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

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.     

Conservation and divergence of Bmp2a, Bmp2b, and Bmp4 expression patterns within and between dentitions of teleost fishes

The diversity of tooth location in teleost fishes provides an excellent system for comparing genetic divergence between teeth in different species (phylogenetic homologs) with divergence between teeth within one species (iterative homologs). We have chosen to examine the expression of three members of the bone morphogenetic protein (Bmp) family because they are known to play multiple roles in tooth development and evolution in tetrapod vertebrates. We characterized expression of Bmp2a, Bmp2b, and Bmp4 during the development of oral and pharyngeal dentitions in three species of teleost fishes, the zebrafish (Danio rerio), Mexican tetra (Astyanax mexicanus), and Japanese medaka (Oryzias latipes). We found that expression in teleosts is generally highly conserved, with minor differences found among both iteratively homologous and phylogenetically homologous teeth. Expression of orthologous genes differs in several ways between the teeth of teleost fishes and those of the mouse, but between these vertebrate groups the summed expression pattern of Bmp genes is highly conserved. Significantly, the toothless oral region of the zebrafish lacks Bmp expression domains found in teleosts with oral teeth, implicating these genes in evolutionary tooth loss. We conclude that Bmp expression has been largely conserved in vertebrate tooth development over evolutionary time, and that loss of Bmp expression is correlated with region-specific loss of the dentition in a major group of fishes.     

Differential expression of orthologous Dlx genes in zebrafish and mice: implications for the evolution of the Dlx homeobox gene family

Dlx homeobox genes of vertebrates are often organised as physically linked pairs in which the two genes are transcribed convergently (tail-to-tail arrangement). Three such Dlx pairs have been found in mouse, human, and zebrafish and are thought to have originated from the duplication of an ancestral gene pair. These pairs include Dlx1/Dlx2, Dlx7/Dlx3, and Dlx6/Dlx5 (the zebrafish orthologue of Dlx5 is named dlx4). Expression patterns of physically linked Dlx genes overlap extensively. Furthermore, orthologous Dlx genes often show highly similar expression patterns. We analysed Dlx expression during the gastrula and early somitogenesis of the mouse and zebrafish. It was found that expression of the mouse Dlx6 gene takes place in the rostral ectoderm and presumptive olfactory and otic placodes with patterns similar to the previously reported expression of the physically linked Dlx5 gene. However, we observed only very weak expression of the mouse Dlx3 gene at the same stage. This contrasts with the expression of dlx genes in zebrafish where dlx3 and dlx7, but not dlx4 and dlx6 are expressed during gastrulation in the rostral ectoderm and presumptive placodes. Thus, Dlx expression patterns at early stages are better conserved between paralogous pairs of physically linked genes than between orthologous pairs. This suggests that early expression of Dlx genes existed prior to the duplications that led to the multiple pairs of physically linked genes but was differentially conserved in different paralogs in zebrafish and mice.     

