n  = 6 glands from three mice were analysed. Morphologically transformed clones are indicated in orange (luminal) and red (basal). The analysed numbers of clones for each time point are indicated. Boxplots mark the 25th and 75th percentiles, the line indicates the median and the whiskers mark the minimum and maximum values. Significance was tested using a two-sided Mann–Whitney test, **** P  < 0.0001. d , Average surviving basal and luminal clone fraction as a function of time normalized to the average number of confetti + cells 14 days after recombination. Each data point shows the average of at least n  = 3 mice per time point. Error bars represent ±s.e.m. From a longitudinal data analysis between the Brca1;Trp53 and WT clones there is no significant difference between the groups (Supplementary Information 2 ). See Supplementary Information 1 for sample sizes, P values and statistics for c and d . Scale bars, 50 µm.

Ductal turnover in small MaSC-descendant units

Next, we questioned the factors that drive field clonalization. The mammary epithelium contains a hierarchy of MaSCs 23 and their short-lived descendants 19 , 21 , 24 , 25 . It is therefore expected that confetti-labelled WT descendants should be lost over time, whereas MaSC-derived clones should spread to their descendants, potentially leading to field clonalization. To test this hypothesis, we quantified the size and position of WT confetti clones over time. Because clones were found to change transiently during the oestrous cycle as a result of growth and regression of side branches 9 , 19 , 21 (Extended Data Fig. 6a–c ), quantifications were made at the oestrus stage to avoid potential inconsistencies. Consistent with a MaSC-descendant hierarchy, we found that the number of surviving WT confetti clones showed a steep decrease in the first few months following induction (Fig. 2d ). Between 10 and 20% of stochastically recombined cells seemed to show long-term renewal capacity, whereas 80–90% were relatively short-lived, consistent with a small MaSC-descendant hierarchy of five to ten cells. With ductal homeostasis supported by repeated small MaSC-descendant units, we reasoned that MaSCs must be distributed widely throughout the ductal network. To test this, we reconstructed the topology of the ductal network (Extended Data Fig. 7a ) and identified for each WT clone its relative position within the tree based on its ‘branch level’, defined as the number of branch points that separate the clone from the main duct close to the nipple (Extended Data Fig. 7b ). This analysis indeed showed that WT clones were scattered uniformly throughout the ductal network, even after 550 days of tracing (Extended Data Fig. 7c,d ).

Altogether, our data showed that the behaviour of WT clones is in line with a model in which the homeostatic renewal of the mammary epithelium is organized in repetitive MaSC-descendant units that are distributed evenly throughout the ductal network. This tissue hierarchy supports a model in which field clonalization is preceded by the loss of most mutations that are acquired in short-lived descendants, followed by the spread of mutations from the randomly distributed MaSCs to their adjacent short-lived progenies.

It remains to be determined how the MaSC populations labelled by our unbiased lineage tracing approach relate to the populations identified through promoter-specific tracing strategies, including those based on Bcl11b , Tspan8 or ProcR expression 29 , 41 , 42 . For example, long-lived, quiescent stem cells have been identified in label-retaining assays, and might have a specialized function under perturbed conditions, such as pregnancy or repair 35 , 41 , 42 .

Clonal spread follows a simple statistical rule

In an MaSC-descendant unit of five to ten cells, a WT clone originating from an MaSC could never exceed around ten cells. However, we observed clones that become hundreds of times larger (Fig. 2c ). In other hierarchically organized epithelial tissues, clones can continually expand through a ‘neutral’ process of stochastic stem cell loss and replacement 43 . In homeostasis, such behaviour finds a signature in the statistical scaling behaviour of clone sizes 44 . However, when analysing the distribution of WT clones, we could not find evidence for such statistical scaling behaviour in individual animals across different time points (Extended Data Fig. 8a,b and Supplementary Information 4 ).

We therefore questioned whether other statistical features of the clone size distribution could provide insight into the underlying dynamics. On the basis of the huge variability of WT clone sizes (Figs. 2c and 3a and Extended Data Fig. 8c ), we questioned whether the distribution of the logarithm of clone size, ln  n , might show evidence of scaling. Notably, defining \(C(w,t)\) as the probability of finding a clone with a size larger than w  = ln  n , we found that, when plotted as a function of \(\left(w-\mu \left(t\right)\right)/\sigma (t)\) , where \(\mu (t)=\langle {\rm{l}}{\rm{n}}\,n\rangle \) denotes the average of the logarithm of clone size and \({\sigma }^{2}(t)=\langle ({\rm{l}}{\rm{n}}\,n-\langle {\rm{l}}{\rm{n}}\,n\rangle {)}^{2}\rangle \) represents its variance, the clone size data collapsed onto a single scaling curve (Fig. 3b , Extended Data Fig. 8d and Supplementary Information 4 ); that is, \(C(w,t)=C((w-\mu (t))/\sigma (t))\) , where the scaling function \(C(x)\) is time independent ( Supplementary Information 4 ). Further, inspection of the distribution showed that the scaling function fits well with a log-normal size dependence, with \(C(x)=(1/2){\rm{e}}{\rm{r}}{\rm{f}}{\rm{c}}(x/\sqrt{2})\) , where erfc denotes the complementary error function. These findings provide evidence of a statistical scaling behaviour of WT clone sizes distinct from that encountered in systems supported by local stochastic stem cell loss and replacement 44 , pointing to a different pattern of homeostatic turnover. Yet, the emergence of a simple and conserved pattern of WT clone size expansion, dependent only on the average and variance, \(\mu (t)\) and \({{\sigma }}^{2}(t)\) , suggested that the variability of WT clone sizes derived from cells conforming to a common statistical rule (Fig. 3c,d and Extended Data Fig. 8e,f ).

a , Cumulative distribution of WT (left) and Brca1;Trp53 (right) luminal confetti clone sizes showing the probability of finding a clone larger than the given size (log scale). To account for the impact of large-scale mouse-to-mouse variability on clone size, the curves are shown for a representative set of individual mice (distributions are shown for all mice in Supplementary Information 4 ). n  ≥ 3 mice per time point. b , Rescaled cumulative distribution of the logarithm of WT (left) and Brca1;Trp53 (right) luminal confetti clone size, ln  n , showing the probability of finding a clone with a size larger than \(({\rm{l}}{\rm{n}}\,n-\mu )/\sigma \) , where \(\mu =\langle {\rm{l}}{\rm{n}}\,n\rangle \) denotes the average of the logarithm of clone size and \({\sigma }^{2}=\langle ({\rm{l}}{\rm{n}}\,n-\langle {\rm{l}}{\rm{n}}\,n\rangle {)}^{2}\rangle \) represents the variance. Points show data from a . Once rescaled, data from different time points collapse onto a single curve that fits well with the scaling function \((1/2){\rm{erfc}}(x/\sqrt{2})\) (cyan dashed line), consistent with a log-normal size dependence. For details of statistical significance tests, see  Supplementary Information 4 . c , Variance of the logarithm clone size, \({\sigma }^{2}(t)\) , as a function of the inferred oestrous cycle number for luminal (black) and basal (blue) WT (left) and Brca1;Trp53 (right) confetti clones. Points show data from individual mice and lines (dashed) show a fit to a linear growth characteristic, as predicted by a minimal model of clonal fate based on stochastic growth and regression (main text and Supplementary Information 4 ). d , Average of the logarithm clone size, μ ( t ), as a function of the inferred oestrous cycle number for luminal (black) and basal (blue) WT (left) and Brca1;Trp53 (right) confetti clones. Points show data collected from individual mice and lines (dashed) show a fit to a linear growth characteristic, as predicted by a minimal model. See Supplementary Information 1 for sample sizes and statistics for a – d .

Oestrous cycle drives loss and replacement of MaSCs

Log-normal clonal distributions typically emerge from statistical processes in which the fate of proximate cells—amplification or loss—is positively correlated 45 . This could arise artefactually as the result of clone fragmentation or merger events 46 . However, the contiguity of WT clonal patches and sparsity of clonal labelling ruled against this possibility (Extended Data Fig. 4c,e ). We therefore considered whether previous observations of cyclic expansion of the pool of MaSCs 19 , 21 and regionally localized bouts of proliferation and cell death following the turnover of side branches during the oestrous cycle (Extended Data Fig. 6a–c ) 9 , 19 , 21 could locally correlate MaSC fate. To test this, we used an 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay throughout at least one oestrous cycle. Even though EdU labelling showed heterogeneity along the ductal network (Fig. 4a ), Ripley’s L function-based cluster analysis of the position of EdU + cells provided evidence for a local correlation in proliferative activity (Fig. 4b and Extended Data Fig. 9a ). This clustered proliferation decreased when we halted the oestrous cycle by ovariectomy (Fig. 4b ).

a , 3D views of mammary ducts showing EdU incorporation over 1 week in oestrous-cycling mice (top, three mice) and after ovariectomy (bottom, five mice), stained for CK8 and SMA. b , Ripley cluster analysis of EdU + cell clusters along mammary ducts in cycling (green) and ovariectomized (black) mice. Data are mean ± s.e.m., five regions per mouse, three cycling mice, five ovariectomized mice. c , Branching dynamics in KikGR mice imaged through a mammary imaging window over 1 week. Representative examples of side branch expansion and regression are shown as indicated, n  = 5 mice. d , Schematic of the repeated skin-flap procedure to visualize the mammary tree using intravital microscopy. e , Top panels show in vivo overviews of the fourth mammary gland of a R26-mTmG mouse at 3 (left) and 6 months of age (right) during oestrus. Bottom panels show outlines of the ductal tree with the main ducts in blue and side branches in red. Representative of four animals. f , Top panels show in vivo confocal images of the ductal area (red box in b ) at 3 (left) and 6 months of age (right). Bottom panels show outlines with the main ducts in blue and side branches in red. g – j , Quantification of segment length of ducts ( g ), tertiary branch length ( h ) and tertiary branch complexity ( i ) at 3 and 6 months of age. j , Difference (Δ) in the number of tertiary branches between 6 and 3 months of age. Data derived from i . Colours indicate different mice, lines connect measurements of the same structures. Significance tested using a paired t -test, two-sided. See Supplementary Information 1 for more sample sizes, P values and statistics. Scale bars, 100 μm ( a ), 500 μm ( c , e , f ).

