Jean-Michel Gibert

Gene duplication at the achaete–scute complex and morphological complexity of the peripheral nervous system in Diptera

The number of achaete-scute genes increased during insect evolution, particularly in the Diptera lineage. Sequence comparison indicates that the four achaete-scute genes of Drosophila result from three independent duplication events. After duplication, the new genes acquired individual expression patterns but, in Drosophila, their products can compensate for one another, which raises the question: why retain all four genes? The complexity of the spatial expression of these genes on the notum increased in the lineage leading to the higher Diptera, allowing the development of stereotyped bristle patterns. This probably coincided in time with gene duplication events, raising the possibility that an increase in gene copy number might have provided the flexibility necessary for more complex transcriptional regulation.

The evolution of engrailed genes after duplication and speciation events

Abstract. Members of the engrailed class encode transcription factors involved in major steps of metazoan development. Few developmental regulatory genes have been studied in such a wide range of animals. Furthermore duplications of an ancestral engrailed gene independently generated multiple engrailed paralogues in several organisms. This offers the opportunity to reconstruct the evolution of the engrailed family and to study the processes involved in the functional diversification following speciation or duplication events. The ancestral function of engrailed is very likely involved in neurogenesis. Recent studies in Drosophila and mice have shown its crucial role in neuronal connectivity and neuromuscular targeting. engrailed was probably recruited very early for a role in segmentation through intercalary evolution. Several new functions were acquired later on in specific phyla. Some duplication events have been followed by the loss of one paralogue, whereas others have led to the functional diversification of the paralogues. The Duplication-Degenerescence-Complementation model recently proposed by Force et al. seems to be the main process involved in functional diversification after duplication events. This does not exclude acquisition of new functions for one or both paralogues after duplication. The acquisition of such new functions principally involves the evolution of cis-regulatory sequences, but evolution of the coding sequence has also been revealed. However, in all engrailed duplications studied, even in ancient chromosomal duplications, the paralogues have kept redundant functions. In fact, selection seems to maintain a certain redundancy between engrailed paralogues.

Barnacle duplicate engrailed genes: divergent expression patterns and evidence for a vestigial abdomen

SUMMARY Cirripedes (barnacles) are crustaceans that are characterized by a very peculiar body plan, in particular by the lack of an abdomen. To study their body plan, we searched for their engrailed gene. We found two engrailed (en.a/en.b) genes in cirripedes. The two engrailed genes of the rhizocephalan barnacle Sacculina carcini are expressed in the posterior compartment of developing segments and appendages. When the neuroectoderm differentiates into epidermis and neuroderm the expression patterns of en.a and en.b diverge dramatically. en.a expression fades in segment epidermis whereas it is subsequently detected ventrally in reiterated putative neural cells. At the same time, en.b expression increases in the epidermis, which makes it a very good segmentation marker. Five tiny en.b stripes are observed between the sixth thoracic segment and the telson. We interpret these stripes as the molecular definition of vestigial abdominal segments, being the remnant of an ancestral state in keeping with the bodyplan of maxillopod crustaceans. engrailed expression is the first molecular evidence for a segmented abdomen in barnacles.

Redundant mechanisms mediate bristle patterning on the Drosophila thorax

The thoracic bristle pattern of Drosophila results from the spatially restricted expression of the achaete-scute (ac-sc) genes in clusters of cells, mediated by the activity of many discrete cis-regulatory sequences. However, ubiquitous expression of sc or asense (ase) achieved with a heterologous promoter, in the absence of endogenous ac-sc expression, and the activity of the cis-regulatory elements, allows the development of bristles positioned at wild-type locations. We demonstrate that the products of the genes stripe, hairy, and extramacrochaetae contribute to rescue by antagonizing the activity of Sc and Ase. The three genes are expressed in specific but overlapping spatial domains of expression that form a prepattern that allows precise positioning of bristles. The redundant mechanisms might contribute to the robustness of the pattern. We discuss the possibility that patterning in trans by antagonism is ancestral and that the positional cis-regulatory sequences might be of recent origin.

engrailed Duplication Events During the Evolution of Barnacles

Abstract. Barnacles (Cirripedia) are a subclass of Crustacea. Their peculiar segmentation pattern (few segments, absence of abdominal segments, and, in the parasitic rhizocephalan, loss of segmentation at the adult stage) prompted us to study the engrailed gene, which encodes a homeodomain transcription factor and is expressed in arthropods in the posterior half of each segment. We searched for engrailed genes by PCR in a representative cross section of the Cirripedia. Eight unambiguous engrailed genes were cloned from five species, three genes belonging to the same species (Elminius modestus). This implies two duplication events. Molecular phylogenies were constructed and a cladistic approach was applied to the most informative sites. The results indicate that the older duplication (en.a/en.b) is probably very ancient and concerns the whole cirripedean subclass, whereas the other (en.a1/en.a2) is specific to the Elminius lineage.

