The fins of actinopterygian can regenerate following amputation. Classical papers have shown that the ray, a structural unit of these fins, might regenerate independent of this appendage. Each fin ray is formed by two apposed contralateral hemirays. A hemiray may autonomously regenerate and segmentate in a position-independent manner. This is observed when heterotopically grafted into an interray space, after amputation following extirpation of the contralateral hemiray or when simply ablated. During this process, a proliferating hemiblastema is formed, as shown by bromodeoxyuridine incorporation, from which the complete structure will regenerate. This hemiblastema shows a patterning of gene expression domain similar to half ray blastema. Interactions between contralateral hemiblastema have been studied by recombinant rays composed of hemirays from different origins on the proximo-distal or dorso-ventral axis of the caudal fin. Dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocianine perchlorate labeling of grafted tissues was used as tissular marker. Our results suggest both that there are contralateral interactions between hemiblastema of each ray, and that hemiblastema may vary its morphogenesis, always differentiating as their host region. These non-autonomous, position-dependent interactions control coordinated bifurcations, segment joints and ray length independently. A morphological study of the developing and regenerating fin of another long fin mutant zebrafish suggests that contralateral hemiblastema interactions are perturbed in this mutant.

We have characterized two new members of the Hedgehog (Hh) family in zebrafish, ihha and dhh, encoding for orthologues of the tetrapod Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh) genes, respectively. Comparison of ihha and Type X collagen (col10a1) expression during skeletal development show that ihha transcripts are located in hypertrophic chondrocytes of cartilaginous elements of the craniofacial and fin endoskeleton. Surprisingly, col10a1 expression was also detected in cells forming intramembranous bones of the head and in flat cells surrounding cartilaginous structures. The expression of col10a1 in both endochondral and intramembranous bones reflects an atypical composition of the extracellular matrix of the zebrafish craniofacial skeleton. In addition, during fin ray regeneration, both ihha and col10a1 are detected in scleroblasts, osteoblast-like cells secreting the matrix of the dermal bone fin ray. The presence of cartilage markers suggests that the dermal fin ray possesses an intermediate phenotype between cartilage and bone.

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.

SUMMARY The even‐skipped related genes (evx) encode homeodomain‐containing transcription factors that play key roles in body patterning and neurogenesis in a wide array of Eumetazoa species. It is thought that the genome of the last common ancestor of Chordata contained a unique evx gene linked to a unique ancestral Hox complex. During subsequent evolution, two rounds of whole genome duplication followed by individual gene losses gave rise to three paralogs: evx1, evx2, and eve1. Then, eve1 was maintained in Actinopterygii lineage but not in Tetrapoda. To explain this discrepancy, we examined the expression patterns of the evx1 homologue, Xhox3, in Xenopus laevis and that of evx1 and eve1 in Danio rerio. We show here that Xhox3 is expressed in a manner that closely reflects the inferred expression pattern of the evx1 gene in the last common ancestor of Vertebrata (i.e., in gastrula, the central nervous system, the posterior gut, and the tip of the growing tail). Zebrafish evx1 and Xenopus Xhox3 are expressed in homologous cell lineages of the central nervous system and of the posterior gut, but evx1 was undetectable in the gastrula and the tail bud. Strikingly, eve1 is the only evx gene of zebrafish to be expressed in these two latter regions. Thus, the ancestral expression pattern of evx1 in vertebrates appears to have been distributed between evx1 and eve1 in zebrafish. We propose that evx1 and eve1 underwent a complementary loss of expression domain in zebrafish that allowed the maintenance of the two paralogs in accordance with the duplication‐degeneration‐complementation model. It is important to note that, in zebrafish, Evx1 and Eve1 have lost most of the protein domain upstream of the homeodomain. In addition, Eve1 has accumulated substitutions in positions that are highly conserved in all other Evx proteins. Thus, the reduction of the expression domain of both evx1 and eve1 in zebrafish appears to be associated with the modification of constraints on the protein sequences, allowing the shortening of both genes and an accelerated substitution rate in eve1.

We have performed a time course analysis of the expression of Sonichedgehog (shh) and patched1 (ptc1) in response to exogenous retinoic acid (RA) application to get some insight into the mechanism(s) underlying the formation of a mirror-image duplication of shh and ptc1 domains of expression in the pectoral fin buds of zebrafish. We have shown that RA exposure during the early stages of pectoral fin development first results in a rapid decrease or complete loss of shh/ptc1 expression. This is followed by reappearance of transcripts in the normal posterior domain, then by a stage-dependent and progressive expansion of the shh domain from the ZPA towards the anterior margin of the bud. Shh transcripts are induced in mesenchymal cells underlying the ventral ectoderm at the base of the bud. Once shh expression is activated in the most anterior cells, the number of shh-expressing cells increases in this region, possibly through an amplification mechanism involving signals from the apical ectodermal ridge. At this time, shh expression disappears from cells centrally located in the bud, resulting in the formation of the two distinct domains. An anterior extension of shh expression is also obtained in syu mutants with impaired shh function, suggesting that shh induction across the fin bud is independent of shh signaling. This study suggests the existence of complex mechanisms controlling the spatial and temporal expression of shh in the developing fin bud.

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 and/or 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.

The dermoskeleton of zebrafish fins is composed of actinotrichia and segmented bony rays, or lepidotrichia, which grow by successive addition of distal segments. The present study shows that evx1, a new zebrafish even-skipped related gene (Thaëron et al., 2000) displays during bony ray morphogenesis, a unique repetitive expression pattern along the proximodistal axis of the fin. Whole-mount in situ hybridization performed on larvae and adult regenerating fins show that evx1 signal appears as parallel dash lines crossing the width of each developing and regenerating rays, in a ladder-like fashion. Cytological studies show that a subpopulation of bone forming cells (scleroblasts) expresses evx1 at the level of the joint between two adjacent segments except in the apical part of the differentiating ray where evx1 expression precedes the formation of the joint. This distal transcription is turned on again only when the latest differentiating segment reached its final size and might label the putative next segment boundary. This suggests the existence of a molecular mechanism controlling the periodic expression of evx1 which could be involved in the establishment of segment boundaries during fin ray morphogenesis, and could play a key role during dermal skeleton patterning.

The even-skipped–related homeobox genes (evx) are widely distributed through animal kingdom and are thought to play key role in posterior body patterning and neurogenesis. We have cloned and analyzed the expression of evx1 in zebrafish (see also Borday et al. (Dev. Dyn. 220 (2001) in press) which displays a dynamic and restricted expression pattern during neurogenesis. In spinal cord, rhombencephalon, and epiphysis, evx1 is expressed in several subsets of emerging interneurones prior to their axonal outgrowth, identified as primary interneurones and a subset of Pax2.1+ commissural interneurones. In the hindbrain, evx1 is expressed in reticulospinal interneurones of rhombomeres 5 and 6 as well as in rhombomere 7 interneurones. The latest emerging evx1+ interneurones in the hindbrain correspond to commissural interneurones. evx1 is also dynamically transcribed during the formation of the posterior gut and the uro-genital system in mesenchymal cells that border the pronephric ducts, the wall of the pronephric duct, and later in the posterior gut and the wall of the uro-genital opening. In larvae, the ano-rectal epithelium and the muscular layer that surrounds the analia-genitalia region remain stained up to 27 days. In contrast other vertebrates, evx1displays no early nor caudal expression in zebrafish.