Essentials of Domestic Animal Embryology PDF

 Domestic Essentials of Animal Embryology Commissioning Editor: Robert Edwards, Joyce Rodenhuis Development Editor: Nicola Lally Project Manager: Nancy Arnott Designer/Design direction: Charles Gray Illustration Manager: Merlyn Harvey Illustrator: Ox...

 Domestic Essentials of Animal Embryology Commissioning Editor: Robert Edwards, Joyce Rodenhuis Development Editor: Nicola Lally Project Manager: Nancy Arnott Designer/Design direction: Charles Gray Illustration Manager: Merlyn Harvey Illustrator: Oxford Illustrators  Domestic Essentials of Animal Embryology By Poul Hyttel University of Copenhagen, Denmark Fred Sinowatz LMU Munich, Germany Morten Vejlsted University of Copenhagen, Denmark With the Editorial Assistance of Keith Betteridge Ontario Veterinary College, University of Guelph, Canada Foreword by Eric W. Overström, Ph.D. Professor and Head Department of Biology & Biotechnology Director, Life Sciences & Bioengineering Center Worcester Polytechnic Institute Worcester, Massachusetts Edinburgh London New York Oxford Philadelphia St Louis Sydney Toronto 2010 First published 2010, © Elsevier Limited. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier’s Rights Department: phone: (+1) 215 239 3804 (US) or (+44) 1865 843830 (UK); fax: (+44) 1865 853333; e-mail: [email protected]. You may also complete your request online via the Elsevier website at http://www.elsevier.com/permissions. ISBN 978-0-7020-2899-1 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress Notice Neither the Publisher nor the Editors assume any responsibility for any loss or injury and/or damage to persons or property arising out of or related to any use of the material contained in this book. It is the responsibility of the treating practitioner, relying on independent expertise and knowledge of the patient, to determine the best treatment and method of application for the patient. The Publisher The Working together to grow publisher’s policy is to use libraries in developing countries paper manufactured from sustainable forests www.elsevier.com | www.bookaid.org | www.sabre.org Printed in China  CONTENTS Contributors vii 12. Development of the Blood Cells, Preface ix Heart and Vascular System....... 182 Foreword xi Poul Hyttel Acknowledgements xiii 13. Development of the Immune System.................. 208 1. History of Embryology.......... 1 Morten Vejlsted Poul Hyttel and Gábor Vajta 14. Development of the 2. Cellular and Molecular Mechanisms Gastro-pulmonary System........ 216 in Embryonic Development....... 13 Poul Hyttel Morten Vejlsted 15. Development of the Urogenital 3. Comparative Reproduction........ 25 System.................. 252 Morten Vejlsted Fred Sinowatz 4. Gametogenesis.............. 32 16. Musculo-skeletal System........ 286 Poul Hyttel Fred Sinowatz 5. Fertilization................ 56 17. The Integumentary System....... 317 Fred Sinowatz Fred Sinowatz 6. Embryo Cleavage and Blastulation... 68 18. Comparative Listing of Developmental Chronology....... 330 Morten Vejlsted Poul Hyttel 7. Gastrulation, Body Folding and Coelom Formation............ 79 19. Teratology................. 338 Morten Vejlsted Fred Sinowatz 8. Neurulation................ 95 20. The Chicken and Mouse as Models of Embryology.......... 383 Fred Sinowatz Palle Serup (chicken) and Ernst-Martin 9. Comparative Placentation........ 104 Füchtbauer (mouse) Morten Vejlsted 21. Assisted Reproduction 10. Development of the Central and Technologies............... 402 Peripheral Nervous System....... 120 Gábor Vajta, Henrik Callesen, Fred Sinowatz Gry Boe-Hansen, Vanessa Hall and Poul Hyttel 11. Eye and Ear................ 163 Fred Sinowatz Index 435 This page intentionally left blank  Contributors Several highly qualified scientists have demonstrated their willingness to contribute to the book project. Keith J. Betteridge BVSc MVSc PhD FRCVS Palle Serup Phd University Professor Emeritus Director of Research Department of Biomedical Sciences Department of Developmental Biology Ontario Veterinary College Hagedorn Research Institute University of Guelph, Ontario Denmark Canada Fred Sinowatz Dr.med vet. Dr.med Dr.habil Gry Boe-Hansen DVM Phd Professor Lecturer Institute of Veterinary Anatomy, Histology and School of Veterinary Science Embryology University of Queensland, Australia LMU Munich, Germany Henrik Callesen DVM PhD DVSc Research Professor Gábor Vajta MD PhD DVSc Department of Genetics and Biotechnology Scientific Director Faculty of Agricultural Sciences, Aarhus University, Cairns Fertility Centre Denmark Australia Adjunct Professor, University of Copenhagen, Ernst-Martin Füchtbauer PhD Dr.habil Denmark Associate Professor Adjunct Professor, James Cook University, Australia Department of Molecular Biology Morten Vejlsted DVM Phd Aarhus University Denmark Assistant Professor Department of Large Animal Sciences Vanessa Hall PhD Faculty of Life Sciences, University of Copenhagen, Post Doc Denmark Department of Basic Animal and Veterinary Sciences Faculty of Life Sciences, University of Copenhagen Denmark Poul Hyttel DVM Phd DVSc Professor Department of Basic Animal and Veterinary Sciences Faculty of Life Sciences, University of Copenhagen Denmark vii This page intentionally left blank  Preface For me, some of the most exciting and glorious spotlight that, in my view, obliges contemporary moments in teaching gross anatomy of the domestic embryologists to participate in scientifically based animals have occurred during days shared with societal debates. open-minded students surrounded by steel tables For a while, ever more sophisticated assisted full of pregnant uteri and fetuses in the dissection reproduction technologies moved the “cutting edge” room. To examine those fetal specimens is to open of embryological research out of the body and into an anatomy book; a book in which each organ and the in-vitro environment. However, the expansion structure is perfectly defined and its developmental of this field to encompass embryonic stem cells has history is perfectly retained – truly the optimal situ- refocused us on the embryo per se; the control of ation for memorable anatomical “Aha-Erlebnishen” stem cell differentiation in vitro will depend abso- for students and teachers alike! lutely on fundamental knowledge of the molecular Embryology has always been a prerequisite for a regulation of developmental processes in vivo. The real understanding of gross anatomy and of the tera- embryo itself has become the key to success – the tology that results from development going awry. circle is closed! Today, however, it is that and much more besides; “Cutting edges” are these days fashioned from an contemporary biomedical research requires embry- amalgam of conventional embryology, genomics, ology (or, rather, developmental biology) to play a transcriptomics and epigenomics applied to the central role in its progress – a role with important investigation of the molecular mechanisms of societal implications. Assisted reproduction tech- developmental biology. Information is growing nologies, for example, are as much applied to exponentially, and to combine in-depth molecular domestic animals as to humans in which vitro fer- understanding with overall holistic embryology is a tilization has become a common step ex soma to challenge – a challenge that becomes crystal clear bridge one generation of mankind to the next. In when writing a textbook on the essentials of domes- the domestic animals, techniques such as cloning by tic animal embryology! For years, teaching the somatic cell nuclear transfer have made possible subject has been hampered by the lack of such a genetic modifications that offer the prospects of textbook and medical embryology textbooks were modifying animals so that they produce valuable used as a poor compromise. By 2006, when McGaedy proteins, serve as models of human diseases, or et al published their welcome textbook on veteri- provide organs for xenotransplantation in the future. nary embryology, we had already embarked on the All of these prospects depend upon a thorough present book with the goal of making our own knowledge of embryology and many of them are research material, collected over several decades, contentious. They put the discipline in an ethical palatable and stimulating for students. Working  Preface towards this goal has been a great experience for us “Aha-Erlebnis” in the wondrous world of and we hope that you the reader will find that we embryology! have accomplished at least part of what we intended. Future improvements, of course, will depend largely Poul Hyttel on feedback and I would much appreciate receiving Vidiekjaer constructive criticism. Valby, Denmark In the meantime, it is my sincere wish that June 3, 2009 reading this book may lead you into at least one My painting (2003) of the open horizon of Skagen, Denmark, where I grew up and was ‘imprinted’. I see embryology in the same light: a vista with infinite potentials that are just waiting to be realized. x  Foreword Over the last 20 years, modern life science research Patton’s 1927 benchmark publication in English, efforts have rapidly advanced our knowledge of the Embryology of the Pig, provided a wonderfully