New Partners in Regulation of Gene Expression: The Enhancer of Trithorax and Polycomb Corto Interacts with Methylated Ribosomal Protein L12 Via Its Chromodomain

Chromodomains are found in many regulators of chromatin structure, and most of them recognize methylated lysines on histones. Here, we investigate the role of the Drosophila melanogaster protein Cortos chromodomain. The Enhancer of Trithorax and Polycomb Corto is involved in both silencing and activation of gene expression. Over-expression of the Corto chromodomain (CortoCD) in transgenic flies shows that it is a chromatin-targeting module, critical for Corto function. Unexpectedly, mass spectrometry analysis reveals that polypeptides pulled down by CortoCD from nuclear extracts correspond to ribosomal proteins. Furthermore, real-time interaction analyses demonstrate that CortoCD binds with high affinity RPL12 tri-methylated on lysine 3. Corto and RPL12 co-localize with active epigenetic marks on polytene chromosomes, suggesting that both are involved in fine-tuning transcription of genes in open chromatin. RNA–seq based transcriptomes of wing imaginal discs over-expressing either CortoCD or RPL12 reveal that both factors deregulate large sets of common genes, which are enriched in heat-response and ribosomal protein genes, suggesting that they could be implicated in dynamic coordination of ribosome biogenesis. Chromatin immunoprecipitation experiments show that Corto and RPL12 bind hsp70 and are similarly recruited on gene body after heat shock. Hence, Corto and RPL12 could be involved together in regulation of gene transcription. We discuss whether pseudo-ribosomal complexes composed of various ribosomal proteins might participate in regulation of gene expression in connection with chromatin regulators.

Cloning and expression of the engrailed.a gene of the barnacle Sacculina carcini.

Abstract Cirripedia (barnacles) constitute a crustacean monophyletic taxon which is very well defined by several synapomorphies. In particular, all cirripedes are composed of six thoracic segments, but are devoid of any complete abdominal segment. This body plan is preserved in the adult in non-parasitic groups, while the parasitic rhizocephalan cirripedes completely lose arthropodian segmentation at the adult stage. These traits make them a particularly favourable model for studying the formation and maintenance of segmental identity. For the above reasons, it seemed worthwhile to look at the segmentation gene engrailed in a cirripede. A complete engrailed.a cDNA was isolated from larvae of the rhizocephalan cirripede Sacculina carcini. Its expression was monitored during larval development by use of the monoclonal antibody MAb4D9 directed against the Drosophila homologous proteins. The Sacculina engrailed.a gene is expressed during the second and third larval stages in stripes within a posterior area corresponding to the presumptive trunk segments. Surprisingly, these stripes appear in a posterior to anterior sequence. Six engrailed.a stripes characterize the thoracic segments of the cirripedean ground plan.

Heterospecific transgenesis in Drosophila suggests that engrailed.a is regulated by POU proteins in the crustacean Sacculina carcini

Abstract. Almost all knowledge of the regulation of segmentation genes in arthropods comes from Drosophila. In order to study the regulation of the segment-polarity gene engrailed in a non-insect arthropod we focussed on putative regulatory regions of the engrailed.a (en.a) gene in the barnacle crustacean Sacculina carcini. In this animal, en.a is expressed in segmental stripes like the engrailed genes of other arthropods. As transgenesis in Sacculina is not possible at present, we have used Drosophila as a test tube. The Sacculina en.a intron is able to induce a specific expression of lacZ in the Drosophila wing imaginal disc. This pattern is not an engrailed-like pattern, but does suggest that some Drosophila transcription factors interact with the Sacculina en.a intron. We show that two Drosophila POU proteins, Nubbin and VVL, and Engrailed itself bind to the Sacculina en.a intron in vitro and that they regulate this expression in vivo. The conservation of POU protein binding sites in metazoans suggests that Sacculina POU proteins could recognize the same sequences. Hence, we looked at the expression of nubbin and vvl homologues in Sacculina larvae. Indeed, their expression patterns are consistent with a putative regulatory function on en.a in segments and appendages. Remarkably, the vvl homologue is expressed in Sacculina in a striking striped pattern that is very different from the vvl pattern in Drosophila embryos, and is complementary to the Sacculinaen.a pattern. These experiments suggest that the Sacculina engrailed.a gene is regulated by POU proteins.