Developmental control genes and morphological evolution
Symposium organized by Michael Akam, Thomas R. Bürglin and Walter Gehring, from September 21 to 27, 1996
André Adoutte, Michael E. Akam, Michalis Averof, Jacques Bièrne, Edoardo Boncinelli, Antonius J. M. Bouwmeester, Sydney Brenner, Denis Duboule, Walter J. Gehring, Peter Gruss, Herbert Jackle, Fotis C. Kafatos, Ginès Morata, Nipam Patel, Heinrich Reichert, Еmili Saló, Rosaria Santis (de), Antonio Simeone
The aim of this meeting was to bring developmental biologists working on different organisms together to discuss the implications of their research with respect to evolution. In the wonderful ambience of Les Treilles a diverse group of biologists gathered to discuss organisms as different as mice and flies, flatworms and seasquirts. Despite this range, we found ourselves talking a common language — and often working with the same genes. Many of the genes that control development are now known to be conserved in the most diverse animal phyla. By comparing the expression and fonction of these genes, we can compare the basic patterns of animal development and start to guess how evolution has tinkered with these patterns at the molecular level, to generate the diversity of animal life.
Perhaps the best known and best studied of these developmental control genes are the homeobox genes, which encode a DNA-binding domain — the homeodomain. Of the many dif ferent classes of homeobox genes one group of particular inter-est are the Нох genes, which are organised in clusters on the chromosome. Figure 1 shows a simplified scheme of the Drosophile aid a vertebrate « consensus » Нох cluster, a composite of the four clusters HoxA, НохB, HoxC and HoxD.
Phylogeny of animals
André Adoutte started off the meeting with an overview of the general phylogeny of Metazoa, i.e. the multicellular animals. Using large 18 secondes rRNA datasets and refined computer algorithms he presented phylogenetic analyses to determine the evolutionary origins of the different animal phyla. Some of his results contrast with current phylogenetic models, winch are often based only on morphological data, or limited datasets. Nevertheless, if cladistic branches are very deep and occur within very short time intervals, even very large amounts of ‘ data will not help to resolve precise branching orders. The salient points from his analysis are paraphrased from his abstract : the Metazoa, including the sponges, are monophyletic (single common ancestor), and the fungi are their most probable sister group (as opposed to the plants). There is a deep split between diploblastic (two germ layers) and triploblastic (three germ layers) organisms. Within the triploblasts, most protostomes fall into one clade and all deuterostomes into another. The protostome clade itself is deeply split, with the monophyletic arthropods on one side, and a poorly resolved ensemble of several other phyla on the other side (i.e. Annelida, Mollusca, Sipuncula, Nemertini, Brachipoda, Bryozoa, etc.). Halanych and coworkers had previously proposed the name Lophotrochozoa for this ensemble of phyla, as most of them display spiral cleavage and/or a Trophophore larva. Several of the pseudocoelomates are clustered within the deuterostome or protostome clade, and not placed as an outgroup of coelomates.
A special focus was placed on the position of the platyhelminths (flatworms). The recent 18 secondes rRNA data placed them within the protostome clade. Even though the cladistic branch is very deep, support for this notion comes from the analysis of a particular subset of homeobox genes, the Нох cluster genes. The Нох genes of platyhelminths are remarkably complex, the number and types of genes being similar to those observed in arthropods. In particular, two genes in the Нох cluster, Ultrabithorax (Ubx) and abdominal-A (abd-A), seem to have corresponding genes in platyhelrninths. André Adoutte proposed to call this group the Ubd-A genes. Lively discussion ensued as to the significance of this, as vertebrates seem to lack some of the features of the Ubd-A genes. Nevertheless, those Нох genes in the Ubd-A equivalent pos!-ion in the vertebrate clusters must have arisen frmrn the some common ancestor. Doubtlessly ail participants agree that more data is needed to resolve rnany of the outstanding phylogenetic questions, although some might never be resolved with absolute certainty.