illus- normal and abnormal processes of domestic animal trated, descriptive account of this often utilized development. As our depth of understanding of the example of mammalian embryonic development. cellular and molecular mechanisms has grown, so A concise descriptive publication of development too has the recognition of the potential for, and in the pig, The Embryonic Pig: A Chronological successful application of, this knowledge to enhance Account, was later published by A.W. Marrable in animal-based food and fiber production. It is during 1971. In 1984, Drew Noden and Alexander de embryo and fetal development, from the formation Lahunta, similarly recognizing the lack of an ade- of competent gametes to parturition, that powerful quate text on domestic species embryology that advancements in molecular genetic manipulation would be useful for veterinary students, published and assisted reproductive technologies are employed, The Embryology of Domestic Animals: Developmental and these efforts have had profound impact on Mechanisms and Malformations. An important con- animal production worldwide. As a result of these tribution to veterinary curricula for many years to advances, there remains an unmet need for a con- follow, this book presented traditional system-by- temporaneous text of domestic animal development system descriptive material on the developmental to support education and training of today’s veteri- anatomy of domestic species including birds, and narians, animal scientists and developmental biolo- the authors also included many relevant experimen- gists. Essentials of Domestic Animal Embryology fulfills tal and clinical case references throughout the book. this need by providing the student, the instructor Unfortunately, a revised edition was not forthcom- and the veterinary practitioner with an in depth ing, and is not currently available. More recently, presentation of the elaborate, chronological proc- the finely detailed German text, Lehrbuch der esses that culminate in formation of functional Embryologie der Haustiere (1991), was published by embryonic structures from the development of Imogen Russe and Fred Sinowatz (current co- gametes through the peri-partum period. As our author). Excellent illustrations and micrographs understanding of the precisely orchestrated proc- characterize this comprehensive embryology refer- esses of animal development advances, and animal ence text of domestic species. Printed only in genomes are further unveiled and analyzed, the German, broad international adoption has been importance of animal development becomes central limited. In 2006, McGaedy and colleagues pub- to understanding and enhancing animal growth, lished Veterinary Embryology, a text targeting the sustaining health and determining the underlying particular needs of the veterinary student. Accord- causes of disease. ingly, the publication of Essentials of Domestic Although there continues to be available a Animal Embryology is particularly timely as it fills a number of quality texts of human embryology (for resource void for those students keen to study and example Langman’s Medical Embryology), focus on a understand the fundamental processes of animal single species remains a serious limitation for vet- development, be they students, instructors, research erinary and animal science audiences and prevents scientists or veterinary practitioners. a thorough view of the wide and distinct variations On reflection, this book project was conceived that exist among domestic animal species, with following from a discussion Poul and I had during particular reference to processes of blastogenesis, a scientific meeting in 2000. As we shared and com- implantation and placentation. Certainly, Bradley pared our experiences teaching animal embryology  Foreword and anatomy to veterinary students, there was and has received formal awards and recognition, as mutual recognition that a modern text in domestic well as continuous accolades from students for his animal development was sorely needed to contrib- passion and dedicated commitment to teaching. ute to the essential academic underpinnings for 21st Under his direction, Poul has assembled a distin- century veterinary and animal science curricula. To guished international group of contributing co- appeal to a more global audience, contributions authors including professor Fred Sinowatz (Munich) were solicited from established co-authors recog- and Dr. Morten Vejlsted (Copenhagen), among nized internationally as experts in the field. Their others. Keith Betteridge, distinguished professor of chapters have been seamlessly woven into a very animal reproduction at the University of Guelph, readable and informative text book. Of particular has provided thorough editorial review of the text, note are the inclusion of Chapters 1, 2, 20 and 21, This first edition presents a logical, contemporane- each of which provides a distinguishing topic per- ous and comprehensive view of the current state of spective, and include a historical account of the knowledge in the field. The format provides succinct study of animal embryology, a discussion of current text information that is well supported by quality cell and molecular-based regulatory mechanisms illustrations, photographs and micrographs. I that govern key developmental processes, compara- believe that the student, instructor and practitioner tive embryology of the chicken and mouse, and a alike will embrace this text as it will prove to be an succinct summary of the advent, development and invaluable resource to further both their education broad application assisted reproductive technolo- and their knowledge in the field of domestic animal gies (ARTs) to enhance production of domestic embryology. animals, respectively. Lead author, professor Poul Hyttel is recognized May 2009      Worcester, Massachusetts, USA internationally as a distinguished research scientist, and a passionate educator and student mentor of Eric W. Overström, Ph.D. animal reproduction and cell biology at the Royal Professor and Head Veterinary and Agricultural University, and most Department of Biology & Biotechnology recently the University of Copenhagen. He has Director, Life Sciences & Bioengineering Center directed both the veterinary anatomy and histology/ Worcester Polytechnic Institute cell biology courses for many years in Copenhagen, Worcester, Massachusetts xii  Acknowledgements Writing this embryology textbook set the authors on undertake a thorough linguistic edit of the com- a long and winding road. The project was initially pleted text. Due to the breadth of Keith’s scientific proposed by Eric Overström in 2000. At that time view, the linguistic affair developed into an inspir- Eric taught a course on developmental biology at ing dialogue about many conceptual subjects of Tufts University, Boston, US, and held a Fulbright embryology. It has been a pleasure to learn from the stipend allowing regular trips to Copenhagen, extreme precision with which Keith has tackled each Denmark. I am indebted to Eric for his enthusiasm step in the process. in initiating the book project which resulted in Several qualified persons have devoted time and many happy hours together in Copenhagen. given extremely valuable comments to the text. I feel enormously privileged to have been able to I would like to thank Marie Louise Grøndahl, Vibeke undertake this book project with cutting edge scien- Dantzer and Kjeld Christensen for their highly tists who share my passion for scholarly university appreciated efforts. life. My co-authors, Fred Sinowatz and Morten Vejl- Images have been an important issue in the pro- sted, have both made extraordinary efforts in writing duction of the book. I would like to thank Jytte their many chapters. In particular, Fred’s astonish- Nielsen and Hanne Marie Moelbak Holm for their ing embryological breadth, ranging from the molec- skilled contribution to the preparation of thousands ular to the gross anatomical levels, has been of sections for light and electron microscopy over indispensable to the setting of our goals, and I am the years as well as for digital processing of the truly grateful that we were allowed to use high micrographs. quality drawings from the previous embryology Finally, I would like to thank Danish Pig textbook that he produced with Imogen Rüsse and Production for a very fruitful collaboration enabling published in German. Other highly qualified con- the collection of thousands of porcine embryos tributors, Gry Boe-Hansen, Henrik Callesen, Ernst- over the years. The data generated from these Martin Füchtbauer, Vanessa Hall, Palle Serup and resources have contributed significantly to the book Gábor Vajta, have each brought their particular and many of the photographs are taken from these expertise to bear on other chapters to ensure that the embryos. text is as up-to-date as possible. I am greatly indebted to all these colleagues who have so generously Poul Hyttel shared their time and ideas with me. Vidiekjaer As non-native English speakers the authors are Valby extremely grateful for the willingness of Keith Bet- Denmark teridge, one of the pioneers of embryo transfer, to June 3, 2009 