Fossil data suggest that limbs evolved from fish fins by sequential elaboration of their distal endoskeleton, giving rise to the autopod close to the tetrapod origin. This elaboration may have occurred by a simultaneous reduction of the distal ectodermal fold of fish fins. Modulation of 5′Hoxd gene transcription, through tetrapod-specific digit enhancers, has been suggested as a possible evolutionary mechanism involved in these morphological transformations. Here, we overexpress hoxd13a in zebrafish to investigate the impact of increasing 5′Hoxd expression during fin development. This overexpression causes increased proliferation, distal expansion of chondrogenic tissue and finfold reduction. In addition, we also show that the tetrapod-specific 5′Hoxd enhancer CsC promotes similar expression in zebrafish fins and mouse limbs. Our results support the idea that modulation of 5′Hoxd gene expression, by acquisition of novel enhancer elements, offered the substrate for the evolution of fins and the origin of tetrapod limbs.

Here we have tested these two conditions in the teleost zebrafish. We find that overexpression of hoxd13a in fins causes distal overgrowth of chondrogenic tissue expressing markers characteristic of autopod development. Moreover, this occurs with a concomitant finfold reduction, a phenotype that reflects the morphological changes expected during fin evolution (). In addition, we evaluated the activity of the tetrapod-specific Hoxd13 enhancer CsC during zebrafish fin development and found comparable domains of activity to the ones described in developing mouse limbs (). Together, these results provide functional evidence supporting the hypothesis that modulation of 5′Hoxd transcription, through the addition of novel enhancer elements to its regulatory machinery, was a key evolutionary mechanism for the distal elaboration of vertebrate appendages.

andhave characterized several cis-acting regulatory elements (CREs) involved in the activation 5′Hoxd gene transcription in mice. Their results indicate that interaction between several CREs is required to ensure the strong expression of 5′Hoxd throughout the autopod (). Interestingly one of these CREs, CsC, is tetrapod-specific and promotes expression in the whole autopod, fully recapitulating the 5′Hoxd expression domains. Therefore, CsC is a potential candidate to be involved the 5′Hoxd modulation required for the evolution of fins. Indeed, the appearance of specific 5′Hoxd enhancers in the tetrapod lineage, such as CsC, seems a plausible explanation for the tetrapod-specific pattern of 5′Hoxd expression. In such an evolutionary scenario, two conditions should meet in the last common ancestor of fish and tetrapods. First, the trans-regulatory machinery operating on this enhancer should be present. Second, the downstream target genes of the 5′Hoxd proteins should be ready to respond to such an increase in 5′Hoxd activity by generating distal endoskeletal structures compatible with the evolution of fins.

These observations raise an interesting possibility: modulation of 5′Hoxd gene transcription during evolution may have been a key evolutionary mechanism that led to the formation of novel distal endoskeleton elements in fishes and tetrapods (). If that is the case, the fish patterns described so far may indicate trackways of this mechanism in its evolving form in which the levels of transcription may not still be sufficient to generate an autopod-like structure.

More recently, a reevaluation of 5′hoxd gene patterns in zebrafish indeed indicated a distal expansion of these genes during late fin development (). It is still debatable if these patterns represent a true novel second phase of expression or just an expansion of the first phase (). Interestingly, fishes at other phylogenetic positions throughout vertebrates evolution, such as chondrichthyans, basal actinopterygians, and lungfishes, also seem to have a late distal phase of 5′Hoxd expression during their fin development (). However, these phases are quite diverse among those fishes and never fully recapitulate the expression found in tetrapods ().

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages.

The first attempt to explain the fin-to-limb transition, from a developmental point of view, was made by. By then, 5′Hoxd genes were known to play an essential role during autopod development (). Therefore, the basic question concerned whether HoxD expression in developing fish fins was different than in tetrapod limbs. Remarkable differences were found when expression patterns from zebrafish and tetrapods 5′Hoxd were compared in developing fins and limbs (). During limb development, tetrapods show two phases of 5′Hoxd gene expression. In a first phase, these genes are restricted to the posterior limb buds. Later, in a second phase, their expression patterns expand distal and anteriorly throughout the presumptive autopod region. Interestingly, this expansion was hardly detectable in zebrafish fins (). This observation led to the proposal that the tetrapod-specific second phase of 5′Hoxd expression would have been required to mediate the transition from fish fins to tetrapod limbs ().

