Development and Evolution1

ASHG AWARDS AND ADDRESSES 2011 William Allan Award: Development and Evolution1

John M. Opitz2,*

tens of thousands of patients and their families, fellow students and fellow humanists, staff, and faculty in Iowa, Wisconsin, Montana, Utah, and Italy, happily so many here today. When pondering my modus operandi over the past half- century (MD, 1959), I must confess to a feeling of unease if not downright false pretense, reinforced by a recent letter (an e-mail) from a colleague in China wanting to ‘‘work in my lab’’ (I never had one) but hesitant because of the ‘‘strangeness’’ of my work and ‘‘what does a Julia Creek Dunnart have to do with anything’’? Trivially one might say, Julia Creek Dunnart, (Figure 1) a marsupial, hence no nursing bras or diaper services; but seriously, what can we learn about the evolution of the eutherian placenta, the bane of every woman who has had eclampsia, toxemia, or lost a baby (and almost her life) to HELLP ‘‘syndrome,’’ or suffered the consequences of maternal-fetal cell trafficking (so beautifully delineated by Diana Bianchi2)? Also, the protherians (duckbilled platypus and echidna), metatherians (marsupials of Australia and the Americas), eutherians, other animals, plants, fungi, bacteria, and archaea are all descendants of a last universal common ancestor (LUCA), which then becomes an organism of exceptional interest and evolvability (Figure 2). But to make this confiteor honest, I must also confess to Dear Judy, Max, Lynn, Colleagues, and Friends, the inclination or plain fact that for decades I have been In so far as this award is not a complete delusion on my practicing medicine, pediatrics, medical (clinical) genetics, part, an act of collective madness on your part, or a case of and developmental genetics and developmental pathology mistaken identity, I should still like to quote a letter from from the perspective of a zoologist (BA Zoology, 1956), a great, far better man than I: having been trained in comparative vertebrate embryology ‘‘I am extremely sorry not to be with you all. As you will (1954), evolution, basic genetics, and the biological basis of probably appreciate, things like being honored are not my sex determination and sex differentiation at the University cup of tea. John’’1 of Iowa by Emil Witschi (Figure 3), a student of Richard Thus, allow me to paraphrase John L. Emery, a giant of Hertwig who, together with his brother Oscar, had studied British pediatric and developmental pathology;1 honors with Ernst Haeckel in Jena (who, in turn, had studied are not my cup of tea either, but I am truly grateful to be with Rudolf Virchow in Wu¨rzburg and then briefly with here today with you all, with profound gratitude for the Johannes Mu¨ller in Berlin before Mu¨ller’s untimely death honor you have bestowed on me, happily ante- rather in 1858). Among other matters, Mu¨ller was renowned for than postmortem. In repeating the Domine non sum his studies on human physiology, echinoderms, female dignus., I am overwhelmed by the totally unmerited genitalia (the Mu¨llerian duct), the smooth shark of Aristotle and undeserved ‘‘amazing’’ grace that has accompanied (a placental animal) and the radiolarians, one-celled marine me over the last 60 years since coming to America due to protozoa he named and which engaged Haeckel, Richard family, teachers, and many friends throughout the world, Hertwig, and Valentin Haecker for decades (Figure 4).

1This article is based on the address given by the author at the meeting of the International Congress of Human Genetics on October 13, 2011, in Montreal, Quebec, Canada. The audio of the original address can be found at the web site of the American Society of Human Genetics 2Departments of Pediatrics (Medical Genetics), Human Genetics, Pathology, and Obstetrics and Gynecology, School of Medicine, University of Utah, Salt Lake City, UT 84108, USA *Correspondence: [email protected] DOI 10.1016/j.ajhg.2011.12.025. Ó2012 by The American Society of Human Genetics. All rights reserved.

392 The American Journal of Human Genetics 90, 392–404, March 9, 2012 with Emil Witschi,5 membership in the American Society of Human Genetics, exposure to the many advances in medical genetics being made at that time, and the chance to attend (with Dr. Witschi) my ﬁrst meeting of the American Society of Human Genetics, then assembling together with the American Society of Zoology in College Park, PA—Dr. Witschi just having been elected its President. It was in Dr. Witschi’s ofﬁce that I had met Charles E. Ford and Josef Warkany; thus was confronted by a choice between genetics or teratology, Madison (Smith and Patau) or Cincinnati (Warkany), a choice made easier after attending the ASHG meeting in College Park and applying to the Cincinnati and Madison programs (to make sure of either), accepting the latter a few minutes before being invited to the former at close to midnight late in June, 1961.