Invertebrates (excluding arthropods)
One such phylogenetic question that may lever be resolved with absolute certainty is the position of nematodes. No fossils exist, and molecular analysis is hampered by their very high rate of sequence evolution This is particularly unfortunate, as our knowledge of C. elegans is so extensive, as Thomas Bürglin made clear. Не reported that all major classes of homeobox genes have been found, although they are usually more divergent when compared to arthropods and vertebrates. Even members of the Bar, sine oculis, rough and Pax-6 homeobox gene classes, which are involved in eye development, are found in this apparently eyeless nematode. On the other hand, the C. elegans Нох cluster has fewer homeobox genes than the Нох clusters of other animais in the protostome and deuterostore branches. These data seem to support the notion that the nematode phylum branched off before the later main split of protostomes and deuterostomes. Despite this long evolutionary distance, the fonctions of the homeobox genes show many conserved features. The POU homeobox gene ceh-6 is expressed and fonctions in a series of neurons in the brain, and in neuroblasts in the ventral nerve cord. Further expression is seen in the excretory сell in the back of the brain and epidermal cells in the vulva and rectum. These expression patterns are reminiscent of vertebrate and fly expression patterns of the orthologous genes, which show expression in varions tissues in the brain, the spinal cord/ventral nerve cord, and epidermal tissues, such as filzkorper and tracheal cells in flies.
The PBC-class homeobox gene ceh–20 is an orthologue of the Drosophila gene extrа–deпticle (exd). It is expressed in the body of the animal, overlapping the expression domains of the Нох cluster genes, again reminisсent of the situation in flies. The homeobox gene ceh–2 is an orthologue of Drosophila emр-spiracles (ems). It is expressed in the anterior of the pharynx. ems plays a role in head development in flies suggesting that the anterior region of the pharynx, where ceh–2 is expressed, might actually be highlу derived head structures. Support for this notion stems from the fast that the anterior and posterior regions of the pharynx develop from сell lineages that diverge already at the 4 сell stage.
Emili Saló from Catalunya returned to the question of Нох genes in platyhelminths. He and his group had isolated seven Нохgenes from the freshwater planarian Dugesia tigrina. These genes correspond to the anterior and central Нох cluster genes (Нох-1 to Нох 6/7/8). An interesting feature of freshwater planarians is their enormous capacity to regenerate. Hence, the expression pattern of the Нох genes was studied during regeneration. In normal wild-type animals, no expression of Нох genes could be detected, although a low level of expression must exist, since cDNAs for Нох genes were isolated from such animais. A generalised feature of the Нох cluster genes in (lies and vertebrates is that the genes are arranged on the chromosome in the same order as they are expressed along the anterior-posterior body axis of the animals. During regeneration, string expression is seen in the region of the healing wound, the blastema. however, the colinearity rule appears now violated, since the genes are all activated in the regenerating zone, irrespective whether the cut was made in the anterior or the posterior of an animal. As the wound healing proceeds the genes are down-regulated again. Even though the genes turn off at different rates, the order does not correlate well with their presumed order in the cluster. When lateral cuts are made, again no differenсe can be seen along the body axis during regeneration; gene activation and subsequent deactivation appears synchronous along the axis. This poses an intriguing puzzlе • how is the identity of the various body regions specified ? Emili Saló plans to examine the expression of these genes in a planarian species that shows normal spiralian development, unlike Dugesia. Further, in collaboration with Edoardo Boncinelli, an orthodenticle (otd) homologue was cloned from Dugesia. When the expression of this gene is examined in regenerating Worms, a very clear result is obtained. Dugesia otd is active in regenerating blastemas only in the anterior region, but not in the posterior region. Thus, other homeobox genes will need to be examined for a better understanding of planarian body patterning.