To find further evidence for local tissue remodelling during the oestrous cycle, we visualized ductal remodelling by repeated rounds of intravital microscopy 47 , 48 . Indeed, over the course of a single cycle, we observed both the local formation and loss of side branches (Fig. 4c ). Imaged over the course of 3 months (more than 12 cycles) using a repeated skin-flap method (Fig. 4d ), the spatial organization of main ducts remained largely unaltered (depicted in blue, Fig. 4e–h ). By contrast, the number of side branches increased over time, as well as their size and morphology, suggesting a small bias towards ‘bursts’ of localized growth over loss (as depicted in magenta, Fig. 4e,f,i,j ). Together, these findings are in line with previous studies 9 , 19 , 21 and support the presence of regional bursts of proliferation linked to oestrous cycle-mediated turnover of side branches.

Modelling local MaSC fate reproduces clonal spread

To test quantitatively whether the observed bouts of local proliferation lead to the observed variation in sizes and temporal growth characteristics of WT clones, we derived a minimal model of MaSC fate in which individual MaSCs become active with some probability on side branch turnover during each oestrous cycle, after which they collectively expand or become altogether lost, with a relative probability that ensures long-term homeostasis (Extended Data Fig. 9b and Supplementary Information 4 ). As well as recapitulating the observed log-normal size dependence of WT clones, this minimal model predicted the dynamics of clone growth, including the emergence of a linear-like increase in the variance and average of the logarithm of clone size as a function of time (Fig. 3c,d ), as well as the average clone size and the decay in the fraction of single-cell clones (Extended Data Fig. 8e,f ). Note that, here, to account for large-scale mouse-to-mouse variation in the clonal dynamics, we used the coscaling of the average and variance of the logarithm of clone size to regress out a timescale, measured in terms of an effective oestrous cycle number, benchmarked against measurements obtained from previous studies (see  Supplementary Information 4 for further details and fit parameters).

From a quantitative fit to the clone size data, we estimated that each MaSC supports just a few short-lived descendants on average, consistent with the pattern of clonal loss observed soon after induction (Fig. 2d ). From a fit to the average growth characteristics, we estimated that each MaSC becomes active, contributing to the formation of a side branch, roughly once per ten oestrous cycles. Indeed, proliferation and apoptosis vary spatially during the oestrous cycle 9 , 19 , 21 and the same cells are not in a proliferative state each cycle 14 , 15 , 49 . Owing to MaSC turnover, the model predicted that over a single cycle, 50% of ‘activated clones’ are lost while the surviving clones expand proportionately (that is, field clonalization), a result consistent with the observed formation and loss of side branches (Fig. 4c ). With such a localized and cooperative pattern of turnover, it is plausible that MaSC fate would be highly correlated spatially (schematic in Extended Data Fig. 6a ). Although neighbouring MaSCs labelled with different confetti colours were extremely rare owing to the sparse labelling, when such events did occur we found that their expansion was highly correlated, even when clones belonged to independent HR + and HR − sublineages (Extended Data Fig. 9c ).

Spread of mutant and WT clones follows similar rules

Having traced the origin of WT clone expansion and field clonalization during the oestrous cycle, we turned to consider the dynamics of mutant confetti clones, quantifying 1,745 Brca1;Trp53 clones in 64 glands from 19 mice at time points from 14 to 225 days after labelling (Fig. 2c and Extended Data Fig. 5e,f ). To focus on non-transformed or early transformed lesions, we excluded the palpable lesions (if present) from our analyses. This analysis showed that the spread of Brca1;Trp53 confetti clones mirrored that of WT clones, showing the same conserved pattern of expansion with a log-normal size dependence signature (Fig. 3a,b , Extended Data Fig. 8c,d and Supplementary Information 4 ), and a corresponding linear-like increase in the average and variance of the logarithm of clone size (Fig. 3c,d and Supplementary Information 4 ). Moreover, the total number of surviving Brca1;Trp53 confetti clones showed a similarly steep decrease in the first few months following induction (Fig. 2d ). A fit to the linear growth characteristics showed that luminal Brca1;Trp53 confetti clones experience a net spreading advantage over WT confetti clones, with a marginal increase in the degree of amplification during the oestrous cycle, suggesting a resistance to loss during regression ( Supplementary Information 4 ), a result that we could confirm in vitro (Extended Data Fig. 9d ).

We then investigated whether the spreading advantage of Brca1;Trp53 confetti clones had a regional dependence, distinguishing between clones localized in the static main ducts and those in the more dynamic side branches. Focusing on the 225-day time point, for WT confetti clones, we found no regional dependence (Extended Data Fig. 9e ). By contrast, although clone numbers were small, the distribution of Brca1;Trp53 confetti clones in side branches showed a bias towards larger clone sizes characterized by a much narrower size distribution when compared to the main ducts (Extended Data Fig. 9e ), which coincided with higher chance of transformation (Extended Data Fig. 9f ). As Brca1;Trp53 cells seem to be more resistant to loss (Extended Data Fig. 9d ), mutant side branches generated during the oestrous cycle may be more durable than those formed by WT cells, enabling clones to spread more readily.

Clonal spread is restrained by the ductal geometry

Within the framework of the minimal (non-spatial) model of clone growth, surviving WT and Brca1;Trp53 clones are predicted to expand in size indefinitely at an exponential rate. Yet, such behaviour must become untenable in a tissue context. Therefore, to investigate how the tissue geometry might influence clone expansion, we developed a spatial model, representing the ductal epithelium as a one-dimensional ‘lattice-like’ ribbon of cells. By modelling the observed localized and cooperative loss and replacement of MaSCs during the turnover of side branches each oestrous cycle (Extended Data Figs. 6a and 9b ), we could reproduce quantitatively the log-normal size distribution (Extended Data Fig. 9g and Supplementary Information 4 ). However, at long times, the lattice model predicted that the expansion of clones should become suppressed at the length scale of the remodelled regions, and the clone size distribution cross over from log-normal to a narrower Gaussian-like dependence, consistent with the dynamics expected for neutral cell competition between neighbouring cells in one dimension (Extended Data Fig. 9h ). Consistently, applied to the experimental data, departures from the log-normal dependence could be observed for both WT and Brca1;Trp53 confetti clones when clone sizes spanned hundreds of cells or more (Extended Data Fig. 9i,j ). Together, these results indicate that the one-dimensional ductal organization provides a geometrical constraint that ultimately limits the range expansion of clones, limiting field clonalization and cancerization.

Pregnancy does not increase spread of mutant clones

Next, we questioned how the fate of mutant clones is perturbed during pregnancy, which is associated with massive tissue remodelling. Following induction of Brca1;Trp53 mutant clones in 8–12-week-old mice, at day 64 post-induction, mice went through a round of pregnancy, lactation and involution (Extended Data Fig. 10a ). Parous Brca1;Trp53 mice were analysed at 120 days post-induction and compared to their nulliparous counterparts. Although pregnancy-driven reshaping of the ductal network leads to massive expansion and involution of localized areas 12 , this did not lead to significant changes in clone sizes in the parous versus nulliparous mice (Extended Data Fig. 10b ). Further inspection of mutant clone sizes in parous and nulliparous mice revealed a striking shift in the clone size distribution, with the former having a relatively low number of large clones, potentially the result of skipping several oestrous cycles (Extended Data Fig. 10b ). In contrast to the nulliparous mice, no transformed clones were observed at 120 days post-induction in the mammary glands of parous Brca1;Trp53 mice (Extended Data Fig. 10b–d ), consistent with the conclusion that clonal expansions make ducts more susceptible to transformation. It also echoes the results of previous studies demonstrating the protective role of early parity in breast cancer 50 . In line with this reasoning, in nulliparous glands mutant clones localized at the dynamic side branches showed a bias towards large sizes and have a proportionately higher propensity to transform (Extended Data Fig. 9e,f ). Such behaviour mirrors that found in humans, in which transformation is also thought to occur predominantly in side branches—the terminal ductal lobular units 51 , 52 .

Abolishing mutant spread reduces transformation

To further test the relation between clone size and transformation susceptibility, we blocked experimentally the expansion of Brca1;Trp53 confetti clones by performing an ovariectomy. If rapid amplification of clone sizes is associated with oestrous cycle-driven side branch turnover, we reasoned that, in its absence, the susceptibility for transformation should be largely abolished 53 . Indeed, at later time points (120 and 225 days), when large-scale expansion of clones becomes evident in non-ovariectomized glands, no extensive spread of WT and mutant clones was observed in ovariectomized glands (Fig. 5a–f and Extended Data Figs. 11 and 12a–d ). By contrast, the surviving clone fraction still decreased (Fig. 5g ), consistent with the turnover of cells within the MaSC-descendant units; although for Brca1;Trp53 clones there was a slower loss rate as compared to non-ovariectomized mice ( P  < 0.05, Supplementary Information 2 ). Notably, there was a trend towards a faster loss of WT over Brca1;Trp53 confetti clones, consistent with a survival advantage of the former over WT neighbours (Fig. 5g and Supplementary Information 2 ). The limited spread of the Brca1;Trp53 confetti clones was accompanied by a near complete loss of susceptibility for transformation (Fig. 5h,i ).

a , Luminal (cyan) and basal (blue) WT confetti clone sizes in the homeostatic gland (left, same as Fig. 2c ), and after ovariectomy (right). b , c , Representative whole-mount confocal images of luminal WT confetti clones 120 days ( b ) and 225 days ( c ) after recombination in ovariectomized mice ( n  ≥ 3 mice per condition). Luminal cells are labelled with ECAD ( b ), basal cells are labelled with SMA ( c ). Images depict 3D rendering of Z -stacks, unless otherwise indicated. d , Luminal (cyan) and basal (blue) Brca1;Trp53 confetti clone sizes in the oestrous-cycling condition (left, same as Fig. 2c ), and after ovariectomy (right). e , f , Representative whole-mount confocal images of luminal Brca1;Trp53 confetti clones 120 ( e ) and 225 days ( f ) after recombination in ovariectomized mice ( n  ≥ 3 mice per condition). Luminal cells labelled with ECAD ( e ), basal cells labelled with SMA ( f ). Images depict 3D rendering of Z -stacks, unless otherwise indicated. g , Surviving clone fraction in ovariectomized mice as function of time normalized to the average number of confetti + cells at 14 days. Error bars represent mean ± s.e.m. h , Non-transformed luminal or basal clones (grey) and transformed luminal clones (orange) in the ovariectomized Brca1;Trp53 confetti mouse model. i , Transformed luminal (L, orange) and basal (B, red) clones as percentages of the total number of luminal or basal clones, respectively, in the cycling and ovariectomized conditions. Each dot indicates an individual mouse. a , d , h , Clone numbers are indicated. a , d , i , Boxplots mark 25th and 75th percentile, the line indicates median and the whiskers mark minimum and maximum values. Significance was tested using a two-sided Mann–Whitney test, **** P  < 0.0001. See Supplementary Information 1 for more sample sizes, P values and statistics. Scale bars, 100 µm.