Madison (1961–1979): Developmental Field Theory At the University of Wisconsin, I completed a residency in pediatrics (the last six months as chief resident), translated much of Overzier, wrote a grant application for fellowship support to the NIH (successful), served two years as fellow during the time David Smith was metamorphosing from an endocrinologist (a student of Lawson Wilkins at Johns Hopkins) into a pediatric morphologist (after a one-year sabbatical, 1964–1965, study embryology with Gian To¨n- dury in Zu¨rich) before his move to Seattle (1966) where Smith continued, until his death, a successful program in ‘‘dysmorphology.’’ And it was in the pediatric genetics clinic that I had the ‘‘eureka’’ experience of seeing in quick succession auto- Figure 1. Representative Metatherian or ‘‘Look Ma, No Placenta’’ somal-dominant and autosomal-recessive radius a/dysgen- Top: koala; bottom Julia Creek dunnart, an Australian marsupial, esis, later supplemented on the basis of actual dissections 71 with two young. From Opitz. Copyright retained by Trudy Nich- of trisomy 18 infants.6 Eureka: different causes resulting olson; used with permission. in identical malformations identifying dysmorphogenetic units of the human embryo, which must also be morpho- The Developmental Field First Noted genetic units under normal circumstances, the ‘‘fields’’ Fortunately, or unfortunately, Witschi’s Development of mentioned by Huettner4 and identified and discussed in Vertebrates3 was not published when I took his course in detail by Spemann,7 and Huxley and DeBeer8 (q.v. espe- 1954; instead, Dr. Witschi assigned us Alfred F. Huettner’s cially their Figure 112, amphibian neurula to show the Fundamentals of Comparative Embryology of the Vertebrates4 main regional fields), Needham,9 (see also Oppenheimer10). with its beautiful illustrations and excellent historical To my knowledge, no complete inventory of all fields introduction where I learned: in the vertebrate body has ever been made, and continued ‘‘This organizer is now better known as the chorda-meso- inability to identify biochemical substances causing dermal field which is the first or primary field to exist in the induction in the 1930s led to disillusionment with and amphibian germ. Secondarily it gives rise to secondary virtual abandonment of work on the developmental organizers or subordinated fields, such as nose, eye, ear, field concept. And when this appeared to be a viable gill and others.. notion in medical genetics in the 1960s and early 1970s, . a piece of information filed away in the back of my Dr. Wiedemann and I made a major effort to determine if mind in 1954 until ready ‘‘to put two and two it had ever been introduced into medicine or human together.’’4 embryology to begin with, all to no avail. One of the last mentions of the developmental field concept in Western 1959 biology seems to have been in 1954 by Curt Stern, a former My final year of medical school afforded the opportu- President of the American Society of Human Genetics.11 nity of training with Hans Zellweger and Jacqueline A. In discussions with Oswaldo Frota-Pessoâ, from Saõ Paulo Noonan (after whom I named the eponymic syndromes), and then visiting professor at the University of Wisconsin completing the chapter on the biologic basis of sex determi- in the mid-1960s, heavy emphasis was placed on the nation and sex differentiation for the Overzier textbook statistical correlations within developmental field defects.

The American Journal of Human Genetics 90, 392–404, March 9, 2012 393 Figure 2. Not Born Yesterday or ‘‘Age Is Relative’’ Attempt at an animal phylogeny with rough dates at bottom showing origin of most animal groups to a pre-Cambrian (pre-542 million years ago) era with the most recent origin for vertebrates and urochordates (our closest chordate relatives) and a somewhat older origin for cephalochordates (amphioxus, previously considered our closest chordate relative). Based on various sources, sequencing data, and personal communications (C. Nielsen).

