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Animal Cloning

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A special issue of Animals (ISSN 2076-2615).

Deadline for manuscript submissions: closed (31 October 2023) | Viewed by 6892

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Dear Colleagues,

Animal cloning, scientifically known as somatic cell nuclear transfer (SCNT), is an advanced reproductive technology which has potential application in various aspects of bioscience and biotechnology, such as livestock breeding, endangered species preservation, organ xenotransplantation, and transgenic animal generation. Recently, SCNT has also shown great potential in epigenetics research, where it is employed to reprogram the somatic cell genome into a totipotent state equivalent to that of the fertilized oocyte.

Although handmade cloning has improved the efficiency of traditional cloning, with the potential for use in commercial settings, the wider application of the technique is still limited due to the high incidence of developmental abnormalities and lower pregnancy rate.

We invite original research papers that address methods and approaches in animal cloning for improving its efficiency, with special interest in handmade cloning, the cryopreservation of cloned embryos, and interspecies cloning.

Dr. Muren Herrid Guest Editor

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• somatic cell nuclear transfer
• pregnancy rate
• cryopreservation
• handmade cloning
• reprogramming
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Article Contents

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The Cloning Debates and Progress in Biotechnology

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Paul L Wolf, George Liggins, Dan Mercola, The Cloning Debates and Progress in Biotechnology, Clinical Chemistry , Volume 43, Issue 11, 1 November 1997, Pages 2019–2020, https://doi.org/10.1093/clinchem/43.11.2019

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The perception by humans of what is doable is itself a great determiner of future events. Thus, the successful sheep cloning experiment leading to “Dolly” by Ian Wilmut and associates at Roslin Institute, Midlothian, UK, compels us to look in the mirror and consider the issue of human cloning. Should it occur, and if not, how should that opposing mandate be managed? If human cloning should have an acceptable role, what is that role and how should it be monitored and supervised?

In the February 27, 1997, issue of Nature , Ian Wilmut et al. reported that they cloned a sheep (which they named “Dolly”) by transferring the nuclear DNA from an adult sheep udder cell into an egg whose DNA had been removed ( 1 ). Their cloning experiments have led to widespread debate on the potential application of this remarkable technique to the cloning of humans. Following the Scottish researchers’ startling report, President Clinton declared his opposition to using this technique to clone humans. He moved swiftly to order that federal funds not be used for such an experiment and asked an independent panel of experts, the National Bioethics Advisory Commission (NBAC), chaired by Princeton University President Harold Shapiro, to report to the White House with recommendations for a national policy on human cloning. According to recommendations by the NBAC, human cloning is likely to become a crime in the US in the near future. The Commission’s main recommendation is to enact federal legislation to prohibit any attempts, whether in a research or a clinical setting, to create a human through somatic cell nuclear transfer cloning.

The concept of genetic manipulation is not new and has been a general practice for more than a century, through practices ranging from selective cross-pollination in plants to artificial insemination in domestic farm animals.

Wilmut and his colleagues made 277 attempts before they succeeded with Dolly. Previously, investigators had reported successful cloning in frogs, mice, and cattle ( 2 )( 3 )( 4 )( 5 ), and 1 week after Wilmut’s report, Don Wolf and colleagues at the Oregon Regional Primate Research Center reported their cloning of two rhesus monkeys by utilizing embryonic cells. The achievement of Wilmut’s team shocked nucleic acid experts, who thought it would be an impossible feat. They believed that the DNA of adult cells could not perform similarly to the DNA formed when a spermatozoa’s genes mingle with those of an ovum.

On July 25, 1997, the Roslin team also reported the production of lambs that contained human genes ( 6 ). Utilizing techniques similar to those they had used in Dolly, they inserted a human gene into the nuclei of sheep cells. These cells were next inserted into the ova of sheep from which the DNA had been removed. The resulting lambs contained the human gene in every cell. In this new procedure the DNA had been inserted into skin fibroblast cells, which are specialized cells, unlike previous procedures in which DNA was introduced into a fertilized ovum. The new lamb has been named “Polly” because she is a Poll Dorset sheep. The goal of this new genetically engineered lamb is for these lambs to produce human proteins necessary for the treatment of human genetic diseases, such as factor VIII for hemophiliacs, cystic fibrosis transmembrane conductance regulator (CFTR) substance for patients with cystic fibrosis, tissue plasminogen activator to induce lysis of acute coronary and cerebral artery thrombi, and human growth factor.

Charles Darwin was frightened when he concluded that humans were not specifically separated from all other animals. Not until 20 years after his discovery did he have the courage to publish his findings, which changed the way humans view life on earth. Wilmut’s amazing investigations have also created worldwide fear, misunderstanding, and ethical shock waves. Politicians and a few scientists are proposing legislation to outlaw human cloning ( 7 ). Although the accomplishment of cloning clearly could provide many benefits to medicine and to conservation of endangered species of animals, politicians and a few scientists fear that the cloning procedure will be abused.

The advantages of cloning are numerous. The ability to clone dairy cattle may have a larger impact on the dairy industry than artificial insemination. Cloning might be utilized to produce multiple copies of animals that are especially good at producing meat, milk, or wool. The average cow makes 13 000 pounds (5800 kg) of milk a year. Cloning of cows that are superproducers of milk might result in cows producing 40 000 pounds (18 000 kg) of milk a year.

Wilmut’s recent success in cloning “Polly” represents his main interest in cloning ( 8 ). He believes in cloning animals able to produce proteins that are or may prove to be useful in medicine. Cloned female animals could produce large amounts of various important proteins in their milk, resulting in female animals that serve as living drug factories. Investigators might be able to clone animals affected with human diseases, e.g., cystic fibrosis, and investigate new therapies for the human diseases expressed by these animals.

Another possibility of cloning could be to change the proteins on the cell surface of heart, liver, kidney, or lung, i.e., to produce organs resembling human organs and enhancing the supply of organs for human transplantation. The altered donor organs, e.g., from pigs, would be less subject to rejection by the human recipient. The application of cloning in the propagation of endangered species and conservation of gene pools has been proposed as another important use of the cloning technique ( 9 )( 10 ).

The opponents of cloning have especially focused on banning the cloning of humans ( 11 ). The UK, Australia, Spain, Germany, and Denmark have implemented laws barring human cloning. Opponents of human cloning have cited potential ethical and legal implications. They emphasize that individuals are more than a sum of their genes. A clone of an individual might have a different environment and thus might be a different person psychologically and have a different “soul.” Cloning of a human is replication and not procreation.

Morally questionable uses of genetic material transfer and cloning obviously exist. For example, infertility experts might be especially interested in the cloning technique to produce identical twins, triplets, or quadruplets. Parents of a child who has a terminal illness might wish to have a clone of the child to replace the dying child. The old stigma, eugenics, also raises its ugly head if infertile couples wish to use the nuclear transfer techniques to ensure that their “hard-earned” offspring will possess excellent genes. Moral perspectives will differ tremendously in these cases. Judgments about the appropriateness of such uses are outside the realm of science.

Opponents of animal cloning are concerned that cloning will negate genetic diversity of livestock. This also applies to human cloning, which could negate genetic diversity of humans. Cloning creates, by definition, a second class of human, a human with a determined genotype called into existence, however benevolently, at the behest of another. The insulation of selection-of-mate is lost, and the second class is created. Few contrasts could be so clear. Selection-of-mate is so imprecise that, at present, would-be parents have to accept a complete new genome for the sake of including or excluding one or a few traits; cloning, in contrast, is the precise determination of all genes. If we acknowledge that the creation of a second class of humans is unethical, then we preempt any argument that some motivations for human cloning may be acceptable.

The opponents of cloning also fear that biotechnically cloned foods might increase the risk of humans acquiring some malignancies or infections such as “mad cow disease,” a prion spongiform dementia encephalopathy (human Jakob–Creutzfeldt disease).

The technological advances associated with manipulation of genetic materials now permit us to envision replacement of defective genes with “good” genes. Although current progress is not sufficient to make this practical today for human diseases, any efforts to stop such research as a result of cloning hysteria would preclude the development of true cures for many hereditary human diseases. Unreasonable restrictions on the use of human tissues in gene transfer research will have the inevitable consequences of delaying, if not preventing, the development of strategies to combat defective genes.

Wise legislation will enable humankind to realize the benefits of gene transfer technologies without risking the horrors that could arise from misuse of these technologies. Our hope is that such wise legislation is what will be enacted. In our view, the controversy surrounding human cloning must not lead to prohibitions that would prevent advances similar to those described here.

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Animal cloning applications and issues

• Reviews and Theoretical Articles
• Published: 22 September 2017
• Volume 53 , pages 965–971, ( 2017 )

Cite this article

• F. Ibtisham 1 ,
• M. M. Fahd Qadir 2 , 3 ,
• M. Xiao 1 &

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Significant progress in the field of biotechnology has allowed for the use of cloning in animals which is being used: to improve genetic makeup, to rescue endangered species, in tissue engineering and to increase farm animal population. Unfortunately, cloning has been met with failure due to a variety of reasons namely early and late abortions, compromised immune systems, circulatory and respiratory problems and a high rate of fetal death. The reasons of these problems are unknown, but may research groups are attempting to understand the underlying molecular and cellular mechanisms involved in cloning efficiency. Atypical epigenetic re-programming appears to be the primary cause of ineffective cloning. Understanding molecular mechanisms involving key regulatory proteins is pivotal in the success of animal cloning. This review shows the current paradigm involving animal cloning efficiency, and also further elucidates applications to improve animal cloning efficiency.

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Ibtisham, F., Fahd Qadir, M.M., Xiao, M. et al. Animal cloning applications and issues. Russ J Genet 53 , 965–971 (2017). https://doi.org/10.1134/S102279541709006X

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Human Cloning: Biology, Ethics, and Social Implications

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• 1 MAGI'S LAB, Rovereto (TN), Italy.
• 2 Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.
• 3 MAGI EUREGIO, Bolzano, Italy.
• 4 MAGISNAT, Peachtree Corners (GA), USA.
• 5 School of Food Science and Environmental Health, Technological University of Dublin, Dublin, Ireland.
• 6 Department of Psychology and Neuroscience, Dalhousie University, Halifax, Nova Scotia, Ca-nada.
• 7 Department of Ophthalmology, Center for Ocular Regenerative Therapy, School of Medicine, University of California at Davis, Sacramento, CA, USA.
• 8 Centre for Bioethics, Department of Philosophy and Applied Philosophy, University of St. Cyril and Methodius, Trnava, Slovakia.
• 9 Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
• 10 nstitute of Ophthalmology, Università Cattolica del Sacro Cuore, Fondazione Policlinico Universitario A. Gemelli-IRCCS, Rome, Italy.
• 11 MAGI BALKANS, Tirana, Albania.
• 12 Department of Biotechnology, University of SS. Cyril and Methodius, Trnava, Slovakia.
• 13 International Centre for Applied Research and Sustainable Technology, Bratislava, Slovakia.
• 14 UOC Neurology and Stroke Unit, ASST Lecco, Merate, Italy.
• 15 Center for Preclincal Research and General and Liver Transplant Surgery Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
• 16 Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy.
• 17 Department of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, Milan, Italy.
• 18 UOC Maxillo-Facial Surgery and Dentistry, Fondazione IRCCS Ca Granda, Ospedale Maggiore Policlinico, Milan, Italy.
• 19 Department of Medical Genetics, Faculty of Medicine, Near East University, Nicosia, Cyprus.
• 20 Department of Medical Genetics, Erciyes University Medical Faculty, Kayseri, Turkey.
• 21 Vascular Diagnostics and Rehabilitation Service, Marino Hospital, ASL Roma 6, Marino, Italy.
• 22 San Francisco Veterans Affairs Health Care System, University of California, San Francisco, CA, USA.
• 23 Univ. Grenoble Alpes, CNRS, Grenoble INP, TIMC-IMAG, SyNaBi, Grenoble, France.
• 24 Department of Biotechnology, University of Tirana, Tirana, Albania.
• 25 Total Lipedema Care, Beverly Hills, California, and Tucson, Arizona, USA.
• 26 Federation of the Jewish Communities of Slovakia.
• 27 Department of Psychological Health and Territorial Sciences, School of Medicine and Health Sciences, "G. d'Annunzio" University of Chieti-Pescara, Chieti, Italy.
• 28 Unit of Molecular Genetics, Center for Advanced Studies and Technology (CAST), "G. d'Annunzio" University of Chieti-Pescara, Chieti, Italy.
• 29 Department of Anatomy and Developmental Biology, University College London, London, UK.
• PMID: 37994769
• DOI: 10.7417/CT.2023.2492

This scholarly article delves into the multifaceted domains of human cloning, encompassing its biological underpinnings, ethical dimensions, and broader societal implications. The exposition commences with a succinct historical and contextual overview of human cloning, segueing into an in-depth exploration of its biological intri-cacies. Central to this biological scrutiny is a comprehensive analysis of somatic cell nuclear transfer (SCNT) and its assorted iterations. The accomplishments and discoveries in cloning technology, such as successful animal cloning operations and advances in the efficiency and viability of cloned embryos, are reviewed. Future improvements, such as reprogramming procedures and gene editing technology, are also discussed. The discourse extends to ethical quandaries intrinsic to human cloning, entailing an extensive contemplation of values such as human dignity, autonomy, and safety. Furthermore, the ramifications of human cloning on a societal plane are subjected to scrutiny, with a dedicated emphasis on ramifications encompassing personal identity, kinship connections, and the fundamental notion of maternity. Culminating the analysis is a reiteration of the imperative to develop and govern human cloning technology judiciously and conscientiously. Finally, it discusses several ethical and practical issues, such as safety concerns, the possibility of exploitation, and the erosion of human dignity, and emphasizes the significance of carefully considering these issues.

Keywords: Human cloning; biology; dignity; ethical considerations; safety; social implications.

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Animal cloning

So Dolly was not the first clone, and she looked like any other sheep, so why did she cause so much excitement and concern? Because she was the first mammal to be cloned from an adult cell, rather than an embryo. This was a major scientific achievement, but also raised ethical concerns. Since 1996, when Dolly was born, other sheep have been cloned from adult cells, as have mice, rabbits, horses and donkeys, pigs, goats and cattle. In 2004 a mouse was cloned using a nucleus from an olfactory neuron, showing that the donor nucleus can come from a tissue of the body that does not normally divide.  

How was Dolly created?

