Cellular dynamics of host - parasitoid interactions: Insights from the encapsulation process in a partially resistant host.

Cotesia typhae is an eastern African endoparasitoid braconid wasp that targets the larval stage of the lepidopteran stem borer, Sesamia nonagrioides, a maize crop pest in Europe. The French host population is partially resistant to the Makindu strain of the wasp, allowing its development in only 40% of the cases. Resistant larvae can encapsulate the parasitoid and survive the infection. This interaction provides a very interesting frame for investigating the impact of parasitism on host cellular resistance. We characterized the parasitoid ovolarval development in a permissive host and studied the encapsulation process in a resistant host by dissection and histological sectioning compared to that of inert chromatography beads. We measured the total hemocyte count in parasitized and bead-injected larvae over time to monitor the magnitude of the immune reaction. Our results show that parasitism of resistant hosts delayed encapsulation but did not affect immune abilities towards inert beads. Moreover, while bead injection increased total hemocyte count, it remained constant in resistant and permissive larvae. We conclude that while Cotesia spp virulence factors are known to impair the host immune system, our results suggest that passive evasion could also occur.

Interference with the retinoic acid signalling pathway inhibits the initiation of teeth and caudal primary scales in the small-spotted catshark Scyliorhinus canicula.

The retinoic acid (RA) pathway was shown to be important for tooth development in mammals, and suspected to play a key role in tooth evolution in teleosts. The general modalities of development of tooth and "tooth-like" structures (collectively named odontodes) seem to be conserved among all jawed vertebrates, both with regard to histogenesis and genetic regulation. We investigated the putative function of RA signalling in tooth and scale initiation in a cartilaginous fish, the small-spotted catshark Scyliorhinus canicula. To address this issue, we identified the expression pattern of genes from the RA pathway during both tooth and scale development and performed functional experiments by exposing small-spotted catshark embryos to exogenous RA or an inhibitor of RA synthesis. Our results showed that inhibiting RA synthesis affects tooth but not caudal primary scale development while exposure to exogenous RA inhibited both. We also showed that the reduced number of teeth observed with RA exposure is probably due to a specific inhibition of tooth bud initiation while the observed effects of the RA synthesis inhibitor is related to a general delay in embryonic development that interacts with tooth development. This study provides data complementary to previous studies of bony vertebrates and support an involvement of the RA signalling pathway toolkit in odontode initiation in all jawed vertebrates. However, the modalities of RA signalling may vary depending on the target location along the body, and depending on the species lineage.

Tooth and scale morphogenesis in shark: an alternative process to the mammalian enamel knot system.

BACKGROUND: The gene regulatory network involved in tooth morphogenesis has been extremely well described in mammals and its modeling has allowed predictions of variations in regulatory pathway that may have led to evolution of tooth shapes. However, very little is known outside of mammals to understand how this regulatory framework may also account for tooth shape evolution at the level of gnathostomes. In this work, we describe expression patterns and proliferation/apoptosis assays to uncover homologous regulatory pathways in the catshark Scyliorhinus canicula. RESULTS: Because of their similar structural and developmental features, gene expression patterns were described over the four developmental stages of both tooth and scale buds in the catshark. These gene expression patterns differ from mouse tooth development, and discrepancies are also observed between tooth and scale development within the catshark. However, a similar nested expression of Shh and Fgf suggests similar signaling involved in morphogenesis of all structures, although apoptosis assays do not support a strictly equivalent enamel knot system in sharks. Similarities in the topology of gene expression pattern, including Bmp signaling pathway, suggest that mouse molar development is more similar to scale bud development in the catshark. CONCLUSIONS: These results support the fact that no enamel knot, as described in mammalian teeth, can be described in the morphogenesis of shark teeth or scales. However, homologous signaling pathways are involved in growth and morphogenesis with variations in their respective expression patterns. We speculate that variations in this topology of expression are also a substrate for tooth shape evolution, notably in regulating the growth axis and symmetry of the developing structure.

Molecular footprinting of skeletal tissues in the catshark Scyliorhinus canicula and the clawed frog Xenopus tropicalis identifies conserved and derived features of vertebrate calcification.