And it was as such a statistical entity that the develop- monkeys. Meckel14 cites the unilateral lack of a lung ‘‘als mental field concept was first put into print again,12 to be Fehler der Urbildung,’’ that is as a primary malformation, redefined later in its original morphogenetic sense. In this but points out that in most snakes the left lung is rudimen- connection it is of interest that nowhere in his Silliman tary or absent; complete cleft of bladder, each receiving one lectures7 or his Nobel lecture13 does Spemann use the ureter, is normal in snakes. He repeats and abstracts from concept of the gene when in fact it was genetic and evolu- the literature several human cases with a tail; fusion of tionary studies that shaped the modern field concept on ribs as in birds and turtles; persistence of the pupillary the basis of heterogeneity, homology, and phylogeneity. membrane beyond the eighth month (as in other mammals All primary malformations are causally heterogeneous; blind for a time after birth); lack of pulmonary lobation as in there are many genes and gene mutations but only a few the cetaceae, birds, and reptiles; cleft of epiglottis (Kehl- final developmental paths under normal circumstances deckel) normal in most (other) mammals; uvula first seen and primary malformations under abnormal ones. It was in monkeys and humans but absent in other animals; Meckel’s great merit to have recognized that primary Zwickelbeine (a delightful old German term for Wormian malformations are not contrary to nature (‘‘. die urspru¨ng- bones) present in hydrocephalus (and presumably in osteo- lichen Fehlbildungen sind nicht wider die Natur’’),14 especially genesis imperfecta) but normal in many animals, with the because so many can be found as normal states in related comment that failure of normal fusion of the skull bones species: consider for a moment palatal nonfusion as the (metopic suture) was present not only in his own father’s normal state in all birds and most reptiles; ‘‘agenesis’’ of and three other skulls but was the normal state in almost the corpus callosum the normal state in monotremes, all reptiles and other mammals; short gut without a differ- marsupials, and reptiles; penoscrotal inversion normal in ence between small and large intestine ‘‘as in most fish lagomorphs and marsupials, as is the bifid female marsupial and several (other) mammals’’; and many others. reproductive tract; cloaca formation normal in mono- Many of the examples cited by Meckel are true atavisms tremes; and loss of thumbs normal in platyrrhine (spider) (Darwin’s reversions), recurrence of developmental and/or

394 The American Journal of Human Genetics 90, 392–404, March 9, 2012 species are capable of developing an identical malformation (e.g., HPE), then they share corresponding developmental field structure and dynamics by virtue of descent with modification from a common ancestor. Because developmental fields are the self-organizing units of the embryo and because permanent evolutionary change of anatomical structure involves changes of field dynamics, the units of development (fields) are identical to the units of evolution (modules; see Schlosser and Wag- ner22, q.v. especially chapter 4). In evolutionary modules, as in developmental fields, processes are:

d Epimorphically hierarchical (from primary field to progenitor field18,23to final, secondary, or epimorphic fields); d Epigenetically complex, nonlinear, and reciprocally interactive but not so interactive as to bring down the entire organism, as Cuvier worried, when evolution begins to tinker with one part; d Spatially coordinated and ordered; d Temporally synchronized, and d Phylogenetically highly constrained into a few final, common developmental paths (many genes, few structures—compared to dogs or fancy pigeons, humans are an anatomically dull lot) and even more Figure 3. My Second Father mutations, yet HPE (or cleft palate, etc.) occurs over Emil Witschi around his 70th birthday. Reproduced from Arch d’Anat microscop et de Morphol Expe´rim.72 Not covered by copy- and over again. right. And if a specific teratogen (e.g., alcohol or jervine/ cyclopamine derived from Veratrum californicum) leads to genetic conditions or abnormal and/or rare conditions in a corresponding malformation, that is HPE in different humans but normal in more or less closely related species species, morphogenetically identical to one of genetic (remember Lord Morton’s mare with ancestral Quagga origin (SHH mutation), then it must have interfered with stripes15). Atavisms were not named as such until 1859 (by the same basic signal transduction pathway, in this case Baudemont, a cattle expert). All atavisms must be presumed cholesterol modification of the sonic hedgehog protein. to be morphogenetic vestigia, that is anomalies of incomplete And because pregnant dog mothers do not have mixed differentiation (e.g., cloaca with urogenital sinus, persistence litters of baby elephants, reindeer, and kittens but gener- of tail, or interdigital web). The converse seems not to be true: ally only puppies and because permanent evolutionary open spina bifida or holoprosencephaly (HPE) are as selec- change of anatomical structure involves coordinated tively disadvantageous in humans as in cats or dogs. change over time of several integrated developmental Homology of anatomical structure (e.g., pectoralis major fields, the modules differ from fields by responding over muscle in mammals and birds) infers corresponding few (artificial selection) or numerous generations (natural morphogenesis by virtue of descent, with modification, selection) with changing morphodynamics, changing from a common ancestor (e.g., reptiles or therapsids) gene/allele frequencies, and changing selective adapta- with prototypic pattern of development involving iden- tions (see Futuyama24, q.v. especially chapter 20). Every- tical signal transduction paths.15–18 thing that develops (including Mu¨ller’s, Haeckel’s and And when Leonardo da Vinci said: ‘‘Facile cosa e` farsi univer- Hertwig’s unicellular radiolarians) has evolved; thus, every- sale.,’’19 (that is ‘‘it is easy to become universal, since all land thing that occurs during morphogenesis, whether normal animals resemble each other in the parts of their body, that or a primary malformation, has been made possible by is, muscles, nerves and bones, and differ only in length evolution. Primary malformations, if also atavisms, might and size’’) he was referring to homology (a term coined in perhaps by regarded as evidence for evolution before the 1837 by Richard Owen),20 the best understood concept rest of the body was ready for it. underlying pre-Darwinian ‘‘descent.’’Here, it is hardly neces- The initial meaning of secondary anomaly as per sary to repeat Walter Fitch’s admonition, ‘‘proper phyloge- Meckel, referred to consequences of intrinsic or extrinsic netic analysis should only be based on homology.’’21 events during or after development, for example female Phylogeneity is the only term I have coined in this larynx in boys without testes, small adrenals in anence- connection as a corollary of homology: if several different phalics, absence of a hand due to amputation, paralysis

The American Journal of Human Genetics 90, 392–404, March 9, 2012 395 Figure 4. Beauty Not Only in the Eye of the Beholder Plate of radiolarians from Haeckel (1862), named as such by Johannes Mu¨ller and recognized by him as rhizopods, unicellular animals with axopods. It is an amazing insight that these organisms, not considered animals by some zoologists, for example the late Lynn Margulis, ‘‘knew’’ how to construct these species-specific ‘‘exoskeletons’’ or cages. From Prestel Verlag, reprinted with permission.73 and/or weakness of lower body with lumbar spina bifida, cord in the ADAM sequence, acephaly/acardia in a mono- and so forth. By inference, it is possible to say that zygotic twin fetus with TRAP sequence25,26. secondary anomalies are also those not effected by evolu- In this connection it is important to remember that tion, for example pseudosyndactyly of fingertips, amputa- during ontogeny of direct-developing organisms such as tions, placenta adherent to the brain with a short umbilical Homo sapiens morphogenesis precedes histogenesis, that

