Peter Holland,homeobox genes,and the developmental basis of animal diversity

In 1867 Alexander Kowalevsky published an account of the development of the cephalochordate Amphioxus lanceolatus (now known as Branchiostoma lanceolatum) (Kowalevsky, 1867). Together with his study of the development of urochordates (Kowalevsky, 1866; 1871), this introduced a new way of thinking about the relationship between the evolution and development of animals, and established the basis for long-standing theories of the evolutionary origin of vertebrates. Some one hundred and fifty years later, cephalochordates and urochordates are again in the spotlight, as molecular biology and genome sequencing promise further revelations about the origin of vertebrates. The work of the 2006 Kowalevsky Medal winner, Peter Holland (Fig. 1), has played a central role in their reinstatement (see Mikhailov and Gilbert (2002) for more details of the history of the Kowalevsky Medal). Here, I profile Peter Holland’s contribution to the rebirth of Evolutionary Developmental Biology in general and the study of homeobox genes and vertebrate origins in particular.     

Peter Holland, homeobox genes and the developmental basis of animal diversity

In 1867 Alexander Kowalevsky published an account of the development of the cephalochordate Amphioxus lanceolatus (now known as Branchiostoma lanceolatum) (Kowalevsky, 1867). Together with his study of the development of urochordates (Kowalevsky, 1866; 1871), this introduced a new way of thinking about the relationship between the evolution and development of animals and established the basis for long-standing theories of the evolutionary origin of vertebrates. Some one hundred and fifty years later, cephalochordates and urochordates are again in the spotlight, as molecular biology and genome sequencing promise further revelations about the origin of vertebrates. The work of the 2006 Kowalevsky Medal winner, Peter Holland has played a central role in their reinstatement. Here, I profile Peter Holland's contribution to the rebirth of Evolutionary Developmental Biology in general and the study of homeobox genes and vertebrate origins in particular.     

From development to evolution: the re-establishment of the "Alexander Kowalevsky Medal"

The Saint Petersburg Society of Naturalists has reinstated the Alexander O. Kowalevsky Medal. This article announces the winners of the first medals and briefly reviews the achievements of A.O. Kowalevsky, the Russian comparative embryologist whose studies on amphioxus, tunicates and germ layer homologies pioneered evolutionary embryology and confirmed the evolutionary continuity between invertebrates and vertebrates. In re-establishing this international award, the Society is pleased to recognize both the present awardees and the memory of Kowalevsky, whose work pointed to that we now call evolutionary developmental biology.     

Some problems of the origin of the vertebrata

A.O. Kowalevsky was the first to examine in 1865–1867 the groups related to ancestors of vertebrates. They were represented by lancelet (Branchiostoma) and tunicates (Ascidia). As Grobben (1908) divided metazoans which are more advanced than coelenterates into protostomes and deuterostomes, searching for remote relatives of vertebrates was performed among deuterostomes. For a long time, enteropneusts were considered to be probable ancestors of vertebrates and chordates as a whole. Subsequently, this concept was replaced by the hypothesis that chordates evolved from aberrant deuterostomes with a calcitic skeleton, which were named Stylophora or Calcichordata (Jeffries, 1986). Based on the data on the homeobox genes, Malakhov (2006) proposed that chordates could have acquired characters of deuterostomes independently of echinoderms. A separate position is occupied by the theory of Sepp (1959), who proposed that vertebrates appeared as a result of “duplication” of marine annelids. The most primitive living vertebrates, Cyclostomata, have a hypophysis which is enclosed in an unusually long canal under the brain and opens in an aperture just anterior to the brain. Cyclostomata, along with Paleozoic armored fishlike forms, compose the most primitive vertebrate group, Agnatha.     