An Ancient Gene Network Is Co-opted for Teeth on Old and New Jaws

Vertebrate dentitions originated in the posterior pharynx of jawless fishes more than half a billion years ago. As gnathostomes (jawed vertebrates) evolved, teeth developed on oral jaws and helped to establish the dominance of this lineage on land and in the sea. The advent of oral jaws was facilitated, in part, by absence of hox gene expression in the first, most anterior, pharyngeal arch. Much later in evolutionary time, teleost fishes evolved a novel toothed jaw in the pharynx, the location of the first vertebrate teeth. To examine the evolutionary modularity of dentitions, we asked whether oral and pharyngeal teeth develop using common or independent gene regulatory pathways. First, we showed that tooth number is correlated on oral and pharyngeal jaws across species of cichlid fishes from Lake Malawi (East Africa), suggestive of common regulatory mechanisms for tooth initiation. Surprisingly, we found that cichlid pharyngeal dentitions develop in a region of dense hox gene expression. Thus, regulation of tooth number is conserved, despite distinct developmental environments of oral and pharyngeal jaws; pharyngeal jaws occupy hox-positive, endodermal sites, and oral jaws develop in hox-negative regions with ectodermal cell contributions. Next, we studied the expression of a dental gene network for tooth initiation, most genes of which are similarly deployed across the two disparate jaw sites. This collection of genes includes members of the ectodysplasin pathway, eda and edar, expressed identically during the patterning of oral and pharyngeal teeth. Taken together, these data suggest that pharyngeal teeth of jawless vertebrates utilized an ancient gene network before the origin of oral jaws, oral teeth, and ectodermal appendages. The first vertebrate dentition likely appeared in a hox-positive, endodermal environment and expressed a genetic program including ectodysplasin pathway genes. This ancient regulatory circuit was co-opted and modified for teeth in oral jaws of the first jawed vertebrate, and subsequently deployed as jaws enveloped teeth on novel pharyngeal jaws. Our data highlight an amazing modularity of jaws and teeth as they coevolved during the history of vertebrates. We exploit this diversity to infer a core dental gene network, common to the first tooth and all of its descendants.     

Japanese medaka Hox paralog group 2: insights into the evolution of Hox PG2 gene composition and expression in the Osteichthyes

Hox paralog group 2 (PG2) genes function to specify the development of the hindbrain and pharyngeal arch-derived structures in the Osteichthyes. In this article, we describe the cDNA cloning and embryonic expression analysis of Japanese medaka (Oryzias latipes) Hox PG2 genes. We show that there are only two functional canonical Hox genes, hoxa2a and b2a, and that a previously identified hoxa2b gene is a transcribed pseudogene, psihoxa2b. The functional genes, hoxa2a and b2a, were expressed in developing rhombomeres and pharyngeal arches in a manner that was relatively well conserved compared with zebrafish (Danio rerio) but differed significantly from orthologous striped bass (Morone saxatilis) and Nile tilapia (Oreochromis niloticus) genes, which, we suggest, may be owing to effects of post-genome duplication loss of a Hox PG2 gene in the medaka and zebrafish lineages. psihoxa2b was expressed at readily detectable levels in several noncanonical Hox expression domains, including the ventral aspect of the neural tube, the pectoral fin buds and caudal-most region of the embryonic trunk, indicative that regulatory control elements needed for spatio-temporal expression have diverged from their ancestral counterparts. Comparative expression analyses showed medaka hoxa2a and b2a expression in the 2nd pharyngeal arch (PA2) beyond the onset of chondrogenesis, which, according to previous hypotheses, suggests these genes function redundantly as selector genes of PA2 identity. We conclude that Hox PG2 gene composition and expression have diverged significantly during osteichthyan evolution and that this divergence in teleosts may be related to lineage-dependent differential gene loss following an actinopterygian-specific whole genome duplication.     

Fgf signaling is required for zebrafish tooth development

We have investigated fibroblast growth factor (FGF) signaling during the development of the zebrafish pharyngeal dentition with the goal of uncovering novel roles for FGFs in tooth development as well as phylogenetic and topographic diversity in the tooth developmental pathway. We found that the tooth-related expression of several zebrafish genes is similar to that of their mouse orthologs, including both epithelial and mesenchymal markers. Additionally, significant differences in gene expression between zebrafish and mouse teeth are indicated by the apparent lack of fgf8 and pax9 expression in zebrafish tooth germs. FGF receptor inhibition with SU5402 at 32 h blocked dental epithelial morphogenesis and tooth mineralization. While the pharyngeal epithelium remained intact as judged by normal pitx2 expression, not only was the mesenchymal expression of lhx6 and lhx7 eliminated as expected from mouse studies, but the epithelial expression of dlx2a, dlx2b, fgf3, and fgf4 was as well. This latter result provides novel evidence that the dental epithelium is a target of FGF signaling. However, the failure of SU5402 to block localized expression of pitx2 suggests that the earliest steps of tooth initiation are FGF-independent. Investigations of specific FGF ligands with morpholino antisense oligonucleotides revealed only a mild tooth shape phenotype following fgf4 knockdown, while fgf8 inhibition revealed only a subtle down-regulation of dental dlx2b expression with no apparent effect on tooth morphology. Our results suggest redundant FGF signals target the dental epithelium and together are required for dental morphogenesis. Further work will be required to elucidate the nature of these signals, particularly with respect to their origins and whether they act through the mesenchyme.     

