An Fgf-Shh positive feedback loop drives growth in developing unpaired fins.

The origin and diversification of appendage types is a central question in vertebrate evolution. Understanding the genetic mechanisms that underlie fin and limb development can reveal relationships between different appendages. Here we demonstrate, using chemical genetics, a mutually agonistic interaction between Fgf and Shh genes in the developing dorsal fin of the channel catfish, Ictalurus punctatus. We also find that Fgf8 and Shh orthologs are expressed in the apical ectodermal ridge and zone of polarizing activity, respectively, in the median fins of representatives from other major vertebrate lineages. These findings demonstrate the importance of this feedback loop in median fins and offer developmental evidence for a median fin-first scenario for vertebrate paired appendage origins.

Differences in developmental potential predict the contrasting patterns of dental diversification in characiform and cypriniform fishes.

Morphological diversification during adaptive radiation may depend on factors external or internal to the lineage. We provide evidence for the latter in characiform fishes (tetras and piranhas), which exhibit extensive dental diversity. Phylogenetic character mapping supported regain of lost teeth as contributing to this diversity. To test for latent potential for dentition that would facilitate its evolutionary expansion, we overexpressed a tooth initiation signal, the tumour necrosis factor pathway ligand ectodysplasin, in a model characiform, the Mexican tetra (Astyanax mexicanus). This manipulation resulted in extensive ectopic dentition, in contrast with its previously reported limited effect in the zebrafish (Danio rerio). Tooth location in the order Cypriniformes, to which the zebrafish belongs, is much more restricted than in characiforms, a pattern that may be explained by differences in the retention of ancestral developmental potential. Our results suggest that differences in evolvability between lineages may lead to contrasting patterns of diversification.

Selection and constraint underlie irreversibility of tooth loss in cypriniform fishes.

The apparent irreversibility of the loss of complex traits in evolution (Dollo's Law) has been explained either by constraints on generating the lost traits or the complexity of selection required for their return. Distinguishing between these explanations is challenging, however, and little is known about the specific nature of potential constraints. We investigated the mechanisms underlying the irreversibility of trait loss using reduction of dentition in cypriniform fishes, a lineage that includes the zebrafish (Danio rerio) as a model. Teeth were lost from the mouth and upper pharynx in this group at least 50 million y ago and retained only in the lower pharynx. We identified regional loss of expression of the Ectodysplasin (Eda) signaling ligand as a likely cause of dentition reduction. In addition, we found that overexpression of this gene in the zebrafish is sufficient to restore teeth to the upper pharynx but not to the mouth. Because both regions are competent to respond to Eda signaling with transcriptional output, the likely constraint on the reappearance of oral teeth is the alteration of multiple genetic pathways required for tooth development. The upper pharyngeal teeth are fully formed, but do not exhibit the ancestral relationship to other pharyngeal structures, suggesting that they would not be favored by selection. Our results illustrate an underlying commonality between constraint and selection as explanations for the irreversibility of trait loss; multiple genetic changes would be required to restore teeth themselves to the oral region and optimally functioning ones to the upper pharynx.

Manipulation of Fgf and Bmp signaling in teleost fishes suggests potential pathways for the evolutionary origin of multicuspid teeth.

Teeth with two or more cusps have arisen independently from an ancestral unicuspid condition in a variety of vertebrate lineages, including sharks, teleost fishes, amphibians, lizards, and mammals. One potential explanation for the repeated origins of multicuspid teeth is the existence of multiple adaptive pathways leading to them, as suggested by their different uses in these lineages. Another is that the addition of cusps required only minor changes in genetic pathways regulating tooth development. Here we provide support for the latter hypothesis by demonstrating that manipulation of the levels of Fibroblast growth factor (Fgf) or Bone morphogenetic protein (Bmp) signaling produces bicuspid teeth in the zebrafish (Danio rerio), a species lacking multicuspid teeth in its ancestry. The generality of these results for teleosts is suggested by the conversion of unicuspid pharyngeal teeth into bicuspid teeth by similar manipulations of the Mexican Tetra (Astyanax mexicanus). That these manipulations also produced supernumerary teeth in both species supports previous suggestions of similarities in the molecular control of tooth and cusp number. We conclude that despite their apparent complexity, the evolutionary origin of multicuspid teeth is positively constrained, likely requiring only slight modifications of a pre-existing mechanism for patterning the number and spacing of individual teeth.

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.

bmp2b and bmp4 are dispensable for zebrafish tooth development.