The abundance of mutant cells in human breast tissue from healthy individuals, and its potential relevance for the initiation and recurrence of breast cancer, have long been recognized 1 , 7 , 8 . Here, by tracing the dynamics of WT and Brca1;Trp53 confetti clones in mouse tissue, we have addressed the cellular basis of field cancerization and the sick lobe theory. In contrast to prevailing models of the mouse mammary gland 29 , 42 , 54 , 55 and human breast tissue 56 , 57 , our results indicate that MaSCs are distributed uniformly along the ductal tree, a finding resonant with a previous study of human breast tissue 34 . Following the induction of WT or mutant clones, only those clones that are ‘rooted’ in the MaSC compartment survive over the short term, with their spread limited to their descendant units. In the absence of the oestrous cycle, the spread of mutant clones beyond these units remains limited, even over the long term. However, during stages of the oestrous cycle, ovarian hormones act on HR + luminal cells, triggering remodelling of side branches leading to localized proliferation and apoptosis of HR + , HR − luminal and basal cells 14 , 15 , 20 , 30 . These bouts of growth and regression drive the local and coordinated expansion and loss of MaSCs (regardless of their HR expression), leading to the elimination of most mutant clones, including those bearing oncogenic driver mutations, whereas those that by chance survive expand exponentially (Extended Data Fig. 12e ). Therefore, any clone, regardless of its size, HR status or proliferation capacity, can by chance either increase or decrease in size at any given oestrous cycle. This explains why the spread of clones negative for the HR (for example, basal or Brca1;Trp53 confetti clones) still depends on the oestrous cycle. This process of field clonalization enables entire ductal subtrees to become predisposed to the development of aberrant ductal lesions and transformation, a behaviour resonant with the abundance of signature genomic aberrations observed in both large transformed and non-transformed Brca1;Trp53 confetti clones. Moreover, consistent with these findings, clinical observations show that the risk of tumorigenesis increases with the number of menstrual cycles, the human variant of the oestrous cycle. In particular, the risk of breast cancer correlates with the age of entry into menopause, with increasing age indicating an enhanced number of cycles 58 , 59 , 60 . Yet, it is essential to emphasize that the dependence of clonal expansion on the oestrous cycle is different at later stages of tumour development. At the stage when Brca1;Trp53 tumours grow invasively, clonal expansion is no longer affected by the tissue remodelling that accompanies the oestrous cycle or limited by the one-dimensional architecture of the ductal network, and thus tumour growth is unaffected by ovariectomy 53 .

All mice used for experiments were adult females from a mixed background, housed under standard laboratory conditions and receiving food and water ad libitum. All experiments were performed in accordance with the guidelines of the Animal Welfare Committees of the Netherlands Cancer Institute and KU Leuven. Sample size was determined using a resource equation approach, mice were randomly assigned to experimental groups and blinding was performed during data analysis. R26R-Confetti het (JAX stock no. 013731) 61 , 62 ; R26-CreERT2 het (JAX stock no. 008463) 63 mice were injected intraperitoneally with tamoxifen (Sigma-Aldrich), diluted in sunflower oil, to activate Cre recombinase. To achieve clonal density labelling (fewer than one MaSC per duct on average), R26R-Confetti het ;R26-CreERT2 het mice were injected with 1 mg of tamoxifen per 25 g of body weight between 10 and 15 weeks of age. Ovariectomies were performed between 10 and 15 weeks of age, at least 7 days before lineage tracing initiation. The third, fourth and fifth mammary glands of R26R-Confetti;Brca1 fl/fl ;Trp53 fl/fl (refs. 31 , 64 ) or R26R-Confetti mice were intraductally injected with recombinant TAT-Cre protein (20 units per gland diluted in 20 µl of PBS, Sigma-Aldrich) between 10 and 15 weeks of age. This TAT-Cre injection resulted in roughly one labelled cell for every 100–200 cells (Extended Data Fig. 4e ). As the confetti construct comprises four distinct colours, there is, on average, one cell labelled with a confetti colour per 400 cells. Considering that a MaSC-progeny unit consists of roughly five to ten cells, a single confetti-labelled cell is induced in one out of 40–80 units. Over time, many clones become extinct, leading to a dilution in the number of clones and making collisions even less likely. For each of the experiments, mice were analysed at different time points after lineage tracing initiation as indicated in Fig. 1b . The injected mammary glands of R26R-Confetti;Brca1 fl/fl ;Trp53 fl/fl mice at the latest time point (225 days) were analysed when one of the injected glands developed a palpable tumour of at least 5 × 5 mm, which was between 200 and 250 days after recombination. Tumour sizes did not exceed 1,500 mm 3 in accordance with the guidelines of the Animal Welfare Committee of the Royal Netherlands Academy of Arts and Sciences, the Netherlands Cancer Institute and KU Leuven. Samples were randomly allocated to the experimental groups, sample size was not determined a priori and investigators were not blinded to experimental conditions, except where indicated. For clonal analysis of the R26R-Confetti;Brca1 fl/fl ;Trp53 fl/fl model, we analysed n  = 4 mice (14 days), n  = 4 mice (64 days), n  = 5 mice (120 days) and n  = 6 mice (225 days). For clonal analysis of the R26R-Confetti model, we analysed n  = 3 mice (14 days), n  = 3 mice (64 days), n  = 5 mice (120 days) and n  = 6 mice (225 days). Clonal analysis of the ovariectomized R26R-Confetti;Brca1 fl/fl ;Trp53 fl/fl and R26R-Confetti het models was performed on at least n  = 3 mice per time point. Adult CAG;;KikGR female mice 65 (RIKEN no. CLSTCDB0201T-117830853340) were used to visualize the short-term dynamics of the mammary gland by repeated imaging through a mammary imaging window as described below. R26-mTmG female mice (JAX no. 007676) 66 were used to visualize the long-term stability of the mammary gland through a repeated skin-flap procedure as described below.

Mammary imaging window implantation, repeated skin-flap procedure and intravital imaging

Mice were anaesthetized using isoflurane (Isovet) inhalation (1.5/2% isoflurane/air mixture). The fourth mammary gland of adult CAG;;KikGR female mice was imaged repeatedly through a mammary imaging window as previously described 47 , 48 . The fourth mammary gland of adult R26-mTmG mice was imaged repeatedly with a skin flap as previously described 47 . To visualize the mammary gland, mice were placed in a facemask within a custom designed imaging box. Isoflurane was introduced through the facemask and ventilated by an outlet on the other side of the box. The imaging box and microscope were kept at 34 °C by a climate chamber surrounding the entire stage of the microscope including the objectives. Imaging was performed on an inverted Leica SP8 Dive system (Leica Microsystems) equipped with four tuneable hybrid detectors, a MaiTai eHP DeepSee laser (Spectra-Physics) and an InSight X3 laser (Spectra-Physics) using the Leica Application Suite X (LAS X) software. All images were collected at 8 bit and acquired with a ×25 water immersion objective with a free working distance of 2.40 mm (HC FLUOTAR L ×25/0.95W VISIR 0.17). For the CAG;;KikGR model, Kikume Green was excited at 960 nm and detected at 490–550 nm. Each imaging session, all visible ducts through the imaging window were imaged using a tiled z scan with ×1–2 zoom and a z -step size of 5–10 μm. For the skin-flap imaging, TdTomato was excited at 1,040 nm and detected at 540–730 nm. All visible ducts were imaged together in one tile z scan with a ×0.75 zoom, a z -step size of 10–20 μm. These parameters allowed to scan large regions of up to 2 cm 2 in less than 3 h. At the end of the first skin-flap imaging session, the skin was closed with a continuous, non-resorbable suture. After 3 months, the skin flap was re-opened for the second imaging session, and the same imaging fields were retraced using the nipple and collagen I structures of the first imaging session as landmarks.

Staging of the mice

To determine the oestrous cycle stage of the mice, a vaginal swab was collected as described 67 . In short, the vagina was flushed using a plastic pipette filled with 50 µl PBS, and the liquid was transferred to a dry glass slide. After air drying, the slide was stained with Crystal Violet and the cell cytology was examined using a light microscope.