This page intentionally left blank  CHAPTER 1 Poul Hyttel and Gábor Vajta History of embryology Embryology, the study of development from fertili- existed, looking much like the gastrula stage of zation to birth, has always intrigued philosophers ontogeny. This hypothesized ancestral metazoan, he and scientists. Universal fascination with the way in thought, gave rise to all multi-celled animals. Such which ‘life’ unfolds has led to spirited discussion of a ‘single straight line’ conception of phylogeny, with the complexities of the process amongst embryo­ all creatures standing on the shoulders of predeces- logists over the years. In 1899, Ernst Haeckel sors in one trajectory, has now been abandoned; (1834–1919) considered that ‘Ontogeny is a short phylogeny divides into a multitude of lines. recapitulation of phylogeny’. In other words, ontog- Many of the ancient Greek philosophers were eny (the development of an organism from the fer- interested in embryology. According to Democritus tilized egg to its mature form) reflects, in a matter (ca. 455–370 BC), the sex of an individual is deter- of days or months, the origin and evolution of a mined by the origin of the sperm: males arising species (phylogeny), a continuing process that is from the right testicle (of course!) and females from measured in millions of years. Although this is not the left. This hypothesis was somewhat modified by entirely true, one has only to look at a 19-day-old Pythagoras, Hippocrates and Galen. However, sheep embryo to understand Haeckel’s viewpoint; gender bias was always evident, for science was the the gill-like pharyngeal arches and the somites, for privilege of men; philosophers mostly positioned example (Fig. 1-1) are common to embryos of all females between man and animals, so males were chordates. Although trained as a physician, Haeckel supposed to originate from the stronger sperm of abandoned his practice after reading The Origin of the right testicle. Species by Charles Robert Darwin (1809–1882) The first real embryologist that we know about published in 1859 (and available at that time for was the Greek philosopher Aristotle (384–322 BC). fifteen shillings!). Always suspicious of teleological In The Generation of Animals (ca. 350 BC) he and mystical explanations of life, Haeckel used described the different ways that animals are born: Darwin’s theories as ammunition for attacking from eggs (oviparity, as in birds, frogs and most entrenched religious dogma on the one hand and invertebrates), by live birth (viviparity, as in placen- elaborating his own views on the other. However, tal animals and some fish) or by production of an in projecting phylogeny into ontogeny, Haeckel egg that hatches inside the body (ovoviviparity, made one mistake: his mechanism of change which occurs in certain reptiles and sharks). It was required that formation of new characters, diagnos- Aristotle who also noted the two major patterns of tic of new species, occur through their addition to a cell division in early development: holoblastic basic developmental scheme. For example, because cleavage in which the entire egg is divided into pro- most metazoans pass through a developmental gressively smaller cells (as in frogs and mammals) stage called a gastrula (a ball of cells with an infold- and the meroblastic pattern in which only that ing that later forms the gut) Haeckel thought that at part of the egg destined to become the embryo one time an organism called a ‘gastraea’ must have proper divides, with the remainder serving nutritive  Essentials of Domestic Animal Embryology Fig. 1-1: Sheep embryo at Day 19 of development. purposes (as in birds). The fetal membranes and the tomical lines. One of the first anatomical descriptions umbilical cord in cattle were described by Aristotle of the pregnant uterus of the pig was by Kopho and he recognized their importance for fetal nutri- (years of birth and death unknown) in the early 13th tion. By sequential studies of fertilized chick eggs, century in his work Anatomio Porci. Kopho worked Aristotle made the very important observation that at the famous medical school of Salerno using pigs the embryo develops its organ systems gradually – as models because human cadavers could not be they are not preformed. This concept of de novo dissected for religious reasons. During the early formation of embryonic structures, which is referred Renaissance, the famous and incomparably artistic to as epigenesis, remained enormously controver- anatomical studies of Leonardo da Vinci (1452– sial for more than 2000 years before becoming fully 1519) included investigation of the pregnant uterus accepted; Aristotle was way ahead of his time. The of a cow. His drawings of a pregnant bicornuate enigma of sexual reproduction also intrigued Aris- uterus and of the fetus and fetal membranes released totle. He realized that both sexes are needed for from the uterus are reproduced in Fig. 1-2. In an conception but felt that the male’s semen did not accompanying drawing, Leonardo depicted a human contribute to conception physically, but by provid- uterus cut open to ‘reveal’, not a human placenta ing an unknown form-giving force that interacts but a multiplex, villous, ruminant placenta (see with menstrual blood in the womb of the female to Chapter 9)! Clearly it had not been possible for him materialize as an embryo. In this case Aristotle was to actually study a human pregnancy. The structures wrong. Ironically, in contrast to the non-acceptance are described in the characteristic mirrored writing of his correct views on epigenesis, his error about of Leonardo, who worked mostly in Florence. conception prevailed for about 2000 years, and it The Renaissance, from the 13th to the 15th century, took a battle of almost a hundred years to correct it! was a great time for anatomical studies and, happily, During those 2000 years, the science of embryo­ coincided with the invention of book printing by logy developed very slowly along descriptive ana- Johann Gutenberg. Thus, the first major publication 2  History of embryology A B Fig. 1-2: Drawings of Leonardo da Vinci. A: Top: A pregnant bicornuate uterus of the cow. Bottom: Fetus and fetal membranes showing the cotyledons of the placenta (see Chapter 9). B: Opened simplex uterus of human showing the fetus with the umbilical cord. Note that the placenta is of the ruminant multiplex type with several placentomes drawn on the cut wall of the uterus and, at the right top, a single cotyledon drawn at a higher magnification displaying its villous surface. 1 3  Essentials of Domestic Animal Embryology on comparative embryology was De Formato Foetu (epigenesis), or they are already present in a mini- in 1600 by the Italian anatomist Hieronymus ature form in the egg (or the sperm when this cell Fabricius of Acquapendente (1533–1619). Fabri- was discovered), a concept referred to as preforma- cius described and illustrated the gross anatomy of tion. We will return to this debate in a moment. embryos and their membranes in that book, but was The new microscopic techniques also prompted not actually the first to do so; another Italian anato- a vigorous search for the mammalian gametes. The mist, Bartolomeo Eustachius (1514–1574), had chicken egg and its initial transformation into a previously published illustrations of dog and sheep chick were obvious, as Aristotle had described, but embryos in 1552. We now recognize the names of what mediated the formation of the embryo in Fabricius, in the term bursa Fabricii (the immuno- mammals? Where was the mammalian egg to be logically competent portion of the bird gut), and of found? Eustachius in the Eustachian tube. One of the earliest and most influential names in The work of Eustachius, Fabricius and others gave the fascinating story of the discovery of the mam- insight into how organs develop from their imma- malian egg was that of William Harvey (1578– ture to mature forms, but left unanswered the basic 1657), personal physician to the English kings enigma of how and where the mammalian embryo James I and Charles I, and famous for his descrip- originates. However, the development of the micro- tion of the circulation of the blood. In 1651, Harvey scope by Zacharias Janssen, a Dutch eyeglass maker, published De Generatione Animalium (Disputations in 1590 ushered in a new era of embryological touching the Generation of Animals) with a famous science to tackle that 2000-year-old question. The frontispiece showing Zeus freeing all creation from Dutch dominance in the optical field at that time an egg bearing the inscription Ex ovo omnia (All may not be just a coincidence; the naval ambitions things come from the egg). However, it should be of their new empire required excellent telescopes realized that, far from advancing 17th century knowl- and lens systems. Janssen’s microscope in its origi- edge of reproduction and embryology, Harvey’s nal form, however, was not really appropriate for observations in some ways impeded progress. From cell and tissue research; it was approximately 2 having studied with Fabricius, Harvey was imbued metres long, achieved only 10 to 20 times magnifi- with Aristotle’s view that the semen provided a force