Bone morphogenetic protein (Bmp) signaling has been shown to play important roles in tooth development at virtually all stages from initiation to hard tissue formation. The specific ligands involved in these processes have not been directly tested by loss-of-function experiments, however. We used morpholino antisense oligonucleotides and mutant analysis in the zebrafish to reduce or eliminate the function of bmp2b and bmp4, two ligands known to be expressed in zebrafish teeth and whose mammalian orthologs are thought to play important roles in tooth development. Surprisingly, we found that elimination of function of these two genes singly and in combination did not prevent the formation of mature, attached teeth. The mostly likely explanation for this result is functional redundancy with other Bmp ligands, which may differ between the zebrafish and the mouse.

Hedgehog signaling is required at multiple stages of zebrafish tooth development.

BACKGROUND: The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood. RESULTS: We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region. CONCLUSION: We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.

Hedgehog signalling controls eye degeneration in blind cavefish.

Hedgehog (Hh) proteins are responsible for critical signalling events during development but their evolutionary roles remain to be determined. Here we show that hh gene expression at the embryonic midline controls eye degeneration in blind cavefish. We use the teleost Astyanax mexicanus, a single species with an eyed surface-dwelling form (surface fish) and many blind cave forms (cavefish), to study the evolution of eye degeneration. Small eye primordia are formed during cavefish embryogenesis, which later arrest in development, degenerate and sink into the orbits. Eye degeneration is caused by apoptosis of the embryonic lens, and transplanting a surface fish embryonic lens into a cavefish optic cup can restore a complete eye. Here we show that sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh) gene expression is expanded along the anterior embryonic midline in several different cavefish populations. The expansion of hh signalling results in hyperactivation of downstream genes, lens apoptosis and arrested eye growth and development. These features can be mimicked in surface fish by twhh and/or shh overexpression, supporting the role of hh signalling in the evolution of cavefish eye regression.

Early life-history features associated with brood parasitism in the cuckoo catfish, Synodontis multipunctatus (Siluriformes: Mochokidae).

The cuckoo catfish, Synodontis multipunctatus, is the only known obligate brood parasite among fishes, exploiting the parental care of mouthbrooding cichlids endemic to Lake Tanganyika. Comparisons of this system to brood parasitism in birds may reveal broader principles that underlie the evolution of this life-history strategy in vertebrates. However, little is known about the features of the cuckoo catfish that enable this species to successfully parasitize cichlids. Here, we examine early ontogeny of the cuckoo catfish and compare it to that of its cichlid hosts as well as a non-parasitic congener. We found that cuckoo catfish embryos develop and hatch in advance of host embryos, and begin feeding on cichlid young just as they start to hatch. Overall timing of ontogeny in the cuckoo catfish was found to be similar to that of the substrate-spawning congener Synodontis lucipinnis, suggesting that more rapid development of the cuckoo catfish relative to cichlids is not a unique adaptation to brood parasitism. However, we found that cuckoo catfish progeny exhibit extensive morphological differences from S. lucipinnis, which may represent adaptations to brood parasitism. These life-history observations reveal both similarities and differences between the cuckoo catfish system and brood parasitism in other lineages. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

The Dlx gene complement of the leopard shark, Triakis semifasciata, resembles that of mammals: implications for genomic and morphological evolution of jawed vertebrates.

Extensive gene duplication is thought to have occurred in the vertebrate lineage after it diverged from cephalochordates and before the divergence of lobe- and ray-finned fishes, but the exact timing remains obscure. This timing was investigated by analysis of the Dlx gene family of a representative cartilaginous fish, the leopard shark, Triakis semifasciata. Dlx genes encode homeodomain transcription factors and are arranged in mammals as three convergently transcribed bigene clusters. Six Dlx genes were cloned from Triakis and shown to be orthologous to single mammalian Dlx genes. At least four of these are arranged in bigene clusters. Phylogenetic analyses of Dlx genes were used to propose an evolutionary scenario in which two genome duplications led to four Dlx bigene clusters in a common ancestor of jawed vertebrates, one of which was lost prior to the diversification of the group. Dlx genes are known to be involved in jaw development, and changes in Dlx gene number are mapped to the same branch of the vertebrate tree as the origin of jaws.

Zebrafish dentition in comparative context.

Studies of the zebrafish (Danio rerio) promise to contribute much to an understanding of the developmental genetic mechanisms underlying diversification of the vertebrate dentition. Tooth development, structure, and replacement in the zebrafish largely reflect the primitive condition of jawed vertebrates, providing a basis for comparison with features of the more extensively studied mammalian dentition. A distinctive derived feature of the zebrafish dentition is restriction of teeth to a single pair of pharyngeal bones. Such reduction of the dentition, characteristic of the order Cypriniformes, has never been reversed, despite subsequent and extensive diversification of the group in numbers of species and variety of feeding modes. Studies of the developmental genetic mechanism of dentition reduction in the zebrafish suggest a potential explanation for irreversibility in that tooth loss seems to be associated with loss of developmental activators rather than gain of repressors. The zebrafish and other members of the family Cyprinidae exhibit species-specific numbers and arrangements of pharyngeal teeth, and extensive variation in tooth shape also occurs within the family. Mutant screens and experimental alteration of gene expression in the zebrafish are likely to yield variant tooth number and shape phenotypes that can be compared with those occurring naturally within the Cyprinidae. Such studies may reveal the relative contribution to trends in dental evolution of biases in the generation of variation and sorting of this variation by selection or drift.

