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.     

Ancient vertebrate conserved noncoding elements have been evolving rapidly in teleost fishes

Vertebrate genomes contain thousands of conserved noncoding elements (CNEs) that often function as tissue-specific enhancers. In this study, we have identified CNEs in human, dog, chicken, Xenopus, and four teleost fishes (zebrafish, stickleback, medaka, and fugu) using elephant shark, a cartilaginous vertebrate, as the base genome and investigated the evolution of these ancient vertebrate CNEs (aCNEs) in bony vertebrate lineages. Our analysis shows that aCNEs have been evolving at different rates in different bony vertebrate lineages. Although 78-83% of CNEs have diverged beyond recognition ("lost") in different teleost fishes, only 24% and 40% have been lost in the chicken and mammalian lineages, respectively. Relative rate tests of substitution rates in CNEs revealed that the teleost fish CNEs have been evolving at a significantly higher rate than those in other bony vertebrates. In the ray-finned fish lineage, 68% of aCNEs were lost before the divergence of the four teleosts. This implicates the "fish-specific" whole-genome duplication in the accelerated evolution and the loss of a large number of both copies of duplicated CNEs in teleost fishes. The aCNEs are rich in tissue-specific enhancers and thus many of them are likely to be evolutionarily constrained cis-regulatory elements. The rapid evolution of aCNEs might have affected the expression patterns driven by them. Transgenic zebrafish assay of some human CNE enhancers that have been lost in teleosts has indicated instances of conservation or changes in trans-acting factors between mammals and fishes.     

Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung‐to‐gas bladder evolutionary transition in ray‐finned fishes

The key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray‐finned fishes is the gas bladder, an air‐filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe‐finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral‐to‐dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin, and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gas bladder development, Nkx2.1 and Sox2 expression are inconsistent with the hypothesis that they regulate the direction of gas bladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gas bladder development, Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of Actinopteri (ray‐finned fishes excluding bichir) but absent from mammalian genomes. We hypothesize that Bmp16 was recruited to regulate gas bladder development in the Actinopteri in place of Bmp4.     

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.     

Consequences of hoxb1 duplication in teleost fish

Vertebrate evolution is characterized by gene and genome duplication events. There is strong evidence that a whole-genome duplication occurred in the lineage leading to the teleost fishes. We have focused on the teleost hoxb1 duplicate genes as a paradigm to investigate the consequences of gene duplication. Previous analysis of the duplicated zebrafish hoxb1 genes suggested they have subfunctionalized. The combined expression pattern of the two zebrafish hoxb1 genes recapitulates the expression pattern of the single Hoxb1 gene of tetrapods, possibly due to degenerative changes in complementary cis-regulatory elements of the duplicates. Here we have tested the hypothesis that all teleost duplicates had a similar fate post duplication, by examining hoxb1 genes in medaka and striped bass. Consistent with this theory, we found that the ancestral Hoxb1 expression pattern is subdivided between duplicate genes in a largely similar fashion in zebrafish, medaka, and striped bass. Further, our analysis of hoxb1 genes reveals that sequence changes in cis-regulatory regions may underlie subfunctionalization in all teleosts, although the specific changes vary between species. It was previously shown that zebrafish hoxb1 duplicates have also evolved different functional capacities. We used misexpression to compare the functions of hoxb1 duplicates from zebrafish, medaka and striped bass. Unexpectedly, we found that some biochemical properties, which were paralog specific in zebrafish, are conserved in both duplicates of other species. This work suggests that the fate of duplicate genes varies across the teleost group.     

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.     

Temporal pattern of loss/persistence of duplicate genes involved in signal transduction and metabolic pathways after teleost-specific genome duplication

Background  

Recent genomic studies have revealed a teleost-specific third-round whole genome duplication (3R-WGD) event occurred in a common ancestor of teleost fishes. However, it is unclear how the genes duplicated in this event were lost or persisted during the diversification of teleosts, and therefore, how many of the duplicated genes contribute to the genetic differences among teleosts. This subject is also important for understanding the process of vertebrate evolution through WGD events. We applied a comparative evolutionary approach to this question by focusing on the genes involved in long-term potentiation, taste and olfactory transduction, and the tricarboxylic acid cycle, based on the whole genome sequences of four teleosts; zebrafish, medaka, stickleback, and green spotted puffer fish.     

Making teeth to order: conserved genes reveal an ancient molecular pattern in paddlefish (Actinopterygii)

Ray-finned fishes (Actinopterygii) are the dominant vertebrate group today (+30 000 species, predominantly teleosts), with great morphological diversity, including their dentitions. How dental morphological variation evolved is best addressed by considering a range of taxa across actinopterygian phylogeny; here we examine the dentition of Polyodon spathula (American paddlefish), assigned to the basal group Acipenseriformes. Although teeth are present and functional in young individuals of Polyodon, they are completely absent in adults. Our current understanding of developmental genes operating in the dentition is primarily restricted to teleosts; we show that shh and bmp4, as highly conserved epithelial and mesenchymal genes for gnathostome tooth development, are similarly expressed at Polyodon tooth loci, thus extending this conserved developmental pattern within the Actinopterygii. These genes map spatio-temporal tooth initiation in Polyodon larvae and provide new data in both oral and pharyngeal tooth sites. Variation in cellular intensity of shh maps timing of tooth morphogenesis, revealing a second odontogenic wave as alternate sites within tooth rows, a dental pattern also present in more derived actinopterygians. Developmental timing for each tooth field in Polyodon follows a gradient, from rostral to caudal and ventral to dorsal, repeated during subsequent loss of teeth. The transitory Polyodon dentition is modified by cessation of tooth addition and loss. As such, Polyodon represents a basal actinopterygian model for the evolution of developmental novelty: initial conservation, followed by tooth loss, accommodating the adult trophic modification to filter-feeding.     

