We describe analysis of zebrafish distal-less-related homeobox genes that may serve as specifiers of positional information in anterior regions of the CNS and in peripheral structures. We isolated three zebrafish genes, dlx2, dlx3, and dlx4, by screening embryonic cDNA libraries. Comparisons of the predicted sequences of the Dlx2, Dlx3, and Dlx4 proteins with distal-less proteins from other species suggest that vertebrate distal-less genes can be divided into four orthologous groups. We observed similarities but also unique features of the expression patterns of the zebrafish dlx genes. Among the three genes, dlx3 alone is expressed during gastrulation. Shortly after gastrulation, cells in the ventral forebrain rudiment express dlx2 and dlx4, but not dlx3, and hindbrain neural crest cells express only dlx2. Presumptive precursor cells of the olfactory placodes express dlx3 and dlx4 but not dlx2. Transcripts of dlx3 and dlx4 are present in overlapping subsets of cells in the auditory vesicle and in cells of the median fin fold, whereas dlx2 is never expressed in the auditory vesicle and only at low levels in localized regions of the median fin fold. Cells of the visceral arches and their primordia express all three dlx genes, but with different developmental time courses. We suggest that combinatorial expression of the dlx genes is part of a homeobox gene code specifying pattern formation or cell fate determination in the forebrain, in peripheral structures of the head, and in the fins.

Pluridisciplinary approaches led to the notion that fin regeneration is an intricate phenomenon involving epithelial-mesenchymal and reciprocal exchanges throughout the process as well as interactions between ray and interray tissue. The establishment of a blastema after fin amputation is the first event leading to the reconstruction of the missing part of the fin. Here, we review our knowledge on the origin of the blastema, its formation and growth, and of the mechanisms that control differentiation and patterning of the regenerate. Our current understanding results from studies of fin regeneration performed in various teleost fish over the past century. We also report the recent breakthroughs that have been

In the first part of this paper we review current knowledge regarding fish scales, focusing on elasmoid scales, the only type found in two model species, the zebrafish and the medaka. After reviewing the structure of scales and their evolutionary origin, we describe the formation of the squamation pattern. The regularity of this process suggests a pre-patterning of the skin before scale initiation. We then summarise the dynamics of scale development on the basis of morphological observations. In the absence of molecular data, these observations support the existence of genetic cascades involved in the control of scale development. In the second part of this paper, we illustrate the potential that scale development offers as a model to study organogenesis mediated by epithelial-mesenchymal interactions. Using the zebrafish (Danio rerio), we have combined alizarin red staining, light and transmission electron microscopy and in situ hybridisation using an anti-sense RNA probe for the sonic hedgehog (

Amputation of the zebrafish caudal fin stimulates regeneration of the dermal skeleton and reexpression of sonic hedgehog (shh)-signaling pathway genes. Expression patterns suggest a role for shh signaling in the secretion and patterning of the regenerating dermal bone, but a direct role has not been demonstrated. We established an in vivo method of gene transfection to express ectopically genes in the blastema of regenerating fins. Ectopic expression of shh or bmp2 in the blastema-induced excess bone deposition and altered patterning of the regenerate. The effects of shh ectopic expression could be antagonized by ectopic expression of chordin, an inhibitor of bone morphogenetic protein (bmp) signaling. We disrupted shh signaling in the regenerating fin by exposure to cyclopamine and found a dose-dependent inhibition of fin outgrowth, accumulation of melanocytes in the distal region of each fin ray, loss of actinotrichia, and reduction in cell proliferation in the mesenchyme. Morphological changes were accompanied by an expansion, followed by a reduction, in domains of shh expression and a rapid abolition of ptc1 expression. These results implicate shh and bmp2b signaling in the proliferation and͞or differentiation of specialized bone-secreting cells in the blastema and suggest shh expression may be controlled by regulatory feedback mechanisms that define the region of bone secretion in the outgrowing fin.T he extent of regenerative capacity varies between species and tissue types. Analysis of the regenerative events is not only informative in its own right but may also provide information pertaining to earlier morphogenetic events, because regeneration often recapitulates development. An example of this is the dermal skeleton component of the zebrafish (Danio rerio) caudal fin, which regenerates rapidly after amputation by processes reminiscent of those occurring during larval stages (for review, see ref. 1), including reexpression of developmental genes (2-6).The dermal skeleton of the zebrafish fin comprises mineralized lepidotrichia (fin rays) and more distal collagenous actinotrichia (Fig. 1A). The lepidotrichia are composed of two segmented hemirays that bifurcate periodically along their proximal-distal axis forming sister-ray branches. After amputation, epithelial cells migrate from the stump to cover the wound region (7, 8), beneath which a blastema containing undifferentiated proliferative mesenchymal cells forms (1). Scleroblasts then differentiate within the blastema at the epithelial͞mesenchymal interface and begin to secrete the matrix that will form the new dermal bone.During regeneration, the signaling molecule sonic hedgehog (shh) involved in patterning of many structures (reviewed in ref. 9), its membrane-receptor patched1 (ptc1) (9), and bone morphogenetic protein 2b (bmp2b), a member of the transforming growth factor-␤ family (10), are all initially reexpressed in a single domain at the distal stump of the amputated ray. Expression is found in a subset of cells in the basal layer of the epider...