cation, and its principal use was to attract an audi- that interacted with the menstrual blood to materi- ence at country fairs! In 1672, the Italian medical alize as an embryo. Harvey set out to understand doctor Marcello Malpighi (1628–1694) published this process by looking for the earliest products of the first microscopic account of chick development, conception in female deer killed during the breed- identifying the neural groove, the somites, and cir- ing season in the course of King Charles I’s hunts in culation of blood in the arteries and veins to and his Royal forests and parks over a 12-year period. In from the yolk. Malpighi also observed that even the the red and fallow deer that he studied, the male’s unincubated chick egg is considerably structured, rut begins in mid-September and so Harvey dis- leading him to think that a preformed version of the sected uteri throughout the months of September to chicken resided in the egg. Later (in 1722), the December. Believing, wrongly, that copulation coin- French ophthalmologist Antoine Maître-Jan (1650– cides with the onset of the rut, Harvey was mystified 1730) pointed out that although the egg examined to find nothing that he recognized as an embryo by Malpighi was technically ‘unincubated’, it had until mid-November, some two months later. This been left sitting in the Bolognese sun in August and forced him to the erroneous, but entirely logical so was certainly not ‘unheated’. Nevertheless, Mal- conclusion that ‘nothing after coition is to be found pighi’s notion of a preformed chicken initiated one in [the] uterus for many days together’. When he of the great debates in embryology that was to last did find a conceptus, that, for Harvey, was the egg: throughout the 17th and 18th centuries. The question ‘Aristotle’s definition of an egg applies to it, namely, was: are the organs of the embryo formed de novo an egg is that out of a part of which an animal is 4  History of embryology 1 begotten and the remainder is the food for that were for a long time forgotten. Had he lived a little which is begotten’. longer (he died tragically early, at the age of 32), the Three factors of veterinary interest had led this discovery of the mammalian egg could probably brilliant man astray. First, he did not appreciate that have avoided a delay of about 150 years! the females did not come into oestrus until early As it was, the first scientist to actually see the October and so his estimates of breeding dates were mammalian egg (which everyone believed to exist, wrong. Second, he dismissed the ovaries (‘female but no one had seen) was the Estonian medical testicles’) as making no contribution to conception doctor Karl Ernst von Baer (1792–1876). He because they failed to swell up as the testes of males opened the ‘Graafian egg’, as the follicle was known do during rut. Third, expecting to find an egg-shaped at that time, and saw with his naked eye a small conceptus, he failed to recognize the ‘purulent yellow point which he released and examined under matter … friable … and inclining to yellow’, which the microscope (Baer, 1827). There, upon a first he observed much earlier after mating and describes glance, Baer was stunned and could hardly believe quite vividly, as being the filamentous blastocyst so that he had found what so many famous scientists characteristic of ruminants. Had Harvey used a lens, including Harvey, de Graaf, Purkinje and others had or conducted his studies on species (like rabbits or failed to find. He was so overcome that he had to horses) with spherical early conceptuses, discovery work up courage to look into the microscope a of the real egg might have been advanced second time. The mammalian egg had been considerably! identified. It is easy to be critical with hindsight of course, How about the spermatozoa? Anton van Leeu- and we should remember that Harvey made impor- wenhoek (1632–1723), a Dutch tradesman and tant contributions to embryology: his descriptions scientist from Delft, was the first to report having of early development were impeccable; he was the seen moving spermatozoa. He constructed a single first to observe the blastoderm of the chick embryo lens microscope that magnified up to about 300 (the small region of the egg containing the yolk-free times. Technically, this microscope was an amazing cytoplasm that gives rise to the embryo proper) and achievement – no bigger than a small postage stamp to indicate that blood islands form before the heart and resembling a primitive micromanipulator. It does; and he was aware of the gradual development was used close to the eye like a magnifying glass. of the embryo, subscribing to the school of epi­ Using this, Leeuwenhoek drew spermatozoa from genesis as did Aristotle. different species. Initially, Leeuwenhoek was reluc- The observations by Harvey and the search for the tant to study sperm and he questioned the propriety mammalian egg were extended by Regnier de Graaf of writing about semen and intercourse. When he (1641–1673) who performed detailed studies of the first focused his microscope on semen, Leeuwen- female reproductive organs, especially the ovary. De hoek discovered what he then took to be globules. Graaf, like his friend Leeuwenhoek, worked in Delft. However, he so disliked the prospect of having to From a comparison of mammalian ovaries with discuss his findings that he quickly turned to other those of chicken, de Graaf considered mammalian matters. Three or four years later, however, in 1677, antral ovarian follicles to be the eggs; an assumption a student from the medical school at Leiden brought he confirmed by tasting! His contribution to science him a specimen of semen in which he had found was later acknowledged by the German medical small animals with tails, which Leeuwenhoek now doctor Theodor Ludwig Wilhelm Bischoff (1807– observed as well. Consequently, Leeuwenhoek 1882) who introduced the nomenclature ‘Graafian resumed his own observations and, in his own follicle’. De Graaf also noted some connection semen (acquired, he stressed, not by sinfully defiling between follicular maturation and the development himself but as a natural consequence of conjugal of oocytes but, without an appropriate microscope, coitus), observed a multitude of ‘animalcules,’ less he could not substantiate this and his observations than a millionth the size of a coarse grain of sand 5  Essentials of Domestic Animal Embryology and with thin, undulating, transparent tails. A no biological size scale at the time of these argu- month later, Leeuwenhoek described these observa- ments because the cell theory of Theodor Schwann tions in a brief letter to Lord Brouncker, president (1810–1882) was not proposed until 1847. Thus of the Royal Society in London. Still uneasy about preformationists could claim, as formulated in 1764 the subject matter, he begged Brouncker not to by the Swiss naturalist and philosophical writer publish it if he thought it would give offence. Leeu- Charles Bonnet (1720–1793), that ‘Nature works as wenhoek’s observations prompted vivid discussions small as it wishes’. Basically, preformation was a and controversies on the significance of these living conservative theory, and it was unable to answer objects. At first, it was commonly held that the tad- some of the questions raised by the limited knowl- pole-like creatures were parasites. Leeuwenhoek edge of genetic variation at that time. It was, for may have been biased towards that view as he was example, known that matings between black and actually engaged in parallel studies of parasites and white parents resulted in babies of intermediate was the first to see Giardia, a protozoan parasite that colour, an outcome incompatible with preforma- infects the gastrointestinal tract. Giardia are flagel- tion in either gamete. lated and may, at poor microscopical resolution, In the late 18th century, Caspar Friedrich Wolff resemble spermatozoa. Other scientists considered (1734–1794), a German embryologist working in that the whirling action of the objects was meant to St. Petersburg, made detailed observations on chick prevent solidification of the semen. In the preforma- embryos that resulted in the first strong case for tion school, however, Leeuwenhoek’s observations epigenesis. He demonstrated how the gut arises encouraged the thought that the sperm head con- from the folding of an originally indifferent flat tained preformed miniatures of babies, foals, calves tissue and interpreted his findings as evidence of etc. and so these scientists became known as the epigenesis when, in 1767, he wrote that ‘When the spermists. formation of the intestine in this manner has been The two mammalian gametes had been identi- duly weighed, almost no doubt can remain, I fied. Instead of forming a common platform for believe, of the truth of epigenesis’. Wolff’s name further endeavours, however, this knowledge lives on in the term ‘Wolffian duct’ for the mesone- prompted a bitter dispute, as to whether the embryo phric duct. arose from the egg or from the sperm! In spite of Wolff’s contribution, the preformation In parallel with the search for the mammalian theory persisted until the 1820s when new tech- gametes, the combat