Developmental Control Genes and Morphological Evolution

Developmental Control Genes and Morphological Evolution
September 21 – 27, 1996
by Michael Akam, Thomas R. Bürglin and Walter J. Gehring


André Adoutte (University de Paris-Sud, Orsay), Michael E. Akam (Wellcome/CRC Institute, Cambridge), Michalis Averof (European Molecular Biology Laboratory (EMBL), Heidelberg), Jacques Bièrne Jacques (Université de Reims-Champagne-Ardenne), Edoardo Boncinelli (Istituto Scientifico H. San Raffaele, Milano), Antonius J. M. Bouwmeester (University of California School of Medicine, Los Angeles), Sydney Brenner (University of Cambridge, Great Britain), Denis Duboule (University de Genève, Suisse), Walter J. Gehring, organizer (Biozentrum, University of Basel, Switzerland), Peter Gruss (Max-Planck Institute of Biophysical Chemistry, Gottingen, Germany), Herbert Jäckle (Max-Planck-Institute of Biophysical Chemistry, Gottingen), Fotis C. Kafatos (EMBL, Heidelberg), Gines Morata (Universidad Autonoma de Madrid, Spain), Nipam Patel (Howard Hughes Medical Institute, Chicago), Heinrich Reichert (University of Basel), Emili Saló (Universidad de Barcelona, Spain), Rosaria De Santis (Stazione Zoologica “Anton Dohrn”, Napoli, Italy), Antonio Simeone (International Institute of Genetics & Biophysics IIGB), Napoli)

Compte rendu

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 function 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 different classes of homeobox genes one group of particular interest are the Hox genes, which are organised in clusters on the chromosome. Figure 1 shows a simplified scheme of the Drosophila and a vertebrate “consensus” Hox cluster, a composite of the four clusters HoxA, HoxB, 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, which 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 Hox cluster genes. The Hox 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 Hox cluster, Ultrabithorax (Ubx) and abdominal-A (abdA), seem to have corresponding genes in platyhelminths. 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 Hox genes in the Ubd-A equivalent position in the vertebrate clusters must have arisen from the same common ancestor. Doubtlessly all participants agree that more data is needed to resolve many of the outstanding phylogenetic questions, although some might never be resolved with absolute certainty.

Invertebrates (excluding arthropods)

One such phylogenetic question that may never 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. He 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 Hox cluster has fewer homeobox genes than the Hox clusters of other animals in the protostome and deuterostome 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 functions of the homeobox genes show many conserved features. The POU homeobox gene ceh-6 is expressed and functions in a series of neurons in the brain, and in neuroblasts in the ventral nerve cord. Further expression is seen in the excretory cell 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 various tissues in the brain, the spinal cord/ventral nerve cord, and epidermal tissues, such as filzkörper and tracheal cells in flies.

The PBC-class homeobox gene ceh-20 is an orthologue of the Drosophila gene extra-denticle (exd). It is expressed in the body of the animal, overlapping the expression domains of the Hox cluster genes, again reminiscent of the situation in flies. The homeobox gene oeh-2 is an orthologue of Drosophila empty 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 highly derived head structures. Support for this notion stems from the fact that the anterior and posterior regions of the pharynx develop from cell lineages that diverge already at the 4 cell stage.

Emili Saló from Catalunya returned to the question of Hox genes in platyhelminths. He and his group had isolated seven Hox genes from the freshwater planarian Dugesia tigrina. These genes correspond to the anterior and central Hox cluster genes (Hox-1 to Hox 6/7/8). An interesting feature of freshwater planarians is their enormous capacity to regenerate. Hence, the expression pattern-of the Hox genes was studied during regeneration. In normal wild-type animals, no expression of Hox genes could be detected, although a low level of expression must exist, since cDNAs for Hox genes were isolated from such animals. A generalised feature of the Hox cluster genes in flies 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, strong expression is seen in the regions 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 difference can be seen along the body axis during regeneration; gene activation and subsequent deactivation appears synchronous along the axis. This poses an intriguing puzzle: 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 Hox genes. Reporting a fruitful collaboration with Walter J. Gehring’s laboratory Jacques Bièrne presented sequence data for Hox 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 ancestor of flies and man – a view strongly disputed by André Adoutte, whose earlier presentation placed them firmly among the prototrochozoa. Nonetheless, we could only be impressed by the versatility of these creatures, whose powers of regeneration seem to be limitless.