Jacques Bièrne introduced the participants of the meeting to his favourite animals — the Nemertean Worms of the genus Lineus. It seems likely that this group of unsegmented protostomes will see renewed interest from molecular biologists, as it appears to be a group that retains many primitive characteristics in the structure of its Нох genes. Reporting a fruitful collaboration with Walter J. Gehring’s laboratory, Jacques Bièrne presented sequence data for Нох genes of at least six paralogy groups. Many sequences resembled those of vertebrates more closely than Drosophila, prompting Jacques Bièrne to describe Nemerteans as the closest living relative to the common ancеstor of flies and man — a view strongly disputed by André Adoutte, whose earlier presentation placed them firmlу among the prototrochozoa. Nonetheless, we could only be impressed by the versatihty of these creatures, whose powers of regeneration seem to be limitless.
Rosaria De Santis introduced another invertebrate, the ascidian Ciona intestinales. Ascidians belong to the Urochordates, i.e., they are relatives of the vertebrates, and during larval stages they display characteristic chordate features. In collaboration with Roberto di Lauro’s laboratory, almost 30 homeobox genes were isolated, belonging to different classes such as Dll, Msh, Nk, and Hox cluster genes. Starting with 7 Нох cluster genes thеу have initiated a chromosomal walk to determine the complete structure of the Нох cluster. To date, 9 Нох genes have been identified and linkage between several of them demonstrated. However, the cluster appears to be fairly large. By current estimates it must be bigger than 450 kilobases. In situ hybridisation of the 10×3 aid 10х5 genes shows expression along the anterior-posterior body axis of the larvae as expected. A homeobox gene of special interest is the orthologue of the NK2 class gene TTF 1. This gene is of particular importance for the development of the thyroid in higher vertebrates. It has been proposed that in ascidians, a special organ in the head, the endostyle, is the evolutionary « precursor » of the thyroid. Indeed, in situ hybridisation with the TTF–1 orthologue shows expression in the endostyle, supporting the phylogenetic link.
A second aspect of ascidian development studied in particular by Rosaria De Santis is the problem of self- nonself discrimination. Ciona intestinalis is a self sterile hermaphrodite; the vitelline envelope contains a barrier preventing fertilisation with sperm from the same animal. This system could be a simple model and an evolutionary precursor to the highly complex discrimination system in the vertebrate immune system. The MHC locus contains heat shock protein genes (HSP70), and it is thought that НSP70 might be the ancestral gene. Rosaria De Santis thus isolated an НSP70 gene from Ciona, and preliminary experiments with antibodies against НSP70 suggest that thеу can block sperm entry.
Michael Akam focused on the Нох family of homeobox genes in the Arthropods. Early genetic models proposed that the diversification of these genes occurred in tandem with the diversification of body plans. Although this model cannot yet be completely ruled out, comparisons of Нох cluster соmрlеxity in a range of arthrоpods suggest that the diversity of arthropod body plans has been achieved with a conserved set of Нох genes, by altering their regulation.
In contrast to this overall conservation, rapid evolutionary changes have affected some genes within the insеct Нох clusters. Comparisons of genes from grasshoppers, beetles and files show that at least two Нох cluster genes are diverging relatively rapidlу, both in sequence and probably also in function. These genes have given rise to the zerknüllt (zen) and fushi-tarazu genes of the Drosophila Antennapedia cornplex — genes that are involved in patterning the early syncytial stages of the Drosophila embryo. Michael Akam showed that in grasshoppers, no equivalent syncytial stage еxists — cellularisation is complete before the embryoniс primordium has coalesced. He proposed that changes in early developmental mechanisms mау have allowed the rapid diversification of these « runaway » Нох genes.