Clone isolation and CNA sequencing

Here, 225 days after Cre mediated recombination, fourth mammary glands were extracted, fixed overnight in 1% paraformaldehyde (PFA), incubated in sucrose overnight and stored in optimal cutting temperature (OCT) at −80 °C. For microdissection of individual clones, OCT blocks were thawed at room temperature in the dark for 30 min and mammary glands removed from OCT and washed in 50 ml of PBS on ice. Mammary glands were dissected under a benchtop fluorescent macroscope (Zeiss) using Dumont forceps and fine scissors, using the clone morphology to distinguish between transformed and untransformed clones. Each dissected clone was washed in 1 ml of PBS on ice for 2–5 h (until the end of the dissection procedure). Per mammary gland, one piece of non-fluorescent tissue from the inguinal lymph node was dissected as internal sequencing control. After washing, pieces were lysed in 70 µl of Arcturus lysis buffer following the instructions of the ThermoFisher Scientific Arcturus PicoPure kit KIT0103. Lysis was carried out in a PCR cycler for 18 h at 65 °C followed by 30 min at 75 °C and holding at 4 °C. Samples were then purified using the Roche FFPE DNA extraction kit 06650767001 50-588-384 following the manufacturer’s instructions with elution in 25 µl of PCR grade water. For DNA sequencing, library preparation was carried out with a KAPA Hyper kit (Roche; KK8504) according to the manufacturer protocol with four PCR cycles, before the samples were sequenced by low-coverage whole genome sequencing. The copy number alteration (CNA) analysis was conducted in R, using QDNAseq with 50 kb bins and the mm10 mouse reference genome. This methodology yielded copy number values from both normal and transformed clones, along with an internal control sample. Normalization was achieved by first converting copy number values to log 2 , then subtracting the internal control sample’s values from those of the normal and transformed tumours. We averaged these adjusted copy number values for each replicate across both clone types (13 early normal clones and 13 early transformed clones). Data visualization was executed using the ggplot2 package in R, with specific emphasis on certain chromosomes. Regarding the late-stage tumours published in ref. 40 , copy number profiling data corresponding to ten Wap-Cre;Brca1 fl / fl ;Trp53 fl/fl (WB1P) female mice harbouring a WB1P late-stage mammary tumour, along with internal control samples (spleen) was used. The CNA sequence analysis included the use of cutadapt for adaptor sequence removal and BWA for sequence alignment (using bwa aln, bwa mem) to the mm10 mouse genome. This procedure mirrored the earlier steps up to plotting with ggplot2, repeated for ten WB1P replicates.

Quantification of the distribution of proliferation

Oestrous-cycling mice and mice that had undergone ovariectomy with a 2-week recovery period (all above 8 weeks of age) received 0.5 mg ml −1 EdU in drinking water (refreshed every second day) for 1 week. 3D imaging was performed on three cycling mice and five ovariectomized mice. Per mouse, one-quarter of the mammary gland was taken for subsequent analysis. Samples were fixed in 4% PFA overnight and stained using the FLASH protocol with FLASH Reagent 2 (ref. 68 ). Before adding the primary antibodies, samples were stained for EdU as follows. Tissues were incubated in 5 ml of 3% bovine serum albumin for 1 h, followed by three washes in PBS for 20 min each. EdU was detected with an Alexa-647 azide. The reaction cocktail for EdU fluorescent labelling was prepared according to the manufacturer’s guidelines using the Click-It EdU imaging kit (ThermoFisher Scientific). Per gland, 0.5 ml of reaction cocktail was added for incubation for 4 h at room temperature with gentle agitation on a nutator. The cocktail was removed, samples washed once in 3% bovine serum albumin in PBS for 20 min, followed by three washes in FLASH blocking buffer for 20 min each. Subsequently, samples were stained with primary and secondary antibodies overnight each. Primary antibodies used were KRT8 (rat, Troma-I, Merck Millipore, 1:800) and αSMA (mouse IgG2a, clone 1A4, ThermoFisher Scientific, 1:600). Secondary antibodies used were donkey antirat Alexa-488 and donkey antimouse Alexa546 (ThermoFisher Scientific, catalogue nos. A21208 and A10036, respectively, 1:400), combined with Hoechst 33342 for nucleus detection. Samples were imaged on an Andor Dragonfly spinning disc system, installed on an inverted Leica DMI8 microscope with an Andor Zyla 4+sCMOS camera using a ×10, 0.45 NA Fluo objective (Leica). Imaging was carried out with a 40 μm disc using 405 nm excitation, a 561 nm optically pumped semiconductor laser and 637 nm diode lasers. Images were visualized with Imaris Viewer using gamma correction, ortho slicers and cutting planes to depict deeper tissue layers. For each mammary gland, distribution of proliferation was quantified in five regions. Ripley analysis using QuPath 69 was performed with a custom-made script (available at https://github.com/BioImaging-NKI/qupath_ripley ). The image was opened in QuPath and a freehand line was drawn by hand to outline the duct for analysis. A multipoint annotation was drawn by hand to mark the positions of proliferating cells along the duct. The script calculated Ripleys K function and normalized it to an unclustered distribution resulting in Ripley’s L function. Data were plotted in GraphPad Prism v.10. For simulations, we have generated clustered and unclustered data in Python.

Whole-mount immunofluorescence staining of mammary glands

The third, fourth and fifth mammary glands were dissected and incubated in a mixture of collagenase I (1 mg ml −1 , Roche Diagnostics) and hyaluronidase (50 μg ml −1 , Sigma-Aldrich) at 37 °C for optical clearance, fixed in periodate–lysine–PFA buffer (1% PFA; Electron Microscopy Science), 0.01 M sodium periodate, 0.075 M l -lysine and 0.0375 M P-buffer (0.081 M Na 2 HPO 4 and 0.019 M NaH 2 PO 4 ; pH 7.4) for 2 h at room temperature, and incubated for at least 3 h in blocking buffer containing 1% bovine serum albumin (Roche Diagnostics), 5% normal goat serum (Monosan) and 0.8% Triton X-100 (Sigma-Aldrich) in PBS. Primary antibodies were diluted in blocking buffer and incubated overnight at room temperature. Secondary antibodies diluted in blocking buffer were incubated for at least 6 h. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (0.1 μg ml −1 ; Sigma-Aldrich) in PBS. Glands were washed with PBS and mounted on a microscopy slide with Vectashield hard set (H-1400, Vector Laboratories). Primary antibodies used were: anti-KRT14 (rabbit, Covance, PRB155P, 1:700), anti-ECAD (rat, eBioscience, 14-3249-82, 1:700), anti-oestrogen receptor (rabbit, no. 13258, Cell Signaling, 1:100), anti-progesterone receptor (rabbit, Clone SP2, MA5-14505, ThermoFisher Scientific, 1:200) and anti-SMA (mouse IgG2a, clone 1A4, Sigma-Aldrich, 1:600). Alexa Fluor 647 and Alexa Fluor 488 Phalloidin were used 1:500 (A-22287 and A-12379, ThermoFisher Scientific) and incubated together with the secondary antibodies. Secondary antibodies used were: goat antirabbit, goat antirat or goat antimouse IgG2a, all conjugated to Alexa-647 (ThermoFisher Scientific, catalogue nos. A21245, A21247 and A21241, respectively, 1:400).

Whole-mount imaging of mammary glands

Imaging of whole-mount mammary glands was performed using an inverted Leica TCS SP8 confocal microscope, equipped with a 405 nm laser, an argon laser, a diode-pumped solid-state laser 561 nm laser and a HeNe 633 nm laser. Different fluorophores were excited as follows: DAPI at 405 nm, cyan fluorescent protein (CFP) at 458 nm, green fluorescent protein (GFP) at 488 nm, yellow fluorescent protein (YFP) at 514 nm, red fluorescent protein (RFP) at 561 nm and Alexa-647 at 633 nm. DAPI was collected at 440–470 nm, CFP at 470–485 nm, GFP at 495–510 nm, YFP at 540–570 nm, RFP at 610–640 nm and Alexa-647 at 650–700 nm. All images were acquired with a ×20 (HCX IRAPO N.A. 0.70 WD 0.5 mm) dry objective using a Z -step size of 1–5 μm (total Z -stack around 200 μm). 3D overview tile scans of the mammary glands were acquired by scanning large tile-scan areas ( xyz ). Next, detailed images were obtained of the individual clones. All images were stitched and processed in the true 3D real-time Rendering LAS X 3D Visualization module (Leica Microsystems) and further processed using ImageJ software ( https://imagej.nih.gov/ij/ ).

Clonal analysis on whole-mount glands

Three-dimensional tile-scan images of whole-mount and fully intact mammary glands were used to manually reconstruct the ductal network by outlining the ducts based on the labelling by ECAD, SMA or KRT14 (between 400 and 600 tiles, ×10 objective, Z -step size of 5–10 µm). After localization of the confetti clones in these 3D overview scans, each clone was imaged in detail with a ×25 water objective using confocal imaging by taking a Z -stack with step size between 1 and 3 µm. On the basis of the overlap with the luminal- or basal-cell-specific labelling and cellular morphology (that is, a cuboidal shape for luminal cells and an elongated shape for basal cell), the labelled confetti cells were identified and annotated in the schematic outline of the mammary tree, including information on their confetti colour (GFP, green; YFP, yellow; RFP, red and CFP, cyan) and their identity: that is, luminal or basal. Regions in which, for technical reasons, the gland could not be visualized well were omitted from analysis (in three out of 160 glands). Clone sizes, referring to the number of cells within each clone, were determined through manual visual inspection of tissue samples, with the quantification performed by eye using detailed Z -stack images and 3D rendering of each individual clone. Using custom-made.NET software (available on request from J.v.R.), the coordinates of the branch points, and the position of the labelled cells in ducts and in ductal ends were scored. To calculate the surviving clone fraction, the total number of clones was determined for each of the indicated lineage tracing time points by analysing the entire mammary gland in three dimensions using our whole-gland imaging approach ( n  = 3 glands per time point of two individual mice). Next, the average numbers of clones identified at 64, 120 and 225 days after lineage tracing initiation were divided by the average number of clones identified 14 days after lineage tracing initiation resulting in the surviving clone fraction as depicted in Figs. 2d and 5g .