Producing an animal clone from an adult cell is obviously much more complex and difficult than growing a plant from a cutting. So when scientists working at the Roslin Institute in Scotland produced Dolly, the only lamb born from 277 attempts, it was a major news story around the world. To produce Dolly, the scientists used the nucleus of an udder cell from a six-year-old Finn Dorset white sheep. The nucleus contains nearly all the cell's genes. They had to find a way to 'reprogram' the udder cells - to keep them alive but stop them growing – which they achieved by altering the growth medium (the ‘soup’ in which the cells were kept alive). Then they injected the cell into an unfertilised egg cell which had had its nucleus removed, and made the cells fuse by using electrical pulses. The unfertilised egg cell came from a Scottish Blackface ewe. When the scientists had managed to fuse the nucleus from the adult white sheep cell with the egg cell from the black-faced sheep, they needed to make sure that the resulting cell would develop into an embryo. They cultured it for six or seven days to see if it divided and  developed normally, before implanting it into a surrogate mother, another Scottish Blackface ewe. Dolly had a white face. From 277 cell fusions, 29 early embryos developed and were implanted into 13 surrogate mothers. But only one pregnancy went to full term, and the 6.6kg Finn Dorset lamb 6LLS (alias Dolly) was born after 148 days.

Why are scientists interested in cloning?

The main reason that the scientists at Roslin wanted to be able to clone sheep and other large animals was connected with their research aimed at producing medicines in the milk of such animals. Researchers have managed to transfer human genes that produce useful proteins into sheep and cows, so that they can produce, for instance, the blood clotting agent factor IX to treat haemophilia or alpha-1-antitrypsin to treat cystic fibrosis and other lung conditions. Cloned animals could also be developed that would produce human antibodies against infectious diseases and even cancers. ‘Foreign’ genes have been transplanted into zebra fish, which are widely used in laboratories, and embryos cloned from these fish express the foreign protein. If this technique can be applied to mammalian cells and the cells cultured to produce cloned animals, these could then breed conventionally to form flocks of genetically engineered animals all producing medicines in their milk. There are other medical and scientific reasons for the interest in cloning. It is already being used alongside genetic techniques in the  development of animal organs for transplant into humans (xenotransplantation). Combining such genetic techniques with cloning of pigs (achieved for the first time in March 2000) would lead to a reliable supply of suitable donor organs. The use of pig organs has been hampered by the presence of a sugar, alpha gal, on pig cells, but in 2002 scientists succeeded in knocking out the gene that makes it, and these ‘knockout’ pigs could be bred naturally. However, there are still worries about virus transmission. The study of animal clones and cloned cells could lead to greater understanding of the development of the embryo and of ageing and  age-related diseases. Cloned mice become obese, with related symptoms such as raised plasma insulin and leptin levels, though their offspring do not and are normal. Cloning could be used to create better animal models of diseases, which could in turn lead to further progress in understanding and treating those diseases. It could even enhance biodiversity by ensuring the continuation of rare breeds and endangered species.  

What happened to Dolly?

Dolly, probably the most famous sheep in the world, lived a pampered existence at the Roslin Institute. She mated and produced normal offspring in the normal way, showing that such cloned animals can reproduce. Born on 5 July 1996, she was euthanased on 14 February 2003, aged six and a half. Sheep can live to age 11 or 12, but Dolly suffered from arthritis in a hind leg joint and from sheep pulmonary adenomatosis, a virus-induced lung tumour to which sheep raised indoors are prone. On 2 February 2003, Australia's first cloned sheep died unexpectedly at the age of two years and 10 months. The cause of death was unknown and the carcass was quickly cremated as it was decomposing. Dolly’s chromosomes were are a little shorter than those of other sheep, but in most other ways she was the same as any other sheep of her chronological age. However, her early ageing may reflect that she was raised from the nucleus of a 6-year old sheep. Study of her cells also revealed that the very small amount of DNA outside the nucleus, in the mitochondria of the cells, is all inherited from the donor egg cell, not from the donor nucleus like the rest of her DNA. So she is not a completely identical copy. This finding could be important for sex-linked diseases such as haemophilia, and certain neuromuscular, brain and kidney conditions that are passed on through the mother's side of the family only.  

Improving the technology

Scientists are working on ways to improve the technology. For example, when two genetically identical cloned mice embryos are combined, the aggregate embryo is more likely to survive to birth. Improvements in the culture medium may also help.  

Ethical concerns and regulation

Most of the ethical concerns about cloning relate to the possibility that it might be used to clone humans. There would be enormous technical difficulties. As the technology stands at present, it would have to involve women willing to donate perhaps hundreds of eggs, surrogate pregnancies with high rates of miscarriage and stillbirth, and the possibility of premature ageing and high cancer rates for any children so produced. However, in 2004 South Korean scientists announced that they had cloned 30 human embryos, grown them in the laboratory until they were a hollow ball of cells, and produced a line of stem cells from them. Further ethical discussion was raised in 2008 when scientists succeeded in cloning mice from tissue that had been frozen for 16 years. In the USA, President Clinton asked the National Bioethics Commission and Congress to examine the issues, and in the UK the House of Commons Science and Technology Committee, the Human Embryology and Fertilisation Authority and the Human Genetics Advisory Commission all consulted widely and advised that human cloning should be banned. The Council of Europe has banned human cloning: in fact most countries have banned the use of cloning to produce human babies (human reproductive cloning). However, there is one important medical aspect of cloning technology that could be applied to humans, which people may find less objectionable. This is therapeutic cloning (or cell nucleus replacement) for tissue engineering, in which tissues, rather than a baby, are created. In therapeutic cloning, single cells would be taken from a person and 'reprogrammed' to create stem cells, which have the potential to  develop into any type of cell in the body. When needed, the stem cells could be thawed and then induced to grow into particular types of cell such as heart, liver or brain cells that could be used in medical treatment. Reprogramming cells is likely to prove technically difficult. Therapeutic cloning research is already being conducted in animals, and stem cells have been grown by this method and transplanted back into the original donor animal. In humans, this technique would revolutionise cell and tissue transplantation as a method of treating diseases. However, it is a very new science and has raised ethical concerns. In the UK a group headed by the Chief Medical Officer, Professor Liam Donaldson, has recommended that research on early human embryos should be allowed. The Human Fertilisation and Embryology Act was amended in 2001 to allow the use of embryos for stem cell research and consequently the HFEA has the responsibility for regulating all embryonic stem cell research in the UK. There is a potential supply of early embryos as patients undergoing in-vitro fertilisation usually produce a surplus of fertilised eggs. As far as animal cloning is concerned, all cloning for research or medical purposes in the UK must be approved by the Home Office under the strict controls of the Animals (Scientific Procedures) Act 1986 . This safeguards animal welfare while allowing important scientific and medical research to go ahead.  

Further information

The Roslin Institute has lots of information about the research that led to Dolly, and the scientific studies of Dolly, as well as links to many other sites that provide useful information on the scientific and ethical aspects of this research. The report of the Chief Medical Officer's Expert Advisory Group on Therapeutic Cloning: Stem cell research: medical progress with responsibility is available from the UK Department of Health , PO Box 777, London SE1 6XH. Further information on therapeutic cloning and stem cell research is available from the Medical Research Council . Interesting illustrated features on cloning have been published by Time , New Scientist . BBC News Online has a Q&A What is Cloning?   IMAGE © THE ROSLIN INSTITUTE

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• Published: 19 September 2024

Immobilization secondary to cell death of muscle precursors with a dual transcriptional signature contributes to the emu wing skeletal pattern

• Eriko Tsuboi   ORCID: orcid.org/0009-0007-5131-6928 1   na1 ,
• Satomi F. Ono   ORCID: orcid.org/0009-0003-4386-3447 1   na1 ,
• Ingrid Rosenburg Cordeiro   ORCID: orcid.org/0000-0001-9950-9939 1   na1   nAff6 ,
• Reiko Yu   ORCID: orcid.org/0009-0005-1709-6170 1 ,
• Toru Kawanishi   ORCID: orcid.org/0000-0001-7038-9769 1 ,
• Makoto Koizumi   ORCID: orcid.org/0000-0002-4662-0382 2 ,
• Shuji Shigenobu   ORCID: orcid.org/0000-0003-4640-2323 3 ,
• Guojun Sheng   ORCID: orcid.org/0000-0001-6759-3785 4 ,
• Masataka Okabe   ORCID: orcid.org/0000-0002-8618-6859 5 &
• Mikiko Tanaka   ORCID: orcid.org/0000-0001-8092-8594 1  

Nature Communications volume  15 , Article number:  8153 ( 2024 ) Cite this article

Metrics details

• Body patterning
• Limb development

Limb reduction has occurred multiple times in tetrapod history. Among ratites, wing reductions range from mild vestigialization to complete loss, with emus ( Dromaius novaehollandiae ) serving as a model for studying the genetic mechanisms behind limb reduction. Here, we explore the developmental mechanisms underlying wing reduction in emu. Our analyses reveal that immobilization resulting from the absence of distal muscles contributes to skeletal shortening, fusion and left-right intraindividual variation. Expression analysis and single cell-RNA sequencing identify muscle progenitors displaying a dual lateral plate mesodermal and myogenic signature. These cells aggregate at the proximal region of wing buds and undergo cell death. We propose that this cell death, linked to the lack of distal muscle masses, underlines the morphological features and variability in skeletal elements due to reduced mechanical loading. Our results demonstrate that differential mobility during embryonic development may drive morphological diversification in vestigial structures.

Introduction

Limb reduction has occurred multiple times throughout tetrapod evolution. Snakes, legless lizards and caecilians have lost their limbs, cetaceans reduced their hindlimbs, and ungulates evolved a reduced number of digits. The reduction of limbs in tetrapods is associated with the diversification of their locomotion styles and habitats. Therefore, revealing the mechanisms by which limb reduction occurs is one of the main themes of tetrapod limb evolution research 1 .

Among birds, the forelimb reductions found in ratites range from the mild vestigialization seen in the ostrich to the complete loss of the forelimb in the extinct moa, and might have occurred multiple times in this clade 2 , 3 . Of the ratites available for developmental approaches, the emu exhibits an extreme forelimb reduction, which is already evident during embryonic as a very reduced forelimb bud 4 . Several groups have addressed mechanisms of forelimb bud reduction during early stages of emu limb development, which has been attributed to decreased proliferation of limb bud progenitors, and also to a heterochrony caused by modulation and/or delayed expression of typical limb patterning genes 2 , 5 , 6 , 7 , 8 , 9 , 10 , including higher variability in expression pattern, such as Grem1 8 . Nevertheless, a limb bud is formed, which develops into a patterned wing 4 .

However, reduction of the actual limb bud does not explain mechanisms taking place during later developmental stages that further shape the emu wings, especially their autopodial region. While three digits are initially specified in the emu autopod 7 , most of the cartilaginous elements of the digits 2 and 4 are resorbed during late foetal development 8 , which is followed by fusion of skeletal elements and joint contractures that are typical of adult emu wings 8 , 11 . These appendages display an extensive osteological variation, including presence or absence of phalanges, shape, bone pneumaticity, and muscle attachment 11 . These differences were not restricted to skeletal elements, as it was found that emus lacked several muscle masses in their wings when compared to volant birds as well 11 .

Interestingly, defects in both bone growth and joint formation can be secondary to a loss of movement during development, which has been extensively studied in both animal models 12 , 13 , 14 , 15 , 16 and clinical examples 17 . Limb skeletal defects have been reported in several strains of muscle-less and muscle dysgenesis mice, while avian in ovo models have been crucial to pinpoint the effects of early or late immobilization on longitudinal skeletal growth and joint cavitation 13 , 14 , 15 , 16 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 . In human patients, a decrease in fetal movement can lead to fetal akinesia deformation sequence, a spectrum of skeletal and joint phenotypes 13 . Furthermore, congenital joint contractures often present with a range of severity, including variation in the affected joints and, to a lesser extent, asymmetric presentation—even when the cause of the disease has been mapped to a specific mutation 17 . The highly variable range of phenotypes found in emu wings led us to hypothesize whether it was not only the result of direct genetic factors but also secondary to extrinsic epigenetic factors—more specifically, to a reduced mechanical stimulation.

Here, we show that emu forelimbs exhibit a high intraindividual asymmetry in skeletal patterns, in addition to a reduction in bone length and contractures of joints. Such morphological features seen in emu forelimbs recapitulate the skeletal phenotype found in both experimental and clinical observations of reduced mechanical loading during limb development. In addition, we observe a lack of distal limb movement in emu embryos resulting from the lack of muscle masses at the distal ends of their wings, a condition that is apparent from the stages of prenatal development. Single-cell RNA sequencing (scRNA-seq) data and expression analysis revealed that a subpopulation of muscle progenitors exhibits a dual myogenic/LPM (lateral plate mesoderm) transcriptional signature. This subpopulation undergoes massive cell death and thereby fails to form distal skeletal muscles, which contributes to the unique morphology of emu forelimbs.