Nipam Patel returned to the relationship between Нох gene regulation and the diversification of segments. The domains of expression of particular Нох genes appear to be quite similar in all insects, but this is not so for the Crustacea, which exhibit much more diverse patterns of segment specialisation. Reporting experiments carried out in collaboration with Michalis Averof, Nipam Patel showed that Нох genes of the Ubx/abd-A c!ass are expressed throughout the thorax in those Crustacea where all the thoracic segments are similar. They infer that this pattern is primitive within the Crustacea. However, in manу crustaceans, one or more of the thoracic appendages have become maxillipeds — modified appendages specialised for feeding, with a morphology somewhat intermediate between that of a thoracic and a gnathal appendage. In these animals, expression of the Ubx/abd-A lix genes is excluded from the maxilliреds. In Malacostracan crustaceans, which may have 0, 1 or 2 maxillipeds, there is an excellent correlation between the extent of Ubx/abdLA expression, and the number of maxillipeds formed. This is the clearest example yet of shifts in Нох gene regulation leading to morphological diversity.
Comparison of crustacean and insect limbs provided another example where molecular markers can help to define the relationships between different morphologies. Crustaceans have branched limbs, whereas most insects have unbranched « uniramous » legs. It has been suggested that the branching originates from an antero-posterior fusion of two primitively uniramous limbs, but by using the Engrailed protein as a molecular marker, Nipam Patel showed that each branch of the crustacean limb has anterior aid posterior domains, with branching in the dorso-ventral plane. This makes it unlikеlу that branched limbs arise by the secondary fusion of uniramous limbs.
The branchiopod crustacean Triops provides another fIce example of diversity in developmental mechanisms. In this animal, segment patterning in the dorsal aid ventral halves of the animal begins in register, but in the posterior part of the animal becomes disjunct, such that a single dorsal segment mау span three or more ventral segments, each with its own stripe of engrailed expression.
The origin of novel morphological structures poses particular problems for the evolutionary biologist. One classic case is the insect wing — a structure that appears to arise from nowhere during the evolution of the insects. Two competing hypotheses have been debated for many years — that the wing derives from an extension of the dorsal body wall, or that it is basal branch of a crustacean limb, the base of which has been incorporated into the body wall. Miсhalis Averof reported an attempt to distinguish between these hypotheses by analysing in Crustacea the expression of two genes that are сharactеristiс of wing development in Drosophila — nubbin and apterous. Both proved to be expressed in a gill branch of the Arteria leg, strongly supporting the leg origin hypothesis. This observation prompted lively debate as to whether molecules dеfinеd the homology of structures, or whether structures could be homologous even if the developmental mechanisms that generate them are quite different.
Ginès Morata confessed himself to be a new convert to evolutionary thinking — but his questions were all the more challenging for that. He described how his studies of limb development in Drosophila provided support for an old morphological hypothesis. His focus was the role of the homeodomain protein extradenticle, one of the few defined cofactors of the Hox proteins. In the trunk, extradenticle allows the very similar Hox proteins Ubx and abd-A to specify different segment morphologies. However, it is not expressed in the distal parts of the appendages. Ginès Morata described how, when he engineered the expression of extradenticle expression throughout the developing leg, distal regions fail to develop. He argued that the leg is built of two domains — proximal leg structures which require the regulatory genes extradenticle and teashirt, and distal leg structures which require a different key regulatory gene,
Distalless (Dll). The boundary between these two domains provides a molecular definition for the distinction between coxopodite and epipodite, domalns proposed by Snodgrass in 1935 on the basis of comparative morphology. Interestingly, in the distal limbs, ectopic expression of any one of several Нох genes leads to the same morphological transformation, presumably because in the absence of extradenticle protein, the cells cannot distinguish between them.
Heinrich Reichert chose a more complex problem for his agenda — to resolve the organisation of the brain in Drosophile, perhaps with the hope of relating this to the neural architecture of vertebrates. Using a suite of homeobox genes as molecular markers, and confocal microscopy to section the brain, he was able to relate the structure of normal and mutant brains, in particular Ems and orthodenticle (otd) mutants, to a segmental groundplan, even in the most anterior regions. Here again, the participants found themselves arguing about the relationship between molecular markers and morphology — do stripes of engrailed positive cells in the brain imply segments, or just lineages of neurons ?