Conserved deployment of genes during odontogenesis across osteichthyans

Odontogenesis has only been closely scrutinized at the molecular level in the mouse, an animal with an extremely restricted dentition of only two types and one set. However, within osteichthyans many species display complex and extensive dentitions, which questions the extent to which information from the mouse is applicable to all osteichthyans. We present novel comparative molecular and morphological data in the rainbow trout (Oncorhynchus mykiss) that show that three genes, essential for murine odontogenesis, follow identical spatial-temporal expression. Thus, at all tooth bud sites, epithelial genes Pitx-2 and Shh initiate the odontogenic cascade, resulting in dental mesenchymal Bmp-4 expression, importantly, including the previously unknown formation of replacement teeth. Significantly, this spatial-temporal sequence is the same for marginal and lingual dentitions, but we find notable differences regarding the deployment of Pitx-2 in the developing pharyngeal dentition. This difference may be highly significant in relation to the theory that dentitions may have evolved from pharyngeal tooth sets in jawless fishes. We have provided the first data on operational genes in tooth development to show that the same signalling genes choreograph this evolutionary stable event in fishes since the osteichthyan divergence 420 Myr ago, with the identical spatial-temporal expression as in mammals.     

A zebrafish forebrain-specific zinc finger gene can induce ectopic dlx2 and dlx6 expression

Identification of the earliest forebrain-specific markers should facilitate the elucidation of molecular events underlying vertebrate forebrain determination and specification. Here we report the sequence and characterization of fez (forebrain embryonic zinc finger), a gene that is specifically expressed in the embryonic forebrain of zebrafish. Fez encodes a putative nuclear zinc finger protein that is highly conserved in Drosophila, zebrafish, Xenopus, mouse, and human. In zebrafish, the expression of fez becomes detectable at the anterior edge of the presumptive neuroectoderm by 70% epiboly. During the segmentation period, its expression is completely restricted to the rostral region of the prospective forebrain. At approximately 24 h postfertilization, fez expression is mostly confined to the telencephalon and the anterior-ventral region of the diencephalon. Although fez expression is present in one-eyed pinhead (oep) and cyclops (cyc) zebrafish mutants, the pattern is altered. Forced expression of fez induces ectopic expression of dlx2 and dlx6, two genes involved in brain development. Knockdown of fez function using a morpholino-based antisense oligo inhibited dlx2 expression in the ventral forebrain. Our studies indicate that fez is one of the earliest markers specific for the anterior neuroectoderm and it may play a role in forebrain development by regulating Dlx gene expression.     

Differential expression of hoxa2a and hoxa2b genes during striped bass embryonic development