The Hox-4.8 gene is localized at the 5′ extremity of the Hox-4 complex and is expressed in the most posterior parts of the body during development.

The main innovation of tetrapod appendages is the autopod, a multifinger extremity that evolved from sarcopterygian fins by sequential expansion and elaboration of their appendicular skeleton (). In tetrapod ancestors, the addition of novel distal endoskeleton elements co-occurred with the reduction of the ectodermal finfold characteristic of fish fins (). Living sarcopterygians, such as the Coelacanths, show a coincident evolutionary tendency, having more distal endoskeleton elements and smaller finfolds than fishes that diverged prior to their radiation ().

The colonization of land by vertebrates represents a major event in the history of life. According to the fossil record, tetrapods were the first vertebrate group with the ability to walk on terrestrial environments, which required profound morphological changes in their locomotory appendages during evolution ().

Prior to endoskeletal differentiation, the molecular mechanisms involved in limb development are mostly conserved in zebrafish fins (). The main differences arise later, with the transcription of actinotrichia mRNAs exclusively in the zebrafish finfold and the differential expression of 5′Hoxd distally (). Given that appropriate levels of 5′Hoxd transcription are required for autopod development, addition of novel 5′Hoxd enhancers during evolution, such as CsC, may have increased 5′Hoxd transcription, promoting the expansion of distal endoskeleton that might have served as substrate for further fin elaboration. For that to occur, the upstream machinery acting on this enhancer must have been established prior the autopod origin in tetrapods. To evaluate this hypothesis, we investigated the activity of the murine CsC enhancer (mCsC) during the development of zebrafish fins. We hypothesized that if mCsC was active in the distal fin, this would indicate that developing fins and limbs share the expression of the CsC transactivators, at least to a certain extent. Further, such a result would support the view that these transactivators were already in place prior to the origin of the autopod in tetrapods. To this end, we generated several independent stable transgenic zebrafish lines carrying mCsC enhancer driving GFP expression in a reporter vector specifically designed to evaluate enhancer activity (). During fish development, mCsC activated GFP expression in the posterior fin mesenchyme at 30 hpf and in the distal margin of the endoskeletal territory 3 days later ( Figure 5 A). Transverse sections throughout these fins further confirmed the strong activation of mCsC in the most distal chondrocytes of the endoskeletal disc and, weaker, in mesenchymal cells surrounding this area at 4 dpf ( Figure 5 B). When compared with the pattern of hoxd13a at different time points, the pattern promoted by mCsC seemed to be initially nested within hoxd13a expression. Thus, at 60 hpf, mCsC was observed restricted to the posterior fin mesenchyme but its activity was displaced distally and anteriorly later ( Figure 5 C). This pattern resembles that of CsC and Hoxd13 in mouse limbs (). In light of these analyses, we suggest that mCsC is sufficient to drive a murine Hoxd13-like pattern in zebrafish fins. Therefore, addition of enhancer modules, such as CsC, may have modulated 5′Hoxd gene expression during evolution in order to increase their distal expression.

(C) Comparison between mCsC activity and hoxd13 expression in zebrafish and mouse. Dashed lines indicate the distal limits of the endoskeletal territory. Left panel shows that expression of GFP in mCsC transgenic zebrafish fin is nested within hoxd13a expressing domain at 60 hpf (arrowheads). Central panel shows simultaneous anterior expansion of mCsC activity and hoxd13a expression throughout the distal portion of the endoskeletal territory at 4 dpf (arrowheads). Note that mCsC activity is detected also within a subdomain of hoxd13a expression at this stage. Schemes on the right are based on. Left scheme indicates relative position of mCsC upstream of Hoxd13 in mice. Schemes on the right represent CsC and Hoxd13 expression in mouse limbs (blue and pink respectively). Note that mCsC is also active in a subdomain of Hoxd13 expression in this organism.

(B) Transverse sections throughout a mCsC transgenic fin expressing GFP at 4 dpf. Left panel shows restriction of GFP mRNA to the distal tip of the endoskeletal territory (arrowheads). Central panel shows a higher magnification of the expressing region (arrowheads). Note GFP expression in chondrocyte-like cells (delimited by a white dashed line) and in surrounding cells. The scheme on the right represents the inferred localization of the expression pattern in the endoskeletal territory (green: chondrocytes and undifferentiated distal mesenchyme).