Conserved expression of<Emphasis Type="Italic"> Hoxa1</Emphasis> in neurons at the ventral forebrain/midbrain boundary of vertebrates

The previously described expression patterns of zebrafish and mouse Hoxa1 genes are seemingly very disparate, with mouse Hoxa1 expressed in the gastrula stage hindbrain and the orthologous zebrafish hoxa1a gene expressed in cell clusters within the ventral forebrain and midbrain. To investigate the evolution of Hox gene deployment within the vertebrate CNS, we have performed a comparative expression analysis of Hoxa1 orthologs in a range of vertebrate species, comprising representatives from the two major lineages of vertebrates (actinopterygians and sarcopterygians). We find that fore/midbrain expression of hoxa1a is conserved within the teleosts, as it is shared by the ostariophysan teleost zebrafish (Danio rerio) and the distantly related acanthopterygian teleost medaka (Oryzias latipes). Furthermore, we find that in addition to the described gastrula stage hindbrain expression of mouse Hoxa1, there is a previously unreported neurula stage expression domain, again located more anteriorly at the ventral fore/midbrain boundary. A two-phase expression profile in early hindbrain and later fore/midbrain is shared by the other tetrapod model organisms chick and Xenopus. We show that the anterior Hoxa1 expression domain is localized to the anterior terminus of the medial longitudinal fasciculus (MLF) in mouse, chick, and zebrafish. These findings suggest that anterior expression of Hoxa1 is a primitive characteristic that is shared by the two major vertebrate lineages. We conclude that Hox gene expression within the vertebrate CNS is not confined exclusively to the segmented hindbrain and spinal cord, but rather that a presumptive fore/midbrain expression domain arose early in vertebrate origins and has been conserved for at least 400 million years.     

Characterization of Hox genes in the bichir,Polypterus palmas

It has been suggested that the increase in the number of Hox genes may have been one of the key events in vertebrate evolution. Invertebrates have one Hox cluster, while mammals have four. Interestingly, the number of Hox gene clusters is greater in the teleost fishes, zebrafish and medaka, than in mouse and human. The greater number of Hox clusters in the teleosts suggests that Hox gene duplication events have occurred during the radiation of ray-finned fishes. The question is when the Hox gene duplication event(s) that lead to seven Hox clusters in the teleosts actually occurred.We have addressed this question by studying the Hox genes in the bichir, Polypterus palmas. A preliminary PCR-estimation of the number of Hox genes suggests that Polypterus has five different Hox9 cognate group genes, which may be an indication of more than four Hox clusters in the bichir.     

Conservation of a vitellogenin gene cluster in oviparous vertebrates and identification of its traces in the platypus genome

Vitellogenin (Vtg) derivatives are the main egg-yolk proteins in most oviparous animal species, and are, therefore, key players in reproduction and embryo development. Conserved synteny and phylogeny were used to identify a Vtg gene cluster (VGC) that had been evolutionarily conserved in most oviparous vertebrates, encompassing the three linked Vtgs on chicken (Gallus gallus) chromosome 8. Tandem arranged homologs to chicken VtgII and VtgIII were retrieved in similar locations in Xenopus (Xenopus tropicalis) and homologous transcribed inverted genes were found in medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), pufferfish (Takifugu rubripes), and Tetrahodon (Tetraodon nigroviridis), while zebrafish (Danio rerio) Vtg3 may represent a residual trace of VGC in this genome. Vtgs were not conserved in the paralogous chromosomal segment attributed to a whole-genome duplication event in the ancestor of teleosts, while tandem duplicated forms have survived the recent African clawed frog (Xenopus laevis) tetraploidization. Orthologs to chicken VtgI were found in similar locations in teleost fish, as well as in the platypus (Ornithorhynchus anatinus). Additional Vtg fragments found suggested that VGC had been conserved in this egg-laying mammal. A low ratio of nonsynonymous-to-synonymous substitution values and the paucity of pseudogene features suggest functional platypus Vtg products. Genomic identification of Vtgs, Apob, and Mtp in this genome, together with maximum likelihood and Bayesian inference phylogenetic analyses, support the existence of these three large lipid transfer protein superfamily members at the base of the mammalian lineage. In conclusion, the establishment of a VGC in the vertebrate lineage predates the divergence of ray-finned fish and tetrapods and the shift in reproductive and developmental strategy observed between prototherians and therians may be associated with its loss, as shown by its absence from the genomic resources currently available from therians.