Mammary epithelial cell sorting and real-time qPCR

The third, fourth and fifth mammary glands of R26R-Confetti;Brca1 fl/fl ;Trp53 fl/fl mice were intraductally injected with recombinant TAT-Cre protein (20 units per gland diluted in 20 µl PBS, produced in-house) between 10 and 13 weeks of age. Then 120 to 180 days after injection, mammary glands were harvested, minced and digested at 37 °C for 30 min in a mixture of collagenase A (2 mg ml −1 , Roche Diagnostics), hyaluronidase (300 μg ml −1 , Sigma-Aldrich) and DNase (1 mg ml −1 ) in DMEM/F12 (Gibco). After 10 min incubation with TripLE (Gibco) at 37 °C cells were strained through a 100 μm cell strainer (Fisher Scientific) to obtain single cells. Cells were spun down for 10 min at 550 RCF (relative centrifugal force) at 4 °C followed by blocking for 15 min on ice in 5 mM EDTA/PBS with 2% sterile filtered normal goat serum (Gibco). CD45-Alexa-647 (clone 30-F11, 03123, Biolegend, 1:200) and EpCAM-APC/Cy7 (clone G8.8, 118218, Biolegend, 1:200) were diluted in 5 mM EDTA/PBS with 2% normal goat serum and incubated for 30–45 min on ice to label the immune population (CD45) and the epithelial population (epithelial cell adhesion molecule). Cells were centrifuged for 5 min at 800 RCF at 4 °C and pushed through a 35 μm cell strainer. The FACS Aria III Special Ordered Research Product (BD Biosciences) was used to sort confetti + and confetti cells, by applying a broad FSC/SSC gate, followed by gates excluding doublets (for the gating strategy, see Extended Data Fig. 1d ). Afterwards, non-immune (AF647 – ; 670/30) confetti-positive (RFP + (YG610/20), GFP + /YFP + (BL530/30), CFP + (V450/50)) and, separately, confetti-negative ((RFP – (YG610/20), GFP – /YFP – (BL530/30), CFP – (V450/50)) epithelial cells (APC/Cy7 + ; 780/60) were collected. Similarly, non-immune (AF647 – ; 670/30) epithelial cells (APC/Cy7 + ; 780/60) were collected from three R26R-Confetti;Brca1 fl/fl ;Trp53 fl/fl mice that had not received TAT-Cre intraductally as a negative control. Data were analysed in FlowJo v.10 for the gating strategy (Extended Data Fig. 1d ). Cells were spun down for 10 min at 800 RCF at 4 °C and DNA was isolated using the PicoPure DNA extraction kit (Applied Biosciences; KIT0103) according to the manufacturer’s instructions. The same method was applied to isolate genomic DNA (gDNA) from K14-Cre;Brca1 fl/fl ;Trp53 fl/fl mammary tumour organoids, representing fully recombined samples as a positive control. gDNA concentration was measured using the DeNovix DS-11 spectrophotometer. DNA was diluted to 50–75 ng ml −1 and used for real-time qPCR using the SYBR Green Master Mix (ThermoFisher Scientific, catalogue no. 4309155) in a QuantStudio 6 Flex Real-Time PCR system using the primers listed in the table below. Reactions contained roughly 75 ng of template gDNA and 1 µM of both forward and reverse primers in 20 µl reaction volume. Expression values were calculated by transforming delta–delta Ct values (2 -ΔΔCt ). Ribosomal Protein L38 (Rpl38) was used as a housekeeping gene. To confirm correct qPCR product amplification, 25 μl of qPCR product of the control samples ( R26R-Confetti;Brca1 fl/fl ;Trp53 fl/fl mice that had not received TAT-Cre intraductally) was loaded on an 2% agarose gel with loading buffer (Bioxline, catalogue no. BIO-37045) and a DNA ladder (Meridian Bioscience, catalogue no. BIO-33056) and run at 80 V for 1.5 h, after which the qPCR product was cut out of the gel and purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, catalogue no. 740609.50) according to the manufacturer’s instructions, and was confirmed by sequencing using the qPCR primers.

Generation of Brca1;Trp53 mutant and WT organoids followed by Ki-67/CC3 staining

The fourth and fifth mammary glands of R26R-Confetti;Brca1 fl/fl ;Trp53 fl/fl mice were intraductally injected with recombinant TAT-Cre protein (20 units per gland diluted in 20 µl PBS, Sigma-Aldrich) between 10 and 15 weeks of age. Then, 64 or 225 days post-induction, mammary glands were harvested and prepared for fluorescence-activated cell sorting (FACS) as described before. Both confetti-positive Brca1 −/− ;Trp53 −/− and non-recombined control cells were seeded in a 24-well plate, 10,000 cells per drop of Cultrex Basement Membrane Extract (Type 2, 3532-010-02, R&D Systems) and cultured in the DMEM/F12 (Gibco) supplemented with iInsulin-transferrin-selenium (100×, catalogue no. 41400045, Gibco), B-27 Supplement (50×, catalogue no. 17504044, Gibco), NAC 1.25 mM ( N -acetyl- l -cysteine, 0.125 M in PBS, catalogue no. 6169116, Biogems), mFGF2 2.5 nM (Fibroblast Growth Factor 2, catalogue no. 100-18B, PeproTech) and mEGF 2 nM (Epidermal Growth Factor, catalogue no. 3165-09, PeproTech). After 2 weeks of culture, organoids were fixed with 4% PFA (catalogue no. 47347, AlfaAesar) for 10–15 min at room temperature inside the Basement Membrane Extract droplet on an orbital shaker at 25 rpm. Afterwards, organoids were washed three times for 10 min with PBS, followed by incubation in permeabilization buffer (5% normal goat serum (catalogue no. 16210072, Gibco) and 0.5% Triton X-100 ((Sigma-Aldrich) in PBS) for 3 h. To stain for cell proliferation and cell death, primary antibodies Ki-67 (rat, clone SolA15, 14-5698-82, eBioscience, 1:100) and Cleaved Caspase-3 (rabbit, Asp175, no. 9661, Cell Signaling Technology, 1:400), respectively, were added in the blocking buffer (5% normal goat serum (Gibco) in PBS), and incubated overnight at 4 °C. Organoids were washed three times for 15 min with PBS and secondary antibodies goat antirabbit IgG Antibody, Alexa Fluor 647 (catalogue no. A21244, Thermo Scientific, 1:400), goat antirat IgG Antibody, Alexa Fluor 647 (catalogue no. A21247, Thermo Scientific, 1:400) were added and incubated for more than 5 h at room temperature covered in aluminium foil on an orbital shaker at 25 rpm. Organoids were washed three times for 15 min with PBS and stained organoids were mounted by adding 200 µl of Vectashield mounting medium (VECTASHIELD HardSet Antifade Mounting Medium, H-1400, Vector Laboratories). Organoid imaging was performed on an inverted Leica SP8 Dive system (Leica Microsystems), in which Alexa-647 secondary antibodies were excited at 635 nm and detected between 660 and 700 nm, and organoids were imaged using brightfield. The Ki-67/CC3 ratio was derived by first calculating the organoid area and Ki-67 + or CC3 + areas using ImageJ software ( https://imagej.nih.gov/ij/ ), then calculating the percentage of Ki-67 or CC3 expressing cells per organoid, followed by calculation of the Ki-67/CC3 ratio for every organoid.

P values and statistical tests performed are included in the figure legends or Supplementary Information 1 . The longitudinal data of the clone fractions (Figs. 2d and 5g ) was analysed using a regression model with a time effect, for which the interaction between time and group was tested. For full details, see Supplementary Information 2 . Details on statistics concerning the mathematical modelling can be found in the  Supplementary Information 4 .

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The raw clonal data are all provided in the source data. DNA sequencing data are available at the European Genome-Phenome Archive (ENA, https://www.ebi.ac.uk/ena/browser/home ) under accession number PRJEB71510 , secondary accession ERP156311 and PRJEB30443 (Sample accession numbers SAMEA5202116 – 5202120, 5202122 – 5202126).  Source data are provided with this paper.

Code availability

The procedures used to fit the parameters of the phenomenological theory to the experimental data are defined in Supplementary Information 4 . The basis of the cell-based model is also defined in Supplementary Information 4 . Stochastic simulations of the cell-based model were made using a dedicated Fortran code and the Mathematica software package. The code for the computational and statistical analyses is deposited on the GitHub repository ( https://github.com/BenSimonsLab/Ciwinska_Nature_2024 ). Data supporting the findings of Fig. 4b and Extended Data Fig. 9a , including computer code for stochastic simulations, are available at https://github.com/BioImaging-NKI/qupath_ripley . NET code for branching analysis used in Extended Data Fig. 7 is available from J.v.R on reasonable request. Code to determine longitudinal data statistics is provided in the Supplementary Information 2 .

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Acknowledgements

We thank the laboratories of van Rheenen and Scheele for critically reading the manuscript, and the Netherlands Cancer Institute (NKI) Animal facility, NKI BioImaging facility and the NKI genomics core facility for their technical support. This work was supported by the Boehringer Ingelheim Foundation (PhD Fellowship to C.L.G.J.S.), a Federation of European Biochemical Societies excellence award (to C.L.G.J.S.), the Research Foundation Flanders (PhD grant fundamental research no. 11L7222N to M.C.), EMBO (postdoctoral fellowship grant nos. ALTF 452-2019 to H.A.M. and ALTF 1035-2020 to C.L.G.J.S.) and the European Research Council (consolidator grant no. 648804 to J.v.R.), the Doctor Josef Steiner Foundation (to J.v.R.), the Netherlands Organization of Scientific Research (NWO) (Vici grant no. 09150182110004 to J.v.R., and Veni grant no. 09150161910151 to H.A.M.) and a joint grant of the Cancer Research UK and KWF Kankerbestrijding (ref. C38317/A24043). B.D.S. acknowledges funding from the Royal Society E.P. Abraham Research Professorship (grant nos. RP\R1\180165 and RSRP\R\231004) and Wellcome (grant nos. 098357/Z/12/Z and 219478/Z/19/Z). We regret that we could not cite all the important contributions in this field due to the constraint of being limited to citing only 60 studies. This research was funded, in part, by the Wellcome Trust (098357/Z/12/Z and 219478/Z/19/Z). For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.