396 The American Journal of Human Genetics 90, 392–404, March 9, 2012 is the establishment of cell lineages, a fact well known to LUCA Meckel, who coined the pregnant phrase ‘‘dass die Form Thus, armchair paleontology provides, vicariously even to vor der Textur entsteht.’’ (form arises before [histologic] clinicians, a surfeit of speculations on LUCA,41 an texture), a fact repeated in Continental embryology books, ‘‘organism’’ of extraordinary interest. Organism in quotes for example: ‘‘Thus. ontogeny of an organism comprises since it is yet unclear if this construct was a syncytium, primarily two aspects; one is morphogenesis, the second, colonial aggregate, or heterogeneous group of cells under histologic differentiation. We may add immediately that variably strong natural selection some 3.5 billion years in higher organisms morphogenesis occurs primarily ago.41 Horizontal gene transfer complicates the task of during the early (beginning), histologic differentiation tracing descent; however, in a post-RNA world it is evident during the end stages of development (translated by that the three domains of life share so many properties J.M.O.).27 (homologies) that common ancestry is the only logical As a consequence it is no surprise that most malformed conclusion;42,43 viruses are probably excluded from the organs and body parts are histologically normal and that tree of life.44,45 None of us has ever seen LUCA, it left no most childhood cancers are defects of cell lineages, not of pre-Cambrian fossils and, like the evanescent embryonic malformed organs—think: retinoblastoma in a normally mammalian neural crest, it vanished except for its descen- formed eye or rhabdomyoma in a normally formed heart dant forms of life. But with modern methods it is possible in tuberous sclerosis. to obtain inferences about its biologic nature;41,46–51 its But, during phylogeny histogenesis precedes morphogenetic constitution and content;52–56 and its protein genesis. Examples that come to mind are Amphioxus molecules.57,58 without eyes but with a frontal eye spot and other rudi- Darwin was prescient: ‘‘.we must likewise admit that all mentary photoreceptor cells and Trichoplax adhaerens the organic beings which have ever lived on this earth may without a heart but with contractile cells in its interior. be descended from some one primordial form. [an] inter- In this connection it must be remembered that the molec- ference chiefly grounded on analogy.’’15 On the same ular machinery to ‘‘make’’ organs and body parts arose page Darwin uses the construction ‘‘their embryological long before the organ. Thus, Amphioxus does not possess homologous. structures,’’ the context making it clear any neural crest structures, but has all major genes/tran- that for anatomy he still uses the French term analogie scription factors to do so.28–30 Similarly, T. adhaerens with but homology for embryology. its astonishingly simple (? diploblastic) body structure and sophisticated molecular ‘‘toolbox.’’31 Antiquity and Evolution of Our Genome Older than 3.5 billion years and even then astonishingly The Delights of Clinical Palaeontology complex, LUCA has been estimated to have had from Thus, without a shovel but with a first-class physical exam- 42455 up to 1,00054 protein-coding DNA genes. But, ination, ‘‘phenotype analysis’’ (a term I translated from because no rates can be determined for LUCA’s gene Haecker32) and advances in molecular biology, we are history, it must be done for its descendants using ‘‘phyloge- enabled to the delights of clinical (armchair) paleontology. nomic’’ methods. David and Alm,59 by using the phyloge- For example, DHCR7 activity is present in plants33 and nomic method ‘‘AnGST,’’ analyzed the phylogeny of animals;34–36 was it therefore present in LUCA? The answer almost 4,000 gene families in the three domains of life probably is no because the biosynthesis of cholesterol and showed that a brief period of Archaean expansion requires 11 atoms of oxygen37 and hence was unlikely and rapid diversification of bacterial lineages some 3.2 before the great oxygenation of the earth some 2.4–2.2 billion years ago gave rise to almost 27% of all modern billion years ago. This suggests an interesting composition gene families (Figure 5). of the cell membrane of LUCA (hence no membrane lipid rafts38) and the truth of Goethe’s dictum that ‘‘we see only The Genomic Hourglass what we know,’’ hence the inability of some to see the face Witschi, as so many of his generation, raised overtly or of infants with the RSH (so-called Smith-Lemli-Opitz) covertly (Witschi ‘‘with candle in the attic’’) on Haeckel’s syndrome staring at them in a farmer’s market basket of Natu¨rliche Scho¨pfungsgeschichte (mine is the 8th edition of dwarf Arabidopsis thaliana. 1889) was a recapitulationist but a nuanced admirer of But then there is the Perrault syndrome type P, which I Haeckel. The first substantial question Witschi asked me was privileged to study with Philip D. Pallister, (still active in March 1951 when my musician uncle introduced me at 92 in Boulder, MT)39 and with Mary-Claire King and her to him in the zoology building in Iowa City, was: ‘‘Und gifted team40 more recently. In this familial case the muta- was ist das biogenetische Grundgesetz?’’ (What is the funda- tion involved HARS2, which encodes mitochondrial his- mental biogenetic law?)60 And right in this first momen- tidyl tRNA synthetase present in prokaryotic eubacteria tous encounter, when I was just 15, he graciously and (Escherichia coli) euarchaea (Thermus thermophilus), yeast generously took the time to introduce me to the wonders (Saccharomyces cerevisiae) and other eukaryotes besides of development as related to evolution, hinting, with humans (Caenorhabditis elegans), and hence must have a smiling wink, that, for example, birds might be some been present in LUCA. sort of dinosaur, at which point my uncle left, muttering,