On some historical and theoretical foundations of the concept of chordates

The concept of chordates arose from the alliance between embryology and evolution in the second half of the nineteenth century, as a result of a theoretical elaboration on Kowalevsky’s discoveries about some fundamental similarities between the ontogeny of the lancelet, a putative primitive fish, and that of ascidians, then classified as molluscs. Carrying out his embryological studies in the light of Darwin’s theory and von Baer’s account of the germ layers, Kowalevsky was influenced by the German tradition of idealistic morphology that was concerned with transformations driven by laws of form, rather than with a gradual evolution occurring by means of variation, selection and adaptation. In agreement with this tradition, Kowalevsky interpreted the vertebrate-like structures of the ascidian larva according to von Kölliker’s model of heterogeneous generation. Then, he asserted the homology of the germ layers and their derivatives in different types of animals and suggested a common descent of annelids and vertebrates, in agreement with Saint-Hilaire’s hypothesis of the unity of composition of body plans, but in contrast with Haeckel’s idea of the Chordonia (chordates). In The Descent of Man Darwin quoted Kowalevsky’s discoveries, but accepted Haeckel’s interpretation of the ascidian embryology within the frame of a monophyletic tree of life that was produced by the fundamental biogenetic law. Joining embryology to evolution in the light of idealistic morphology, the biogenetic law turned out to be instrumental in bringing forth different evolutionary hypotheses: it was used by Haeckel and Darwin to link vertebrates to invertebrates by means of the concept of chordates, and by Kowalevsky to corroborate the annelid theory of the origin of vertebrates. Yet, there was still another interpretation of Kowalevsky’s discoveries. As an adherent to empiricism and to Cuvier’s theory of types, von Baer asserted that these discoveries did not prove convincingly a dorsal position of the nervous system in the ascidian tadpole larva; hence, they could not support a homology between different animal types suggesting a kinship between ascidians and vertebrates.     

Windows of the brain: towards a developmental biology of circumventricular and other neurohemal organs

We review the anatomical and functional features of circumventricular organs in vertebrates and their homologous neurohemal organs in invertebrates. Focusing on cyclostomes (lamprey) and urochordates (ascidians), we discuss the evolutionary origin of these organs as a function of their cell type specification and morphogenesis.     

Molecular phylogeny and divergence times of deuterostome animals

The phylogenetic relationships among deuterostome animals have been debated for many years, and a diversity of hypotheses have been proposed based on both morphological and molecular data. Here we have assembled sequences of 217 nuclear-encoded proteins to address specific questions concerning their relationships and times of origin. We recovered significant support for urochordates as the closest relative of vertebrates with an analysis of 59 proteins (17,400 amino acids) and suggest that the basal position of urochordates found in previous molecular studies may have been the result of long-branch attraction biases. Our results also support Ambulacraria, the pairing of hemichordates with echinoderms (nine proteins; 2,382 amino acids), and Cyclostomata, the pairing of lampreys with hagfish (25 proteins; 6,895 amino acids). In addition, 325 shared proteins (102,110 amino acids) were obtained from the complete genomes of six vertebrates and a urochordate for phylogenetic analysis and divergence time estimation. An evolutionary timescale was estimated using a local (Bayesian) molecular clock method. We found that most major lineages of deuterostomes arose prior to the Cambrian Explosion of fossils (approximately 520 MYA) and that several lineages had originated before periods of global glaciation in the Precambrian.     

Developmental genetics in primitive chordates

Recent advances in the study of the genetics and genomics of urochordates testify to a renewed interest in this chordate subphylum, believed to be the most primitive extant chordate relatives of the vertebrates. In addition to their primitive nature, many features of their reproduction and early development make the urochordates ideal model chordates for developmental genetics. Many urochordates spawn large numbers of transparent and externally developing embryos on a daily basis. Additionally, the embryos have a defined and well-characterized cell lineage until the end of gastrulation. Furthermore, the genomes of the urochordates have been estimated to be only 5-10% of the size of the vertebrates and to have fewer genes and less genetic redundancy than vertebrates. Genetic screens, which are powerful tools for investigating developmental mechanisms, have recently become feasible due to new culturing techniques in ascidians. Because hermaphrodite ascidians are able to self-fertilize, recessive mutations can be detected in a single generation. Several recent studies have demonstrated the feasibility of applying modern genetic techniques to the study of ascidian biology.     