Transgenic analysis of Dlx regulation in fish tooth development reveals evolutionary retention of enhancer function despite organ loss.

It has been considered a "law" that a lost structure cannot reappear in evolution. The common explanation, that genes required for the development of the lost structure degrade by mutation, remains largely theoretical, however. Additionally, the extent to which this mechanism applies to systems of repeated parts, where individual modules are likely to exhibit few unique aspects of genetic control, is unclear. We investigated reversibility of evolution in one such system, the vertebrate dentition, using as a model loss of oral teeth in cypriniform fishes, which include the zebrafish. This evolutionary event, which occurred > 50 million years ago, has not been reversed despite subsequent diversification of feeding modes and retention of pharyngeal teeth. We asked whether the cis-regulatory region of a gene whose expression loss parallels cypriniform tooth loss, Dlx2b, retains the capacity for expression in oral teeth. We first created a zebrafish reporter transgenic line that recapitulates endogenous dlx2b expression. We then showed that this zebrafish construct drives reporter expression in oral teeth of the related characiform Astyanax mexicanus. This result, along with our finding that Dlx genes are required for normal tooth development, suggests that changes in trans-acting regulators of these genes were responsible for loss of cypriniform oral teeth. Preservation of oral enhancer function unused for > 50 million years could be the result of pleiotropic function in the pharyngeal dentition. If enhancers of other genes in the tooth developmental pathway are similarly preserved, teeth lost from specific regions may be relatively easy to reacquire in evolution.

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.

Developmental genetic mechanisms of evolutionary tooth loss in cypriniform fishes.

The fossil record indicates that cypriniform fishes, a group including the zebrafish, lost oral teeth over 50 million years ago. Despite subsequent diversification of feeding modes, no cypriniform has regained oral teeth, suggesting the zebrafish as a model for studying the developmental genetic basis of evolutionary constraint. To investigate the mechanism of cypriniform tooth loss, we compared the oral expression of seven genes whose mammalian orthologs are involved in tooth initiation in the zebrafish and the Mexican tetra, Astyanax mexicanus, a related species retaining oral teeth. The most significant difference we found was an absence in zebrafish oral epithelium of expression of dlx2a and dlx2b, transcription factors that are expressed in early Astyanax odontogenic epithelium. Analysis of orthologous genes in the Japanese medaka (Oryzias latipes) and a catfish (Synodontis multipunctatus) suggests that expression was lost in cypriniforms, rather than gained in Astyanax. Treatment of Astyanax with an inhibitor of Fibroblast growth factor (Fgf) signaling produced a partial phenocopy of the zebrafish oral region, in that oral teeth, and expression of dlx2a and dlx2b, were lost, whereas shh and pitx2, genes whose expression is present in zebrafish oral epithelium, were unaffected. We hypothesize that a loss of Fgf signaling to oral epithelium was associated with cypriniform tooth loss.

Zebrafish furin mutants reveal intricacies in regulating Endothelin1 signaling in craniofacial patterning.

Endothelin1 (Edn1) signaling promotes ventral character to the facial skeleton. In zebrafish edn1 mutants, the ventral jaw structures are severely reduced and fused to their dorsal counterparts, with a loss of joints that normally form at an intermediate dorsal-ventral position. Loss of function at another locus, sturgeon, also yields joint losses, but only mild reductions in the ventral jaw structures. We show that sturgeon encodes one of two orthologs of Furin present in zebrafish, and that both furin genes may function partially redundantly to activate Edn1 signaling. Supporting this hypothesis, early expression of edn1-dependent genes is downregulated in sturgeon (furinA) mutants. Later in development, expression of most of these genes recovers to near wild-type levels in furinA mutants but not in edn1 mutants. The recovery explains the less severe furinA mutant skeletal phenotype and suggests that late gene expression is dependent on a critical level of Edn1 signaling not present in the more severe edn1 mutants. However, expression defects in the intermediate joint-forming domains in both mutants persist, explaining the joint losses observed later in both mutants. We further show that in both mutants the arches fail to correctly undergo ventral elongation before skeletogenesis begins and propose a model in which this failure is largely responsible for the loss of an Edn1-dependent compartmentation of the arch into the intermediate and ventral domains.