Author information

These authors contributed equally: Marta Ciwinska, Hendrik A. Messal, Hristina R. Hristova

These authors jointly supervised this work: Benjamin D. Simons, Colinda L. G. J. Scheele, Jacco van Rheenen

Authors and Affiliations

VIB-KULeuven Centre for Cancer Biology, Department of Oncology, Leuven, Belgium

Marta Ciwinska & Colinda L. G. J. Scheele

Division of Molecular Pathology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, the Netherlands

Hendrik A. Messal, Hristina R. Hristova, Catrin Lutz, Laura Bornes, Nathalia S. M. Langedijk, Stefan J. Hutten, Jos Jonkers & Jacco van Rheenen

Centre for Molecular Medicine, UMC Utrecht, Utrecht, the Netherlands

Theofilos Chalkiadakis & Stefan Prekovic

Bioimaging Facility, The Netherlands Cancer Institute, Amsterdam, the Netherlands

Rolf Harkes

Biostatistics Centre and Department of Psychosocial Research and Epidemiology, The Netherlands Cancer Institute, Amsterdam, the Netherlands

Renée X. Menezes

Gurdon Institute, University of Cambridge, Cambridge, UK

Benjamin D. Simons

Cambridge Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK

Department of Applied Mathematics and Theoretical Physics, Centre for Mathematical Sciences, University of Cambridge, Cambridge, UK

The Netherlands Cancer Institute, Amsterdam, the Netherlands

Jelle Wesseling, Jos Jonkers, Jacco van Rheenen, Esther H. Lips, Marjanka Schmidt, Lodewyk F. A. Wessels & Proteeti Bhattacharjee

Baylor College of Medicine, Houston, TX, USA

Alastair M. Thompson

University of Cambridge, Cambridge, UK

Serena Nik-Zainal & Helen R. Davies

King’s College London, London, UK

Elinor J. Sawyer

MD Anderson Cancer Center, Houston, TX, USA

Andrew Futreal & Nicholas E. Navin

Duke University School of Medicine, Durham, NC, USA

E. Shelley Hwang

Kansas University Medical Center, Kansas City, KS, USA

Fariba Behbod

University of Birmingham, Birmingham, UK

Independent Cancer Patients’ Voice, London, UK

Hilary Stobart

Patient Advocates in Research, Danville, CA, USA

Deborah Collyar

DCIS 411, San Diego, CA, USA

Donna Pinto

Borstkankervereniging Nederland, Utrecht, the Netherlands

Ellen Verschuur & Marja van Oirsouw

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Grand Challenge PRECISION consortium

• Jelle Wesseling
• , Alastair M. Thompson
• , Serena Nik-Zainal
• , Elinor J. Sawyer
• , Helen R. Davies
• , Andrew Futreal
• , Nicholas E. Navin
• , E. Shelley Hwang
• , Jos Jonkers
• , Jacco van Rheenen
• , Fariba Behbod
• , Esther H. Lips
• , Marjanka Schmidt
• , Lodewyk F. A. Wessels
• , Daniel Rea
• , Proteeti Bhattacharjee
• , Hilary Stobart
• , Deborah Collyar
• , Donna Pinto
• , Ellen Verschuur
•  & Marja van Oirsouw

Contributions

C.L.G.J.S. and J.v.R. conceived the study and designed experiments. C.L.G.J.S., with the help of M.C., H.A.M., H.R.H., L.B. and N.S.M.L., performed experiments and analyses. S.J.H. and C.L. performed intraductal injections, supervised by J.J. R.H. wrote the Ripley’s K script. T.C. and S.P. supported the analysis of chromosomal aberrations. R.X.M. designed and performed the longitudinal data analysis. B.D.S. performed theoretical and statistical analyses. C.L.G.J.S., B.D.S. and J.v.R wrote the paper, which was approved by all authors.

Corresponding authors

Correspondence to Benjamin D. Simons , Colinda L. G. J. Scheele or Jacco van Rheenen .

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Competing interests.

The authors declare no competing interests.

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Nature thanks Mohamed Bentires-Alj, Alexander Anderson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended data fig. 1 stochastic recombination in the brca1 fl/fl ;trp53 fl/fl ;r26r-confetti mouse model..

a , Quantification of the total number of Brca1;Trp53 confetti clones after TAT-Cre mediated recombination in at least 4 different 4th mammary glands derived from different mice. Number of clones was determined by using large tilescans (xyz) of the entire mammary glands (whole-mount) labelled with SMA (basal cells) or ECAD (luminal cells). Luminal clones are depicted in cyan, basal clones are depicted in blue. b , qPCR for the Brca1 allele in sorted confetti-positive cells and sorted confetti-negative cells derived from TAT-Cre recombined Brca1 fl/fl ;Trp53 fl/fl ;R26R-Confetti mammary glands and a positive control ( Brca1 Δ /Δ ) normalized to a non-recombined control ( Brca1 F/F ). The recombined samples demonstrate that the vast majority of confetti-positive cells have fully recombined Brca1 alleles. n  = 3 biological replicates. Each dot represents a biological replicate and error bars indicate s.e.m. c , qPCR for the Trp53 allele in sorted confetti-positive cells and sorted confetti-negative cells derived from TAT-Cre recombined Brca1 fl/fl ;Trp53 fl/fl ;R26R-Confetti mammary glands and a positive control ( Trp53 Δ /Δ ) normalized to non-recombined control ( Trp53 F/F ). The recombined samples demonstrate that the vast majority of confetti-positive cells have fully recombined Trp53 alleles. Note that the confetti-negative cells show some loss of the Trp53 allele as well. Each dot represents a biological replicate and error bars indicate s.e.m. n  = 3 biological replicates. d , Representative plots depicting the gating strategy to sort the Confetti positive and negative mammary epithelial cells from TAT-Cre recombined Brca1;Trp53 ; R26R-Confetti mammary glands (left panels) and mammary epithelial cells from non-recombined mammary glands (right panels). Sorted cells were used to determine Brca1 and Trp53 gene levels by qPCR in Extended Data Fig. 1b and c . Numbers in panels indicate order of gating. The Brca1;Trp53 mammary cells in the RFP-CFP- gate in panel 5 were selected to sort GFP_YFP+ and Confetti- cells in panel 6. e , 3D rendering of a Z-stack confocal image of a whole-mount Brca1;Trp53 confetti mammary gland 225 days after recombination labelled with SMA, confetti clones are represented in their respective colours. Brca1;Trp53 mutant confetti clones are distributed throughout the mammary ductal tree and span large areas of the ducts without changing the ductal morphology. Note the recruitment of SMA-positive stromal cells near the GFP clone in panel 1. Scale bar represents 1 mm (overview image) and 100 µm (panel 1 and 2). Representative image of n  = 6 mice.

Extended Data Fig. 2 Characterization of Brca1;Trp53 confetti clones using antibody labelling in intact mammary glands.

a , b , Whole-mount confocal images (3D rendering of a Z-stack in top panels and a representative 2D section of the Z-stack in the bottom panel) of luminal Brca1;Trp53 confetti cells. Luminal confetti cells show overlap with E-cadherin (ECAD) labelling ( a ) and no overlap with alpha-smooth muscle actin (SMA) expression ( b ) depicted in white. Scale bars represent 100 µm. c, d , Whole-mount confocal images (3D rendering of a Z-stack in top panels and a representative 2D section of the Z-stack in the bottom panel) of basal Brca1;Trp53 confetti cells. Basal confetti cells show no overlap with E-cadherin (ECAD) labelling ( c ) and overlap with alpha-smooth muscle actin (SMA) expressing cells ( d ) depicted in white. Scale bars represent 100 µm. e , f , Representative whole-mount confocal images (3D rendering of a Z-stack) showing luminal ( e ) and basal ( f ) Brca1;Trp53 confetti cells that expanded within the ducts leading to clonal fields of mutant cells without morphological transformation of the ducts. Ducts are labelled with SMA, depicted in white. Scale bars represent 100 µm (e, left top panel) or 1 mm (other panels). g , Tumour growth dynamics of palpable transformed lesions within the Brca1;Trp53 confetti model after recombination. h , Representative whole-mount confocal image (3D rendering of a Z-stack) showing a Brca1;Trp53 luminal confetti clone that expanded within the ducts leading to transformation of the ductal morphology (hyperbranching), including local invasion (white arrows denote Brca1;Trp53 RFP cells within the stroma). Ducts are labelled with SMA, depicted in white. Scale bar represents 1 mm. All images represent n  ≥ 4 biological repeats (mice).

Extended Data Fig. 3 Genomic alterations in Brca1;Trp53 confetti clones and end stage tumours.

a , DNA copy number profiles in untransformed (top) and transformed (middle) Brca1;Trp53 confetti clones 225 days after Cre-recombination, and in Brca1;Trp53 end-stage tumours (bottom). b , Example of genomic events that occur early in Brca1;Trp53 tumorigenesis. DNA copy number profiles of chromosome 12 showing genomic losses that are found in early transformed and untransformed Brca1;Trp53 clones, as well as in end-stage tumours. c , Example of genomic events occurring late in Brca1;Trp53 tumorigenesis. DNA copy number plots of chromosome 6 showing copy number changes that are unique to end-stage Brca1;Trp53 tumours, but not found in early clones. All plots show averages of 3 mice (225d timepoint: 13 transformed clones and 13 untransformed clones) and 10 mice (end-stage tumours).