Understanding the evolutionary emergence and subsequent diversification of the vertebrate skeleton requires a comprehensive view of the diverse skeletal cell types found in distinct developmental contexts, tissues, and species. To date, our knowledge of the molecular nature of the shark calcified extracellular matrix, and its relationships with osteichthyan skeletal tissues, remain scarce. Here, based on specific combinations of expression patterns of the Col1a1, Col1a2, and Col2a1 fibrillar collagen genes, we compare the molecular footprint of endoskeletal elements from the chondrichthyan Scyliorhinus canicula and the tetrapod Xenopus tropicalis. We find that, depending on the anatomical location, Scyliorhinus skeletal calcification is associated to cell types expressing different subsets of fibrillar collagen genes, such as high levels of Col1a1 and Col1a2 in the neural arches, high levels of Col2a1 in the tesserae, or associated to a drastic Col2a1 downregulation in the centrum. We detect low Col2a1 levels in Xenopus osteoblasts, thereby revealing that the osteoblastic expression of this gene was significantly reduced in the tetrapod lineage. Finally, we uncover a striking parallel, from a molecular and histological perspective, between the vertebral cartilage calcification of both species and discuss the evolutionary origin of endochondral ossification.

Altered retinoic acid signalling underpins dentition evolution.

Small variations in signalling pathways have been linked to phenotypic diversity and speciation. In vertebrates, teeth represent a reservoir of adaptive morphological structures that are prone to evolutionary change. Cyprinid fish display an impressive diversity in tooth number, but the signals that generate such diversity are unknown. Here, we show that retinoic acid (RA) availability influences tooth number size in Cyprinids. Heterozygous adult zebrafish heterozygous for the cyp26b1 mutant that encodes an enzyme able to degrade RA possess an extra tooth in the ventral row. Expression analysis of pharyngeal mesenchyme markers such as dlx2a and lhx6 shows lateral, anterior and dorsal expansion of these markers in RA-treated embryos, whereas the expression of the dental epithelium markers dlx2b and dlx3b is unchanged. Our analysis suggests that changes in RA signalling play an important role in the diversification of teeth in Cyprinids. Our work illustrates that through subtle changes in the expression of rate-limiting enzymes, the RA pathway is an active player of tooth evolution in fish.

Heterogeneous conservation of Dlx paralog co-expression in jawed vertebrates.

BACKGROUND: The Dlx gene family encodes transcription factors involved in the development of a wide variety of morphological innovations that first evolved at the origins of vertebrates or of the jawed vertebrates. This gene family expanded with the two rounds of genome duplications that occurred before jawed vertebrates diversified. It includes at least three bigene pairs sharing conserved regulatory sequences in tetrapods and teleost fish, but has been only partially characterized in chondrichthyans, the third major group of jawed vertebrates. Here we take advantage of developmental and molecular tools applied to the shark Scyliorhinus canicula to fill in the gap and provide an overview of the evolution of the Dlx family in the jawed vertebrates. These results are analyzed in the theoretical framework of the DDC (Duplication-Degeneration-Complementation) model. RESULTS: The genomic organisation of the catshark Dlx genes is similar to that previously described for tetrapods. Conserved non-coding elements identified in bony fish were also identified in catshark Dlx clusters and showed regulatory activity in transgenic zebrafish. Gene expression patterns in the catshark showed that there are some expression sites with high conservation of the expressed paralog(s) and other expression sites with events of paralog sub-functionalization during jawed vertebrate diversification, resulting in a wide variety of evolutionary scenarios within this gene family. CONCLUSION: Dlx gene expression patterns in the catshark show that there has been little neo-functionalization in Dlx genes over gnathostome evolution. In most cases, one tandem duplication and two rounds of vertebrate genome duplication have led to at least six Dlx coding sequences with redundant expression patterns followed by some instances of paralog sub-functionalization. Regulatory constraints such as shared enhancers, and functional constraints including gene pleiotropy, may have contributed to the evolutionary inertia leading to high redundancy between gene expression patterns.

Pattern and polarity in the development and evolution of the gnathostome jaw: both conservation and heterotopy in the branchial arches of the shark, Scyliorhinus canicula.