A novel amphioxus cadherin that localizes to epithelial adherens junctions has an unusual domain organization with implications for chordate phylogeny

Although data are available from only vertebrates, urochordates, and three nonchordate animals, there are definite differences in the structures of classic cadherins between vertebrates plus urochordates and nonchordates. In this study we examined structural diversity of classic cadherins among bilaterian animals by obtaining new data from an amphioxus (Cephalochordata, Chordata), an acorn worm (Hemichordata), a sea star (Echinodermata), and an oyster (Mollusca). The structures of newly identified nonchordate cadherins are grouped together with those of the known sea urchin and Drosophila cadherins, whereas the structure of an amphioxus (Branchiostoma belcheri) cadherin, designated BbC, is differently categorized from those of other known chordate cadherins. BbC is identified as a cadherin by its cytoplasmic domain whose sequence is highly related to the cytoplasmic sequences of all known classic cadherins, but it lacks all of the five repeats constituting the extracellular homophilic-binding domain of other chordate cadherins. The ectodomains of BbC match the ectodomains found in nonchordate cadherins but not present in other chordate cadherins. We show that the BbC functions as a cell-cell adhesion molecule when expressed in Drosophila S2 cells and localizes to adherens junctions in the ectodermal epithelia in amphioxus embryos. We argue that BbC is the amphioxus homologue of the classic cadherins involved in the formation of epithelial adherens junctions. The structural relationships of the cadherin molecules allow us to propose a possibility that cephalochordates might be basal to the sister-groups vertebrates and urochordates.     

Characterization of microRNAs in cephalochordates reveals a correlation between microRNA repertoire homology and morphological similarity in chordate evolution

SUMMARY Cephalochordates, urochordates, and vertebrates comprise the three extant groups of chordates. Although higher morphological and developmental similarity exists between cephalochordates and vertebrates, molecular phylogeny studies have instead suggested that the morphologically simplified urochordates are the closest relatives to vertebrates. MicroRNAs (miRNAs) are regarded as the major factors driving the increase of morphological complexity in early vertebrate evolution, and are extensively characterized in vertebrates and in a few species of urochordates. However, the comprehensive set of miRNAs in the basal chordates, namely the cephalochordates, remains undetermined. Through extensive sequencing of a small RNA library and genomic homology searches, we characterized 100 miRNAs from the cephalochordate amphioxus, Branchiostoma japonicum , and B. floridae . Analysis of the evolutionary history of the cephalochordate miRNAs showed that cephalochordates possess 54 miRNA families homologous to those of vertebrates, which is threefold higher than those shared between urochordates and vertebrates. The miRNA contents demonstrated a clear correlation between the extent of miRNA overlapping and morphological similarity among the three chordate groups, providing a strong evidence of miRNAs being the major genetic factors driving morphological complexity in early chordate evolution.     

Metaphylogeny of 82 gene families sheds a new light on chordate evolution

Achieving a better comprehension of the evolution of species has always been an important matter for evolutionary biologists. The deuterostome phylogeny has been described for many years, and three phyla are distinguishable: Echinodermata (including sea stars, sea urchins, etc...), Hemichordata (including acorn worms and pterobranchs), and Chordata (including urochordates, cephalochordates and extant vertebrates). Inside the Chordata phylum, the position of vertebrate species is quite unanimously accepted. Nonetheless, the position of urochordates in regard with vertebrates is still the subject of debate, and has even been suggested by some authors to be a separate phylum from cephalochordates and vertebrates. It was also the case for agnathans species -myxines and hagfish- for which phylogenetic evidence was recently given for a controversial monophyly. This raises the following question: which one of the cephalochordata or urochordata is the sister group of vertebrates and what are their relationships? In the present work, we analyzed 82 protein families presenting homologs between urochordata and other deuterostomes and focused on two points: 1) testing accurately the position of urochordata and cephalochordata phyla in regard with vertebrates as well as chordates monophyly, 2) performing an estimation of the rate of gene loss in the Ciona intestinalis genome. We showed that the urochordate phyla is the vertebrate sister group and that gene loss played a major role in structuring the urochordate genome.     