between the schools of epigen- niques for tissue staining and microscopy allowed a esis and preformation became more and more further advance in the science of embryology. Three intense. The latter had the backing of 18th century friends, Christian Pander (1794–1865), Karl Ernst science, religion and philosophy for several reasons. von Baer, and Martin Heinrich Rathke (1793– First, if the body is prefigured and just needs to be 1860), all of whom came from the Baltic region and unrolled, no extra mysterious force is needed to studied in Germany, formulated concepts of great initiate embryonic development. This was a reli- relevance for contemporary embryology. Pander giously convenient point of view, paying proper expanded the observations made by Wolff and also, respect to God’s creation of mankind. Second, if the despite studying the chick embryo for only some 15 body is prefigured in the germ cells, a further genera- months before becoming a palaeontologist, discov- tion will already exist prefigured in the germ cells of ered the germ layers (Pander, 1817). The overall the next, rather like Russian nested Matryoshka term ‘germ layers’ is derived from the Latin germen dolls. This concept was also convenient, ensuring (‘bud’ or ‘sprout’) whereas the three individual that the forms of species would remain constant. layers are of Greek origin: ectoderm from ectos The fact that at a certain point Matryoshka dolls (‘outside’) and derma (‘skin’), mesoderm from mesos cannot get any smaller would seem like an obvious (‘middle’), and endoderm from endon (‘within’). objection to the concept today. However, there was Pander also noted that organs were not formed from 6  History of embryology 1 a single germ layer. A remarkable feature of Pander’s pharyngeal arches common to the development of book from 1817 is the quality of the illustrations these animals. ‘Rathke’s pouch’ – the ectodermal drawn by the German anatomist and artist Eduard contribution to the pituitary gland – commemorates Joseph d’Alton (1772–1840); they beautifully him. depict details that had not yet been defined (Fig. In addition to identifying the mammalian ovum, 1-3). This classical work underlines the necessity for von Baer extended Pander’s observations on chick precise observational skills in embryology. embryos and described the notochord for the first Rathke studied comparative embryology in frogs, time. Moreover, von Baer again appreciated the salamanders, fish, birds, and mammals and pointed common principles that direct initial embryological out the similarities in development among all these development regardless of species; in 1828 he wrote vertebrate groups. He described for the first time the ‘I have two small embryos preserved in alcohol that I forgot to label. At present I am unable to determine the genus to which they belong. They may be lizards, small birds, or even mammals’. Staining and microscopy techniques continued to improve during the 19th century and allowed for more detailed observations on the initial cleavage stages by the German biologist Theodor Ludwig Wilhelm von Bischoff (1807–1882) in the rabbit, and by the Swiss anatomist and physiologist Rudolph Albert von Kölliker (1817–1905) in man and various domestic animals. Kölliker also pub- lished the first textbook on embryology in man and higher animals in 1861. Thanks to the contributions of Pander, von Baer and Rathke, the preformation school in its radical form ceased in the 1820s. However, the concept survived for another 80 years in the sense that a certain group of scientists regarded the cells of the early cleavage stage embryo to represent right and left halves of the body as it took form. This implied that the information for building the body is segre- gated regionally in the egg. In 1893, August Weis- mann (1834–1914) proposed his germ cell plasm theory as an extension of this idea. Based on the sparse knowledge of fertilization available at that time, he was far-sighted enough to propose that the egg and the sperm provided equal chromosomal contributions, both quantitatively and qualitatively, to the new organism. Moreover, he postulated that chromosomes carried the inherited potentials of this new organism, which was remarkable at that time considering that the chromosomes had not yet been identified as the carriers of inherited matter. Fig. 1-3: Drawing of a Day 2 chick embryo by Eduard However, Weismann thought that not all informa- Joseph d’Alton displayed in Pander (1817). tion on the chromosomes passed into every cell of 7  Essentials of Domestic Animal Embryology the embryo. Rather, different parts of information the nucleus from one of the embryo cells to slip over went to different cells, explaining their differentia- into the egg cytoplasm on the other side. Spemann tion. Weismann clearly understood the principle of then promptly tightened the noose completely, how traits are inherited through fertilization, but he physically breaking the ball of cytoplasm and its was wrong about the mechanisms of differentiation. new nucleus away from the remains of the 16-cell Weismann’s differentiation theory was put to the embryo. From this single cell grew a normal sala- test practically by the German embryologist Wilhelm mander embryo, proving that the nucleus from an Roux (1850–1924) who had already, in 1888, pub- early embryonic cell was able to direct the complete lished the results of experiments in which individual growth of a salamander. Spemann had created the cells of 2- and 4-cell frog embryos were destroyed first clone by nuclear transfer. Spemann published by a hot needle. As predicted by Weismann’s theory, his results in his 1938 book ‘Embryonic Develop- Roux observed the formation of embryos in which ment and Induction’ in which he called for the ‘fan- only one side developed normally. These results tastical experiment’ of cloning from differentiated or inspired another German embryologist, Hans Adolf adult cells and theoretically paved the way for the Eduard Driesch (1867–1941) to perform experi- cloning by somatic cell nuclear transfer that we ments using cell separation instead of Roux’s cell know today. Unfortunately, Spemann saw no practi- destruction technique. To his enormous surprise, cal way of realizing such an experiment at that time. Driesch obtained results that were quite different Spemann was awarded the Nobel Prize for Physiol- from those of Roux. Using separated cells from early ogy or Medicine in 1935 for his discovery of the cleavage stage sea urchin embryos he demonstrated effect now known as embryonic induction – the that each of the cells was able to develop into a influence exercised by various parts of the embryo small but complete embryo and larva (Driesch, that directs the development of groups of cells into 1892). He repeated the same experiment with 4-cell particular tissues and organs. The works of Driesch embryos and obtained similar results; the larvae and Spemann finally put an end to the concept that were smaller but otherwise looked completely inherited information is divided among the cells of normal. the developing embryo. The final evidence against the Roux-Weismann The whereabouts of inherited materials had still theory was provided by the elegant experiments not been determined in the late 19th and early 20th published by yet another German embryologist, century when a group of American embryologists set Hans Spemann (1869–1941). Originally, just like out to discover whether inheritance resided in the Driesch, he had set out to support the theory with cytoplasm or the nucleus of the fertilized egg. his experiments on salamanders. However, by sepa- Edmund Beecher Wilson (1856–1939) was of the rating the cells of early cleavage stage embryos with opinion that the nucleus is the carrier while Thomas a ligature (a hair taken from his newborn son’s Hunt Morgan (1866–1945) thought the cytoplasm head), he soon found that the separated cells were to be responsible. Wilson allied himself with the each able to form a small embryo – they were German biologist Theodor Heinrich Boveri (1862– totipotent. In 1928, Spemann conducted the first 1915) working at the Naples Zoological Station. nuclear transfer experiment, transferring the nucleus Boveri had produced major support for the chromo- of a salamander embryo cell into a cell without a somal hypothesis of inheritance by fertilizing sea nucleus. Using a hair as a noose, as he had done in urchin eggs with two spermatozoa. At first cleavage, his 1902 splitting of the salamander embryo, such eggs produced four mitotic poles and divided Spemann tightened the noose around a newly ferti- into four cells instead of two. Subsequently, Boveri lized egg cell, forcing the nucleus to one side and separated the cells and demonstrated that they cytoplasm to the other. Next, he waited as the side developed abnormally, each in its own particular with the nucleus divided and grew into a 16-cell way, due to the fact that they carried different chro- embryo. Then he loosened the noose, and allowed mosomes. Hence, Boveri claimed that each chromo- 8  History of embryology 1 some is distinct and controls different vital processes. of the Brachyury gene (Wilkinson et al., 1990). Wad- Wilson