High intraindividual left-right variation is found in the adult skeletal pattern of the distal emu wings

Previously, we and others have shown that a great range of interindividual variation exists in the digital pattern of adult emu forelimbs 8 , 11 . Understanding whether the variation is present within limbs of the same individual, or is consistent between left and right limbs, would help elucidate the source of variation itself. Here, a comparison between the pair of forelimbs of eight adult emu specimens revealed an intraindividual, left–right asymmetric skeletal pattern (Fig.  1a ; Supplementary Fig.  1a ). The variation included different status regarding fusion of the ulna and radius, fusion of autopodial skeletal elements, fusion of joints and bone pneumaticity between left and right limbs of the same individual (Fig.  1a ). Furthermore, the humeral, ulnar and metacarpal lengths were relatively asymmetric in all examined emu forelimbs compared to chicken forelimbs (Fig.  1b ; Supplementary Fig.  1b ). Thus, there is a high degree of intra- and interindividual variability intrinsic to emu wing skeletal elements.

a Three-dimensional renderings from CT images of the distal part of adult emu forelimb skeletons and transverse sections taken from the emu limb at the level of the dashed line. The left and right limbs of two specimens are shown. Arrowheads indicate the fusions. 3-4, digits 3-4; 4*, rudiment of digit 4; d3, metacarpal of digit 3; r, radiale; R, radius; U, ulna. Scale bars, 1 cm. b Ratio of left to right bone length (coefficient of variation: 2.23%, 3.60 %, 1.93%, 4.24%, 0.59%, 3.54% for chicken humerus, emu humerus, chicken ulna, emu ulna, chicken metacarpus, emu metacarpus, respectively). Mean ± SEM. n  = 6 (chicken), n  = 8 (emu). c Immunostaining with MF20 in forelimbs of chicken and emu embryos. Although the formation of muscles was recognized in autopodial regions of stage 35 ( n  = 4) and 37 ( n  = 5) chicken embryos, no autopodial muscles and only a few or no zeugopodial muscles were observed in the forelimbs of emu embryos at the same stages (EMR, EIL, Anc on the dorsal side and the flexor carpi ulnaris (FCU) on the ventral side at stage 35 ( n  = 3); EMR, Anc on the dorsal side and FCU on the ventral side ( n  = 1), Anc only ( n  = 1), no muscles ( n  = 1)). Brackets indicate autopodial regions. 2-4, digits 2-4; Anc, anconeus; EDC, extensor digitorum communis; EIL, extensor indicis longus; EML, extensor medius longus; EMR, extensor metacarpi radialis. Scale bars, 1 mm. d Rate of distal movements (see Materials and Methods for details). n  = 5 (chicken), n  = 4 (emu). Mean ± SEM. Welch’s two tailed t-test. ** p  = 0.0023. e Ratio of left to right bone length of chicken embryos treated with PBS ( n  = 7) or DMB (n = 13) from E10 to E18 (coefficient of variation: 1.39%, 7.08%, 1.17%, 4.66%, 1.37%, 5.93% for control humerus, immobilized humerus, control ulna, immobilized ulna, control metacarpus, immobilized metacarpus, respectively). f Chicken embryos were treated with PBS or DMB from E6 (stage 28) to E14 (stage 39). Safranin O staining of the wrist joints of chicken and emu embryos and Alcian blue staining of forelimbs of emu embryos. The control panel is flipped horizontally. The distance between the ulna and the distal carpal/metacarpal of digit 3 (yellow lines) was measured in control chickens ( n  = 4), immobilized chickens ( n  = 4) and emu embryos ( n  = 3). Mean ± SEM. Welch’s two tailed t-test. *** p  = 0.0003. dc, distal carpal; d3, metacarpal of digit 3; U, ulna. Scale bars, 500 μm.

Developing emu forelimbs receive less mechanical input due to the absence of distal muscle masses

Studies in chicken and mouse embryos revealed that longitudinal bone growth of the limb and normal joint cavity formation require mechanical input as a result of embryonic muscle contraction 13 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 . Importantly, a marked reduction in the number of muscle masses is found in adult emu wings, especially towards its distal part 11 , although the remaining muscles display a typical skeletal muscle morphology (Supplementary Fig.  1c ). A normal gross morphology of the forelimb muscle masses has been reported in stage 30 emu forelimbs 7 ; however, it remains unclear when muscle reduction occurs during autopodial development, which takes place at later stages. To address this question, we analyzed the process of muscle formation in developing forelimb buds of emu embryos and compared them to those in chicken embryos (Fig.  1c ; Supplementary Fig.  2a ). At stage 30, both chicken and emu display the typical avian forelimb structure containing three digits 7 . While no autopodial muscle mass could be detected at this stage (Supplementary Fig.  2a ), they became evident at stages 33, 35 and 37 (Fig.  1c ; Supplementary Fig.  2a ). In contrast, digits 2 and 4 of emu wings displayed various degrees of vestigialization, which takes place up to stage 37 8 . No autopodial muscles were observed, and most zeugopodial muscles were missing from developing emu forelimb buds up to stage 37 (Fig.  1c ; Supplementary Fig.  2a ). By recording embryonic movement in ovo , we also found that the distal part of forelimb buds showed hardly any movement in emu embryos at stage 39, while embryonic muscle contraction was already evident in the distal forelimb buds in chicken embryos or the proximal portion of emu wings at the same stage (Fig.  1d ; Supplementary Fig.  2b ). These results demonstrate that the distal part of the emu forelimb receives less mechanical input during its development in connection to the absence of distal muscle formation.

Loss of mobility recapitulates characteristic features of emu forelimbs

Much research has been devoted to understanding the effect of muscle contraction on the growth of limb bones and joint cavity formation in avian limbs 13 , 15 , 16 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 . Although the decreased mechanical load secondary to the absence of distal muscle could explain the left–right variation seen in the emu forelimb skeleton, to our knowledge, there have been no reports demonstrating the effect of immobilization specifically on intraindividual left–right asymmetric patterning of limbs. Here, we will address these questions by first focusing on the effect of late-stage immobilization (E10-18) on the longitudinal growth of individual skeletal elements, followed by examining how immobilization starting at pre-cavitation stages (E6-E10) could affect both growth and joint cavitation asymmetrically.

Firstly, we induced rigid paralysis in chicken embryos under conditions known to lead to a reduction in the length of hindlimbs after joint cavitation occurs 13 , 24 , 25 , 26 , 27 , 28 , 29 and examined the length of skeletal elements, joint cavity formation and left–right patterning of forelimbs (Supplementary Fig.  3 ). The lengths of both the right and left humerus, ulna and metacarpus of digit 3 were significantly reduced in chicken embryos immobilized between E10-18 (Supplementary Fig.  3a, b ). Pharmacologically induced immobility also led to drastic intraindividual variation in the length of the left and right humerus, ulna and metacarpus, compared to controls (Fig.  1e ). These results suggest that mobility is necessary for symmetric limb patterning and growth.

Intriguingly, even upon immobilization at a post cavitation stage, two out of thirteen immobilized chickens showed an asymmetric pattern at the wrist joint with fusion of cartilaginous fusion of limb elements (Supplementary Fig.  3c ). To understand further the effect of mechanical inputs on symmetrical growth and patterning of autopodial skeletal elements, we have immobilized chicken embryos from E6 (Supplementary Figs.  3 d, 5 and 6 ), when muscle masses had developed, and forelimb movements were apparent (Supplementary Fig.  4a, b ). The total length of skeletal elements of digits (metacarpus and phalanges) was significantly reduced compared to that in controls for all digits (Supplementary Fig.  5b ). In addition, the reduction in digit length was relatively asymmetric in digits 2 and 4, while the reduction in digit 3 was relatively symmetric (Supplementary Fig.  5c ). Of note, emu forelimbs exhibit severe reduction of digits 2 and 4 as well, but not of digit 3 (Fig.  1a ; Supplementary Fig.  1a ).

As previously described 12 , immobilization of the chicken embryo at pre-cavitation stages led to a failure of proper joint cavity formation at the wrist region; we have observed that the cartilaginous fusion of skeletal elements was also asymmetric (Fig.  1f ; Supplementary Fig.  6 ). Thinning or absence of the joint cavity at the wrist is one of the characteristic features of the emu forelimb skeletal pattern 8 (Fig.  1a ). Immobilization significantly reduced the distance between the ulna and the distal carpal in chicken embryos (Fig.  1f ). Similar to immobilized chickens, the distance between the ulna and the metacarpus of digit 3 was also narrow in emu embryos at the same stage of development (Fig.  1f ). These results indicate that loss of mobility from early stages recapitulated characteristic features of emu forelimbs, such as reduction or loss of the joint cavity at the wrist caused by asymmetric fusion of skeletal elements.

Taken together, these results reveal that the lack of distal muscle formation in emu embryos, when compared to chicken, led to a non-experimental immobilization phenotype that closely resembles experimental immobilization phenotypes in chicken. Specifically, we have shown that the reduction in bone length, increased susceptibility of the digits 2 and 4 to growth reduction when compared to digit 3, joint fusion and/or joint cavitation defects had an intraindividual asymmetric presentation in both emu and immobilized chicken forelimbs.

A population with dual myogenic and LPM cell signature is recognized in emu wings

Our findings suggest that the lack of distal muscle masses in comparison with chicken recapitulated, at least partially, the distinctive skeletal pattern of the emu wing. However, the underlying developmental reasons for the reduction of muscle in emu forelimbs are still not clear. To explore this, we first studied in detail the developmental process of forelimb muscles in emu embryos. In amniotes, muscle precursors delaminated from the ventral dermomyotome extensively migrate toward the distal part of the limb bud and differentiate into skeletal muscles. The delamination of muscle precursors in emu takes place in stages equivalent to chicken 9 ; however, they migrate in a diffuse pattern and are not divided into dorsal and ventral muscle masses when compared to the emu hindlimb 6 or to chicken forelimbs 33 . The migratory muscle precursors 33 are characterized by the expression of Lbx1 , which encodes a ladybird homeobox transcription factor 34 , thought to be involved in controlling their migration 35 , 36 , 37 , 38 . cMet , which encodes a tyrosine kinase receptor, is expressed in the ventral dermomyotome as well as in muscle precursors migrating toward limb buds 39 , 40 , 41 . In addition, it has been previously reported that the emu forelimb bud co-opted Nkx2.5 expression 7 , and Nkx2.5 -positive cells become skeletal muscle in forelimbs 7 , 9 . Thus, we examined the expression pattern of Lbx1 , cMet as well as Nkx2.5 in developing emu wing buds (Fig.  2 ; Supplementary Figs.  7 and 8 ). In chickens, the invasion of Lbx1 -positive migratory muscle precursors into the forelimb bud became apparent at stage 18 33 (Supplementary Fig.  7a ). In emu, transcripts of Lbx1 and cMet were enriched in the lateral region of the dermomyotome, and their distribution in the forelimb field became detectable at stage 18 (Fig.  2a ). The expression of Nkx2.5 was seen only in the anterior edge of the forelimb field at the same stages (Fig.  2a ). By stage 19, Lbx1/cMet -positive migratory muscle precursors invaded into the dorsal one half to one-third of the forelimb bud region, and Nkx2.5 expression was observed to the central region of the anterior three-quarters of limb buds (Fig.  2a, b ). At stage 22, expression of Nkx2.5 was seen from the cervical to the forelimb level in the lateral plate mesoderm (Supplementary Fig.  7b ). By stage 23, cMet expression was observed in migratory muscle precursors spread throughout the limb buds, and Nkx2.5 expression was seen in the ventral two-thirds of the forelimb bud (Fig.  2b ). To observe gene expression in more detail, in situ hybridization using RNAscope probes was also used. It was found that cMet transcripts were sparsely distributed in proximal muscle precursors derived from the dermomyotome and were more densely distributed in the medial part of forelimb buds, where Nkx2.5 transcripts were observed, at stages 19 and 23 (Fig.  2c ; Supplementary Fig.  8 ). At stage 25, the expression of Lbx1 and Nkx2.5 was observed in the proximal region and the subapical region, and the expression of MyoD , a key regulator of myogenic differentiation, overlapped with these cells at the subapical region (Fig.  2b ). Co-localization of cMet and Nkx2.5 transcripts were confirmed by using RNAscope probes, and MyoD transcripts also co-localized at the subapical region (Fig.  2d ; Supplementary Fig.  8 ). Subsequently, the expression of Nkx2.5 was detected in the skeletal muscles (Supplementary Fig.  7c ), as previously reported 7 , 9 . Our results and the results of others indicate that muscle progenitors of emu wings are positive for both Lbx1 and cMet , marker genes for somite-derived myogenic cells, as well as for Nkx2.5 , a gene typically expressed in the vertebrate cardiac mesoderm 42 .

a Expression of Lbx1 , cMet and Nkx2.5 (arrowheads) in forelimb fields (FL) of emu embryos (stage 18 Lbx1 ( n  = 6), cMet ( n  = 2), Nkx2.5 ( n  = 4); stage 19 Nkx2.5 ( n  = 2)). Scale bars, 500 μm. b Expression of Lbx1 , cMet , Nkx2.5 and MyoD (arrowheads) in serial sections of emu forelimb buds at mid- (mid. FL) or posterior level (post. FL). A dotted circle indicates Nkx2.5 negative region. Panels of stages 19 and 23 sections were flipped horizontally. (stage 19 Lbx1 ( n  = 6), cMet ( n  = 4), Nkx2.5 ( n  = 6); stage 23 cMet ( n  = 2), Nkx2.5 ( n  = 2); stage 25 Lbx1 ( n  = 3), Nkx2.5 ( n  = 3), MyoD ( n  = 2)). dm, dermomyotome; D, dorsal; V, ventral. Scale bars, 50 μm. c , c’ , c” Distribution of cMet and Nkx2.5 transcripts in developing emu forelimb buds at stage 23 ( n  = 4). Arrows indicate the cMet transcripts in muscle precursors derived from the dermomyotome. Arrowheads indicate co-locaized transcripts of cMet and Nkx2.5 . d , d’ Distribution of cMet , Nkx2.5 and MyoD transcripts in emu forelimb buds at stage 25 ( n  = 3). Note that transcripts of cMet and Nkx2.5 (arrowheads) are present in cell clusters, in which MyoD expression is also detected (arrows). White cells are blood cells (asterisks), not stained cells.