Phylogenies of Developmentally Important Proteins Do Not Support the Hypothesis of Two Rounds of Genome Duplication Early in Vertebrate History

It has been proposed that two rounds of duplication of the entire genome (polyploidization) occurred early in vertebrate history (the 2R hypothesis); and the observation that certain gene families important in regulating development have four members in vertebrates, as opposed to one in Drosophila, has been adduced as evidence in support of this hypothesis. However, such a pattern of relationship can be taken as support of the 2R hypothesis only if (1) the four vertebrate genes can be shown to have diverged after the origin of vertebrates, and (2) the phylogeny of the four vertebrate genes (A–D) exhibits a topology of the form (AB) (CD), rather than (A) (BCD). In order to test the 2R hypothesis, I constructed phylogenies for nine protein families important in development. Only one showed a topology of the form (AB) (CD), and that received weak statistical support. In contrast, four phylogenies showed topologies of the form (A) (BCD) with statistically significant support. Furthermore, in two cases there was significant support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes: in one case there was significant support for duplication of the vertebrate genes at least prior to the divergence of vertebrates and urochordates, and in one case there was weak support for duplication of the vertebrate genes prior to the divergence of deuterostomes and protostomes. Taken together with other recently published phylogenies of developmentally important genes, these results provide strong evidence against the 2R hypothesis. Received: 22 December 1997 / Accepted: 5 October 1998     

Retinoic acid signaling and the evolution of chordates

In chordates, which comprise urochordates, cephalochordates and vertebrates, the vitamin A-derived morphogen retinoic acid (RA) has a pivotal role during development. Altering levels of endogenous RA signaling during early embryology leads to severe malformations, mainly due to incorrect positional codes specifying the embryonic anteroposterior body axis. In this review, we present our current understanding of the RA signaling pathway and its roles during chordate development. In particular, we focus on the conserved roles of RA and its downstream mediators, the Hox genes, in conveying positional patterning information to different embryonic tissues, such as the endoderm and the central nervous system. We find that some of the control mechanisms governing RA-mediated patterning are well conserved between vertebrates and invertebrate chordates, such as the cephalochordate amphioxus. In contrast, outside the chordates, evidence for roles of RA signaling is scarce and the evolutionary origin of the RA pathway itself thus remains elusive. In sum, to fully understand the evolutionary history of the RA pathway, future research should focus on identification and study of components of the RA signaling cascade in non-chordate deuterostomes (such as hemichordates and echinoderms) and other invertebrates, such as insects, mollusks and cnidarians.     

Kowalevsky, comparative evolutionary embryology, and the intellectual lineage of evo-devo

Alexander Kowalevsky was one of the most significant 19th century biologists working at the intersection of evolution and embryology. The reinstatement of the Alexander Kowalevsky Medal by the St. Petersburg Society of Naturalists for outstanding contributions to understanding evolutionary relationships in the animal kingdom, evolutionary developmental biology, and comparative zoology is timely now that Evo-devo has emerged as a major research discipline in contemporary biology. Consideration of the intellectual lineage of comparative evolutionary embryology explicitly forces a reconsideration of some current conceptions of the modern emergence of Evo-devo, which has tended to exist in the shadow of experimental embryology throughout the 20th century, especially with respect to the recent success of developmental biology and developmental genetics. In particular we advocate a sharper distinction between the heritage of problems and the heritage of tools for contemporary Evo-devo. We provide brief overviews of the work of N. J. Berrill and D. T. Anderson to illustrate comparative evolutionary embryology in the 20th century, which provides an appropriate contextualization for a conceptual review of our research on the sea urchin genus Heliocidaris over the past two decades. We conclude that keeping research questions rather than experimental capabilities at the forefront of Evo-devo may be an antidote to any repeat of the stagnation experienced by the first group of evolutionary developmental biologists over one hundred years ago and acknowledges Kowalevsky's legacy in evolutionary embryology.     