Here, we report the cloning and expression analysis of two previously uncharacterized paralogs group 2 Hox genes, striped bass hoxa2a and hoxa2b, and the developmental regulatory gene egr2. We demonstrate that both Hox genes are expressed in the rhombomeres of the developing hindbrain and the pharyngeal arches albeit with different spatio-temporal distributions relative to one another. While both hoxa2a and hoxa2b share the r1/r2 anterior boundary of expression characteristic of the hoxa2 paralog genes of other species, hoxa2a gene expression extends throughout the hindbrain, whereas hoxa2b gene expression is restricted to the r2-r5 region. Egr2, which is used in this study as an early developmental marker of rhombomeres 3 and 5, is expressed in two distinct bands with a location and spacing typical for these two rhombomeres in other species. Within the pharyngeal arches, hoxa2a is expressed at higher levels in the second pharyngeal arch, while hoxa2b is more strongly expressed in the posterior arches. Further, hoxa2b expression within the arches becomes undetectable at 60hpf, while hoxa2a expression is maintained at least up until the beginning of chondrogenesis. Comparison of the striped bass HoxA cluster paralog group 2 (PG2) genes to their orthologs and trans-orthologs shows that the striped bass hoxa2a gene expression pattern is similar to the overall expression pattern described for the hoxa2 genes in the lobe-finned fish lineage and for the hoxa2b gene from zebrafish. It is notable that the pharyngeal arch expression pattern of the striped bass hoxa2a gene is more divergent from its sister paralog, hoxa2b, than from the zebrafish hoxa2b gene. Overall, our results suggest that differences in the Hox PG2 gene complement of striped bass and zebrafish affects both their rhombomeric and pharyngeal arch expression patterns and may account for the similarities in pharyngeal arch expression between striped bass hoxa2a and zebrafish hoxa2b.     

Ectoderm,endoderm, and the evolution of heterodont dentitions

Mammalian dentitions consist of different shapes/types of teeth that are positioned in different regions of the jaw (heterodont) whereas in many fish and reptiles all teeth are of similar type (homodont). The process by which heterodont dentitions have evolved in mammals is not understood. In many teleosts teeth develop in the pharynx from endoderm (endodermal teeth), whereas mammalian teeth develop from the oral ectoderm indicating that teeth can develop (and thus possibly evolve) via different mechanisms. In this article, we compare the molecular characteristics of pharyngeal/foregut endoderm with the molecular characteristics of oral ectoderm during mouse development. The expression domains of Claudin6, Hnf3β, α‐fetoprotein, Rbm35a, and Sox2 in the embryonic endoderm have boundaries overlapping the molar tooth‐forming region, but not the incisor region in the oral ectoderm. These results suggest that molar teeth (but not incisors) develop from epithelium that shares molecular characteristics with pharyngeal endoderm. This opens the possibility that the two different theories proposed for the evolution of teeth may both be correct. Multicuspid (eg. molars) having evolved from the externalization of endodermal teeth into the oral cavity and monocuspid (eg. incisors) having evolved from internalization of ectodermal armour odontodes of ancient fishes. The two different mechanisms of tooth development may have provided the developmental and genetic diversity on which evolution has acted to produce heterodont dentitions in mammals. genesis 48:382–389, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular cloning and expression of the col2a1a and col2a1b genes in the medaka, Oryzias latipes

The Col2a1 gene is expressed in notochord, otic vesicle, cartilaginous tissue and the anlage of endochondral bone during development in higher vertebrates. Type II collagen, a homotrimeric product of the Col2a1 gene, functions as a key regulatory protein for cartilage development and endochondral ossification. In medaka and zebrafish, a single homolog of the col2a1 gene has been identified. However, it is necessary to note that many genes are duplicated in teleost fishes. To clarify function of col2a1 genes in teleost fishes and to further understand the process of cartilage development and endochondral ossification, we cloned and mapped the gene loci of two col2a1 orthologs in medaka. The proteins encoded by both medaka col2a1a and col2a1b genes were highly conserved (85.3% and 82.6%) relative to human COL2A1, but synteny was not observed. We also examined the expression patterns of col2a1a and col2a1b during embryonic development. Whole-mount insitu hybridization data suggests that expression patterns of both medaka co2a1a and col2a1b genes are similar to that of zebrafish co2a1 in the early embryonic stages. In medaka, the two col2a1 genes show a closely correlated pattern of spatial and temporal expression. In late embryonic stages, however, there were differences in both expression patterns in the pectoral fin. This study is the first report of two homologs of col2a1 in teleosts and also the first examination of col2a1a and col2a1b expression patterns in this group.     

Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.