While skeletal muscle cells and limb bud cells are derived from the paraxial mesoderm and the somatic layer of the lateral plate mesoderm (LPM), respectively, the cardiac mesoderm is derived from the splanchnic layer of the LPM. Thus, these results raised the possibility that the emu muscle progenitors possess atypical markers, including ones typically linked to the LPM identity. To investigate gene expression in an unbiased manner and at single cell resolution, we performed single-cell RNA sequencing (scRNA-seq) using the Chromium platform and paired-end Illumina next-generation sequencing of the dissociated cells from the emu trunk tissue at the forelimb level at stage 20/21 and the forelimb bud at stage 25. Cells obtained from stage 20/21 trunk tissues ( n  = 2696) and stage 25 forelimb buds ( n  = 7363) were used for analyses after quality control of datasets using the Seurat package to remove potential doublets and low-quality reads. Multidimensional reduction through tSNE clustering led to the unbiased identification of the muscle clusters, which contained cells enriched for markers of the somite-derived myogenic cells, such as Pax3 43 , cMet , and Lbx1 as well as markers of skeletal muscle progenitors, such as TnnT3 ( Troponin T3, Fast Skeletal Type ), MyoD1 and Myog ( myogenin ) from stages 20/21 trunk tissues at the forelimb level (Fig.  3a, b ; Supplementary Figs.  9 and 10 ; Supplementary Data  1 ). Interestingly, in this muscle cluster, 6.2% of cells expressing both Pax3 and Hand2 , which is a general marker of the lateral plate mesodermal cells 44 , 45 , 46 , 47 , were identified (21/339 cells; Fig.  3c ). We also identified the muscle clusters expressing the myogenic markers from stage 25 forelimb buds (Fig.  3d, e ; Supplementary Fig.  11 ; Supplementary Data  2 ). In the muscle cluster of the stage 25 forelimb buds (Fig.  3d, e ), 19.2% of muscle progenitors positive for both Pax3 and Hand2 were identified (97/505 cells; Fig.  3f ). In this Pax3  + / Hand2 + subpopulation of cells, significantly higher levels of expressions of other somatic LPM cell markers, such as Prrx1 and Tbx5 48 , 49 , 50 , 51 , 52 , 53 , compared to the Pax3  + / Hand2 - progenitors were detected (Fig.  3g ; Supplementary Data  3 ). In addition, the expression of MyoD1 and Tnnt3 was detected in this Pax3  + / Hand2 + subpopulation (Fig.  3g ). These results suggest that the Pax3  + / Hand2 + cells found during emu limb development have a dual transcriptional signature of the somite-derived myogenic cell and the LPM cell.

a, d , tSNE plots of body trunk at the forelimb level of stage 20/21 emu embryo data (a) and stage 25 emu forelimb buds (d), respectively. b, e , Dot plots of subcluster marker gene expression of stage 20/21 data (b) and stage 25 data (e), respectively. Dot color represents the average expression level, and dot size represents the percentage of cells expressing marker genes. c, f , Venn diagram showing number of cells expressing Pax3 and/or Hand2 in the muscle cluster of stage 20/21 data (c) and stage 25 data (f), respectively. g , Violin plots showing the expression levels of Pax3, Lbx1, MyoD1, Tnnt3, Hand2, Prrx1 , and Tbx5 in Pax3  + / Hand2- cells, and Pax3  + / Hand2+ cells in the muscle cluster of stage 25 emu forelimb data. The two-sided Wilcoxon rank sum-test was used for statistical test. The exact p -values are indicated.

To determine whether such subpopulation of muscle cells with a dual transcriptional signature is a unique feature of emu forelimbs, we analyzed a publicly available single-cell RNA-seq dataset obtained from stage 24 and 27 chicken forelimb buds 54 and compared them with data from stage 25 emu forelimb buds. In stage 25 emu forelimb buds, we identified clusters of muscle cells enriched with LPM cell markers (Supplementary Fig.  12 ). In contrast, in stage 24 and 27 chicken forelimb buds, Hand2 expression was not observed in the extracted cluster of muscle cells, and the expression of other LPM markers were not enriched (Supplementary Fig.  13 ; Supplementary Data  4 - 5 ). This suggests that muscle progenitors with an LPM transcriptional signature are distinctive to emu forelimb buds.

Next, we used in situ hybridization chain reaction (HCR) technique to examine the distribution of the subpopulation of cells with the dual Pax3  + / Hand2 + identity in developing emu forelimb buds (Fig.  4 ; Supplementary Fig.  14 ). At stage 19, transcripts of Pax3 were detected in the dermomyotome and those of Hand2 were detected in the forelimb mesoderm (Fig.  4a ; Supplementary Fig.  14a ), but co-localization of Pax3 and Hand2 transcripts was not identified in the forelimb buds. By stage 23, although a few cells begin to exhibit a dual transcriptional signature, most Pax3 + somite-derived myogenic cells and Hand2 + forelimb mesenchyme can be distinguished from each other (Fig.  4b ; Supplementary Fig.  14b ). However, by stage 25, Pax3 transcripts were co-localized with Hand2 transcripts in a population of cells aggregated at the proximal region of forelimb buds (Fig.  4c, d ; Supplementary Fig.  14c, d ), and continued to be observed at the same region until stage 27 (Supplementary Fig.  15 ). This cell population contained extensive pyknotic nuclei when compared with the surrounding cells (Fig.  4c’, e ; Supplementary Fig.  15 ). In contrast, in developing chicken forelimb buds, Pax3 + dermomyotome and muscle progenitors were clearly distinguishable from Hand2 + mesenchymal cells, and unlike in emu forelimb buds, cell aggregation was not observed in proximal region (Fig.  4f–h ; Supplementary Fig.  16 ). The cell population with pyknotic nuclear feature in emu limb buds appeared to be entrapped at the proximal region by forming aggregates, and most importantly, some of their nuclei were condensed or fragmented (Fig.  4c, d ; Supplementary Fig.  15 ). At this stage, MF20-positive muscle cells were recognized among Pax3 + migrating muscle progenitors; however, such a strong MF20 staining was either absent or only sparsely recognized in the aggregated cells (Fig.  4d , Supplementary Figs.  14 c and 15 ). Taken together, in emu forelimb buds, a subpopulation of muscle progenitors exhibits a dual Pax3 + somite-derived myogenic cell/ Hand2  + LPM cell signature and can be located forming aggregates at the proximal region during emu wing development.

a – e HCR for Pax3 and Hand2 and immunostaining for MF20 in emu forelimb buds at stages 19 ( a , n  = 2), 23 ( b , n  = 3) and 25 ( c – e , n  = 3). c’ Enlarged images indicated in ( c ). Arrowheads indicate fragmented nuclei. Note that transcripts of both Pax3 and Hand2 were observed in aggregated cells (arrows). d , Note that MF20 signals (arrows) were not detected in the aggregated cell population (a dotted circle). e HCR for Pax3 and Hand2 in the aggregated cell population at the proximal region of stage 25 emu forelimb buds (arrows). Note that transcripts of Pax3 and Hand2 are co-localized. Arrowheads indicate fragmented nuclei. f – h HCR for Pax3 and Hand2 in chicken forelimb buds at stages 19 ( f , n  = 3), 23 ( g , n  = 4) and 25 ( h , n  = 2). h’ Enlarged images indicated in ( h ). Unlike emu forelimb buds, no aggregated muscle progenitors are observed at the proximal part of chicken forelimb buds at stages 23 or 25. Scale bars, 100 μm ( a – d , c ’, h ’), 20 μm ( e ), 200 μm ( f – h ). Panels of ( a ), ( c , c ’) and ( g ) are flipped horizontally.

A population of muscle progenitors with dual LPM/myogenic cell underwent cell death during wing development

Fragmentation of nuclei seen in the Pax3  + / Hand2 + cell population is a characteristic feature of apoptotic cells. In accordance with this prediction, scRNA-seq data showed that the expression of BCL2 Antagonist/Killer 1 ( BAK1 ), encoding a pro-apoptotic protein, Caspase-10 ( CASP10 ), encoding a cysteine peptidase responsible for the activation of the apoptotic cascade, and Apoptotic Peptidase Activating Factor 1 ( APAF1 ), encoding a cytoplasmic protein that assembles into the apoptosome upon cytochrome c binding, were recognized in the Pax3  + / Hand2 + cell population, albeit not statistically upregulated at expression level (Fig.  5a ; Supplementary Data  3 ). Of note, most cells actively undergoing apoptosis have been eliminated from our scRNA-seq during cell preparation steps (density gradient centrifugation excludes apoptotic bodies) or bioinformatics quality control (exclusion of cells with mtDNA content > 15%). Nevertheless, it remained unclear whether the cells exhibiting nuclear fragmentation were congruent with those identified through scRNA-seq analysis. Therefore, to investigate this further, apoptosis assays were conducted using TUNEL staining, along with immunostaining for cleaved caspase-3 and the oxidative damage marker 8-oxoguanine, on sections of emu forelimb buds (Fig.  5b–d ; Supplementary Fig.  17 ). In this population, cells positive for TUNEL, caspase-3, and 8-oxoguanine were highly abundant (Fig.  5b–d ). Additionally, some TUNEL-positive cells in the proximal region were surrounded by MF20-positive cells (Supplementary Fig.  17 ), suggesting that these dying cells were among muscle progenitors. These findings indicate that a distinct population of muscle progenitor cells, characterized by a dual LPM/myogenic cell signature, undergoes cell death.

a Violin plots showing the expression levels of Bak1 , Casp10 and Apaf1 in Pax3  +  /Hand2 - cells, or Pax3  +  /Hand2 + cells in the muscle cluster of stage 25 emu forelimb data. b – d TUNEL staining ( b , n  = 3), immunostaining for active caspase-3 ( c , n  = 2) and immunostaining for 8-oxoguanine ( d , n  = 2) in the aggregated cell population at the proximal region of stage 25 emu forelimb buds. b’ – d’ Enlarged images indicated in ( b – d ). Panels of ( b , b’ ) and ( c , c’ ) are flipped horizontally. Scale bars, 50 μm. e Schematic model of forelimb development in emu embryos. Migratory muscle precursors ( Pax3  + , Lbx1  + , cMet  + ) delaminated from the ventral edge of the dermomyotome and begin to migrate into the forelimb mesenchyme ( Hand2  + , Prrx1  + , Tbx5  + ). Subsequently, a subpopulation of muscle precursors with a dual somite-derived myogenic cell ( Pax3  + , Lbx1  + , cMet  + ) /LPM cell ( Hand2  + , Prrx1  + , Tbx5  + ) appears and aggregates at the proximal part of forelimb buds. This aggregated cell population undergoes cell death and thereby failing to form majority of muscles. Impaired formation of limb muscles seems to be at least partially responsible for the asymmetric reduction or fusion of distal skeletal elements. Our results and those of others 2 , 5 , 6 , 7 , 8 , 9 suggest that multiple mechanisms contribute to the unique emu wing morphology. See text for details.

Here, we show that emu forelimbs exhibit left–right asymmetric skeletal patterns, in addition to a reduction in longitudinal bone growth in the limbs and contractures of wrist joints. The observed morphological traits in emu forelimbs can be attributed to a reduction of mechanical loading from embryonic movements, which in turn is due to the absence of muscles at the distal parts of their forelimbs. Impaired skeletal muscles of emu forelimbs are, at least partially, attributed to massive cell death of a population of muscle progenitors exhibiting a dual LPM/myogenic cell signature.

During myogenesis, muscle progenitors/myoblasts take one of three distinct fates 55 . While the majority of myoblasts fuse to form multinucleated muscle fibers/myotubes, there is a second population of cells located between the basal membrane and the sarcolemma of the muscle fibers that becomes myogenic stem cells/satellite cells. Furthermore, a third population of muscle progenitors that fails to differentiate into muscle fibers undergo programmed cell death 55 . In emu forelimb buds, some of Pax3  + / Hand2 - migrating muscle progenitors expressed high levels of myosin heavy chain (MF20), indicating differentiation into muscle fibers. On the other hand, MF20 signals were hardly detectable in a subpopulation of cells exhibiting a dual LPM/myogenic cell signature, suggesting that these cells might have failed to differentiate into muscle fibers and undergo other ‘default’ states, such as cell death. It is reasonable to assume that muscle progenitors with a LPM transcriptional signature may not be able to form muscle fibers which usually form by a fusion of cells in a homogenous population of myoblasts. Interestingly, the differentiation of myoblasts into muscle fibers requires activation of caspase-3 56 , as well as cytochrome c and Apaf-1 57 , suggesting that the mitochondrial death pathway can promote cell fusion as well as cell death. Caspase-dependent non-lethal differentiation has been reported not only in myoblasts, but in a variety of cell types 58 , 59 , however, the mechanisms of how some cell types avoid cell death under the presence of caspase is largely unknown 57 . The duration and intensity of caspase-3/7 activity is shown to be critical to determine whether mouse embryonic stem cells differentiate into cardiomyocytes or undergo cell death 60 . In emu forelimb buds, a subpopulation of cells exhibiting a dual LPM/myogenic cell signature displayed a strong activity of caspase-3, along with oxidative damages of DNA and fragmented nuclei, suggesting caspase-dependent apoptosis occurred. Given that caspase-dependent apoptotic signals are required for differentiation of myoblast into muscle fibers, it is conceivable that cells with a dual LPM/myogenic cell signature fail to differentiate into muscle fibers due to excess level of caspase activity for differentiation, and consequentially, underwent cell death before migrating toward the distal region.

Multiple factors have been associated with the morphological features of emu forelimbs. In the early limb bud of emu embryos, the width of Grem1 expression domain, which indicates the amount of digit progenitors regulated by the SHH/GREM1/AER-FGF system 61 , 62 , 63 , 64 , 65 , exhibits individual variation 8 . Recent work showed that regulatory changes lead to the lower expression of Fgf10 and a concomitant failure to express genes related to cell proliferation in the early emu forelimb 9 . Such alterations in gene expression, including a reduction in Fgf8 expression in the apical ectodermal ridge, might contribute to the individual variation in Grem1 expression seen in the early limb bud. The reduction in limb proliferation due to the lower expression of Fgf10 can explain the heterochrony observed in emu forelimb outgrowth 6 , 9 . Loss of embryonic mobility also resulted in a late-stage reduction in the forelimb growth (Fig.  2b ), as previously shown for hindlimbs of both chicken and crocodiles 29 . Thus, the lack of movement of the distal part of the forelimb might also be a source of the heterochronic development found at later stages in emu forelimbs 6 in regard to their skeletal development. Massive cell death of muscle progenitors is correlated with the reduction of muscle masses in the emu forelimb, which became evident at later stages (Fig.  1c ) as well as in adult wings 11 . The resulting decrease in mechanical stimulation, essential for osteoblast differentiation 66 , likely results in a scarcity of specified skeletal progenitors. This scarcity contributes to a range of skeletal alteration, such as the reduction and fusion of skeletal elements, reflecting the skeletal abnormalities observed subsequent to the loss of mobility during embryonic and fetal stages 12 . These data suggest an integrated model in which multiple mechanisms contribute to the extreme reduction and unique phenotype of the flightless emu wing.

We propose that the morphological features seen in emu forelimbs are, at least partially, attributed to a reduction in mechanical loading from embryonic movements, which is caused by the absence of distal muscles. Impaired development of forelimb muscles seems to be associated with the massive cell death of muscle progenitors with a dual LPM/myogenic cell signature. These progenitor cells accumulate at the proximal region, thereby losing their motility, and eventually undergoes cell death (Fig.  5e ).