(A) Enhancer activity of mouse CsC (mCsC) shown by the expression of GFP in the posterior half of the fin buds (Fb) at 30 hpf and surrounding the distal margin of the endoskeletal disc 4 dpf. Bracket line delimits the distal border of this territory. The scheme on the right represents the main domain of mCsC activity in zebrafish fins at 4 dpf (green).

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages.

Two recent reports indicate that proximal-distal limb patterning depends on diffusible signals and growth (). Thus, distal digit identity is acquired only if sufficient growth takes place within the developing limb to displace cells beyond the influence of flank-derived signals, which promote proximal fates. Interestingly, increased distal cell proliferation in fins was also proposed to be involved in the evolution of fins potentiating the development of novel distal endoskeletal structures (). As shown above, the enlarged distal chondrogenic tissue observed in hoxd13a overexpressing fins was associated with a distally extended cyclin d1 expression domain ( Figures 2 C and 4 A ), a gene that promotes cell proliferation (). To further evaluate the impact that hoxd13a modulation might have in cell proliferation during fin development, we compared BrdU incorporation in control and hoxd13a-overexpressing fins at 150 hpf ( Figure 4 B). BrdU was combined with DAPI staining to determine nuclear morphology. Although cell proliferation was quite restricted to the tip of the endoskeletal territory in control wild-type fins at 150 hpf (30 ± 8 BrdU cells/fin, n = 8; Figure 4 B), it was drastically increased in hoxd13a-overexpressing fins (111 ± 15 BrdU cells/fin, n = 11; Figure 4 B). BrdU positive cells were detected in the distal endoskeletal territory, where a mass of densely packed cells was observed proliferating and folding. Interestingly, the domains of increased BrdU incorporation coincide with the areas of cyclin d1 expression (compare Figures 4 A and 4B) and where hoxd13a was expressed at similar stages ( Figure 2 A). These results suggest that zebrafish has retained the capacity to respond to a sustained expression of hoxd13 in the fins by increasing cell proliferation distally ( Figure 4 C).

(C) Schematic representation summarizing cell proliferation levels in wild-type and hoxd13a-overexpressing fins. Chondrocytes are represented in blue and BrdU staining in red.

(B) Blue (DAPI) corresponds to cell nucleus and pink are proliferating cells (anti-BrdU). In WT fins, BrdU labeled cells are detected mainly at the distal end of the endoskeletal disc (arrow). Confocal planes throughout hoxd13a overexpressing fins show proliferation in the distal endoskeletal territory (white arrowheads) and in the mass of cells folding from it (arrowheads). Note similarities of the cyclin d1 expression and BrdU labeled cells distribution (yellow arrows mark equivalent domains).

(A) cyclin d1 expression is restricted to the distal end of the endoskeletal plate in WT (arrow). In hoxd13a-overexpressing fins, cyclin d1is observed throughout the expanded endoskeleton (arrowheads) and in the mass of cells folding from it (arrowheads).

meis1 regulates cyclin D1 and c-myc expression, and controls the proliferation of the multipotent cells in the early developing zebrafish eye.

Finally, we examined the expression of sonic hedgehog (shha), a gene that controls anteroposterior (AP) polarity during limb development (). Shh-deficient mouse limbs present severe distal truncation lacking the development of digits (). In the hoxd13a overexpressing fins, shha appeared upregulated (100%, n = 12, Figure 3 D), which was somehow expected given that Shh is trans-regulated by Hoxd13 during limb development (). Therefore, modulation of shh through Hoxd13 may have been also important for fin evolution and our results suggest that this trans-regulation might be conserved in fish fins. These results suggest that mesenchymal cells of the distal fin, which are affected by the overexpression of hoxd13a, change their identity resembling the ones in the distal limb prior to autopod formation ( Figure 3 E).