Mark Q. Martindale: shedding new light on developmental diversity

The Saint-Petersburg Society of Naturalists awarded the 2009 "Alexander Kowalevsky Medal" to Mark Q. Martindale, Professor of Organismal Biology at the University of Hawaii and Director of the Kewalo Marine Laboratory, Honolulu. This international award inaugurated first in 1910 was re-established only in 2001. In memory of Alexander Onufrievich Kowalevsky, it is awarded to outstanding zoologists and embryologists who have made great contributions to the field of embryology and developmental biology from an evolutionary perspective. Mark Q. Martindale has worked on a wide range of animals, mostly marine species, in contrast to many evo-devo researchers who often use a single "well-established" model organism. His work demonstrates how the insights gained by studying less "popular" animal taxa not only complement, but also significantly enrich our knowledge of the evolution of metazoan body plans and of the events that have led to the current animal diversity.     

The caspase family in urochordates: distinct evolutionary fates in ascidians and larvaceans

BACKGROUND INFORMATION: Caspases are cysteine proteases that mediate apoptosis (programmed cell death) initiation and execution. Apoptosis is a conserved mechanism shared by all metazoans, although its physiological function and complexity show considerable taxon-dependent variations. To gain insight into the caspase repertoire of putative ancestors to vertebrates, we performed exhaustive genomic searches in urochordates, a sister taxon to vertebrates in which ascidians and appendicularians display chordate characters at early stages of their development. RESULTS: We identified the complete caspase families of two ascidians (Ciona intestinalis and C. savignyi) and one larvacean (Oikopleura dioica). We found in ascidian species an extremely high number of caspase genes (17 for C. intestinalis and 22 for C. savignyi), deriving from five founder gene orthologues to human pro-inflammatory, initiator and executioner caspases. Although considered to be sibling species, C. intestinalis and C. savignyi only share 11 orthologues, most of the additional genes resulting from recent mass duplications. A sharply contrasted picture was found in O. dioica, which displayed only three caspase genes deriving from a single founder gene distantly related to caspase 3/7. The difference between ascidian and larvacean caspase repertoires is discussed in the light of their developmental patterns and life cycles. CONCLUSIONS: The identification of caspase members in two ascidian species delineates five founder genes that bridge the gap between vertebrates and Ecdysozoa (arthropods and nematodes). Given the amazing diversity among urochordates, determination and comparison of the caspase repertoires in species from orders additional to Enterogona (ascidians) and Oikopleuridae might be highly informative on the evolution of caspase-dependent physiological processes.     

Organizing chordates with an organizer

Understanding how the chordate body plan originated and evolved is still controversial. The discovery by Spemann and Mangold in 1924 of the vertebrate organizer and its inductive properties in patterning the AP and DV axis was followed by a long gap until the 1960s when scientists started characterizing the molecular events responsible for such inductions. However, the evolutionary origin of the organizer itself remained obscure until very recently; did it appear together with the origin and radiation of vertebrates, or was it a chordate affair? A recent study by Yu and collaborators, 1 which analyses the expression of several organizer-specific genes in amphioxus together with recent phylogenetic data that reversed the position of invertebrate extant chordates (e.g. urochordates and cephalochordates), indicates that the organizer probably appeared in early chordates. It likely had separate signalling centres generating BMP and Wnt signalling gradients along the DV and AP axis. The organizer was then lost in the urochordate lineage, most probably as an adaptation to a rapid and determinate development. BioEssays 29:619–624, 2007. © 2007 Wiley Periodicals, Inc.     