Mechanical loading from embryonic movements, which can be influenced by a range of environmental factors, has been suggested to contribute to the establishment of various skeletal design of limbs 13 , 15 , 16 , 29 , 31 . The locomotory system displays a remarkable integration between its muscular and skeletal components, allowing for coordinated phenotypic changes throughout evolution. The interdependency of these systems can be identified at several organizational levels. For example, at a molecular level, the mechanotransducer and transcriptional regulator YAP regulates the development of both the skeleton 67 , 68 and muscle 69 . At a cellular level, cells with dual muscle and LPM identity were identified during mouse limb development and play an important role in integrating the skeletal and muscle compartments at the myotendinous junctions 69 , 70 , 71 . And finally, at organismal level, the “two-legged goat” example reveals how a change in behavior led to morphological traits that phenocopied several evolutionary novelties found in bipedal mammals, in a remarkable example of plasticity that is only possible when the development of muscular and skeletal compartments is strongly coordinated 72 .

Species-specific traits can arise not only from distinct load bearing on the adult skeleton, but also from differential motility affecting skeletal proportions and joint formation during development 31 . In a contrasting example of morphological evolution driven by a naturally occurring immobilization, the intrinsic muscles of jerboa feet disappear by a muscle atrophy mechanism without evidence of cell death 73 . Our results demonstrate that differential embryonic muscle contractions, which can be altered both genetically and epigenetically, may underlie the morphological diversification that has occurred during vertebrate evolution, including in vestigial structures such as the emu wing.

Data reporting

No statistical methods were used to predetermine the sample size. The experiments were not randomized, and the investigators were not blinded to allocation during the experiments and outcome assessment.

White Leghorn chicken ( Gallus gallus ) eggs were incubated at 37.5 °C and staged 74 . Fertilized emu ( Dromaius novaehollandiae ) eggs were purchased from Kakegawa Kachoen and Okhotsk Emu Pasture, incubated at 36 °C and staged as described in ref. 4 . Wing samples of adult chickens were purchased post-mortem from a commercial supplier. Wing samples of adult emu were provided post-mortem from Tokyo Nodai Bioindustry Corporation. All animal work was performed in accordance with the guidelines for animal experiments of the Tokyo Institute of Technology, Kumamoto University, and The Jikei University School of Medicine, and the experimental protocols were approved by the committees of Tokyo Institute of Technology, and Kumamoto University. The sex of the animals was unknown.

Manipulation of embryo movement

Rigid paralysis 27 was induced in chicken embryos with decamethonium bromide (DMB), as previously described in ref. 29 with slight modifications. Briefly, embryos were immobilized between either E10-17 or E6-10. On the first day, 100 μl of sterile filtered 5 mg/ml DMB in 100 μl phosphate-buffered saline (PBS) was injected through the egg window onto the chorioallantoic membrane to induce immobilization. On each subsequent day, 100 μl of 1 mg/ml DMB in PBS was administered to maintain paralysis. Embryos were monitored by viewing through the egg window on each day of treatment and any DMB-treated embryos that were not fully paralyzed were excluded from the study. Control animals were administered only vehcle (PBS). The embryos were euthanized one day after the treatment and fixed in 4% paraformaldehyde. To quantify the rate of distal movements in the wings of embryos (Supplementary Fig.  1d ), we recorded the behavior of stage 39 chicken and emu embryos incubated at 37.5 °C and 36 °C, respectively, through the window of the eggshell. Counts were conducted only when the embryos exhibited active movement, defined as any instance where the head, arms or legs were actively moving for more than 10 s. The rate of distal movements per individual was calculated as the distal movement count divided by the proximal movement count in five minutes of total active movement time. Proximal movement corresponded to extension and flexion of the elbow joint; distal movement corresponded to any wrist or phalangeal movement (Supplementary Fig.  2b ).

Probe synthesis and in situ hybridization

Total RNA was extracted from stage 25–26 chickens, and stage 25 emu embryos using RNeasy kit (Qiagen). cDNA was synthesized by reverse transcription and used as a template for PCR. The construct used to synthesize RNA probes were based on the pBlueScript II SK(+), using In-Fusion HD Cloning Kits (Clontech). The primers used to amplify most of the gene fragments were designed with the forward (5′-ATCGATAAGCTTGAT…−3′) and reverse (5′-CTGCAGGAATTCGAT…−3′) prefixes complementary to the ends of the linearized vector, which were followed by the specific sequences of the primers that were based on the indicated published sequences: chicken Lbx1 (Ensembl, ENSGALG00000034189), 5′-CTGCGCTTCAACTTTTGCTC-3′ and 5′-GGTTCTGGAACCAGGTGAT-3′; chicken MyoD (GenBank accession number, NM_204214), 5′-CTTCTATGACGACCCGTGC-3′ and 5′-GTCTTGGAGCTTGGCTGAA-3′; emu cMet (Ensembl, ENSDNVG00000012888), 5′-AGGAGCCATGGACAATGCAA-3′ and 5′-CGAATGGACCTCTTCCTC-3′; emu GlobinA (Ensembl, ENSDNVT00000001340.1), 5′-GAGCTGCAACCATG-3′ and 5′-TGGCTGCTCGCTG-3′. For emu Nkx2.5 (Ensembl, ENSDNVG00000013910.1), the open reading frame (ORF) of emu Nkx2.5 was amplified using specific primers (5′-CCCACCGCAATGTTTCCTAGCCCT-3′ and 5′-CTACCAGGCTCGGATCCCGTGCA-3′) and cloned into the Eco RV site of the pBlueScript SK(−) (pBSK- Nkx2.5 ). For emu Lbx1 (Ensembl, ENSDNVG00000014711), emu genomic DNA was used as a template for PCR. The 5′ and 3′ fragments of emu Lbx1 were amplified using the specific primers with the sequences complementary to the ends of the linearized pBlueScript II SK(+) (5′ fragment, 5′-ATCGATAAGCTTGATGTGGGCTTCAACTTTTGCTC-3′ and 5′-CTGCAGGAATTCGATGAGCCCCTTGAAGGTCTT-3′; 3′ fragment, 5′-ATCGATAAGCTTGATACCTATTCGTGGCCATGTCG-3′ and 5′-CTGCAGGAATTCGATCCGATGGGGCTGAGTGTAA-3′) and cloned into the vector using an In-Fusion HD Cloning Kit. The first exon sequence was obtained by fusing the DNA duplex synthesized using the oligonucleotides (5′-AGACCTTCAAGGGGCTCGAAGTGAGCGTGCTGCAGGCGGAATTCCTGCAGCCC-3′ and 5′-GGGCTGCAGGAATTCCGCCTGCAGCACGCTCACTTCGAGCCCCTTGAAGGTCT-3′) into the Hind III site (3′ side) of the 5′ fragment. The full-length of emu Lbx1 was obtained by cloning of the first exon sequence using specific primers with Hind III sequence (5′-GGTATCGATAAGCTTGATGTGGGCTTCAACTTTTG-3′ and 5′-GGAGACGCTGCGATTCGCCTGCAGCACGCT −3′), into the Hind III site of the vector including the second exon sequence, which was obtained by fusing the fragment amplified using the following primers by using In-Fusion HD Coning Kits (5′-GGTATCGATAAGCTTAATCGCAGCGTCTCCCCTGC-3′ and 5′-CGACATGGCC ACGAATAGGT-3′) to the 5′ side of the 3′ fragment.

For in situ hybridization, embryos were fixed overnight in 4% paraformaldehyde in phosphate-buffered saline, dehydrated in a graded methanol series and stored in 100% methanol at −20 °C. Whole-mount and section in situ hybridization were carried out as described 75 , 76 . For RNAscope in situ hybridization, probes for emu cMet (targeting 827–1801 bp of XM_026097341.1), emu Nkx2.5 (targeting 2-1564 bp of XM_026122666.1), and emu MyoD (targeting 31–1358 bp of XM_026092794.1) were designed commercially by the manufacturer (Advanced Cell Diagnostics, Inc.). RNAscope in situ hybridization was performed using the RNAscope Fluorescent Multiplex Reagent Kit according to the manufacturer’s instructions. HCR in situ hybridization was conducted according to the manufacturer’s instructions. HCR probes targeting emu Pax3 (XM_026109761.1) and Hand2 (XM_026122869.1) were purchased commercially from Molecular Instruments, Inc. Images were obtained using an LSM 780 confocal microscope (Zeiss).

Whole-mount immunostaining was performed as previously described 77 . Supernatant containing the monoclonal anti-MYH1E antibody was used to detect against myosin heavy chain (MF-20 hybridoma clone, DSHB). Goat anti-mouse IgG, HRP-conjugated antibodies (HAF007, R&D Systems) were used at a concentration of 1:200. Section immunostaining was performed as described in ref. 78 . Briefly, cryosections were incubated overnight with 1:1000 anti-cleaved caspase-8 antibody (9664, Cell signaling technology) or 1:100 anti-8-oxoguanine antibody (ab 64548, Abcam) at 4 °C. Sections were washed and incubated overnight with 1:1000 goat anti-rabbit IgG Alexa Fluor-488 conjugated antibody (ab150077, Abcam) or goat anti-mouse IgG Alexa Fluor-488 conjugated antibody (Invitrogen) at 4 °C and then washed. For TUNEL staining, cryosections were stained using TUNEL Mix (In situ Cell Death Kit, Roche) according to the manufacturer’s protocol. The sections were then incubated overnight with 1:400 sheep anti-fluorescein-AP antibody (Roche) at 4 °C and then washed. For Safranin-O staining, sections of embryos were prepared as described 79 . Briefly, embryos were fixed with 4% paraformaldehyde, dehydrated in ethanol and acetone, and embedded in Technovit 8100 resin (Heraues-Kulzer, Wehrheim, Germany). Sections were cut at a thickness of 8 μm and stained with Weigert’s iron hematoxylin, Safranin-O and Fast Green as described in ref. 18 . The narrowest parts of the interspace distance between the ulna and the distal carpus (chicken) or metacarpus (emu) were measured using cellSens software (Olympus).

Skeleton staining

Skeletons were stained according to standard protocols using Alcian blue for cartilage and Alizarin red for bone 80 . The length of individual bones was measured using Fiji 81 .

Computed tomography imaging

Computed tomography (CT) imaging of adult emu forelimb skeletons was performed by a micro-CT system (Latheta LCT-200, Hitachi Aloka Medical Ltd., Tokyo, Japan). Acquired slice data were rendered as three-dimensional images using VGStudio MAX2.0 software (Volume Graphics GmbH., Heidelberg, Germany). Fusion of skeletal elements was evaluated by RadiAnt DICOM viewer ( www.radiantviewer.com/ ).

Single-cell RNA sequencing and analysis

Trunk tissue at the forelimb level from four stage 20/21 emu embryos, and from fifteen forelimb buds from stage 25 emu embryos, were collected, pooled and then dissociated into single cells by enzymatic digestion as previously described 82 , with modifications. Briefly, limb buds were treated with 2000 U dispase II (Wako) in Ca 2+ - and Mg 2+ -free Tyrode’s solution (CMF-Tyrode) at 4 °C for 50 min and transferred to Dulbecco’s modified Eagle’s medium (DMEM) containing 1% fetal bovine serum (FBS), when their ectodermal sheets were removed by peeling off with a pair of forceps. Then, the remaining limb tissues or trunk tissues were transferred to Cellbanker 1 plus (Nippon Zenyaku Kogyo) and stored at −80 °C. Thawed tissues were washed in DMEM containing 0.04% bovine serum albumin (BSA), and incubated in CMF-Tyrode at 37 °C for 40 min. Softened tissues were dissociated into a single cell suspension by pipetting in DMEM containing 0.04% BSA. The suspension was first filtered using pluriStrainer-Mini 100 μm (pluriSelect), and the debris was removed using Debris Removal Solution (Miltenyi Biotec) according to the manufacturer’s instructions. Then, cells were filtered again using pluriStrainer-Mini 40 μm (pluriSelect), suspended in DMEM containing 2% BSA and proceeded immediately to cell encapsulation, indexing and transcriptome library prepration, as described below.

The single-cell transcriptome data of emu embryos were generated at the Functional Genomics Facility of the National Institute for Basic Biology (NIBB) in Okazaki, Japan. For scRNAseq library construction, barcoded single-cell cDNA libraries were synthesized using 10x Genomics Chromium Single Cell 3’ Reagent Kits v3.1 (Dual Index) according to the manufacturer’s instructions. Libraries were sequenced on an Illumina Hiseq platform at a depth of 117,418 and 48,543 mean reads per cell for stage 20/21 and stage 25 samples, respectively. The single-cell transcriptome data of chicken forelimbs were published previously 54 . Chicken forelimb single cell RNA sequence data of stage 24 (SRA accession numbers: SRR14570167 to SRR14570174) and stage 27 (SRA accession numbers: SRR14570175 to SRR14570178) were downloaded from the Sequence Read Archive (SRA).

Raw sequencing data were processedand aligned with a emu reference genome assembly droNov1 (GCF_003342905.1) or a chicken reference genome assembly GRCg6a (Ensembl release 105) using Cell Ranger software version 7.1.0 (10x Genomics). Cell Ranger filtered outputs files for each dataset were processed using R package Seurat v4.3.0 83 . Following the standard pre-processing workflow of Seurat, low-quality cells and potential cell doublets were removed from the dataset. Gene expression was log normalized with NormalizedData and scaled with ScaleData using all genes. PCA-reduction was performed with RunPCA and nearest neighbor graph was constructed using the 50 PCs with FindNeighbors, and then clusters were identified using FindClusters. Cluster were assigned specific identities based on differentially expressed genes. To visualize these datasets, tSNE (t-distributed Stochastic Neighbor Embedding) was performed with RunTSNE using 1:50 dims as input. Differentially expressed genes for each cluster of emu and chicken data were provided by using the FindAllMarkers, which performs Wilcoxon rank-sum tests. Differential expression testing between Pax3  +  /Hand2+ cells and Pax3  +  /Hand2- cells from the muscle cluster of stage 25 emu was conducted using the FindMakers function, which applies Wilcoxon rank-sum tests.

Statistical analysis

All statistical analyses, except for Figs.  3 g and 5a , were performed with Prism 8.0.2 (GraphPad). n numbers represent the numbers of limbs, and measurements are represented as the mean ± SEM. All measurements were taken from distinct samples. Two-parameter comparisons between measurements of biological samples assumed unequal variances, thus Welch’s unpaired t -test was used. The coefficient of variation was calculated as the ratio of the standard deviation to the mean. Statistical details can be found in the legends. Significance was defined at P  ≤ 0.05 (* p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001). For differentially expressed genes in Figs.  3 g and 5a , the two-sided Wilcoxon rank-sum tests were used for statistical tests ( p -val adjusted < 0.05).