The acquisition of jaws constitutes a landmark event in vertebrate evolution, one that in large part potentiated their success and diversification. Jaw development and patterning involves an intricate spatiotemporal series of reciprocal inductive and responsive interactions between the cephalic epithelia and the cranial neural crest (CNC) and cephalic mesodermal mesenchyme. The coordinated regulation of these interactions is critical for both the ontogenetic registration of the jaws and the evolutionary elaboration of variable jaw morphologies and designs. Current models of jaw development and evolution have been built on molecular and cellular evidence gathered mostly in amniotes such as mice, chicks and humans, and augmented by a much smaller body of work on the zebrafish. These have been partnered by essential work attempting to understand the origins of jaws that has focused on the jawless lamprey. Chondrichthyans (cartilaginous fish) are the most distant group to amniotes within extant gnathostomes, and comprise the crucial clade uniting amniotes and agnathans; yet despite their critical phylogenetic position, evidence of the molecular and cellular underpinnings of jaw development in chondrichthyans is still lacking. Recent advances in genome and molecular developmental biology of the lesser spotted dogfish shark, Scyliorhinus canicula, make it ideal for the molecular study of chondrichthyan jaw development. Here, following the 'Hinge and Caps' model of jaw development, we have investigated evidence of heterotopic (relative changes in position) and heterochronic (relative changes in timing) shifts in gene expression, relative to amniotes, in the jaw primordia of S. canicula embryos. We demonstrate the presence of clear proximo-distal polarity in gene expression patterns in the shark embryo, thus establishing a baseline molecular baüplan for branchial arch-derived jaw development and further validating the utility of the 'Hinge and Caps' model in comparative studies of jaw development and evolution. Moreover, we correlate gene expression patterns with the absence of a lambdoidal junction (formed where the maxillary first arch meets the frontonasal processes) in chondrichthyans, further highlighting the importance of this region for the development and evolution of jaw structure in advanced gnathostomes.

The homology of odontodes in gnathostomes: insights from Dlx gene expression in the dogfish, Scyliorhinus canicula.

BACKGROUND: Teeth and tooth-like structures, together named odontodes, are repeated organs thought to share a common evolutionary origin. These structures can be found in gnathostomes at different locations along the body: oral teeth in the jaws, teeth and denticles in the oral-pharyngeal cavity, and dermal denticles on elasmobranch skin. We, and other colleagues, had previously shown that teeth in any location were serially homologous because: i) pharyngeal and oral teeth develop through a common developmental module; and ii) the expression patterns of the Dlx genes during odontogenesis were highly divergent between species but almost identical between oral and pharyngeal dentitions within the same species. Here we examine Dlx gene expression in oral teeth and dermal denticles in order to test the hypothesis of serial homology between these odontodes. RESULTS: We present a detailed comparison of the first developing teeth and dermal denticles (caudal primary scales) of the dogfish (Scyliorhinus canicula) and show that both odontodes develop through identical stages that correspond to the common stages of oral and pharyngeal odontogenesis. We identified six Dlx paralogs in the dogfish and found that three showed strong transcription in teeth and dermal denticles (Dlx3, Dlx4 and Dlx5) whereas a weak expression was detected for Dlx1 in dermal denticles and teeth, and for Dlx2 in dermal denticles. Very few differences in Dlx expression patterns could be detected between tooth and dermal denticle development, except for the absence of Dlx2 expression in teeth. CONCLUSIONS: Taken together, our histological and expression data strongly suggest that teeth and dermal denticles develop from the same developmental module and under the control of the same set of Dlx genes. Teeth and dermal denticles should therefore be considered as serial homologs developing through the initiation of a common gene regulatory network (GRN) at several body locations. This mechanism of heterotopy supports the 'inside and out' model that has been recently proposed for odontode evolution.

Evolution of repeated structures along the body axis of jawed vertebrates, insights from the Scyliorhinus canicula Hox code.

The Hox gene family encodes homeodomain-containing transcription factors involved in the patterning of structures composed of repeated elements along the antero-posterior axis of Bilateralia embryos. In vertebrate, Hox genes are thought to control the segmental identity of the rhombomeres, the branchial arches, and the somites. They are therefore thought to have played a key role in the morphological evolution of structures like the jaw, girdles, and vertebrae in gnathostomes. Thus far, our knowledge about the expression patterns of the Hox genes, the Hox code, has been mainly restricted to osteichthyans species and little is known about chondrichthyans. Recently, we identified 34 Hox genes clustered in three complexes (HoxA, HoxB, and HoxD) in the dogfish (Scyliorhinus canicula) genome suggesting that in sharks most, if not all, genes belonging to the HoxC complex are lost. To gain insights into the evolution of gnathostome Hox transcription, we present here expression patterns along the anteroposterior axis for all Hox genes known in the dogfish. A comparison of these patterns with those of osteichthyans shows that the expression patterns of the Hox genes in serially homologous compartments such as the branchial arches, the hindbrain, and the somites underwent only subtle changes during the evolution of gnathostomes. Therefore, the nested expression of Hox genes in these structures, the Hox code, is a ground plan, which predates the morphological diversification of serially homologous structures along the body axis.