Rosaria De Santis introduced another invertebrate, the ascidian Ciona intestinalis. 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 Hox cluster genes they have initiated a chromosomal walk to determine the complete structure of the Hox cluster. To date, 9 Hox 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 Hox3 and Hox5 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 NK-2 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 HSP70 might be the ancestral gene. Rosaria De Santis thus isolated an HSP70 gene from Ciona, and preliminary experiments with antibodies against HSP70 suggest that they can block sperm entry.


Michael Akam focused on the Hox 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 Hox cluster complexity in a range of arthropods suggest that the diversity of arthropod body plans has been achieved with a conserved set of Hox genes, by altering their regulation.

In contrast to this overall conservation, rapid evolutionary changes have affected some genes within the insect Hox clusters. Comparisons of genes from grasshoppers, beetles and flies show that at least two Hox cluster genes are diverging relatively rapidly, 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 complex — genes that are involved in patterning the early syncytial stages of the Drosophila embryo. Michael Akam showed that in grasshoppers, no equivalent syncytial stage exists – cellularisation is complete before the embryonic primordium has coalesced. He proposed that changes in early developmental mechanisms may have allowed the rapid diversification of these “runaway” Hox genes.

Nipam Patel returned to the relationship between Hox gene regulation and the diversification of segments. The domains of expression of particular Hox 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 Hox genes of the Ubx/abd-A class 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 many 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 Hox genes is excluded from the maxillipeds. In Malacostracan crustaceans, which may have 0, 1 or 2 maxillipeds, there is an excellent correlation between the extent of Ubx/abd-A expression, and the number of maxillipeds formed. This is the clearest example yet of shifts in Hox 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 and posterior domains, with branching in the dorso-ventral plane. This makes it unlikely that branched limbs arise by the secondary fusion of uniramous limbs.
The branchiopod crustacean Triops provides another nice example of diversity in developmental mechanisms. In this animal, segment patterning in the dorsal and ventral halves of the animal begins in register, but in the posterior part of the animal becomes disjunct, such that a single dorsal segment may 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. Michalis Averof reported an attempt to distinguish between these hypotheses by analysing in Crustacea the expression of two genes that are characteristic of wing development in Drosophilanubbin and apterous. Both proved to be expressed in a gill branch of the Artemia leg, strongly supporting the leg origin hypothesis. This observation prompted lively debate as to whether molecules defined 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, domains proposed by Snodgrass in 1935 on the basis of comparative morphology. Interestingly, in the distal limbs, ectopic expression of any one of several Hox 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 Drosophila, 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 Hox cluster might have evolved through unequal crossing-over. It might explain why the central Hox 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-class homeobox gene Pax-6. In vertebrates this gene has also been shown to be involved in eye development (see talk by Peter Gruss). To test if Pax-6 is not only required for eye formation in flies, but also sufficient, Pax-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 Pax-6 gene, showing the functional conservation. Cloning and expression analysis of Pax-6 homologues from Lineus and Dugesia (in collaboration with Emili Saló) revealed that also in these organisms Pax-6 is expressed in the primitive eye spots. Thus Pax-6 can be regarded as the master control gene for eye development. Evidence was found that a possible target for Pax-6 is another gene important for eye development, sine oculis (so). 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 also a role in eye development. It appears that toy first induces ey, which turns on a cascade of genes leading to eye development.