Extended Data Fig. 4 Recombination of wild-type confetti clones with TAT-Cre or tamoxifen results in similar labelling efficiencies.

a , Schematic representation of the R26R-Confetti construct (left), which was recombined sporadically through an intraperitoneal injection with a low dose of tamoxifen in the presence of R26-CreERT2 (R26R-Confetti het ;R26-CreERT2 het mouse model), which is the gold standard method, or through intraductal injection of TAT-Cre recombinant protein. b , Confocal overview image of a whole-mount 4th mammary gland derived from a R26R-Confetti het ;R26-CreERT2 het adult female mouse 14 days after tracing initiation by tamoxifen-mediated recombination. Zooms show ducts containing single confetti-labelled cells of both basal and luminal origin, representative of the initial labelling density after tracing initiation. Ductal tree is stained with alpha-smooth muscle actin (SMA) depicted in white, which marks the basal cell layer. Scale bar left image represents 1 mm, scale bar right images represents 100 µm. c , Quantification of the confetti-positive cell fraction 14 days after tamoxifen-mediated recombination. Each dot represents the fraction of recombined cells in a randomly selected area within each mammary gland of approximately 1 ×1 mm, n  = 6 glands derived from 6 different mice. Basal cells are normalized to the total number of basal cells in the selected area (blue dots) and luminal cells are normalized to the total number of luminal cells in the selected area (cyan dots). Error bar represents mean ± s.d. d , Confocal overview image of a whole-mount 4th mammary gland derived from a R26R-Confetti het adult female mouse, 14 days after tracing initiation recombined by the TAT-Cre intraductal injection method. Zooms show ducts containing single confetti-labelled cells of both basal and luminal origin, representative of the initial labelling density after tracing initiation. Ductal tree is stained with E-cadherin (ECAD) depicted in white, which marks the luminal cell layer. Scale bar left image represents 1 mm, scale bar right images represents 100 µm. e , Quantification of the confetti-positive cell fraction 14 days after TAT-Cre-mediated recombination for basal (blue dots) and luminal (cyan dots) cells. Each dot represents the fraction of recombined cells in a randomly selected area within each mammary gland of approximately 1 ×1 mm, n  = 3 glands derived from 3 different mice. Error bar represents mean ± s.d. Note that recombination efficiencies of luminal and basal cell populations are similar between the tamoxifen- and TAT-Cre-induced recombination techniques. Both induction methods result in the recombination of approximately one labelled cell for every 100–200 cells. As the Confetti construct comprises four distinct colours, there is, on average, one cell labeled with a confetti colour per 400–800 cells. Considering that a MaSC-progeny unit consists of approximately 5 to 10 cells, a single confetti-labeled cell is induced in 1 out of 40–80 units. Importantly, over time, many clones become extinct (Fig. 2d ), leading to a dilution in the number of clones and making collisions even less likely.

Extended Data Fig. 5 Wild-type and Brca1;Trp53 basal and luminal confetti cells form large cohesive clones spanning multiple ducts and branch points.

a , b , Representative whole-mount confocal images of wild-type luminal confetti clones ( a ) and wild-type basal confetti clones ( b ) showing extensive field clonalization within the existing ductal structure. Ducts are labelled with alpha-smooth muscle actin (SMA), confetti fluorophores are represented in their respective colours. Scale bars represent 100 µm. Images in a and b represent n  ≥ 3 biological repeats (mice). c , Representative whole-mount confocal images (3D rendering of Z-stacks) of basal Brca1;Trp53 confetti clones at different timepoints after recombination showing clonal expansion within the ductal tree over a period of 225 days. Ducts are labelled with SMA. d , Representative whole-mount confocal images of wild-type basal confetti clones showing extensive field clonalization within the existing ductal structure over a period of 225 days. Ducts are labelled with Keratin 14 (KRT14). c , d , Persisting clones form cohesive clusters of cells spanning multiple ducts and branch points. Scale bars represent 100 µm. e , f , Quantification of clone sizes in the Brca1;Trp53 and wild-type confetti conditions at different timepoints after tracing initiation for the luminal ( e ) and basal ( f ) clones separately. The number of quantified clones is indicated within the graph, transformed clones are shown in orange (luminal) and red (basal). Boxplots mark the 25th and 75th percentile, line indicates the median, and whiskers mark the minimum and maximum values. Significance was tested using a two-sided Mann-Whitney test, * P  < 0,05, ** P  < 0.01, **** P  < 0.0001. Same data as depicted in Fig. 2c . See Supplementary Information  1 for more sample sizes, P values and statistics for e and f .

Extended Data Fig. 6 Wild-type clone sizes transiently increase during each oestrous cycle.

a , Schematic depicting the cellular basis of the cell-based model of mammary epithelial turnover (see also Extended Data Fig. 9b ). Note that, during one round of oestrous cycle, some clones are collectively lost (e.g., yellow and green clone), while others expand (e.g., blue and red clones). b , Quantification of wild-type confetti + clone sizes during oestrus (O) and dioestrus (D) stage 120 days after lineage tracing initiation, demonstrating a temporary increase of clone sizes during dioestrus stage. n  = 3 mice for oestrus stage (300 luminal clones, 101 basal clones) and n  = 3 mice for dioestrus stage (43 luminal clones, 9 basal clones). Error bars represent mean ± s.d. Significance was tested using a two-sided Mann-Whitney test, **** P  < 0.0001. c , Representative whole-mount confocal images of wild-type confetti + clones 120 days after lineage tracing initiation during oestrus (left panels) and dioestrus stage (right panels). Both luminal (top panels) and basal clones (bottom panels) show an increase in clone size during dioestrus stage. Confetti-labelled cells are depicted in their respective colour, and the mammary ducts are labelled with Keratin 14 (KRT14) or Phalloidin in white. Scale bars represent 100 µm.

Extended Data Fig. 7 Spatial and size distribution of wild-type confetti clones in the mammary gland.

a , Representative confocal overview image of a whole mount 5th mammary gland after 550 days of lineage tracing, illustrating the distribution of wild-type confetti clones within the ductal tree. Images depict 3D-rendering of Z-stacks, with the confetti labelled cells in their respective colour and the mammary ducts labelled with Keratin 14 (KRT14) shown in white. Scale bars represent 1 mm (left panel), 100 µm (panel 1 and 2), and 50 µm (panel 3 and 4). Representative image of n  = 8 glands from 4 biological repeats (mice). b , Branch levels are defined as the number of branch points starting from the main duct close to the nipple. c , Quantification of the wild-type confetti clone size by branch level. Each dot represents a clone, cyan dots for luminal clones and blue dots for basal clones. Line indicates linear regression of the luminal and basal clone sizes with R 2 , slope and 95% confidence interval of the slope indicated in the graph, n  = 6 glands from 3 mice. d , Number of wild-type confetti clones represented by bars for each branch level after 550 days of tracing in n  = 6 glands from 3 mice.

Extended Data Fig. 8 Wild-type and Brca1;Trp53 confetti clones follow a log-normal distribution.

a , b , Cumulative distribution of luminal ( a ) and basal ( b ) wild-type confetti clone size as a function of the scaled clone size n / ⟨ n ⟩ , where ⟨ n ⟩ denotes the average clone size. To account for the impact of large-scale mouse-to-mouse variability in clone size, curves are shown for a representative set of individual mice (shown in Fig. 3a, b ) with corresponding distributions shown for all mice in  Supplementary Information 4 . Note that the data does not show evidence for collapse towards a statistical scaling behavior, as would be predicted for clonal dynamics based on local stochastic stem cell loss and replacement (see main text and Supplementary Information 4 ). n  ≥ 3 mice per time point. c , Cumulative distribution of wild-type (left) and Brca1;Trp53 (right) basal confetti clone size showing the probability of finding a clone larger than the given size (log scale) across time points. To account for the impact of large-scale mouse-to-mouse variability in clone size, the curves are shown for a representative set of individual mice with corresponding distributions shown for all mice in  Supplementary Information 4 . n  ≥ 3 mice per time point. d , Rescaled cumulative distribution of the logarithm of wild-type (left) and Brca1;Trp53 (right) basal confetti clone size, ln n , showing the probability of finding a clone with a size larger than \(({\rm{l}}{\rm{n}}\,n-\mu )/\sigma \) , where \(\mu =\langle {\rm{l}}{\rm{n}}\,n\rangle \) denotes the average of the logarithm of clone size and \({\sigma }^{2}=\langle ({\rm{l}}{\rm{n}}\,n-\langle {\rm{l}}{\rm{n}}\,n\rangle {)}^{2}\rangle \) represents the variance. Points show data from panel ( d ). Once rescaled, data from different time points collapse onto a single curve that fits well with the scaling function \((1/2){\rm{erfc}}(x/\sqrt{2})\) (dashed line), consistent with a log-normal size dependence (see main text). For details of statistical significance tests, see  Supplementary Information 4 . e , Average luminal clone size as a function of the inferred oestrous cycle number for wild-type confetti clones. Points show data from individual mice and line shows the theoretical prediction of the model. (Note that the average of the logarithm of clone size is not the same as the logarithm of the average.) f , Fraction of single-cell luminal wild-type confetti clones as a function of inferred oestrous cycle number. Points show data and line (dashed) shows theoretical prediction of the model based on the fits in Fig. 3c, d . Bars in e and f denote mean values +/− SEM. For details of the model, the model fits, and the inference of oestrous cycle number, see main text and Supplementary Information 4 .

Extended Data Fig. 9 Fits and predictions of the phenomenological theory of the turnover of the mammary gland epithelium.