Development of oral and pharyngeal teeth in the medaka (Oryzias latipes): comparison of morphology and expression of eve1 gene.

Gnathostome teeth are one of the most promising models for developmental evolutionary studies, they are the most abundant organ in the fossil record and an excellent example of organogenesis. Teeth have a complex morphology and are restricted to the mouth in mammals, whereas actinopterygian teeth have a simple morphology and are found in several locations, notably on pharyngeal bones. Morphological and developmental similarities support the hypothesis that oral and pharyngeal teeth are serially homologous. Gene expression data from the mouse and some teleosts have shown that the gene families involved in pharyngeal odontogenesis are also involved in oral tooth formation, with the notable exception of the evx gene family. Here, we present a complete description of early odontogenesis in the medaka (Oryzias latipes), which has both oral and pharyngeal dentition. We show that oral and pharyngeal teeth share deep developmental similarities. In the medaka, like in the zebrafish, eve1 is the only evx gene expressed during odontogenesis. In each forming tooth, regardless of its location, eve1 transcription is activated in the placode, then becomes restricted to the inner dental epithelium and is activated in the dental mesenchyme during early differentiation, and finally ceases at late differentiation. Thus eve1 expression is not specific to pharyngeal teeth development as was previously suggested. Because it permits direct comparisons between oral and pharyngeal teeth by molecular, development and functional studies, the medaka is an excellent model to develop further insights into the evolution of odontogenesis in gnathostomes.

Posterior hoxa genes expression during zebrafish bony fin ray development and regeneration suggests their involvement in scleroblast differentiation.

Expression of two zebrafish developmental posterior hoxa genes, hoxa11b and hoxa13b, was studied by in situ hybridization during pectoral and caudal fin development and regeneration. Expression was restricted to cells of the bony rays region. During fin development, molecular cytological analysis revealed that a subpopulation of mesenchymal cells expressed these two hoxa genes during their early differentiation in the subapical region of the developing ray. These cells were identified as differentiating dermal bone making cells (scleroblasts). During fin regeneration, hoxa11b and hoxa13b genes are both induced in undifferentiated cells of the distalmost blastema region (DMB) and the proliferating zone (PZ) and later in differentiating bone-forming cells. In addition, the transient regionalization of the hoxa13b expression pattern in differentiated bone-forming cells along the proximodistal axis of the regenerating ray suggests that hoxa13b could participate in ray patterning. This study is the first to establish a correlation between hoxa gene expression and dermal bone cell differentiation.

Comparison of even-skipped related gene expression pattern in vertebrates shows an association between expression domain loss and modification of selective constraints on sequences.

The even-skipped related genes (evx) encode homeodomain-containing transcription factors that play key roles in body patterning and neurogenesis in a wide array of Eumetazoa species. It is thought that the genome of the last common ancestor of Chordata contained a unique evx gene linked to a unique ancestral Hox complex. During subsequent evolution, two rounds of whole genome duplication followed by individual gene losses gave rise to three paralogs: evx1, evx2, and eve1. Then, eve1 was maintained in Actinopterygii lineage but not in Tetrapoda. To explain this discrepancy, we examined the expression patterns of the evx1 homologue, Xhox3, in Xenopus laevis and that of evx1 and eve1 in Danio rerio. We show here that Xhox3 is expressed in a manner that closely reflects the inferred expression pattern of the evx1 gene in the last common ancestor of Vertebrata (i.e., in gastrula, the central nervous system, the posterior gut, and the tip of the growing tail). Zebrafish evx1 and Xenopus Xhox3 are expressed in homologous cell lineages of the central nervous system and of the posterior gut, but evx1 was undetectable in the gastrula and the tail bud. Strikingly, eve1 is the only evx gene of zebrafish to be expressed in these two latter regions. Thus, the ancestral expression pattern of evx1 in vertebrates appears to have been distributed between evx1 and eve1 in zebrafish. We propose that evx1 and eve1 underwent a complementary loss of expression domain in zebrafish that allowed the maintenance of the two paralogs in accordance with the duplication-degeneration-complementation model. It is important to note that, in zebrafish, Evx1 and Eve1 have lost most of the protein domain upstream of the homeodomain. In addition, Eve1 has accumulated substitutions in positions that are highly conserved in all other Evx proteins. Thus, the reduction of the expression domain of both evx1 and eve1 in zebrafish appears to be associated with the modification of constraints on the protein sequences, allowing the shortening of both genes and an accelerated substitution rate in eve1.