The zebrafish caudal fin provides a simple model to study molecular mechanisms of dermal bone regeneration. We previously showed that misexpression of Bone morphogenetic protein 2b (Bmp2b) induces ectopic bone formation within the regenerate. Here we show that in addition to bmp2b and bmp4 another family member, bmp6, is involved in fin regeneration. We further investigated the function of BMP signaling by ectopically expressing the BMP signaling inhibitor Chordin which caused: (1) inhibition of regenerate outgrowth due to a decrease of blastema cell proliferation and downregulation of msxb and msxC expression and (2) reduced bone matrix deposition resulting from a defect in the maturation and function of bone-secreting cells. We then identified targets of BMP signaling involved in regeneration of the bone of the fin rays. runx2a/b and their target col10a1 were downregulated following BMP signaling inhibition. Unexpectedly, the sox9a/b transcription factors responsible for chondrocyte differentiation were detected in the non-cartilaginous fin rays, sox9a and sox9b were not only differentially expressed but also differentially regulated since sox9a, but not sox9b, was downregulated in the absence of BMP signaling. Finally, this analysis revealed the surprising finding of the expression, in the fin regenerate, of several factors which are normally the signatures of chondrogenic elements during endochondral bone formation although fin rays form through dermal ossification, without a cartilage intermediate.

The early development of teleost paired fins is strikingly similar to that of tetrapod limb buds and is controlled by similar mechanisms. One early morphological divergence between pectoral fins and limbs is in the fate of the apical ectodermal ridge (AER), the distal epidermis that rims the bud. Whereas the AER of tetrapods regresses after specification of the skeletal progenitors, the AER of teleost fishes forms a fold that elongates. Formation of the fin fold is accompanied by the synthesis of two rows of rigid, unmineralized fibrils called actinotrichia, which keep the fold straight and guide the migration of mesenchymal cells within the fold. The actinotrichia are made of elastoidin, the components of which, apart from collagen, are unknown. Here we show that two zebrafish proteins, which we name actinodin 1 and 2 (And1 and And2), are essential structural components of elastoidin. The presence of actinodin sequences in several teleost fishes and in the elephant shark (Callorhinchus milii, which occupies a basal phylogenetic position), but not in tetrapods, suggests that these genes have been lost during tetrapod species evolution. Double gene knockdown of and1 and and2 in zebrafish embryos results in the absence of actinotrichia and impaired fin folds. Gene expression profiles in embryos lacking and1 and and2 function are consistent with pectoral fin truncation and may offer a potential explanation for the polydactyly observed in early tetrapod fossils. We propose that the loss of both actinodins and actinotrichia during evolution may have led to the loss of lepidotrichia and may have contributed to the fin-to-limb transition.

Cell proliferation and cell movement during early regeneration of zebrafish caudal fins were examined by injecting BrdU and Di-I, respectively. In normal fins of adult fish, a small number of proliferating cells are observed in the epidermis only. Shortly following amputation, epithelial cells covered the wound to form the epidermal cap but did not proliferate. However, by 24 hr, epithelial cells proximal to the level of amputation were strongly labeled with BrdU. Label incorporation was also detected in a few mesenchymal cells. Proliferating cells in the basal epithelial layer were first observed at 48 hr at the level of the newly formed lepidotrichia. At 72 hr, proliferating mesenchymal cells were found distal to the plane of amputation whereas more proximal labeled cells included mainly those located between the lepidotrichia and the basal membrane. When BrdU-injected fins were allowed to regenerate for longer periods, labeled cells were observed in the apical epidermal cap, a location where cells are not thought to proliferate. This result is suggestive of cell migration. Epithelial cells, peripheral to the rays or in the tissue between adjacent rays, were labeled with Di-I and were shown to quickly migrate towards the site of amputation, the cells closer to the wound migrating faster. Amputation also triggered migration of cells of the connective tissue located between the hemirays. Although cell movement was induced up to seven segments proximal from the level of amputation, cells located within two segments from the wound provided the main contribution to the blastema. Thus, cell proliferation and migration contribute to the early regeneration of zebrafish fins.

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