Reporting summary

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

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its Supplementary Information files. The raw sequencing data generated in this study have been deposited in the DDBJ Sequence Read Archive under accession numbers: DRA017391 and DRA014432 ; bioproject: PRJDB13845 and PRJDB16987 . The publicly available chicken datasets, published before 54 , were retrieved from Sequence Read Archive (SRA) under the accession numbers SRR14570167 to SRR14570174 ( https://www.ncbi.nlm.nih.gov/sra/SRX10913414 [accn]) and SRR14570175 to SRR14570178 ( https://www.ncbi.nlm.nih.gov/sra/SRX10913415 [accn]).  Source data are provided with this paper.

Code availability

All code used to reproduce the Supplementary Fig. presented in this paper is publicly available through GiHub. The corresponding DOI is https://doi.org/10.5281/zenodo.11127881 .

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Acknowledgements

We thank Dr. Q. Zhou for providing wing samples of emu embryos, Drs. J. Miyazaki, T. Ogura and A. P. McMahon for plasmids, Tokyo Nodai Bioindustry Corporation for providing wing samples of adult emu, Kakegawa Kachoen and Okhotsk Emu Pasture for providing emu eggs, Dr. T. Hirasawa for valuable comments, Mr. A. Irifune and Dr. M. Uesaka for technical assistance, and Biotechnology Center of Tokyo Institute of Technology for sequencing services. This work was supported by JSPS KAKENHI Grant Numbers JP20H03301, and JP17KT0106, MEXT KAKENHI Grant Number JP18H04818, NIBB Collaborative Research Program (21-357), Astellas Foundation for Research on Metabolic Disorders, Mitsubishi Foundation and Yamada Science Foundation to M.T.

Author information

Ingrid Rosenburg Cordeiro

Present address: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden

These authors contributed equally: Eriko Tsuboi, Satomi F. Ono, Ingrid Rosenburg Cordeiro.

Authors and Affiliations

School of Life Science and Technology, Tokyo Institute of Technology, B-17, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa, Japan

Eriko Tsuboi, Satomi F. Ono, Ingrid Rosenburg Cordeiro, Reiko Yu, Toru Kawanishi & Mikiko Tanaka

Laboratory Animal Facilities, The Jikei University School of Medicine, 3-25-8 Nishi-shimbashi, Minato-ku, Tokyo, Japan

Makoto Koizumi

Trans-Omics Facility, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, Aichi, Japan

Shuji Shigenobu

International Research Center for Medical Sciences, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, Japan

Guojun Sheng

Department of Anatomy, The Jikei University School of Medicine, 3-25-8 Nishi-shimbashi, Minato-ku, Tokyo, Japan

Masataka Okabe

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E.T., I.R.C., S.F.O. and M.T. designed the project and wrote the manuscript. E.T. analyzed the length and patterns of skeletal elements and muscles, I.R.C. examined gene expression, S.F.O. examined the movement of embryos, and performed scRNA-seq analyses, S.F.O. and T.K. captured confocal images, M.T. performed histology, immunology and examined gene expression by using DIG-, RNA scope- and HCR-probes, R.Y. constructed plasmids, S.S. provided sequencing research infrastructures, G.S. provided plasmids related to blood differentiation and emu embryos. M.K. took CT images, M.O. assisted with CT scanning.

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Tsuboi, E., Ono, S.F., Cordeiro, I.R. et al. Immobilization secondary to cell death of muscle precursors with a dual transcriptional signature contributes to the emu wing skeletal pattern. Nat Commun 15 , 8153 (2024). https://doi.org/10.1038/s41467-024-52203-x

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• v.112(29); 2015 Jul 21

Cloning humans? Biological, ethical, and social considerations

Author contributions: F.J.A. wrote the paper.

There are, in mankind, two kinds of heredity: biological and cultural. Cultural inheritance makes possible for humans what no other organism can accomplish: the cumulative transmission of experience from generation to generation. In turn, cultural inheritance leads to cultural evolution, the prevailing mode of human adaptation. For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments. Nevertheless, natural selection persists in modern humans, both as differential mortality and as differential fertility, although its intensity may decrease in the future. More than 2,000 human diseases and abnormalities have a genetic causation. Health care and the increasing feasibility of genetic therapy will, although slowly, augment the future incidence of hereditary ailments. Germ-line gene therapy could halt this increase, but at present, it is not technically feasible. The proposal to enhance the human genetic endowment by genetic cloning of eminent individuals is not warranted. Genomes can be cloned; individuals cannot. In the future, therapeutic cloning will bring enhanced possibilities for organ transplantation, nerve cells and tissue healing, and other health benefits.

Chimpanzees are the closest relatives of Homo sapiens , our species. There is a precise correspondence bone by bone between the skeletons of a chimpanzee and a human. Humans bear young like apes and other mammals. Humans have organs and limbs similar to birds, reptiles, and amphibians; these similarities reflect the common evolutionary origin of vertebrates. However, it does not take much reflection to notice the distinct uniqueness of our species. Conspicuous anatomical differences between humans and apes include bipedal gait and an enlarged brain. Much more conspicuous than the anatomical differences are the distinct behaviors and institutions. Humans have symbolic language, elaborate social and political institutions, codes of law, literature and art, ethics, and religion; humans build roads and cities, travel by motorcars, ships, and airplanes, and communicate by means of telephones, computers, and televisions.

Human Origins

The hominin lineage diverged from the chimpanzee lineage 6–7 Ma, and it evolved exclusively in the African continent until the emergence of Homo erectus , somewhat before 1.8 Ma. Shortly after its emergence in tropical or subtropical Africa, H. erectus spread to other continents. Fossil remains of H. erectus (sensu lato) are known from Africa, Indonesia (Java), China, the Middle East, and Europe. H. erectus fossils from Java have been dated at 1.81 ± 0.04 and 1.66 ± 0.04 Ma and from Georgia at 1.6–1.8 Ma ( 1 ). Anatomically distinctive H. erectus fossils have been found in Spain, deposited before 780,000 y ago, the oldest in southern Europe ( 2 ).

The transition from H. erectus to H. sapiens occurred around 400,000 y ago, although this date is not well determined owing to uncertainty as to whether some fossils are erectus or archaic forms of sapiens. H. erectus persisted for some time in Asia, until 250,000 y ago in China and perhaps until 100,000 ago in Java, and thus was contemporary with early members of its descendant species, H. sapiens. Fossil remains of Neandertal hominids ( Homo neanderthalensis ), with brains as large as those of H. sapiens , appeared in Europe earlier than 200,000 y ago and persisted until 30,000 or 40,000 y ago ( 3 , 4 ).

There is controversy about the origin of modern humans. Some anthropologists argue that the transition from H. erectus to archaic H. sapiens and later to anatomically modern humans occurred consonantly in various parts of the Old World. Proponents of this “multiregional model” emphasize fossil evidence showing regional continuity in the transition from H. erectus to archaic and then modern H. sapiens . Most anthropologists argue instead that modern humans first arose in Africa somewhat before 100,000 y ago and from there spread throughout the world, eventually replacing elsewhere the preexisting populations of H. erectus , H. neanderthalensis, and archaic H. sapiens . The African origin of modern humans is supported by a wealth of recent genetic evidence and is therefore favored by many evolutionists ( 2 , 4 ).

We know about these matters in three ways: by comparing living primates, including humans, with each other; by discovery and investigation of fossil remains of primates that lived in the past; and by comparing their DNA, proteins, and other molecules. DNA and proteins give us the best information about how closely related we are to each of the primates and those to each other. However, to know how the human lineage changed in anatomy and behavior over time as our ancestors became more and more human-like, we have to study fossils and the tools they used and made, as well as other remnants of their activities ( 2 , 5 ).

Humans live in groups that are socially organized and so do other primates. However, other primate societies do not approach the complexity of human social organization. A distinctive human social trait is culture, which may be understood as the set of nonstrictly biological human activities and creations. Culture includes social and political institutions, ways of doing things, religious and ethical traditions, language, common sense and scientific knowledge, art and literature, technology, and in general all of the creations of the human mind. The advent of culture has brought with it cultural evolution, a superorganic mode of evolution superimposed on the organic mode, that has become the dominant mode of human evolution. Cultural evolution has come about because of cultural inheritance, a distinctively human mode of achieving adaptation to the environment ( 2 , 6 , 7 ).

There are in mankind two kinds of heredity: the biological and the cultural. Biological inheritance in humans is very much like that in any other sexually reproducing organism; it is based on the transmission of genetic information encoded in DNA from one generation to the next by means of the sex cells. Cultural inheritance, on the other hand, is based on transmission of information by a teaching-learning process, which is in principle independent of biological parentage. Culture is transmitted by instruction and learning, by example and imitation, through books, newspapers, radio, television, and motion pictures, through works of art, and through any other means of communication. Culture is acquired by every person from parents, relatives, and neighbors and from the whole human environment. Acquired cultural traits may be beneficial but also toxic; for example, racial prejudice or religious bigotry.

Biological heredity is Mendelian or vertical; it is transmitted from parents to their children, and only inherited traits can be transmitted to the progeny. (New mutations are insignificant in the present context.) Cultural heredity is Lamarckian: acquired characters can be transmitted to the progeny. However, cultural heredity goes beyond Lamarckian heredity, because it is horizontal and oblique and not only vertical. Traits can be acquired from and transmitted to other members of the same generation, whether or not they are relatives, and also from and to all other individuals with whom a person has contact, whether they are from the same or from any previous or ensuing generation.

Cultural inheritance makes possible for people what no other organism can accomplish—the cumulative transmission of experience from generation to generation. Animals can learn from experience, but they do not transmit their experiences or their discoveries (at least not to any large extent) to the following generations. Animals have individual memory, but they do not have a “social memory.” Humans, on the other hand, have developed a culture because they can transmit cumulatively their experiences from generation to generation.

Cultural inheritance makes possible cultural evolution, a new mode of adaptation to the environment that is not available to nonhuman organisms. Organisms in general adapt to the environment by means of natural selection, by changing over generations their genetic constitution to suit the demands of the environment. However, humans, and humans alone, can also adapt by changing the environment to suit the needs of their genes. (Animals build nests and modify their environment also in other ways, but the manipulation of the environment by any nonhuman species is trivial compared with mankind's manipulation.) For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments.

To extend its geographical habitat, or to survive in a changing environment, a population of organisms must become adapted, through slow accumulation of genetic variants sorted out by natural selection, to the new climatic conditions, different sources of food, different competitors, and so on. The discovery of fire and the use of shelter and clothing allowed humans to spread from the warm tropical and subtropical regions of the Old World to the whole Earth, except for the frozen wastes of Antarctica, without the anatomical development of fur or hair. Humans did not wait for genetic mutants promoting wing development; they have conquered the air in a somewhat more efficient and versatile way by building flying machines. People travel the rivers and the seas without gills or fins. The exploration of outer space has started without waiting for mutations providing humans with the ability to breathe with low oxygen pressures or to function in the absence of gravity; astronauts carry their own oxygen and specially equipped pressure suits. From their obscure beginnings in Africa, humans have become the most widespread and abundant species of mammal on earth. It was the appearance of culture as a superorganic form of adaptation that made mankind the most successful animal species.

Cultural adaptation has prevailed in mankind over biological adaptation because it is a more effective mode of adaptation; it is more rapid and it can be directed. A favorable genetic mutation newly arisen in an individual can be transmitted to a sizeable part of the human species only through innumerable generations. However, a new scientific discovery or technical achievement can be transmitted to the whole of mankind, potentially at least, in less than one generation. Witness the rapid spread of personal computers, iPhones, and the Internet. Moreover, whenever a need arises, culture can directly pursue the appropriate changes to meet the challenge. On the contrary, biological adaptation depends on the accidental availability of a favorable mutation, or of a combination of several mutations, at the time and place where the need arises ( 2 , 6 , 7 ).

Biological Evolution in Modern Humans

There is no scientific basis to the claim sometimes made that the biological evolution of mankind has stopped, or nearly so, at least in technologically advanced countries. It is asserted that the progress of medicine, hygiene, and nutrition have largely eliminated death before middle age; that is, most people live beyond reproductive age, after which death is inconsequential for natural selection. That mankind continues to evolve biologically can be shown because the necessary and sufficient conditions for biological evolution persist. These conditions are genetic variability and differential reproduction. There is a wealth of genetic variation in mankind. With the trivial exception of identical twins, developed from a single fertilized egg, no two people who live now, lived in the past, or will live in the future, are likely to be genetically identical. Much of this variation is relevant to natural selection ( 5 , 8 , 9 ).

Natural selection is simply differential reproduction of alternative genetic variants. Natural selection will occur in mankind if the carriers of some genotypes are likely to leave more descendants than the carriers of other genotypes. Natural selection consists of two main components: differential mortality and differential fertility; both persist in modern mankind, although the intensity of selection due to postnatal mortality has been somewhat attenuated.

Death may occur between conception and birth (prenatal) or after birth (postnatal). The proportion of prenatal deaths is not well known. Death during the early weeks of embryonic development may go totally undetected. However, it is known that no less than 20% of all ascertained human conceptions end in spontaneous abortion during the first 2 mo of pregnancy. Such deaths are often due to deleterious genetic constitutions, and thus they have a selective effect in the population. The intensity of this form of selection has not changed substantially in modern mankind, although it has been slightly reduced with respect to a few genes such as those involved in Rh blood group incompatibility.

Postnatal mortality has been considerably reduced in recent times in technologically advanced countries. For example, in the United States, somewhat less than 50% of those born in 1840 survived to age 45, whereas the average life expectancy for people born in the United States in 1960 is 78 y ( Table 1 ) ( 8 , 10 ). In some regions of the world, postnatal mortality remains quite high, although there it has also generally decreased in recent decades. Mortality before the end of reproductive age, particularly where it has been considerably reduced, is largely associated with genetic defects, and thus it has a favorable selective effect in human populations. Several thousand genetic variants are known that cause diseases and malformations in humans; such variants are kept at low frequencies due to natural selection.

Percent of Americans born between 1840 and 1960 surviving to ages 15 and 45

Reprinted from ref. 8 .