The American Journal of Human Genetics 90, 392–404, March 9, 2012 397 Figure 5. ‘‘Better’’ for Gain or for Loss? Slightly modiﬁed from David and Alm59. Colors: Red, average rate of gene birth. Blue: gene duplication; green: horizontal gene transfer; yellow: gene loss. No rate can be established for LUCA except to estimate that at approximately 3.7 BYA it was still accruing more than it was losing. Please see legend to their Figure 1 in the original publication for further details as well as the supplementary information (especially supplemental Figure 9). Reprinted by permission from Macmillan Publishers.

‘‘damn grackles.’’ To my astonishment, Witschi also taught names I did not note], and I am altogether not able to me, with the markings on red-wing blackbird and Balti- determine to which class they belong. They could be more oriole eggs, that bird eggs are laid broad end first. lizards, small birds or very young mammals. So similar is He was proud of the trove of primary sources on Mu¨ller, head and trunk formation in these animals’’ (p. 221, trans- Haeckel, and the Hertwig brothers he kept in a small locked lated by J.M.O.).16 cabinet to the left of his desk. Darwin made much of the study of embryos as evidence Haeckel wrote ‘‘Ontogeny recapitulates phylogeny,’’ for descent; however, in the first two editions of the Origin hence, the oversimplified diagrams of the striking morpho- of the Species.15 he attributed the above passage to Agassiz, logic similarity of vertebrate embryos at roughly compa- and while correcting the attribution in the third he did not rable stages of development (Figure 6) and the severe translate from the original but used Huxley’s 1853 para- criticism leveled at Haeckel while alive and after his death phrase, going beyond von Baer’s conclusions to bolster (for one example see Gould61). And yet he was onto a truth his conviction that this resemblance of embryonic stages already enunciated by von Baer: ‘‘The embryos of was due to community of descent.10 mammals, birds, lizards and snakes, probably also of Nevertheless, the ‘‘hourglass’’ model (stage during later turtles, in earlier stages [Zusta¨nden, literally ‘‘conditions’’] gastrulation of greatest morphologic similarity regardless are extraordinarily similar to each other, in general [im of dramatic differences in early cleavage and later Ganzen] as in the development of their parts, so that it is ontogeny) has entered mainstream embryology as the often possible to distinguish these embryos only according ‘‘pharyngula’’ (Figure 7). Modern molecular methods to size. I own two small embryos in alcohol which I have in fact validated the genomic hourglass in insects, neglected to label [die Namen zu notieren, literally whose that is six sequenced Drosophila species separated up to

398 The American Journal of Human Genetics 90, 392–404, March 9, 2012 Figure 6. Haeckel’s Point Essentially Correct From Ernst-Haeckel-Haus museum guide (1990) with mammalian embryos (from left to right): B, Beutelthier (marsupial, Didelphys, opossum); S, Schwein (Sus. pig); C, Deer (Capreolus); R, Rind (Bos, cow); H, Hund (Canis, dog); F, Fledermaus (Rhinolophus, literally ‘‘ﬂitting mouse,’’ bat); L, Kaninchen (Lepus, rabbit); M, Mensch (Homo, man). From the original publication: sixth edition of Haeckel’s Anthropogenie und Entwicklungsgeschichte des Menschen (1905).