a , Simulation of unclustered and clustered data analyzed with the Ripley’s K analysis leading to the Ripley’s L function. Two datasets were used for the analysis; 50 points from a uniform random distribution and 50 points from a normal distribution were generated for the clustered simulation, 100 random points from a uniform random distribution for the unclustered simulation. Details on the code and data can be found at https://github.com/BioImaging-NKI/qupath_ripley . b , Schematic depicting spatial model of ductal turnover (for details, see  Supplementary Information 4 ). The mammary ductal epithelium is represented as a one-dimensional lattice. During the oestrous cycle, random non-overlapping domains of size l cells are turned over so that the central domain of l /2 cells are lost and replaced by the stochastic expansion of the 2 × l /4 neighboring sites. Through iterations of this process, clones are continuously lost, while others expand. Once clones extend beyond the size of the activated domain, l , their further expansion proceeds as a process of stochastic expansion and contraction on the clone boundary. c , 3D-rendering of confocal Z-stacks (overview image) and single Z-plane (zoom images) showing full labelling of two luminal lineages in the same part of the mammary gland after 550 days of lineage tracing; a PR + clone labelling all PR + luminal cells in this region (confetti RFP), and a PR − clone labelling all PR − in this part of the mammary gland (confetti YFP). PR + luminal cells are shown in white, confetti YFP cells are shown in green. Scale bars represent 50 µm (overview image) and 10 µm (zoom images). Representative example of n  = 3 mice. d , Quantification of ratio between Ki-67 +  (proliferative) and cleaved caspase3 + (CC3) cells within organoids derived from wild-type (WT) or Brca1;Trp53 confetti+ mammary epithelial cells, 64 and 225 days post-recombination. Each dot represents an organoid ( n  = 33 organoids for WT condition, n  = 30 organoids for the Brca1;Trp53 64 days condition, and n  = 74 organoids for the Brca1;Trp53 225 days condition. Violin plots depict distribution of data points, horizontal lines denote median, 1st and 3rd quartile. Significance was tested using a two-sided Mann Whitney Test, *** P  < 0.0005, **** P  < 0.0001, ns P  = 0.1572. e , Quantification of luminal (cyan dots) and basal (blue dots) wild-type confetti clones (left) and Brca1;Trp53 confetti clones (right) in the ducts and side branches, represented on a logarithmic scale. For each timepoint at least n  = 6 glands from 3 mice were analyzed. Morphologically transformed clones are indicated in orange (luminal clones) and red (basal clones). Boxplots mark the 25th and 75th percentile, line indicates the median, and whiskers mark the minimum and maximum values. Significance was tested using a two-sided Mann-Whitney test, **** P  < 0.0001. f , Transformed luminal (L, orange) and basal (B, red) clones in the ducts and side branches as percentage of the total number of luminal or basal clones respectively. Each dot indicates an individual mouse and boxplots mark the 25th and 75th percentile, line indicates the median, and whiskers mark the minimum and maximum values. Significance was tested using a two-sided Mann-Whitney test, ** P  < 0.01. g , Cumulative distribution of the logarithm of clone size, ln n , obtained from the spatial cell-based model in ( b ) showing the probability of finding a clone with a size larger than \(({\rm{l}}{\rm{n}}\,n-\mu )/\sigma \) , where μ and \({\sigma }^{2}\) are obtained from a least-square fit of the data for n < l /2 to the log-normal size dependence, \((1/2){\rm{erfc}}(x/\sqrt{2})\) (dashed line) (cf. Fig. 3b and Extended Data Fig. 8d ). Here, each lattice site is associated with a renewing MaSC with a total domain size of l =1000 lattice sites. The points show the results of stochastic simulation of the spatial model (averaged over an ensemble of 1000 realizations of the model on a periodic lattice of 10 6 sites) for different numbers of oestrous cycles. In line with the quantitative analysis of the experimental data, the activation rate of domains is taken as 0.1 per oestrous cycle, with a loss probability set by the model of 0.5. For further details of the spatial model, see  Supplementary Information 4 . The code can be obtained from https://github.com/BenSimonsLab/Ciwinska_Nature_2024 . Note that, at large time scales, the data departs from a log-normal size dependence. h , When plot on a log scale, the cumulative distribution of clone size shows the suppression at size scales in excess of the domain size l /2, a manifestation of the constraints imposed by the one-dimensional geometry of the ductal network. Points show the results of stochastic simulation and lines show the corresponding fits to the log-normal size dependence obtained from the fits in panel g. i , j , Cumulative distribution of luminal clone size for wild-type ( i ) and Brca1;Trp53 ( j ) confetti clones for mice showing the largest effective oestrous cycle number from the 225 and 64 day time points, respectively (see  Supplementary Information 4 ). Points show data and lines show the least-squares fits to a log-normal size dependence at small clone sizes. The respective colours are matched to the data shown in  Supplementary Information 4 . Note that, when plot on a log scale, the data reveals a departure from a log-normal size dependence, with a suppression at the largest clone sizes, mirroring the behavior of the spatial model ( h ). See Supplementary Information  1 for more sample sizes, P values and statistics for e and f .

Extended Data Fig. 10 Pregnancy and lactation do not increase the spread of Brca1;Trp53 mutant clones.

a , Schematic depicting the experimental timeline of the pregnancy and lactation experiments in induced Brca1;Trp53 confetti glands. b , Quantification of Brca1;Trp53 confetti clone sizes in nulliparous (left) and parous (right) glands, 120 days after recombination and lineage tracing initiation represented on a logarithmic scale. For each timepoint, at least n  = 3 glands from 3 different mice were analyzed. Boxplots mark the 25th and 75th percentile, line indicates the median, and whiskers mark the minimum and maximum values. Significance was tested using a two-sided Mann-Whitney test, **** P  < 0.0001. c , Transformed luminal (L, orange) and basal (B, red) clones as a percentage of the total number of luminal or basal clones respectively. Each dot indicates an individual mouse and boxplots mark the 25th and 75th percentile, line indicates the median, and whiskers mark the minimum and maximum values. n  = 5 mice (nulliparous) and n  = 3 mice (parous). d , Representative whole-mount confocal images of Brca1;Trp53 confetti clones in parous glands (one pregnancy-involution cycle), 120 days after recombination. Luminal cells are labelled with E-cadherin (ECAD), basal cells are labelled with alpha-smooth muscle actin (SMA). Images depict 3D-rendering of Z-stacks. Scale bars represent 100 µm. Representative image of n  = 3 biological repeats (mice). See Supplementary Information  1 for more sample sizes, P values and statistics for b, c .

Extended Data Fig. 11 Ovariectomy abolishes field clonalization and cancerization of basal and luminal cell clones.

a , b , Clone size quantification of luminal ( a ) and basal ( b ) wild-type confetti clones in the homeostatic gland (left), and after ovariectomy (right) represented on a logarithmic scale. Ovariectomy abolishes clonal expansion and field clonalization. Same data as Fig. 5a , but now with basal and luminal clones presented in separate graphs. For each timepoint at least n  = 6 glands from 3 different mice were analyzed. Analyzed number of clones for each timepoint are indicated in the graphs. Boxplots mark the 25th and 75th percentile, line indicates the median, and whiskers mark the minimum and maximum values. Significance was tested using a two-sided Mann-Whitney test, *** P  < 0.001, **** P  < 0.0001. c , d , Clone size quantification of luminal ( c ) and basal ( d ) Brca1;Trp53 confetti clones in the presence of oestrous cycling (left) and after ovariectomy (right) represented on a logarithmic scale. Ovariectomy abolishes clonal expansion and field cancerization. Same data as Fig. 5d , but now with basal and luminal clones presented in separate graphs. For each timepoint at least n  = 6 glands from 3 different mice were analyzed. Analyzed number of clones for each timepoint are indicated in the graphs. Boxplots mark the 25th and 75th percentile, line indicates the median, and whiskers mark the minimum and maximum values. Significance was tested using a two-sided Mann-Whitney test, *** P  < 0.001, **** P  < 0.0001. e , f , Representative whole-mount confocal images of basal wild-type confetti clones 120 days ( e ) and basal and luminal wild-type confetti clones 225 days ( f ) after recombination in ovariectomized condition. Luminal cells are labelled with E-cadherin (ECAD) ( e ), basal cells are labelled with alpha-smooth muscle actin (SMA) ( f ). Images depict 3D-rendering of Z-stacks, unless otherwise indicated. Scale bars represent 100 µm, except for the scale bar in 2D section ( e ) which represents 10 µm. Representative images of n  = 3 biological repeats (mice). g , h , Representative whole-mount confocal images of basal Brca1;Trp53 confetti clones 120 days ( g ) and 225 days ( h ) after recombination in ovariectomized condition. Luminal cells are labelled with E-cadherin (ECAD) ( g ), basal cells are labelled with alpha-smooth muscle actin (SMA) ( h ). Images depict 3D-rendering of Z-stacks, unless otherwise indicated. Scale bars represent 100 µm. Representative images of n  = 3 biological repeats (mice). See Supplementary Information  1 for more sample sizes, P values and statistics for a-d .

Extended Data Fig. 12 Tissue protection mechanisms against field cancerization in the mammary gland.

a , b , Cumulative distribution of luminal ( a ) and basal ( b ) clone size of ovariectomized wild-type confetti mice showing the probability (log scale) of finding a clone larger than the given size across a range of time points. c , d , Cumulative distribution of luminal ( c ) and basal ( d ) clone size of ovariectomized Brca1;Trp53 confetti mice showing the probability (log scale) of finding a clone larger than the given size across a range of time point. e , Model depicting how tissue protection mechanisms drive field cancerization in the mammary gland. The mammary ductal epithelium confers several layers of protection against field cancerization by mutant cells. Protection mechanism #1: The ductal epithelial network is supported by a short, lineage-restricted MaSC-descendant cell hierarchy. As a result, the majority of mutant cells will be lost through homeostatic tissue turnover, and only a few mutations rooted in the stem cell compartment can survive in the medium term. Protection mechanism #2: Local stem cell loss and replacement driven by the oestrous cycle leads to large-scale elimination of the majority of mutant stem cell clones over time. This large-scale clonal loss occurs at the expense of an accelerated (exponential-like) expansion of the minority of clones that survive, allowing them to colonize large areas of the epithelium. Protection mechanism #3: Once clones extend beyond the size of regions activated during the oestrous cycle, their expansion becomes limited by the one-dimensional geometry of the ducts, a phenomenon that is particularly effective in restricting mutant clone expansion.

Supplementary information

Supplementary information 1.

Overview of sample sizes, P values and statistical tests.

Reporting Summary

Supplementary information 2.

Longitudinal data statistics. Statistical analysis code used to compare the different groups in Figs. 2d and 5g.

Supplementary Information 3

Genomic alterations in Brca1;Trp53 confetti clones. Individual DNA copy number profiles of each 225d Brca1;Trp53 clone and chromosome. The profiles are sorted by clone transformation status as indicated.

Supplementary Information 4

This file contains a Supplementary Note and Sections 1–5 including Figs. 1–10 and Tables.

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Ciwinska, M., Messal, H.A., Hristova, H.R. et al. Mechanisms that clear mutations drive field cancerization in mammary tissue. Nature 633 , 198–206 (2024). https://doi.org/10.1038/s41586-024-07882-3

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Received : 13 December 2022

Accepted : 26 July 2024

Published : 04 September 2024

Issue Date : 05 September 2024

DOI : https://doi.org/10.1038/s41586-024-07882-3

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