It might seem at first that selection due to differential fertility has been considerably reduced in industrial countries as a consequence of the reduction in the average number of children per family that has taken place. However, this is not so. The intensity of fertility selection depends not on the mean number of children per family, but on the variance in the number of children per family. It is clear why this should be so. Assume that all people of reproductive age marry and that all have exactly the same number of children. In this case, there would not be fertility selection whether couples all had very few or all had very many children. Assume, on the other hand, that the mean number of children per family is low, but some families have no children at all or very few, whereas others have many. In this case, there would be considerable opportunity for selection—the genotypes of parents producing many children would increase in frequency at the expense of those having few or none. Studies of human populations have shown that the opportunity for natural selection often increases as the mean number of children decreases. An extensive study published years ago showed that the index of opportunity for selection due to fertility was four times larger among United States women born in the 20th century, with an average of less than three children per woman, than among women in the Gold Coast of Africa or in rural Quebec, who had three times or more children on average ( Table 2 ) ( 8 , 11 ). There is no evidence that natural selection due to fertility has decreased in modern human populations.

Mean number of children per family and index of opportunity for fertility selection I f , in various human populations

I f is calculated as the variance divided by the square of the mean number of children. The opportunity for selection usually increases as the mean number of children decreases. Reprinted from ref. 8 .

Natural selection may decrease in intensity in the future, but it will not disappear altogether. As long as there is genetic variation and the carriers of some genotypes are more likely to reproduce than others, natural selection will continue operating in human populations. Cultural changes, such as the development of agriculture, migration from the country to the cities, environmental pollution, and many others, create new selective pressures. The pressures of city life are partly responsible for the high incidence of mental disorders in certain human societies. The point to bear in mind is that human environments are changing faster than ever owing precisely to the accelerating rate of cultural change, and environmental changes create new selective pressures, thus fueling biological evolution.

Natural selection is the process of differential reproduction of alternative genetic variants. In terms of single genes, variation occurs when two or more alleles are present in the population at a given gene locus. How much genetic variation exists in the current human population? The answer is “quite a lot,” as will be presently shown, but natural selection will take place only if the alleles of a particular gene have different effects on fitness; that is, if alternative alleles differentially impact the probability of survival and reproduction.

The two genomes that we inherit from each parent are estimated to differ at about one or two nucleotides per thousand. The human genome consists of somewhat more than 3 billion nucleotides ( 12 ). Thus, about 3–6 million nucleotides are different between the two genomes of each human individual, which is a lot of genetic polymorphism. Moreover, the process of mutation introduces new variation in any population every generation. The rate of mutation in the human genome is estimated to be about 10 −8 , which is one nucleotide mutation for every hundred million nucleotides, or about 30 new mutations per genome per generation. Thus, every human has about 60 new mutations (30 in each genome) that were not present in the parents. If we consider the total human population, that is 60 mutations per person multiplied by 7 billion people, which is about 420 billion new mutations per generation that are added to the preexisting 3–6 million polymorphic nucleotides per individual.

That is a lot of mutations, even if many are redundant. Moreover, we must remember that the polymorphisms that count for natural selection are those that impact the probability of survival and reproduction of their carriers. Otherwise, the variant nucleotides may increase or decrease in frequency by chance, a process that evolutionists call “genetic drift,” but will not be impacted by natural selection ( 2 , 12 , 13 ).

Genetic Disorders

More than 2,000 human diseases and abnormalities that have a genetic causation have been identified in the human population. Genetic disorders may be dominant, recessive, multifactorial, or chromosomal. Dominant disorders are caused by the presence of a single copy of the defective allele, so that the disorder is expressed in heterozygous individuals: those having one normal and one defective allele. In recessive disorders, the defective allele must be present in both alleles, that is, it is inherited from each parent to be expressed. Multifactorial disorders are caused by interaction among several gene loci; chromosomal disorders are due to the presence or absence of a full chromosome or a fragment of a chromosome ( 14 , 15 ).

Examples of dominant disorders are some forms of retinoblastoma and other kinds of blindness, achondroplastic dwarfism, and Marfan syndrome (which is thought to have affected President Lincoln). Examples of recessive disorders are cystic fibrosis, Tay-Sachs disease, and sickle cell anemia (caused by an allele that in heterozygous condition protects against malaria). Examples of multifactorial diseases are spina bifida and cleft palate. Among the most common chromosomal disorders are Down syndrome, caused by the presence of an extra chromosome 21, and various kinds due to the absence of one sex chromosome or the presence of an extra one, beyond the normal condition of XX for women and XY for men. Examples are Turner’s syndrome (XO) and Klinefelter’s syndrome (XXY) ( 16 ).

The incidence of genetic disorders expressed in the living human population is estimated to be no less than 2.56%, impacting about 180 million people. Natural selection reduces the incidence of the genes causing disease, more effectively in the case of dominant disorders, where all carriers of the gene will express the disease, than for recessive disorders, which are expressed only in homozygous individuals. Consider, for example, phenylketonuria (PKU), a lethal disease if untreated, due to homozygosis for a recessive gene, which has an incidence of 1 in 10,000 newborns or 0.01%. PKU is due to an inability to metabolize the amino acid phenylalanine with devastating mental and physical effects. A very elaborate diet free of phenylalanine allows the patient to survive and reproduce if started early in life. The frequency of the PKU allele is about 1%, so that in heterozygous conditions it is present in more than 100 million people, but only the 0.01% of people who are homozygous express the disease and are subject to natural selection. The reduction of genetic disorders due to natural selection is balanced with their increase due to the incidence of new mutations.

Let’s consider another example. Hereditary retinoblastoma is a disease attributed to a dominant mutation of the gene coding for the retinoblastoma protein, RB1, but it is actually due to a deletion in chromosome 13. The unfortunate child with this condition develops a tumorous growth during infancy that, without treatment, starts in one eye and often extends to the other eye and then to the brain, causing death before puberty. Surgical treatment now makes it possible to save the life of the child if the condition is detected sufficiently early, although often one or both eyes may be lost. The treated person can live a more or less normal life, marry, and procreate. However, because the genetic determination is dominant (a gene deletion), one half of the progeny will, on the average, be born with the same genetic condition and will have to be treated. Before modern medicine, every mutation for retinoblastoma arising in the human population was eliminated from the population in the same generation owing to the death of its carrier. With surgical treatment, the mutant condition can be preserved, and new mutations arising each generation are added to those arisen in the past (refs. 17 and 18 ; www.abedia.com/wiley/index.html ).

The proportion of individuals affected by any one serious hereditary infirmity is relatively small, but there are more than 2,000 known serious physical infirmities determined by genes. When all these hereditary ailments are considered together, the proportion of persons born who will suffer from a serious handicap during their lifetimes owing to their heredity is more than 2% of the total population, as pointed out above (refs. 15 , 16 , and 19 ; www.abedia.com/wiley/index.html ).

The problem becomes more serious when mental defects are taken into consideration. More than 2% of the population is affected by schizophrenia or a related condition known as schizoid disease, ailments that may be in some cases determined by a single mutant gene. Another 3% or so of the population suffer from mild mental retardation (IQ less than 70). More than 100 million people in the world suffer from mental impairments due in good part to the genetic endowment they inherited from their parents.

Natural selection also acts on a multitude of genes that do not cause disease. Genes impact skin pigmentation, hair color and configuration, height, muscle strength and body shape, and many other anatomical polymorphisms that are apparent, as well as many that are not externally obvious, such as variations in the blood groups, in the immune system, and in the heart, liver, kidney, pancreas, and other organs. It is not always known how natural selection impacts these traits, but surely it does and does it differently in different parts of the world or at different times, as a consequence of the development of new vaccines, drugs, and medical treatments, and also as a consequence of changes in lifestyle, such as the reduction of the number of smokers or the increase in the rate of obesity in a particular country.

Genetic Therapy

Where is human evolution going? Biological evolution is directed by natural selection, which is not a benevolent force guiding evolution toward sure success. Natural selection brings about genetic changes that often appear purposeful because they are dictated by the requirements of the environment. The end result may, nevertheless, be extinction—more than 99.9% of all species that ever existed have become extinct. Natural selection has no purpose; humans alone have purposes and they alone may introduce them into their evolution. No species before mankind could select its evolutionary destiny; mankind possesses techniques to do so, and more powerful techniques for directed genetic change are becoming available. Because we are self-aware, we cannot refrain from asking what lies ahead, and because we are ethical beings, we must choose between alternative courses of action, some of which may appear as good and others as bad.

The argument has been advanced that the biological endowment of mankind is rapidly deteriorating owing precisely to the improving conditions of life and to the increasing power of modern medicine. The detailed arguments that support this contention involve some mathematical exercises, but their essence can be simply presented. Genetic changes (i.e., point or chromosome mutations) arise spontaneously in humans and in other living species. The great majority of newly arising mutations are either neutral or harmful to their carriers; only a very small fraction are likely to be beneficial. In a human population under the so-called “natural” conditions, that is, without the intervention of modern medicine and technology, the newly arising harmful mutations are eliminated from the population more or less rapidly depending on how harmful they are. The more harmful the effect of a mutation, the more rapidly it will be eliminated from the population by the process of natural selection. However, owing to medical intervention and, more recently, because of the possibility of genetic therapy, the elimination of some harmful mutations from the population is no longer taking place as rapidly and effectively as it did in the past.

Molecular biology has introduced in modern medicine a new way to cure diseases, namely genetic therapy, direct intervention in the genetic makeup of an individual. Gene therapy can be somatic or germ line. Germ-line genetic therapy would seek to correct a genetic defect, not only in the organs or tissues impacted, but also in the germ line, so that the person treated would not transmit the genetic impairment to the descendants. As of now, no interventions of germ-line therapy are seriously sought by scientists, physicians, or pharmaceutical companies.

The possibility of gene therapy was first anticipated in 1972 ( 20 ). The possible objectives are to correct the DNA of a defective gene or to insert a new gene that would allow the proper function of the gene or DNA to take place. In the case of a harmful gene, the objective would be to disrupt the gene that is not functioning properly.

The eminent biologist E. O. Wilson (2014) has stated, many would think somewhat hyperbolically, that the issue of how much to use genetic engineering to direct our own evolution, is “the greatest moral dilemma since God stayed the hand of Abraham” ( 21 ).

The first successful interventions of gene therapy concerned patients suffering from severe combined immunodeficiency (SCID), first performed in a 4-y-old girl at the National Institutes of Health in 1990 ( 22 ), soon followed by successful trials in other countries ( 23 ). Treatments were halted temporarily from 2000 to 2002 in Paris, when 2 of about 12 treated children developed a leukemia-like condition, which was indeed attributed to the gene therapy treatment. Since 2004, successful clinical trials for SCID have been performed in the United States, United Kingdom, France, Italy, and Germany ( 24 , 25 ).

Gene therapy treatments are still considered experimental. Successful clinical trials have been performed in patients suffering from adrenoleukodystrophy, Parkinson’s disease, chronic lymphocytic leukemia, acute lymphocytic leukemia, multiple myeloma, and hemophilia ( 26 , 27 ). Initially, the prevailing gene therapy methods involved recombinant viruses, but nonviral methods (transfection molecules) have become increasingly successful. Since 2013, US pharmaceutical companies have invested more than $600 million in gene therapy ( 28 ). However, in addition to the huge economic costs, technical hurdles remain. Frequent negative effects include immune response against an extraneous object introduced into human tissues, leukemia, tumors, and other disorders provoked by vector viruses. Moreover, the genetic therapy corrections are often short lived, which calls for multiple rounds of treatment, thereby increasing costs and other handicaps. In addition, many of the most common genetic disorders are multifactorial and are thus beyond current gene therapy treatment. Examples are diabetes, high blood pressure, heart disease, arthritis, and Alzheimer’s disease, which at the present state of knowledge and technology are not suitable for gene therapy.

If a genetic defect is corrected in the affected cells, tissues, or organs, but not in the germ line, the ova or sperm produced by the individual will transmit the defect to the progeny. A deleterious gene that might have been reduced in frequency or eliminated from the population, owing to the death or reduced fertility of the carrier, will now persist in the population and be added to its load of hereditary diseases. A consequence of genetic therapy is that the more hereditary diseases and defects are cured today, the more of them will be there to be cured in the succeeding generations. This consequence follows not only from gene therapy but also from typical medical treatments.

The Nobel laureate geneticist H. J. Muller eloquently voiced this concern about the cure, whether through genetic therapy or traditional medical treatment, of genetic ailments. “The more sick people we now cure and allow them to reproduce, the more there will be to cure in the future.” The fate toward which mankind is drifting is painted by Muller in somber colors. “The amount of genetically caused impairment suffered by the average individual…must by that time have grown….[P]eople’s time and energy…would be devoted chiefly to the effort to live carefully, to spare and to prop up their own feebleness, to soothe their inner disharmonies and, in general, to doctor themselves as effectively as possible. For everyone would be an invalid, with his own special familial twists….” (ref. 29 ; Fig. 1 ).

The bionic human, on the cover of Science : an image that could represent how H. J. Muller anticipates the human condition, a few centuries hence, showing the accumulation of physical handicaps as a consequence of the medical cure of hereditary diseases. Image by Cameron Slayden and Nathalie Cary; reprinted with permission from AAAS.

It must be pointed out that the population genetic consequences of curing hereditary diseases are not as immediate (“a few centuries hence”) as Muller anticipates. Consider, as a first example, we look at the recessive hereditary condition of PKU. The estimated frequency of the gene is q = 0.01; the expected number of humans born with PKU is q 2 = 0.0001, 1 for every 10,000 births. If all PKU individuals are cured all over the world and all of them leave as many descendants, on the average, as other humans, the frequency of the PKU allele will double after 1/q = 1/0.01 = 100 generations. If we assume 25 y per generation, we conclude that after 2,500 y, the frequency of the PKU allele will be q = 0.02, and q 2 = 0.0004, so that 4 of every 10,000 persons, rather than only 1, will be born with PKU.

In the case of dominant lethal diseases, the incidence is determined by the mutation frequency of the normal to the disease allele, which is typically of the order of m = 10 −6 –10 −8 , or between one in a million and one in one hundred million. Assuming the highest rate of m = 10 −6 , the incidence of the disease after 100 generations will become 1 for every 10,000 births. It would therefore seem likely that much earlier than 2,500 y, humans are likely to find ways of correcting hereditary ailments in the germ line, thereby stopping their transmission.

It must be pointed out that, although the proportion of individuals affected by any one serious hereditary infirmity is relatively small, there are many such hereditary ailments, which on the aggregate make the problem very serious. The problem becomes more serious when mental defects are taken into consideration. As pointed out above, more than 100 million people in the world suffer from mental impairments due in good part to the genetic endowment they inherited from their parents.