and Nettie Maria Stevens (1861–1912), one dington, on the other hand, addressed the causal of the first American women to be recognized for link between embryology and genetics by isolating her contribution to science, extended the work of several genes that caused wing malformations in Boveri. They demonstrated the relationship between fruit flies. Moreover, his interpretation and visual chromosomes and sex: XO or XY embryos devel- conception of ‘the epigenetic landscape’ affecting oped into males and XX embryos into females initial cell differentiation in the embryo still surfaces (Wilson, 1905; Stevens, 1905a,b). For the first time during contemporary presentations on embryonic a particular phenotypical characteristic was clearly stem cells and their differentiation (Waddington, correlated with a property of the nucleus. Eventu- 1957, Fig. 1-4). ally, Morgan found mutations that correlated with sex and with the X chromosome. This persuaded him that his earlier view that inheritance was through the cytoplasm was wrong and that genes are physically linked to one another on the chromo- somes. Consequently, a group of embryologists had laid a cornerstone to the discovery that the chromo- somes in the cell nucleus are responsible for the development of inherited characteristics. In the early 20th century, embryology and genetics were not considered separate sciences. They diverged in the 1920s when Morgan redefined genetics as the science studying the transmission of inherited traits, distinguishing it from embryology, the science stud- ying the expression of those traits. This division did not occur without hostility; geneticists considered the embryologists old-fashioned while embryolo- gists looked upon geneticists as being uninformed about how organisms actually develop! Fortunately, we nowadays see a rapprochement of genetics and embryology in a very fruitful symbiosis. Two of the scientists who advocated synergism between embry- ology and genetics in the early days were Salome Gluecksohn-Schoenheimer (now Gluecksohn- Waelsch; 1907–2007) and Conrad Hal Waddington (1905–1975). Gluecksohn-Schoenheimer received her doctorate in Spemann’s laboratory, but fled Hit- ler’s Germany for the United States. Her far-sighted research demonstrated that mutations in the Brachy- ury gene of the mouse caused aberrant development of the posterior portion of the embryo, and she localized the defect to the notochord (Gluecksohn- Fig. 1-4: Top: CH Waddington’s depiction of the epigenetic Schoenheimer, 1938, 1940), providing another landscape with the ball representing a cell and the valleys representing different avenues of differentiation. Bottom: example of the close link between embryology and A less commonly depicted view behind the epigenetic genetics. Interestingly, it took 50 years for her results landscape illustrating how the tension of different genes to be confirmed by DNA hybridization after cloning control the fate of the ball. From Waddington (1957) 9  Essentials of Domestic Animal Embryology The question of totipotency, initially addressed clones never progressed beyond the formation of by Driesch and Spemann, was later revisited at a the neural tube. However, when serial nuclear trans- finer level. Thus, whereas Driesch and Spemann had fers were made from the cloned embryos to other proved the totipotency of the cells of the early cleav- enucleated eggs, it was possible to generate numer- age stage embryo, experiments in the 1950s by ous tadpoles; the genomic totipotency of somatic Robert Briggs (1911–1983) and Thomas King cells had been proven. It should be noted though (1921–2000) tested the totipotency of the nucleus or that the frog experiments never managed to close rather the genome. Their nuclear transfer model was the developmental circle by producing an adult an exact realization of the ‘fantastic experiment’ pro- organism by transferring a somatic cell nucleus from posed by Hans Spemann, although they had never another adult organism. heard about his suggestion. To accomplish their Closing the circle did not happen until the nuclear objective they had to develop methods by which transfer technique was transposed to mammals by they could remove the genome of an egg without the Danish veterinarian Steen Malte Willadsen destroying it (enucleation), pick up a donor nucleus working in Cambridge during the 1980s. Willadsen of another cell, and transfer that nucleus to the enu- (1986) succeeded in transferring not just the nucleus, cleated egg. As their approach was extremely unor- but the entire cell from sheep morula-stage embryos thodox, their first grant application to the National to enucleated eggs by electrical cell fusion. His work Cancer Institute was refused as a ‘hare-brained’ idea. resulted in the first mammal to be born after cloning However, they eventually obtained some support, by nuclear transfer. In 1996, this technology was and after months of experimentation they produced taken one step further by Keith H Campbell, the first blastocyst from nuclear transfer. Their initial working at the Roslin Institute in Scotland in a success was short-lived. In their enthusiasm, they research group headed by Ian Wilmut. Campbell et gathered the complete staff of the institute to show al. (1996) succeeded in producing lambs following them the blastocyst. After numerous looks into the transfer of nuclei of cultured cells, harvested from microscope, followed by applause and congratula- the inner cell mass, to enucleated eggs. Key to this tions, they re-checked the dish and found only a success was Campbell’s meticulous cell cycle experi- completely destroyed embryo. Fortunately, although mentation that demonstrated the need for a certain the first embryo died, the nuclear transfer system degree of synchrony of cell cycle between the donor worked; in 1952, Briggs and King successfully dem- nucleus and the recipient cytoplasm. The ability to onstrated that donor nuclei from frog blastula stages clone mammals from cultured cells represented a could direct the development of complete tadpoles major breakthrough in biomedical science, facilitat- when transferred into enucleated eggs. This research ing genetic manipulation of the cells prior to nuclear further paved the way for the somatic cell nuclear transfer and opening an avenue for production of transfer that is nowadays used for cloning of transgenic animals (animals in which a foreign gene, mammals. Briggs and King also discovered that the transgene, has been added). Consequently, when cells from later stages (tailbud-stage tadpoles Angelika Schnieke, working in the same group of for example), were used as nuclear donors, normal researchers, was able to announce the birth of the development did not occur unless the nuclei came transgenic sheep Polly, a lamb cloned from cultured from the germ cells. Thus, somatic cells appeared to fetal sheep fibroblasts into which the gene for human lose their ability to direct development as their clotting factor IX had been inserted with a promoter degree of differentiation increased. This point was that would allow for expression of the transgene in later pursued by John B. Gurdon who worked with the mammary gland (Schnieke et al., 1997). It was another frog species, Xenopus, rather than Briggs and from that event that the concept of ‘biopharming’ King’s Rana. Gurdon et al. (1975) found that when (production of valuable proteins in transgenic nuclei of cultured skin cells from adult frogs were animals) emerged. The report on Polly, however, transferred into enucleated eggs, development of the was preceded by another from the Roslin group; a 10  History of embryology 1 publication that stunned not only the scientific com- Baer, K.E.V. (1827): De ovi mammalium et hominis genesi. munity, but all layers of society, world-wide. That Voss, Leipzig. Bonnet, C. (1764): Contemplation de la Nature. Marc- was the report of the birth of the cloned lamb Dolly Michel Ray, Amsterdam. (Wilmut et al. 1997). Dolly was created by Wilmut Briggs, R. and King, T.J. (1952): Transplantation of living and his group by transferring cultured mammary nuclei from blastula cells into enucleated frogs’ eggs. Proc. Natl. Acad. Sci. USA 38:455–464. gland cells from a 6-year-old ewe into enucleated Campbell, K.H., McWhir, J., Ritchie, W.A. and Wilmut I. eggs. Again, the success depended on control of the (1996): Sheep cloned by nuclear transfer from a cultured cell cycle; the mammary gland nuclear donor cells cell line. Nature 380:64–66. were kept under culture conditions that suppressed Darwin, C. (1859): On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races their mitotic activity, provoked a state of cellular in the Struggle for Life. John Murray, London. senescence, and locked them at the G1 state of the Fabricius, H. of Aquapendente (1600): De formato foetu. cell cycle, or G0. Research since then has resulted in Pasquala, Padova. the cloning of many animals of many species Gilbert, S.F. (2003): Developmental biology. 