Herbert Jäckle’s talk provided a striking example of how studies carried out without any reference to evolution can have profound effects on our thinking about evolutionary processes. He described studies of the segmentation gene hierarchy in Drosophila, focusing on the dissection of regulatory elements that interpret positional information in the blastoderm 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 Jäckle 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 proteins, which have been exploited by the metazoa to form a huge family of gene regulators. He 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 ones. Functional sites are not necessarily homologous (related by descent), but analogous. By comparing two closely related zinc finger genes in Drosophila, spalt and spalt-related, 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 frog Xenopus laevis. One of the signals responsible for the inductive events is BMP4, a relative of the Drosophila factor decapentaplegic (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 BMP4. A second factor, noggin, has an even higher affinity to BMP4. Thus, both these factors can be viewed as negative regulators of BMP4. 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 BMP4 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 arose 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, which is expressed in a 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 Drosophila eyeless gene reported on by Walter J. Gehring. This gene is required not only for eye formation but also for the development of the nose and forebrain structures, as well as ventral structures. Mutations in mouse Pax6 cause the Small eye (Sey) phenotype, however fragments of protein are still present in these mutations. Large deletions of Sey cause death at implantation. Is Pax-6 involved in the signaling? In knock-out mice, the optic vesicle still forms, thus Pax-6 is not required for its initial formation. However, the subsequent inductive events, i.e. lens placode and lens retina formation depend on Pax-6.
Another “fly gene” involved in eye development is sine oculis. Several vertebrate orthologues were isolated, and one of these genes, Six3, appears to be the functional homologue of so. 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 eyeless in flies, Six3 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 eye.
Peter Gruss proceeded to present evidence for a role of the gene Pax2, a paired domain gene, in eye development. Initial Pax2 expression is confined to the ventral optic vesicle and is later found in regions that contribute to the optic nerve and optic chiasm. In Pax2 mutant mice defects are seen in axonal outgrowth. The optic tracts remain ipsilateral in mutant brains, since the optic chiasm fails to form properly. Clearly, Pax2 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 detailed expression analysis of the genes Emx1 and Emx2, both orthologues of Drosophila ems, and Otx1 and Otx2, orthologues of otd. At day 10 of mouse development, these genes are expressed in continuous regions, contained within each other. Otx2 is expressed in the full fore- and midbrain, Otx1 in a subset, Emx2 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 Emx2 gene, EMX2. 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 EMX2 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 function diverged after the duplication event. A homozygous knock-out of Otx1 (Otx1-/-) 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 (Otx2-/+) showed weaker phenotypes, i.e. variable penetrance of craniofacial malformations. 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 after birth. This phenotype is more serious than Otx1+/-, Otx2+/- (25 % viability), which in turn is more serious than Otx1+/+, Ot2+/-. Thus, to get through very early development, a single copy of Otx2 is sufficient, while for gross brain development one copy each of Otx1 and Otx2, or two copies of Otx2 are sufficient. When Otx2-/- is replaced by Otx1+/+ (giving 4 copies of Otx1+) partial rescue results. Gastrulation 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-specific 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 Hox cluster. A particular feature of the vertebrate Hox 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. To 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 Hoxd-9 between the Evx-2 and the Hoxd-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 Hox cluster in a colinear fashion. Further, specific regulatory elements within the Hoxd-10 to Hoxd-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 Hoxd-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 animals, he almost shot a priest instead of a boar. Fortunately, his scientific career did not 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, HoxB, HoxC and HoxD. The cluster in the centre is a hypothetical reconstruction of how the original duster might have looked at the point when protostomes and deuterostomes diverged in evolution. Lines between the individual homeobox genes represent the inferred relationships


Andre Adoutte – A parallel look at evolution of metazoa and that of the Hox-genes complex
Michael Akam – Diversity of developmental mechanisms in insects
Michael Averof – The evolutionary origin of insect wings from ancient respiratory appendages
Jacques BièrneLineus sanguineus is the closest relative to the last common ancestor of arthropods and chordates
Edoardo Boncinelli – Role of Otx and Emx genes in the developing brain and cerebral cortex
Antonius J. M. Bouwmeester – Role of Chordin and Cerberus in patterning of Xenopus embryo
Thomas Bürglin – Evolutionary conservation of C. elegans homeobox genes
Rosalia De Santis – From gametes to embryo: Ascidians as a classical model for modern developmental biology
Denis Duboule – Hox genes and the evolution of morphologies
Walter Gehring – Genetic control of the morphogenesis and evolution of the eyes
Peter Gruss – Genes involved in the development of the vertebrate visual system
Herbert Jäckle – From gradients to stripes: Early pattern formation in Drosophila
Fotis Kafatos – The evolution of transcriptional regulation: Some facts and some speculations
Ginés Morata – Hox genes and the development and evolution of the arthropod limb
Nipam Patel – Evolution of arthropod segmentation and body patterning
Heinrich Reichert – Development and evolution of the brain: Insights in insects
Emili Saló – Planarian Hox gene expression bears no obvious relation to axial polarity during regeneration
Antonio Simeone – A functional study of the Otx1 and Otx2 genes in mouse

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