Zebrafish fgf24 functions with fgf8 to promote posterior mesodermal development.

Fibroblast growth factor (Fgf) signaling plays an important role during development of posterior mesoderm in vertebrate embryos. Blocking Fgf signaling by expressing a dominant-negative Fgf receptor inhibits posterior mesoderm development. In mice, Fgf8 appears to be the principal ligand required for mesodermal development, as mouse Fgf8 mutants do not form mesoderm. In zebrafish, Fgf8 is encoded by the acerebellar locus, and, similar to its mouse otholog, is expressed in early mesodermal precursors during gastrulation. However, zebrafish fgf8 mutants have only mild defects in posterior mesodermal development, suggesting that it is not the only Fgf ligand involved in the development of this tissue. We report here the identification of an fgf8-related gene in zebrafish, fgf24, that is co-expressed with fgf8 in mesodermal precursors during gastrulation. Using morpholino-based gene inactivation, we have analyzed the function of fgf24 during development. We found that inhibiting fgf24 function alone has no affect on the formation of posterior mesoderm. Conversely, inhibiting fgf24 function in embryos mutant for fgf8 blocks the formation of most posterior mesoderm. Thus, fgf8 and fgf24 are together required to promote posterior mesodermal development. We provide both phenotypic and genetic evidence that these Fgf signaling components interact with no tail and spadetail, two zebrafish T-box transcription factors that are required for the development of all posterior mesoderm. Last, we show that fgf24 is expressed in early fin bud mesenchyme and that inhibiting fgf24 function results in viable fish that lack pectoral fins.

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.

Development and evolution of craniofacial patterning is mediated by eye-dependent and -independent processes in the cavefish Astyanax.

We studied the development and evolution of craniofacial features in the teleost fish, Astyanax mexicanus. This species has an eyed surface dwelling form (surface fish) and many different cave dwelling forms (cavefish) with various degrees of reduced eyes and pigmentation. The craniofacial features we examined are the tooth-bearing maxillary bones, the nasal and antorbital bones, the circumorbital bones, and the opercular bones, all of which show evolutionary modifications in different cavefish populations. Manipulations of eye formation by transplantation of the embryonic lens, by lentectomy, or by removing the optic vesicle showed that eye-dependent and -independent processes change both the surface fish and cavefish craniofacial skeletons. The size of the olfactory pits, which the nasal and antorbital bones define, and the size and positioning of the circumorbital bones were found to correlate with eye development. For the six suborbital bones (SO1-6), the relationship with the developing eye appears to be due to ossification initiated from foci in the suborbital canal of cranial neuromasts, whose patterning is also highly correlated with the presence or absence of an eye. By contrast, we found that the number of maxillary teeth, the number of SO3 bone elements, the positioning of SO4-6 with respect to the opercular bone, and the shape of the opercular bone are not dependent on eye formation and vary among different cavefish populations. The results suggest that evolution of the cavefish craniofacial skeleton is controlled by multiple developmental events, some a direct consequence of eye degeneration and others unrelated to loss of the eye.

Genomic structure and functional control of the Dlx3-7 bigene cluster.

The Dlx genes are involved in early vertebrate morphogenesis, notably of the head. The six Dlx genes of mammals are arranged in three convergently transcribed bigene clusters. In this study, we examine the regulation of the Dlx3-7 cluster of the mouse. We obtained and sequenced human and mouse P1 clones covering the entire Dlx3-7 cluster. Comparative analysis of the human and mouse sequences revealed several highly conserved noncoding regions within 30 kb of the Dlx3-7-coding regions. These conserved elements were located both 5' of the coding exons of each gene and in the intergenic region 3' of the exons, suggesting that some enhancers might be shared between genes. We also found that the protein sequence of Dlx7 is evolving more rapidly than that of Dlx3. We conducted a functional study of the 79-kb mouse genomic clone to locate cis-element activity able to reproduce the endogenous expression pattern by using transgenic mice. We inserted a lacZ reporter gene into the first exon of the Dlx3 gene by using homologous recombination in yeast. Strong lacZ expression in embryonic (E) stage E9.5 and E10.5 mouse embryos was found in the limb buds and first and second visceral arches, consistent with the endogenous Dlx3 expression pattern. This result shows that the 79-kb region contains the major cis-elements required to direct the endogenous expression of Dlx3 at stage E10.5. To test for enhancer location, we divided the construct in the mid-intergenic region and injected the Dlx3 gene portion. This shortened fragment lacking Dlx7-flanking sequences is able to drive expression in the limb buds but not in the visceral arches. This observation is consistent with a cis-regulatory enhancer-sharing model within the Dlx bigene cluster.