Walter J. Gehring first presented his model for how the Нох cluster might have evolved through unequal crossing-over. It might explain why the central Нох genes have been most conserved in evolution (98 % identical in the homeodomain) . Then Walter J. Gehring turned to eye development. The Drosophila eyeless (ey) mutation has been shown to correspond to the paired-ckiss homeobox gene Рax-6. In vertebrates this gene has also been shown to be involved in eye development (see talk by Peter Gruss) . То test if Рах-6 is not only required for eye formation in flies, but also sufficient, Рax-6 was expressed ectopically in other tissues. Strikingly, eyes could be induced on wings, legs and antenna. While hardly anybody had believed this to be possible, Walter J. Gehring remembered his old experiments : in long-term culture wing imaginal discs could spontaneously generate eye structures, suggesting that a minimal change can alter the fate drastically. The ectopic eyes are fully functional, although they do not connect to the brain. Further, ectopic eyes can be induced with the vertebrate Рax-6 gene, showing the functional conservation. Cloning and expression analysis of Рах-6 homologues from Lineas and Dugesia (in collaboration with Emili Saló) revealed that also in these organisme Рах-6 is expressed in the primitive eye spots. Thus Рaх-6 can be regarded as the master control gene for eye development. Evidence was found that a possible target for Рax-6 is another gene important for eye development, sine oculis (s0). Since highly conserved orthologues exist in vertebrates, this would indicate also conservation of whole genetic circuits.
In collaboration with Meinrad Busslinger, a second gene with high similarity to ey was identified, twin–of–eyeless (toy): toy is expressed significantly earlier than ey in the presumptive eye/brain region in the blastoderm stage. toy can also induce ectopic eyes, suggesting 2150 a role in eye development. It appears that toy first induces ey, which turns on a cascade of genes leading to eye development.
Herbert Jackle’s talk provided a striking example of how studies carried out without any reference to evolution сan have profound effects on our thinking about evolutionary processes. Не described studies of the segmentation gene hierarchy in Drosophila, focusing on the dissection of regulatory elements that interpret positional information in the bastoderm syncytium. Individual enhancer elements are microcomputers of remarkable complexity — one 900 base regulatory element in the hairy gene drives a single stripe of expression, yet contains more than sixty binding sites for a total of at least seven different transcription factors. Herbert Jackle pointed out that activation of a gene never occurs in response to a single factor binding at a single site. With this complexity, the possibilities for evolutionary tinkering to modify the regulation of gene activity are endless.
Echoing these sentiments, Fotis C. Kafatos explored the reasons for the evolutionary flexibility of zinc finger proteilns, which have been exploited by the metazoa to form a huge family of gene regulators. Hе pointed out that the zinc finger proteins are modular, with a single finger binding to a triplet nucleotide motif. DNA recognition by each finger is permissive, allowing a population of sequences to be recognised by each protein. At a single gene, individual targets for recognition come and go as base substitution generates new matches and destroys old oves. Functional sites are not necessarily homologous (related by descent), but analogous. By comparing two closely related zinc finger genes in Drosophila, spalt and spaltrelated, he showed how duplicated genes may have overlapping but not identical functions — a common characteristic that perhaps allows both to be retained by selection.