40 million years ago at Sander’s62 phylotypic stage,63 and ploid,68 awaited confirmation from ever more efficient in zebrafish, chick, and mouse.64 This work shows, respec- sequencing of many genomes, given we already knew of tively, the lowest gene expression divergence at the one Hox cluster in Drosophila and four in mouse and hu- extended germ band stage, the most ancient gene expres- mans. Confirmation came, to me most gratifyingly, from sion at pharyngula, and the most modern gene expression a ‘‘humble’’ ancient beast namely Kowalewski’s Amphi- at the earliest and latest stages of development (Figure 8).65 oxus (Branchiostoma lanceolatum, or B. floridae69). Their Haldane66 seems to have been the first to put forward the ancestry in the chordate lineage might date to pre- view ‘‘that in related organisms homologous structures Cambrian times; their anatomy is simple, apparently are developed through the action of homologous genes, bilaterally symmetrical (not really), without brain, eyes, and that therefore genic homology is more fundamental or neural crest but with a notochord and well-docu- than morphologic morphology.’’ mented embryogenesis. Analysis of its genome compared to that of a urochordate Ohno Confirmed (tunicate, e.g., Ciona intestinalis), sea urchin, teleost, cyclo- In the 1960s the late and greatly gifted Susumu Ohno stome, fruitfly, and human appears to confirm two genome (whom I first met in Witschi’s office) began to call duplications shortly before or after the divergence of the attention to traces of former apparent genome duplica- lampreys and hagfish by auto- (not allo-) tetraploidy tion(s) in our genetic constitution,67 suggesting, a.o., from an original set of approximately 17 linkage groups/ that such duplications enabled efficient evolutionary chromosomes (Figure 9).69 modifications, co-opting duplicates of old genes into What extraordinarily stimulating times for a ‘‘medical new roles. Ohno’s startling proposal of two genome zoologist’’ raised on a human chromosome complement duplications, making early vertebrates possibly octo- of 48 and yet privileged to be involved, if only peripherally,

The American Journal of Human Genetics 90, 392–404, March 9, 2012 399 Figure 7. Different Vertabrates, Similar Pharyngula, Pace Haeckel Masterpiece in color of graphic art (comparable to the best of Witschi, who began his career as an artist in Munich before World War I, or of Haeckel who wanted to abandon medicine and biology to become an artist). Showing in the center circle in diagrammatic form the pharyngula, here for frog, chick, pig, and human, with radically divergent cleavage patterns and postgastrulation development. From Hinrichsen,74 Figure 8-1, p. 149, used with permission. in the revolutions beginning in the late 1950s in cytoge- and infants in Utah are not autopsied. This is not just netics, biochemistry, molecular biology/genetics, and cutting corners by the medical profession but a gross taxonomy/phylogenetics. Von Baer16 was right: Simplex cultural misperception of the great value of an appropriate, est sigillum veritatis. thorough autopsy to the parents and their families (‘‘My baby has suffered enough’’; ‘‘You are not going to cut on The Last Frontier her’’? ‘‘Study of the brain does not allow an open casket Lastly, a matter of great concern to me and speaking now as funeral!’’—false, etc.). Often the NICU personnel also a member of a pathology department, specifically the Divi- misses a point: although they have had the privilege of sion of Pediatric Pathology at Primary Children’s Medical learning from the infant while alive, their attitude at death Center (PCMC), where autopsies are performed on all maybe ‘‘no need to do an autopsy,’’ we know she died of infants and children who die at PCMC, all fetuses delivered Down syndrome, hence depriving the pathology residents at the University Hospital with an autopsy permit, or those of the chance to learn something about the developmental referred from outside hospitals. Beginning my work in this pathology of trisomy 21, or 18, or 13, etc. field with Enid Gilbert (now Gilbert-Barness) in 1970 in After some three dozen years in this field, I’ve also come Madison and continuing for at least 12 years in Helena, I to appreciate the necessity of involving human genetics at am now privileged to leave the dissections to others, but every such autopsy. I go most carefully over the external, internal, and skeletal A brief example. A recent (2011) boy fetus, born prema- phenotype and placenta for any signs of congenital turely to a young primigravid woman but surviving for anomaly or high recurrence risk. And just knowing what a while with numerous complications, mostly pulmonary, is in the neonatal ICU and coming up for delivery at the until withdrawal of life support, came to autopsy with University Hospital, I know that many malformed fetuses permit ‘‘chest only.’’ Without a geneticist that might