Human cloning may refer to “therapeutic cloning,” particularly the cloning of embryonic cells to obtain organs for transplantation or for treating injured nerve cells and other health purposes. Human cloning more typically refers to “reproductive cloning,” the use of somatic cell nuclear transfer (SCNT) to obtain eggs that could develop into adult individuals.

Human cloning has occasionally been suggested as a way to improve the genetic endowment of mankind, by cloning individuals of great achievement, for example, in sports, music, the arts, science, literature, politics, and the like, or of acknowledged virtue. These suggestions seemingly have never been taken seriously. However, some individuals have expressed a wish, however unrealistic, to be cloned, and some physicians have on occasion advertised that they were ready to carry out the cloning ( 30 ). The obstacles and drawbacks are many and insuperable, at least at the present state of knowledge.

Biologists use the term cloning with variable meanings, although all uses imply obtaining copies more or less precise of a biological entity. Three common uses refer to cloning genes, cloning cells, and cloning individuals. Cloning an individual, particularly in the case of a multicellular organism, such as a plant or an animal, is not strictly possible. The genes of an individual, the genome, can be cloned, but the individual itself cannot be cloned, as it will be made clear below.

Cloning genes or, more generally, cloning DNA segments is routinely done in many genetics and pharmaceutical laboratories throughout the world ( 12 , 31 ). Technologies for cloning cells in the laboratory are seven decades old and are used for reproducing a particular type of cell, for example a skin or a liver cell, in order to investigate its characteristics.

Individual human cloning occurs naturally in the case of identical twins, when two individuals develop from a single fertilized egg. These twins are called identical, precisely because they are genetically identical to each other.

The sheep Dolly, cloned in July 1996, was the first mammal artificially cloned using an adult cell as the source of the genotype. Frogs and other amphibians were obtained by artificial cloning as early as 50 y earlier ( 32 ).

Cloning an animal by SCNT proceeds as follows. First, the genetic information in the egg of a female is removed or neutralized. Somatic (i.e., body) cells are taken from the individual selected to be cloned, and the cell nucleus (where the genetic information is stored) of one cell is transferred with a micropipette into the host oocyte. The egg, so “fertilized,” is stimulated to start embryonic development ( 33 ).

Can a human individual be cloned? The correct answer is, strictly speaking, no. What is cloned are the genes, not the individual; the genotype, not the phenotype. The technical obstacles are immense even for cloning a human’s genotype.

Ian Wilmut, the British scientist who directed the cloning project, succeeded with Dolly only after 270 trials. The rate of success for cloning mammals has notably increased over the years without ever reaching 100%. The animals presently cloned include mice, rats, goats, sheep, cows, pigs, horses, and other mammals. The great majority of pregnancies end in spontaneous abortion ( 34 ). Moreover, as Wilmut noted, in many cases, the death of the fetus occurs close to term, with devastating economic, health, and emotional consequences in the case of humans ( 35 ).

In mammals, in general, the animals produced by cloning suffer from serious health handicaps, among others, gross obesity, early death, distorted limbs, and dysfunctional immune systems and organs, including liver and kidneys, and other mishaps. Even Dolly had to be euthanized early in 2003, after only 6 y of life, because her health was rapidly decaying, including progressive lung disease and arthritis ( 35 , 36 ).

The low rate of cloning success may improve in the future. It may be that the organ and other failures of those that reach birth will be corrected by technical advances. Human cloning would still face ethical objections from a majority of concerned people, as well as opposition from diverse religions. Moreover, there remains the limiting consideration asserted earlier: it might be possible to clone a person’s genes, but the individual cannot be cloned. The character, personality, and the features other than anatomical and physiological that make up the individual are not precisely determined by the genotype.

The Genotype and the Individual

The genetic makeup of an individual is its genotype. The phenotype refers to what the individual is, which includes not only the individual’s external appearance or anatomy, but also its physiology, as well as behavioral predispositions and attributes, encompassing intellectual abilities, moral values, aesthetic preferences, religious values, and, in general, all other behavioral characteristics or features, acquired by experience, imitation, learning, or in any other way throughout the individual’s life, from conception to death. The phenotype results from complex networks of interactions between the genes and the environment.

A person’s environmental influences begin, importantly, in the mother’s womb and continue after birth, through childhood, adolescence, and the whole life. Impacting behavioral experiences are associated with family, friends, schooling, social and political life, readings, aesthetic and religious experiences, and every event in the person’s life, whether conscious or not. The genotype of a person has an unlimited number, virtually infinite, of possibilities to be realized, which has been called the genotype’s “norm of reaction,” only one of which will be the case in a particular individual ( 37 ). If an adult person is cloned, the disparate life circumstances experienced many years later would surely result in a very different individual, even if anatomically the individual would resemble the genome’s donor at a similar age.

An illustration of environmental effects on the phenotype, and of interactions between the genotype and the environment, is shown in Fig. 2 ( 38 ). Three plants of the cinquefoil, Potentilla glandulosa , were collected in California—one on the coast at about 100 ft above sea level (Stanford), the second at about 4,600 ft (Mather), and the third in the Alpine zone of the Sierra Nevada at about 10,000 ft above sea level (Timberline). From each plant, three cuttings were obtained in each of several replicated experiments, which were planted in three experimental gardens at different altitudes, the same gardens from which the plants were collected. The division of one plant ensured that all three cuttings planted at different altitudes had the same genotype; that is, they were genetic clones from one another. ( P. glandulosa , like many other plants, can be reproduced by cuttings, which are genetically identical.)

Interacting effects of the genotype and the environment on the phenotype of the cinquefoil Pontentilla glandulosa . Cuttings of plants collected at different altitudes were planted in three different experimental gardens. Plants in the same row are genetically identical because they have been grown from cuttings of a single plant; plants in the same column are genetically different but have been grown in the same experimental garden. Reprinted with permission from ref. 13 .

Comparison of the plants in any row shows how a given genotype gives rise to different phenotypes in different environments. Genetically identical plants (for example, those in the bottom row) may prosper or not, even die, depending on the environmental conditions. Plants from different altitudes are known to be genetically different. Hence, comparison of the plants in any column shows that in a given environment, different genotypes result in different phenotypes. An important inference derived from this experiment is that there is no single genotype that is best in all environments.

The interaction between the genotype and the environment is similarly significant, or even more so, in the case of animals. In one experiment, two strains of rats were selected over many generations; one strain for brightness at finding their way through a maze and the other for dullness ( Fig. 3 ; ref. 39 ). Selection was done in the bright strain by using the brightest rats of each generation to breed the following generation, and in the dull strain by breeding the dullest rats of every generation. After many generations of selection, the descendant bright rats made only about 120 errors running through the maze, whereas dull rats averaged 165 errors. That is a 40% difference. However, the differences between the strains disappeared when rats of both strains were raised in an unfavorable environment of severe deprivation, where both strains averaged 170 errors. The differences also nearly disappeared when the rats were raised with abundant food and other favorable conditions. In this optimal environment, the dull rats reduced their average number of errors from 165 to 120. As with the cinquefoil plants, we see ( i ) that a given genotype gives rise to different phenotypes in different environments and ( ii ) that the differences in phenotype between two genotypes change from one environment to another—the genotype that is best in one environment may not be best in another.

Results of an experiment with two strains of rats: one selected for brightness and the other for dullness. After many generations of selection, when raised in the same environment in which the selection was practiced (normal), bright rats made about 45 fewer errors than dull rats in the maze used for the tests. However, when the rats were raised in an impoverished (restricted) environment, bright and dull rats made the same number of errors. When raised in an abundant (stimulating) environment, the two strains performed nearly equally well. Reprinted with permission from ref. 13 .

Cloning Humans?

In the second half of the 20th century, as dramatic advances were taking place in genetic knowledge, as well as in the genetic technology often referred to as “genetic engineering,” some utopian proposals were advanced, at least as suggestions that should be explored and considered as possibilities, once the technologies had sufficiently progressed. Some proposals suggested that persons of great intellectual or artistic achievement or of great virtue be cloned. If this was accomplished in large numbers, the genetic constitution of mankind would, it was argued, considerably improve.

Such utopian proposals are grossly misguided. It should be apparent that, as stated above, it is not possible to clone a human individual. Seeking to multiply great benefactors of humankind, such as persons of great intelligence or character, we might obtain the likes of Stalin, Hitler, or Bin Laden. As the Nobel Laureate geneticist George W. Beadle asserted many years ago: “Few of us would have advocated preferential multiplication of Hitler’s genes. Yet who can say that in a different cultural context Hitler might not have been one of the truly great leaders of men, or that Einstein might not have been a political villain” ( 8 ). There is no reason whatsoever to expect that the genomes of individuals with excellent attributes would, when cloned, produce individuals similarly endowed with virtue or intelligence. Identical genomes yield, in different environments, individuals who may be quite different. Environments cannot be reproduced, particularly several decades apart, which would be the case when the genotype of the persons selected because of their eminent achievement might be cloned.

Are there circumstances that would justify cloning a person, because he or she wants it? One might think of a couple unable to have children, or a man or woman who does not want to marry, or of two lesbian lovers who want to have a child with the genotype of one in an ovum of the other, or of other special cases that might come to mind ( 40 ). It must be, first, pointed out that the cloning technology has not yet been developed to an extent that would make possible to produce a healthy human individual by cloning. Second, and most important, the individual produced by cloning would be a very different person from the one whose genotype is cloned, as belabored above.

Ethical, social, and religious values will come into play when seeking to decide whether a person might be allowed to be cloned. Most people are likely to disapprove. Indeed, many countries have prohibited human cloning. In 2004, the issue of cloning was raised in several countries where legislatures were also considering whether research on embryonic stem cells should be supported or allowed. The Canadian Parliament on March 12, 2004 passed legislation permitting research with stem cells from embryos under specific conditions, but human cloning was banned, and the sale of sperm and payments to egg donors and surrogate mothers were prohibited. The French Parliament on July 9, 2004 adopted a new bioethics law that allows embryonic stem cell research but considers human cloning a “crime against the human species.” Reproductive cloning experiments would be punishable by up to 20 y in prison. Japan’s Cabinet Council for Science and Technology Policy voted on July 23, 2004 to adopt policy recommendations that would permit the limited cloning of human embryos for scientific research but not the cloning of individuals. On January 14, 2001, the British government amended the Human Fertilization and Embryology Act of 1990 by allowing embryo research on stem cells and allowing therapeutic cloning. The Human Fertilization and Embryology Act of 2008 explicitly prohibited reproductive cloning but allowed experimental stem cell research for treating diabetes, Parkinson’s disease, and Alzheimer’s disease ( 41 , 42 ). On February 3, 2014, the House of Commons voted to legalize a gene therapy technique known as mitochondrial replacement, or three-person in vitro fertilization, in which mitochondria from a donor’s egg cell contribute to a couple’s embryo ( 43 ). In the United States, there are currently no federal laws that ban cloning completely ( 42 ). Thirteen states (Arkansas, California, Connecticut, Iowa, Indiana, Massachusetts, Maryland, Michigan, North Dakota, New Jersey, Rhode Island, South Dakota, and Virginia) ban reproductive cloning, and three states (Arizona, Maryland, and Missouri) prohibit use of public funds for research on reproductive cloning ( 44 ).

Therapeutic Cloning

Cloning of embryonic cells (stem cells) could have important health applications in organ transplantation, treating injured nerve cells, and otherwise. In addition to SCNT, the method discussed above for cloning individuals, another technique is available, induced pluripotent stem cells (iPSCs), although SCNT has proven to be much more effective and less costly. The objective is to obtain pluripotent stem cells that have the potential to differentiate in any of the three germ layers characteristic of humans and other animals: endoderm (lungs and interior lining of stomach and gastrointestinal tract), ectoderm (nervous systems and epidermal tissues), and mesoderm (muscle, blood, bone, and urogenital tissues). Stem cells, with more limited possibilities than pluripotent cells, can also be used for specific therapeutic purposes ( 45 ).

Stem cell therapy consists of cloning embryonic cells to obtain pluripotent or other stem cells that can be used in regenerative medicine, to treat or prevent all sorts of diseases, and for the transplantation of organs. At present, bone marrow transplantation is a widely used form of stem cell therapy; stem blood cells are used in the treatment of sickle cell anemia, a lethal disease when untreated, which is very common in places where malaria is rife because heterozygous individuals are protected against infection by Plasmodium falciparum , the agent of malignant malaria. One of the most promising applications of therapeutic cloning is the growth of organs for transplantation, using stem cells that have the genome of the organ recipient. Two major hurdles would be overcome. One is the possibility of immune rejection; the other is the availability of organs from suitable donors. Another regenerative medical application that might be anticipated is the therapeutic growth of nerve cells. There are hundreds of thousands of individuals throughout the world paralyzed from the neck down and confined for life to a wheelchair as a consequence of damage to the spinal cord below the neck, often as a consequence of a car accident or a fall, that interrupts the transmission of nerve activity from the brain to the rest of the body and vice versa. A small growth of nerve cells sufficient to heal the wound in the spinal cord would have enormous health consequences for the wounded persons and for society.

At present, the one gene therapy modification of the embryo that can be practiced is mitochondrial replacement (MR), legalized in the United Kingdom by the House of Commons on February 3, 2014 ( 43 ), as mentioned earlier. Mutations in the mitochondrial DNA of about 1 in 6,500 individuals account for a variety of severe and often fatal conditions, including blindness, muscular weakness, and heart failure ( 46 ). With MR, the embryo possesses nuclear DNA from the mother and father, as well as mtDNA from a donor female who has healthy mtDNA. However, MR remains technically challenging, with a low rate of success. One complicating issue is that mtDNA replacement is not 100% successful; disease-causing mutant mtDNA persists in the developing embryo and may account for eventual diseases due to heteroplasmy, at least in some tissues. A second issue of concern is that mtDNA disorders often appear late in life. It remains unknown whether the benefits of MR as currently practiced may persist in advanced age.

The author declares no conflict of interest.

This paper results from the Arthur M. Sackler Colloquium of the National Academy of Sciences, “In the Light of Evolution IX: Clonal Reproduction: Alternatives to Sex,” held January 9–10, 2015, at the Arnold and Mabel Beckman Center of the National Academies of Sciences and Engineering in Irvine, CA. The complete program and video recordings of most presentations are available on the NAS website at www.nasonline.org/ILE_IX_Clonal_Reproduction .

This article is a PNAS Direct Submission.