7th edn. Sinauer Associates, Sunderland, Massachusetts. (including cattle, mice, goats, pigs, cats, rabbits, Gluecksohn-Schoenheimer, S. (1938): The development of horses, dogs, and rats) and has also demonstrated two tailless mutants in the house mouse. Genetics that bringing the nuclear donor cells into quiescence 23:573–584. is not a necessity. Gluecksohn-Schoenheimer, S. (1940): The effect of an early lethal (t0) in the house mouse. Genetics In its combination with genetics, embryology is 25:391–400. an exponentially developing science; how it will Gurdon, J.B., Laskey, R.A. and Reeves, O.R. (1975): The continue to develop, only time can tell. As a subject, developmental capacity of nuclei transplanted from embryology made its way into the curriculum of keratinized cells of adult frogs. J. Embryol. Exp. Morphol. 34:93–112. veterinary medicine in the mid 19th century when it Harvey, W. (1651): Excitationes de generatione animalium. became incorporated into the teaching of anatomy. Elzevier, Amsterdam. In 1924, the first textbook on veterinary embryology Kölliker, A. (1881): Entwiklungsgeschichte des Menschen und der höhere Thiere. Engelmann, Leipzig. was published by Zeitzschmann and others (though Maître-Jan, A. (1722): Observations sur la formation du not many) have appeared since. Embryology of the Pig poulet. L. d’Houdry, Paris. by Bradley M Patten deserves special mention as it Malpighi, M. (1672): De formatione pulli in ovo (London). has been an admirable resource and inspiration for Reprinted in HB Adelmann ‘Marcello Malpighi and the evolution of embryology’. Cornell University Press, the authors of this book. Likewise, the ‘bible’ of Ithaca, NY, 1966. comparative embryology Developmental Biology by Pander, H.C. (1817/18): Beitrage zur Entwiklungsgeschichte Scott F. Gilbert we have found admirable for its des Hühnchens in Eye. Brönner, Wüzburg. breadth of coverage and for its inimitable style. Patten, B.M. (1948): Embryology of the pig. 3rd edn. Blakiston Company, New York, Toronto. Because embryology was originally based upon Roux, W. (1888): Contributions to the developmental the anatomical descriptive tradition and also entered mechanisms of the embryo. On the artificial production the curricula of medicine and veterinary medicine of half embryos by destruction of one of the first two as part of anatomy, its nomenclature is Latin- and blastomeres and the later development (postgeneration) of the missing half of the body. In B.H. Willier and J.M. Greek-based. For ease of reading, however, we have Oppenheimer (eds.) 1974 ‘Foundations of experimental anglicized some terms rather than use their strict embryology’, Hafner, New York, pp. 2-37. Latin or Greek forms. Schnieke, A., Schnieke, A.E., Kind, A.J., Ritchie, W.A., Mycock, K., Scott, A.R., Ritchie, M., Wilmut, I., Colman, A. and Campbell, K.H. (1997): Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science FURTHER READING 278:2130–2134. Schwann, T. and Schleyden, M.J. (1847): Microscopical Aristotle (ca. 350 BC): The generation of animals. A.L. Peck researches into the accordance in the structure and (trans.). eBooks@Adelaide, 2004 (see http://etext.library. growth of animals and plants. London: Printed for the adelaide.edu.au/a/aristotle/generation/). Sydenham Society. 11  Essentials of Domestic Animal Embryology Stevens, N.M. (1905a): Studies in spermatogenesis with Willadsen, S.M. (1986): Nuclear transplantation in sheep special reference to the ‘accessory chromosome’. Carnegie embryos. Nature 320:63–65. Institute of Washington, Washington. D.C. Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. and Stevens, N.M. (1905b): A study of the germ cells of Aphis Campbell, K. (1997): Viable offspring from fetal and rosae and Aphis oenotherae. J. Exp. Zool. 2:371–405, adult mammalian cells. Nature 385:810–814. 507–545. Wilson, E.B. (1905): The chromosome in relation to the Waddington, C.H. (1957): The strategy of the genes. Geo determination of sex in insects. Science 22:500–502. Allen & Unwin, London. Wolf, K.F. (1767): De formatione intestorum praecipue. Weismann, A. (1893): The germ-plasm: A theory of Novi Commentarii Academine Scientarum Imperialis heredity. Translated by W. Newton Parker and Petropolitanae 12:403–507. H. Ronnfeld. Walter Scott Ltd., London. Wilkinson, D.G., Bhatt, S. and Herrmann, B.G. (1990): Expression pattern of the mouse T-gene and its role in mesoderm formation. Nature 343:657–659. 12  CHAPTER 2 Morten Vejlsted Cellular and molecular mechanisms in embryonic development the size of the blastomeres halves with each divi- INITIAL EMBRYONIC DEVELOPMENT sion. Such unique cell divisions are referred to as AND GESTATIONAL PERIODS cleavages (Chapter 6). Then, coincident with the cells having reached a certain minimal size through Embryonic development encompasses all the proc- cleavage divisions, a phase of cell growth is intro- esses whereby a single cell – the fertilized egg or duced into the cell cycles, with daughter cells zygote – gives rise to first an embryo, then a fetus growing to approximately the size of the mother which at birth has the capacity to adapt to post-natal cell. This more constant cell size, together with life. These processes occur as a continuum but, for abundant cell proliferation and deposition of extra- convenience, they may be arbitrarily divided into cellular material, then leads to an increase in the size successive periods. Thus, intra-uterine development of the embryo as a whole. Both cell proliferation is often divided into an embryonic period, where and cell growth are principles of general cell biology all major organ systems are established, and a fetal and will not be dealt with in further detail in this period, which consists primarily of growth and book. organ refinement (see Chapter 18). Development of The blastomeres eventually form a small mul- the organism does not stop with birth, however; berry-like cluster of cells referred to as the morula organs continue to grow and mature at least until (Fig. 2-1; see Chapter 6). Initially, the morula is puberty and many tissues need continuous replen- characterized by bulging individual blastomeres; ishment throughout life. Aging and death may later, outer cells will adhere tightly to each other and therefore also be included the natural developmen- form a more uniform surface of the morula. This tal process of the organism. process is referred to as compaction. The outer cells The embryonic period is initiated at fertilization develop into the trophectoderm. Subsequently, when the oocyte, covered by the zona pellucida, is during the process of blastulation, a fluid-filled penetrated by the fertilizing spermatozoon resulting cavity, the blastocyst cavity, develops inside the tro- in the formation of the one-celled zygote, in which phectoderm, and the inner cells, forming the inner a maternal pronucleus (from the oocyte) and a cell mass (ICM), gather at one pole of the embryo paternal pronucleus (from the spermatozoon) which is now known as a blastocyst. The trophec- develop (Fig. 2-1; see Chapter 5). At the first post- toderm will participate in placenta formation fertilization mitosis the zygote develops into the (see Chapter 9) while the ICM gives rise to the 2-cell embryo. These two, and subsequent early embryo proper. The blastocyst expands, hatches embryonic cells, are for a period referred to as from the zona pellucida, and later, towards the end blastomeres. During the initial mitotic divisions of of blastulation, the ICM forms an internal and exter- blastomeres, the increase in the number is not nal cell layer, referred to as the hypoblast and epi- accompanied by any increase in the volume; the blast, respectively, to establish the bilaminar overall volume of the embryo remains constant as embryonic disc. Formation of the disc is, in  Essentials of Domestic Animal Embryology Fig. 2-1: Initial development of the mammalian embryo. A: Zygote; B: 2-cell embryo; C: 4-cell embryo; D: Early morula; E: Compact morula; F: Blastocyst; G: Expanded blastocyst; H: Blastocyst in the process of hatching from the zona pellucida; I: Ovoid blastocyst with embryonic disc; J: A Elongated blastocyst; K: Embryonic disc in the process of 7 gastrulation. 