Evolutionary embryology resurrected in Japan with a new molecular basis: Nori Satoh and the history of ascidian studies originating in Kyoto during the 20th century

This article briefly summarizes the scientific contributions of Nori Satoh, the winner of the 2005 edition of the Kowalevsky Medal, to Developmental Biology and especially to Evo-Devo with his 30 years of research on tunicates - a primitive chordate species. His research began with his pure developmental interest in the clock mechanism of cell differentiation and later expanded into various aspects of evolutionary and developmental phenomena. He is not only known as a founder of molecular biology-based tunicate studies, but also for his world-wide service to education and his prestigious publications in international scientific journals.     

Evolution of developmental roles of Pax2/5/8 paralogs after independent duplication in urochordate and vertebrate lineages

Background

Gene duplication provides opportunities for lineage diversification and evolution of developmental novelties. Duplicated genes generally either disappear by accumulation of mutations (nonfunctionalization), or are preserved either by the origin of positively selected functions in one or both duplicates (neofunctionalization), or by the partitioning of original gene subfunctions between the duplicates (subfunctionalization). The Pax2/5/8 family of important developmental regulators has undergone parallel expansion among chordate groups. After the divergence of urochordate and vertebrate lineages, two rounds of independent gene duplications resulted in the Pax2, Pax5, and Pax8 genes of most vertebrates (the sister group of the urochordates), and an additional duplication provided the pax2a and pax2b duplicates in teleost fish. Separate from the vertebrate genome expansions, a duplication also created two Pax2/5/8 genes in the common ancestor of ascidian and larvacean urochordates.

Results

To better understand mechanisms underlying the evolution of duplicated genes, we investigated, in the larvacean urochordate Oikopleura dioica, the embryonic gene expression patterns of Pax2/5/8 paralogs. We compared the larvacean and ascidian expression patterns to infer modular subfunctions present in the single pre-duplication Pax2/5/8 gene of stem urochordates, and we compared vertebrate and urochordate expression to infer the suite of Pax2/5/8 gene subfunctions in the common ancestor of olfactores (vertebrates + urochordates). Expression pattern differences of larvacean and ascidian Pax2/5/8 orthologs in the endostyle, pharynx and hindgut suggest that some ancestral gene functions have been partitioned differently to the duplicates in the two urochordate lineages. Novel expression in the larvacean heart may have resulted from the neofunctionalization of a Pax2/5/8 gene in the urochordates. Expression of larvacean Pax2/5/8 in the endostyle, in sites of epithelial remodeling, and in sensory tissues evokes like functions of Pax2, Pax5 and Pax8 in vertebrate embryos, and may indicate ancient origins for these functions in the chordate common ancestor.

Conclusion

Comparative analysis of expression patterns of chordate Pax2/5/8 duplicates, rooted on the single-copy Pax2/5/8 gene of amphioxus, whose lineage diverged basally among chordates, provides new insights into the evolution and development of the heart, thyroid, pharynx, stomodeum and placodes in chordates; supports the controversial conclusion that the atrial siphon of ascidians and the otic placode in vertebrates are homologous; and backs the notion that Pax2/5/8 functioned in ancestral chordates to engineer epithelial fusions and perforations, including gill slit openings.     

The origin of vertebrates: a hypothesis based on kidney development

The habitat of the earliest vertebrates (craniates) is still being debated. Marine as well as freshwater habitats and anadromous behaviour have been proposed. In contrast, an estuarine origin of vertebrates is suggested here, based on ontogenetic, comparative anatomical and functional data. This approach should resolve inconsistencies between the probable existence of glomeruli in the vertebrate ancestors and the marine habitat of all related extant groups (e.g. urochordates and cephalochordates). The kidney, as the main osmoregulatory organ, must have been developed according to the environmental prerequisites even in stem vertebrates. In the absence of fossil evidence only deductions from contemporary animals are possible. These data indicate that ancestral stem vertebrates probably had well-developed glomeruli, and were capable of at least some ion-exchange between urine and the body. However, they were probably unable to cope with a strong osmotic gradient with respect to their environment. The conclusion is that these animals were osmoconformers at around 300–350 mOsm and therefore were restricted to brackish water.  © 2007 The Linnean Society of London, Zoological Journal of the Linnean Society , 2007, 150 , 435–441.