Mélanie Debiais-Thibaud

Expression of Dlx genes during the development of the zebrafish pharyngeal dentition: evolutionary implications

SUMMARY In order to investigate similarities and differences in genetic control of development among teeth within and between species, we determined the expression pattern of all eight Dlx genes of the zebrafish during development of the pharyngeal dentition and compared these data with that reported for mouse molar tooth development. We found that (i) dlx1a and dlx6a are not expressed in teeth, in contrast to their murine orthologs, Dlx1 and Dlx6; (ii) the expression of the six other zebrafish Dlx genes overlaps in time and space, particularly during early morphogenesis; (iii) teeth in different locations and generations within the zebrafish dentition differ in the number of genes expressed; (iv) expression similarities and differences between zebrafish Dlx genes do not clearly follow phylogenetic and linkage relationships; and (v) similarities and differences exist in the expression of zebrafish and mouse Dlx orthologs. Taken together, these results indicate that the Dlx gene family, despite having been involved in vertebrate tooth development for over 400 million years, has undergone extensive diversification of expression of individual genes both within and between dentitions. The latter type of difference may reflect the highly specialized dentition of the mouse relative to that of the zebrafish, and/or genome duplication in the zebrafish lineage facilitating a redistribution of Dlx gene function during odontogenesis.

Evolution of Hox Gene Clusters in Gnathostomes: Insights from a Survey of a Shark (Scyliorhinus canicula) Transcriptome

It is now well established that there were four Hox gene clusters in the genome of the last common ancestor of extant gnathostomes. To better understand the evolution of the organization and expression of these genomic regions, we have studied the Hox gene clusters of a shark (Scyliorhinus canicula). We sequenced 225,580 expressed sequence tags from several embryonic cDNA libraries. Blast searches identified corresponding transcripts to almost all the HoxA, HoxB, and HoxD cluster genes. No HoxC transcript was identified, suggesting that this cluster is absent or highly degenerate. Using Hox gene sequences as probes, we selected and sequenced seven clones from a bacterial artificial chromosome library covering the complete region of the three gene clusters. Mapping of cDNAs to these genomic sequences showed extensive alternative splicing and untranslated exon sharing between neighboring Hox genes. Homologous noncoding exons could not be identified in transcripts from other species using sequence similarity. However, by comparing conserved noncoding sequences upstream of these exons in different species, we were able to identify homology between some exons. Some alternative splicing variants are probably very ancient and were already coded for by the ancestral Hox gene cluster. We also identified several transcripts that do not code for Hox proteins, are probably not translated, and all but one are in the reverse orientation to the Hox genes. This survey of the transcriptome of the Hox gene clusters of a shark shows that the high complexity observed in mammals is a gnathostome ancestral feature.

In vivo and in vitro assessment of cardiac β-adrenergic receptors in larval zebrafish ( Danio rerio )

SUMMARY β-Adrenergic receptors (βARs) are crucial for maintaining the rate and force of cardiac muscle contraction in vertebrates. Zebrafish (Danio rerio) have one β1AR gene and two β2AR genes (β2aAR and β2bAR). We examined the roles of these receptors in larval zebrafish in vivo by assessing the impact of translational gene knockdown on cardiac function. Zebrafish larvae lacking β1AR expression by morpholino knockdown displayed lower heart rates than control fish, whereas larvae deficient in both β2aAR and β2bAR expression exhibited significantly higher heart rates than controls. These results suggested a potential inhibitory role for one or both β2AR genes. By using cultured HEK293 cells transfected with zebrafish βARs, we demonstrated that stimulation with adrenaline or procaterol (a β2AR agonist) resulted in an increase in intracellular cAMP levels in cells expressing any of the three zebrafish βARs. In comparison with its human βAR counterpart, zebrafish β2aAR expressed in HEK293 cells appeared to exhibit a unique binding affinity profile for adrenergic ligands. Specifically, zebrafish β2aAR had a high binding affinity for phenylephrine, a classical α-adrenergic receptor agonist. The zebrafish receptors also had distinct ligand binding affinities for adrenergic agonists when compared with human βARs in culture, with zebrafish β2aAR being distinct from human β2AR and zebrafish β2bAR. Overall, this study provides insight into the function and evolution of both fish and mammalian β-adrenergic receptors.

The relationship between dlx and gad1 expression indicates highly conserved genetic pathways in the zebrafish forebrain

The Dlx genes encode a family of transcription factors important for the development of the vertebrate forebrain. These genes have very similar expression domains during the development of the telencephalon in mice and play a role in γ‐aminobutyric acid (GABAergic) interneuron differentiation. We have used triple fluorescent in situ hybridization to study the relative expression domains of the dlx and gad1 genes in the zebrafish telencephalon and diencephalon. We also generated transgenic zebrafish with regulatory elements from the zebrafish dlx1a/2a locus. The zebrafish dlx regulatory elements recapitulated dlx expression in the forebrain and mimicked the relationship between the expression of the dlx genes and gad1. Finally, we show that a putative enhancer located downstream of dlx2b can also activate reporter gene expression in a tissue‐specific manner similar to endogenous dlx2b expression. Our results indicate the dlx genes are regulated by an evolutionarily conserved genetic pathway and may play a role in GABAergic interneuron differentiation in the zebrafish forebrain. Developmental Dynamics 239:2298–2306, 2010.