1: Inner cell mass; 2: Trophectoderm; 3: 8 Epiblast; 4: Hypoblast; 5: Embryonic disc; 6: Amniotic folds; 9 7: Ectoderm; 8: Mesoderm; 9: Endoderm. K B 6 domestic animal species, associated with the removal of the trophectoderm covering the epiblast (see Chapter 6). Along with this, the overall shape of the C embryo changes from spherical to ovoid and further J to tubular and filamentous except in the horse. The time period from fertilization to completion of blas- tulation lasts about 10 to12 days in pigs, sheep, goat, 5 and cat, 14 days in cattle and horses, and 16 days in the dog. D During the subsequent phase of the embryonic period, the bilaminar embryonic disc is transformed into a trilaminar embryonic disc, through the process of gastrulation (Fig. 2-1; see Chapter 7). Gastrulation leads to formation of the three somatic germ layers, ectoderm, mesoderm and endoderm, E I and formation of the primordial germ cells, the progenitors of the germ cell lineage. Early during the 1 3 process of gastrulation, a portion of the trophecto- 2 derm located around the embryonic disc, together with its underlying extra-embryonic mesoderm, 4 forms amniotic folds that finally fuse and enclose F the disc in the amniotic cavity. Following gastrulation, the three somatic germ layers further differentiate into various cell types and finally form the outline of most organ systems, thereby defining the end of the embryonic period H (Fig. 2-2). The increasing number of cell types G derived from the initial three somatic cell lineages formed during the process of gastrulation obviously requires intricate regulation of cell behaviour and organization of cells in order to create a complete organism with its many different, but interdepend- ent, tissues and organs. Different mechanisms 14  Cellular and molecular mechanisms in embryonic development 2 Zygote Blastomeres Trophectoderm Inner cell mass Hypoblast Mammary Sweat Sebaceous Epiblast glands glands glands Primordial Hair Outer Endoderm germ cells Mesoderm Ectoderm epithelium Hooves, claws of body Intermediate Lateral Paraxial Neural mesoderm mesoderm mesoderm tube Auditory Proctodeal vesicle epithelium Gametes Splanchnic Somatic Myotomes Dermatomes Optic Gonads mesoderm mesoderm Sclerotomes vesicle Lens Dermis Retina Inner ear Anal canal Pronephros Limb Limb Axial Axial ofk skin Stomodeal Epiphysis skeleton muscles muscles skeleton epithelium Posterior Anterior Neural pituitary pituitary crest Spinal Brain Cornea Oral Parameso- Mesonephros Trunk cord Cranial motor epithelium nephric Pleura neural nerves ducts Metanephros Parietal Pericardium crest Schwann Spinal motor Schwann Peritoneum cells nerves cells Pleura Mesonephric Varietal Sensory Peritoneum Vagina ducts nerves Uterus Mesenteries Pigment Uterine tubes Dentine Enamel Hemangioblastic cells Cranial of teeth of teeth Ductus epididymis tissue neural Ductus deferens crest Blood cells Sympathetic Cephalic Allantois Endothelium ganglia connective Gut Adrenal: of blood tissue and bones Urinary cortex vessels bladder medulla Epicardium Trachea Myocardium Heart Lungs Endocardium Wall of Outflow tract Pancreas respiratory Pharynx Walls of aortic tract arches I Middle ear Auditory tube Liver Digestive Wall of II Tonsils tract gut Stroma of pharyngeal Thyroid Pharyngeal III Thymus pouch derivatives pouches Inf. parathyroids IV Sup. parathyroids Post. branchial bodies Fig. 2-2: Differentiation of the derivatives of the zygote into the tissues of the body. 15  Essentials of Domestic Animal Embryology regulating embryonic development should not be Induction looked at in isolation but, again for convenience, the concomitant processes of differentiation, pat- Within the embryo, cells are often induced to dif- terning and morphogenesis will be outlined sepa- ferentiate through cell-to-cell signalling. Interaction rately below. at close range between two or more cells is termed The embryonic period is followed by the fetal proximate interaction or induction. During devel- period, lasting until term, with growth, maturation opment, induction between cells within a tissue, or and remodelling of the organ systems. The develop- within different tissues, is pivotal for the organiza- ment of each organ system, collectively referred to tion of differentiating cells into their respective as ‘special embryology’, will be covered in subse- tissues and organs. In order for induction to occur, quent chapters. In general, most embryonic loss however, cells to be induced (the potential respond- occurs early in the embryonic period. This is also ers) have to be competent or receptive to the induc- the time when the embryo is most susceptible to tive signals. Competence, manifested through teratogens (see Chapter 19). expression of cell-surface receptors for example, is often present only during a certain critical period. If not induced within this critical period, a competent DIFFERENTIATION cell may undergo programmed cell death, apopto- sis, instead of differentiation. Apoptosis is to be The adult mammalian body is composed of more regarded as a normal mechanism of embryonic than 230 different cell types, all originating from a development. single cell, the fertilized egg or zygote. The process The first embryologist to investigate induction whereby specialized cell types develop from less spe- was Hans Spemann (see Chapter 1). In amphibian cialized is known as cell differentiation. In general, embryos, he noted that lens formation in the surface an event preceding cell differentiation is cell com- ectoderm was achieved only when a close interac- mitment which, in turn, may be divided into a tion between the optic vesicle from the developing labile, reversible phase referred to as cell specifica- brain and the surface ectoderm was allowed to occur tion followed by an irreversible one called cell (Fig. 2-3; see Chapter 11). Spemann was able to determination. Once a cell is ‘determined’, its fate remove the optic vesicle before this interaction is fixed and it will irrevocably differentiate. occurred and show that, as a consequence, no lens- Cell differentiation is ultimately regulated formation was seen in the overlying ectoderm. Inter- through differential gene expression. Like a stream estingly, if the optic vesicle was placed beneath the running down the side of a mountain with its flow trunk surface ectoderm, it was not possible to induce branching many times before it reaches the bottom, lens formation in the overlying ectoderm. In other so do embryonic cells differentiate from common words, the trunk surface ectoderm was not compe- origins to gradually form specialized cell types (Figs. tent for lens induction. 1-4, 2-2). And just as a leaf dropped onto the stream Another example of induction is the generation follows one path only, so does a particular cell of the nervous system through the induction of neu- follow a single line of differentiation. However, for roectoderm (Fig. 2-4, see Chapter 8). Early ectoder- both the leaf and the cell, numerous decisions are mal cells may either differentiate into surface taken along the way to the final destination. Hence, ectoderm (and thence the epidermis), or neuroecto- cell differentiation during embryonic development derm (thence forming the nervous system). Initially, involves many branch points where lineages divide early ectodermal cells are uncommitted (or naïve) and sequential decisions on differentiation are and competent to differentiate in either direction. taken. As outlined above, these decisions are at first However, signals from the notochord (an important reversible (cell specification), but later become irre- midline structure in vertebrate species appearing versible (cell determination). during gastrulation; see Chapter 7) induce overlying 16  Cellular and molecular mechanisms in embryonic development 2 (1) Normal induction of lens by optic (3) Optic vesicle is vesicle removed; Head no lens is induced (4) Tissue other than optic vesicle is implanted; no induction occurs (2) Optic vesicle cannot induce Trunk ectoderm that is not competent Fig. 2-3: Induction of lens formation as studied by Hans Spemann. Under normal circumstances, the neural ectoderm of the optic vesicle induces lens formation in the competent surface ectoderm of the head (1). An ectopically placed optic vesicle cannot induce lens formation in incompetent ectoderm of the trunk (2). If the optic vesicle is removed, no lens formation is induced (3). Other tissues transplanted beneath the competent surface ectoderm of the head do not induce lens formation (4). ectodermal cells to differentiate into neuroecto- involved in embryonic cell patterning (see below). derm. In particular the signalling molecule known Four major families of these signalling molecules as Sonic hedgehog (Shh) is pivotal in this process. are particularly important: Ectodermal cells outside the embryonic midline receive no inductive signals and form surface ecto- The Fibroblast Growth Factor (FGF) family, derm by default. Experiments have shown that: if which includes more than 20 related proteins. the notochord is removed, only surface ectoderm The Hedgehog family, including proteins will form; placing notochordal tissue outside the encoded by the Sonic hedgehog (Shh), Desert embryonic midline will induce the formation of hedgehog (Dhh), and Indian hedgehog (Ihh) neuroectoderm instead of surface ectoderm; and genes. midline ectoderm removed from the influence of The Wingless (Wnt) family, containing at least underlying notochord will form surface ectoderm. 15 members, all of which interact with This example illustrates the fact that during cell transmembrane receptors known as Frizzled specification the fate of cell differentiation can be proteins. altered by changing the position of the cell within The Transforming Growth Factor-β (TGF-β) the embryo. Once cell determination has occurred, superfamily, which includes at least 30 molecules this plasticity is lost. with members such as the TGF-β, Activin, and Signalling molecules, such as Sonic hedgehog, Bone Morphogenetic Protein (BMP) families as that act between cells within a close range are well as the proteins Nodal, Glial-Derived referred to as paracrine factors or morphogens. Neurotrophic Factor (GDNF), Inhibin, and Many of these, involved in induction, are also Müllerian Inhibitory Substance (MIS). 17  Essentials of Domestic Animal Embryology Differentiation Shh Pax6 Pax3 Pax7 Patterning