400 The American Journal of Human Genetics 90, 392–404, March 9, 2012 Figure 8. Genomic Hourglass Figure 1 from Prud’homme and Gompel65. Developmental hourglass in Drosophila (Sander’s extended germ band stage) and three vertebrates (zebrafish, chick, and mouse). At phylotypic stage (i.e., least morphologically dissimilar in six sequenced Drosophila spp. separated by up to 40 million years) and pharyngula stage (most similar at gastrulation) showing in Drosophila lowest gene expression divergence and maximal conservation of the oldest gene set, and in the vertebrates most modern genes expressed at earliest and latest embryonic stages. Diagram based on Kalinka et al.63 and Domazet-Lozo and Tautz.64 Reprinted by permission from Macmillan Publishers. have yielded the diagnosis: bronchopulmonary dysplasia basis of phenotype and necessitating a laborious process of prematurity—no recurrence risk, unless, of course, her of revisiting the autopsy permit to go through the dia- next premature infant also was oxygen-dependent for a phragm for liver biopsy to allow ultrastructural studies while. This fetus was evaluated genetically antemortem and at least electron-microscopic confirmation of diagnosis without causal diagnosis. At autopsy I was rushing to and (I cell disease, 25% recurrence risk). How long ago it was fro between fetus and films (several fractures) before the that Enid Gilbert and I did the first two autopsies on LG ‘‘penny finally dropped,’’ suggesting a diagnosis on the and JG with this diagnosis,70 and yet it seems only

Figure 9. Ohno Victorious! Ohno Supported Two genome duplications around the time of divergence of cyclostomes and hagﬁsh from the ancestor of ﬁsh, amphibians, and amni- otes; see text; based on Furlong and Holland68 and Putnam et al.69 and several other sources.

The American Journal of Human Genetics 90, 392–404, March 9, 2012 401 yesterday that I saw in the clinic the faces of these wonder- 2. Bianchi, D.W. (2000). Fetomaternal cell trafficking: a new ful children who taught me so much, ante- and post- cause of disease? Am. J. Med. Genet. 91, 22–28. mortem, put to diagnostic use again almost 40 years later. 3. Witschi, E. (1956). Development of Vertebrates (Philadelphia: Thus, radical cultural change is required in and out of WB Saunders). hospital to help us speak for those unable to speak for 4. Huettner, A.F. (1953). Fundamentals of Comparative themselves (yet, so eloquent!) by increasing the autopsy Embryology of the Vertebrates, Revised Edition (New York: MacMillan). rate as high as possible (given the precarious reimburse- 5. Witschi, E., and Opitz, J.M. (1963). Fundamental aspects of ment rates and means at this time) and involving (inter- intersexuality. In Intersexuality, C. Overzier, ed. 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Essays in the History of Embry- ology and Biology (Cambridge, MA: MIT Press). logically and (phylo)genetically, it is not so much different 11. Stern, C. (1954). Two or three bristles. Am. Sci. 42, 213–247. from the development of the Julia Creek Dunnart. For 12. Opitz, J.M., Herrmann, J., and Dieker, H. (1969). The study of every human gene you study will you please ask yourself: malformation syndromes in man. Birth Defects Orig Artic Ser Present in how many domains of life? And if in all three, V(2), 1–10. will it not be possible to establish a LUCA database so 13. Spemann, H. (1935). Spemann’s Nobel lecture: Les Prix Nobel that we, all together, can finally figure out what LUCA en 1935 (Stockholm: Imprim Roy., Norsted and So¨ner). looked like? Admittedly, not a very prepossessing ancestor, 14. Meckel, J.F. (1812). Handbuch der pathologischen Anatomie, but THE ancestor from whom we all descended ultimately, Volume 1 (Leipzig: Reclam). including our self-awareness! 15. Darwin, C. (1968). The Origin of Species by Means of Natural Awareness of descent from a common ancestor is the most Selection or the Preservation of Favored Races in the Struggle ennobling feeling in biology. How delighted I am to be for Life. With Introduction and Bibliography by J.W. Burrow, a Radiolarian relative soon to be done while they, so Reprint of, First Edition (London: John Murray/Penguin much less complex and phylogenetically much, much Books). ¨ older than I, will continue to survive. 16. Von Baer, K.E. (1828). Uber Entwickelungsgeschichte der Thiere. Beobachtung und Reflexion Part I. (Ko¨nigsberg: Borntrager). Acknowledgments 17. Gilbert, S.F. (2010). Developmental biology, Ninth Edition (Sunderland, MA: Sinauer). To Susan and Emma (!); all the patients and their families, some 18. Davidson, E.H. (2006). The regulatory genome (Amsterdam: maintaining contact over a half-century, the most important AP/Elsevier). teachers in my professional life; academic teachers, among so 19. 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