Antonius J.-M. Bouwmeester presented the latest data from the laboratory of Eddy De Robertis. The main research focus lies on inductive events in the early development of the &og Xenopus laevis. One of the signals responsible for the inductive events is ВМР4, a relative of the Drosophila factor decaрentaрlegic (dpp). A new molecule involved in this signalling pathway was isolated by ‘the De Robertis laboratory, chordin. Chordin is also a secreted molecule and it was demonstrated that it interacts physically with ВМР4. A second factor, noggin, has an even higher affinity to ВМР4. Thus, both these factors can be viewed as negative regulators of ВМР4. Chordin is related to the Drosophila gene short-gastrulation (sog). If sog is injected into Xenopus embryos, double axis are induced as if chordin had been injected, suggesting functional homology. In Xenopus embryos, chordin is expressed in the neurogenic ectoderm in the dorsal side, while ВМР4 is secreted from the ventral area ; in flies sog is expressed in the ventral region, while dpp is expressed in the dorsal region. This « inversion » of developmental control genes lends support to the old hypothesis that deuterostomes arise from protostomes by simply « swimming on their back ».
Many other secreted factors play important roles during these early developmental stages. In a differential screen, a novel molecule was identified, cerberus. It is a secreted molecule, winch is expressed in а very narrow window during gastrulation. Expression analysis and injection experiments suggest that cerberus plays a role in the anterior endoderm. A second gene identified in the screen was frezzled, a gene involved in the wnt signalling pathway. Coinjection experiments demonstrated that Frezzled most like interacts directly with Wnt8.
Peter Gruss reported on inductive events in the mouse embryo that occur at a later stage during the development of the visual system. Recent evidence suggests that lens formation is a three step process : a) competence, an autonomous process within the ectoderm, b) bias, provided by the anterior plate, and c) final determination, induced by the optic vesicle. One key gene involved in this process is Pax6, the orthologue of the Drosophiles eyеless gene reported on by Walter J. Gehring. This gene is required not only for eye formation but also for the development of the pose and forebrain structures, as well as ventral structures. Mutations in mouse Рax6cause the Small eye (Sey) phenotype, however fragments of protein are still present in these mutations. Large deletions of Sey cause death at Implantation. Is Рax–6 involved in the signaling ? In knock-out mice, the optic vesicle still forms, thus Рax-6 is not required for its initial formation. However, the subsequent inductive events, i.e. lens placode and lens retina formation depend on Рcх–6.
Another « fly gene » involved in eye development is sine осиlis. Several vertebrate orthologues were isolated, and one of these genes, Six3, appears to be the funcional homologue of s0. Six3 is expressed early in the anterior neural plate and later in the region of the optic recess, the optic vesicles and the hypothalamus. In an experiment similar to the ectopic expression of eyеless in flies, Six 3 was ectopically expressed under a widely acting promoter. A striking transformation results : a lens is formed in or near the optic vesicle. This demonstrates that Six3 is also a key player in the development of the vertebrate еуe.
Peter Gruss proceeded to present evidence for a role of the gene Рах2, a paired domain gene, in eye development. Initial Рах2 expression is confined to the ventral optic vesicle and is later found in regions that contribute to the optic nerve and optic chiasm. In Рах2 mutant mice defects are seen in axonal outgrowth. The optic tracts remain ipsilateral in mutant brains, since the optic chiasm fails to form properly. Сlearlу, Рах2 is a major player for the proper development of optic nerve trajectories.
Edoardo Boncinelli and Antonio Simeone continued the theme of the development of the mammalian brain. Edoardo Boncinelli presented a dеtailеd expression analysis of the genes Ernx1 and Етх2, both orthologues of Drosophila ers, and Otx1 and Otx2, orthologues of otd. At day 10 of mouse development, these genes are expressed in continuons regions, contained within each other. Otx2 is expressed in the full fore-and midbrain, Otx1 in a subset, Етх2 in the forebrain and Emx1 only in part of the forebrain. Vertebrate brain development is a complex process, in which these genes appear to play an important role.
This is highlighted by the occurrence of rare mutations in the human Етх2 gene, ЕМХ2. These sporadic cases of schizencephaly are characterised by a full-thickness cleft within the cerebral hemispheres. Large regions of the cerebral hemispheres can be missing. The results of such lesions are mental retardation, seizures, hypotonia, spasticity, inability to walk or speak, and blindness. Clearly, the gene ЕMX2 plays an important role in the development of the human cerebral cortex.