Expression of the dlx gene family during formation of the cranial bones in the zebrafish (Danio rerio): Differential involvement in the visceral skeleton and braincase

We have used dlx genes to test the hypothesis of a separate developmental program for dermal and cartilage bones within the neuro‐ and splanchnocranium by comparing expression patterns of all eight dlx genes during cranial bone formation in zebrafish from 1 day postfertilization (dPF) to 15 dPF. dlx genes are expressed in the visceral skeleton but not during the formation of dermal or cartilage bones of the braincase. The spatiotemporal expression pattern of all the members of the dlx gene family, support the view that dlx genes impart cellular identity to the different arches, required to make arch‐specific dermal bones. Expression patterns seemingly associated with cartilage (perichondral) bones of the arches, in contrast, are probably related to ongoing differentiation of the underlying cartilage rather than with differentiation of perichondral bones themselves. Whether dlx genes originally functioned in the visceral skeleton only, and whether their involvement in the formation of neurocranial bones (as in mammals) is secondary, awaits clarification. Developmental Dynamics 235:1371–1389, 2006.

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

SUMMARY 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.

Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling

One of the goals of evolutionary developmental biology is to link specific adaptations to changes in developmental pathways. The dentition of cypriniform fishes, which in contrast to many other teleost fish species possess pharyngeal teeth but lack oral teeth, provides a suitable model to study the development of feeding adaptations. Here, we have examined the involvement of retinoic acid (RA) in tooth development and show that RA is specifically required to induce the pharyngeal tooth developmental program in zebrafish. Perturbation of RA signaling at this stage abolished tooth induction without affecting the development of tooth‐associated ceratobranchial bones. We show that this inductive event is dependent on RA synthesis from aldh1a2 in the ventral posterior pharynx. Fibroblast growth factor (FGF) signaling has been shown to be critical for tooth induction in zebrafish, and its loss has been associated with oral tooth loss in cypriniform fishes. Pharmacological treatments targeting the RA and FGF pathways revealed that both pathways act independently during tooth induction. In contrast, we find that in Mexican tetra and medaka, species that also possess oral teeth, both oral and pharyngeal teeth are induced independently of RA. Our analyses suggest an evolutionary scenario in which the gene network controlling tooth development obtained RA dependency in the lineage leading to the cypriniforms. The loss of pharyngeal teeth in this group was cancelled out through a shift in aldh1a2 expression, while oral teeth might have been lost ultimately due to deficient RA signaling in the oral cavity.—Gibert, Y., Bernard, L., Debiais‐Thibaud, M., Bourrat, F., Joly, J.‐S., Pottin, K., Meyer, A., Retaux, S., Stock, D. W., Jackman, W. R., Seritrakul, P., Begemann, G., Laudet, V. Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling. FASEB J. 24, 3298–3309 (2010). www.fasebj.org

Low divergence in Dlx gene expression between dentitions of the medaka (Oryzias latipes) versus high level of expression shuffling in osteichtyans

SUMMARY Serially homologous structures are believed to originate from the redeployment of a genetic cascade in different locations of the body. Serial homologs may diverge at the genetic and morphological level and acquire developmental independency (individualization). Teeth are repeated units that form dentitions found on different bones of the oral–pharyngeal cavity in gnathostomes and provide a good model to study such processes. Previous comparisons of dlx gene expression patterns between mouse oral teeth and zebrafish pharyngeal teeth showed a high level of divergence. Furthermore, these genes are differentially expressed in different teeth of the zebrafish, and in the mouse they are responsible for tooth identity (incisors vs. molars). We examined the potential divergence of dlx gene expression between oral and pharyngeal teeth by examining the expression pattern in the development of the first generation teeth of the medaka and comparing it with data from the zebrafish and the mouse. Out of the seven medaka dlx genes, five are expressed during odontogenesis compared with six in both the zebrafish and the mouse. The only difference observed between oral and pharyngeal teeth in the medaka is an earlier expression of dlx5a in the oral dental epithelium. The subset of dlx genes expressed in the medaka, zebrafish, and mouse is slightly different but their detailed expression patterns are highly divergent. Our results demonstrate a low constraint on dlx gene expression shuffling in the odontogenic cascade within osteichtyans but the non‐individualization of oral and pharyngeal dentitions in the medaka.

A cost-effective straightforward protocol for shotgun Illumina libraries designed to assemble complete mitogenomes from non-model species

The mitogenome is an inescapable tool in conservation biology studies. Yet, its routine sequencing may remain tricky despite next-generation sequencing technologies. An enrichment step is often necessary but not always straightforward depending on the initial DNA quality or quantity. Furthermore, the availability of close mitochondrial DNA reference sequences for non-model species limits the primer design for long-range PCR or bait synthesis. Here we propose an easy and cost-effective protocol without enrichment step for building and sequencing multiplexed Illumina libraries from small quantities of either high-quality or degraded genomic DNA. We validated the approach through the successful assembly of the complete mitogenome of 60 bats and 7 tunicates. Our protocol allows the sequencing and assembly of mitochondrial genomes from non-model species with sufficient coverage for applications in conservation genetics.

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.