Antonio Simeone proceeded with an in-depth analysis of the mouse genes Otx1 and Otx2. Given that Otx1 and Otx2 must have arisen by duplication from a single ancestral gene, their analysis can provide insights how gene fonction diverged alter the duplication event. A homozygous knock-out of Otx1 (0tx1-/-) shows that Otx1 is required for terminal differentiation events in the cortex, mesencephalon and cerebellum, but the mice are viable. In contrast, analysis of a Otx2 knock-out (Otx2-/-) revealed that it is required early on for proper development of the epiblast and the rostral neuroectoderm, as development arrests at gastrulation. A single copy of the Otx2 knock-out over a wild-type copy (Оtx2 -/+) showed weaker phenotypes, i.e. variable penetrance of craniofacial malformatiоns. Given the overlapping expression pattern, the question of redundancy and gene dosage was addressed by making various combinations of the knock-outs. Otx1-/–, Otx2+/- mice show dramatic malformations of the brain with ensuing lethality alter birth. This phenotype is more serious than Otx1+/-, Otx2+/- (25 % viabilitу), which in turn is more serious than Otx1+/+, Otx2+/-. Thus, to get through very early development, a single copy of Otx2 is suffiсient, while for gross brain development one copy each of Otx1 and Otx2, or two copies of Otx2 are suffiсient. When Otx2–/– is replaced by Otx1+/+ (giving 4 copies of 0tx1+) partial rescue results. Gastrulatiоn occurs quite normally, however the rostral brain remains small, and the mice die at 13 days. Thus there is virtually no difference for early development between the Otx1 and Otx2 proteins, while later brain-speсifiс functions require the distinct gene products.
Last but not least, Denis Duboule, though not the last speaker, reported on the last, i.e. the most posterior genes of the vertebrate Нох cluster. A particular feature of the vertebrate Нох clusters is that the genes are activated in a temporal sequence that reflects their linear order in the clusters. Denis Duboule’s hypothesis is that the sequential activation is a key component of the colinearity. То examine how such temporal regulation is achieved and if higher order chromatin structure plays a role in these processes, Denis Duboule undertook the task of placing reporter constructs of Hoxd–11 or Нохd-9 between the Evx–2 and the Нохd-13 genes in the Hoxd cluster. These experiments showed that there must be high-order regulatory mechanisms that are responsible for the temporal regulation of the Нох сluster in a colinear fashion. Further, specific regulatory elements within the Hoxd–10 to Нохd-13 region are shared between several genes and are responsible for the expression in the distal limb and in genital domains. Evx-2, located next to Нoxd-13, is also under the control of these enhancer elements, and gene knock-out experiments of Evx-2 demonstrated that it also plays a role in digit development. Given that these genes play a role in digit development, Denis Duboule examined their expression patterns in zebrafish to elucidate how possibly the fins transformed to limbs. While early expression in fin buds looks similar to early expression in mammalian limb buds (expression in the posterior in a nested fashion), the later expression phase in the digital area is not seen, suggesting that digit development might be a new invention in higher vertebrates.
The meeting was held in a very amiable spirit and it was a rewarding experience for all participants, not only because of the science, but also because of the magnificent efforts of the staff of Les Treilles and the unique ambience of the place. Encounters with wild boars enhanced the atmosphere further, and it prompted one participant to confess that, in the past, while hunting for these impressive animais, he almost shot a priest instead of a boar. Fortunately, his scientific career did rot suffer from this incidence.
Schematic representation of the Drosophila melanogaster Hox cluster and a « generic » vertebrate Hox cluster, which is a composite derived from the Hox clusters HoxA, НохВ, HoxC and HoxD. The cluster in the centre is a hypothetical reconstruction of how the original cluster might have looked at the point when protostomes and deuterostomes diverged in evolution. Lines between the individual homeobox genes represent the inferred relationships.