We then further examined other markers in fins clearly affected by increased hoxd13a function at 120 hpf ( Figure 3 ). Both cyp26b1 and pea3 genes were distally expanded in the hoxd13a-GR overexpressing fins ( Figures 3 A and 3B; 80% and 100%, n = 30 and 10, respectively). Transverse sections throughout these fins ( Figure 3 A) showed cyp26b1 expression surrounding the endoskeleton that extended toward the tip of the affected fin, as observed with AB/PAS/hematoxylin stained sections (compare with Figure 1 D). Opposite results were found for the expression of fgf8a, which typically marks the signaling activity of the finfold (). In affected fins, the expression domain of this gene was much narrower than in controls (80%, n = 15, Figure 3 C). Therefore, we suggest that overexpression of hoxd13a negatively affects the development of the finfold structure and also its signaling activity.

(E) Schematic representations of the observed phenotypes. Blocks of graded blue represent the domain where characteristic distal limb markers (hox13b, pea3, and cyp26b1) are expressed in wild-type and in hoxd13a overexpressing fins.

(C) fgf8a expression is confined to a narrower distal domain in injected fins. Dashed lines indicate the approximate plane of the section shown bellow.

(A and B) cyp26b1 (A) and pea3 (B) is extended distally in hoxd13a overexpressing fins. Dashed lines in (A) indicate the approximate plane of the sections shown bellow. In these sections distal cyp26b1 expression can be observed surrounding the differentiated chondrocytes (yellow dashed line) in injected fins.

Each panel shows expression pattern of a gene at 120 hpf, in which wild-type controls (WT + Dex) and Dex-treated hoxd13a-GR injected fins are on left and right columns, respectively. Stages of development are indicated at left of each panel. Arrows point to the WT expression and arrowheads indicate expression induced by hoxd13a-GR injections. Ed, endoskeletal disc; Ff, finfold.

In addition, when we evaluate hsp70:hoxd13-GFP and col2a1:hoxd13-GFP transgenic fins with reduced finfolds at 3–4 dpf, we found distally expanded expression domains of hoxa13b and cyclin d1 (75%, n = 28) and and1 downregulation (100%, n = 15) not observed in control embryos exposed to heat shock treatment ( Figure 2 E).

In order to evaluate whether finfold reduction in hoxd13a-overexpressing fins was indeed indicative of structural changes in this region, we also examined the expression profile of and1 throughout development, a gene that encodes for a structural component of the finfold (). No differences in and1 expression were observed between hoxd13a-GR injected and wild-type embryos equally treated with Dex at 48 hpf ( Figure 2 D, n = 50). We later found a progressive reduction of and1 expression in the finfold in hoxd13a Dex-treated individuals at 60 (35%, n = 50) and 90 hpf (42%, n = 50). By 120 hpf, when we specifically selected fins with clear reduction of the finfold, we detected drastic downregulation of and1 in all fins evaluated (n = 15). No alterations were observed in wild-type or hoxa4-GR injected fins neither in the presence or absence of the hormone ( Figure S3 ). Because both hoxa13b and cyclin d1 were already upregulated in a significative number of injected embryos at 48 hpf by the time changes in and1 were not yet visible (compare Figures 2 B–2D), we suggest that hoxd13a overexpression affects primarily the fin endoskeletal component having a later effect in finfold development.

We also monitored the expression pattern of cyclin d1 throughout development, a gene expressed in the distal endoskeleton and known to promote cell proliferation (). In wild-type fins treated with Dex, we found that cyclin d1 was expressed in a narrow territory immediately under the AER between 48 to 90 hpf ( Figure 2 C). However, in Dex-treated embryos injected with hoxd13a-GR, cyclin d1 was considerably upregulated throughout the entire fin mesenchyme in 40% of fins at 48 hpf (n = 50), resembling the expression pattern of hoxd13a and hoxa13b found in injected fins ( Figures 2 A–2C). The levels of cyclin d1 expression remained high in 64% of fins at 60 hpf (n = 50) and 62% of fins at 90 hpf (n = 50, Figure 2 C). We then analyzed the expression pattern of cyclin d1 in a pool of fins clearly affected by hoxd13a overexpression at 120 hpf (n = 12). In all these fins, cyclin d1 expression was clearly expanded toward the distal region where extra-chondrogenesis took place (compare Figures 2 C and 1 D–1F). These effects were not observed in fins overexpressing the hoxa4a-GR construct and treated with Dex ( Figure S3 ).

meis1 regulates cyclin D1 and c-myc expression, and controls the proliferation of the multipotent cells in the early developing zebrafish eye.

The expression of hoxa13b at 48 hpf appeared expanded to most fin mesenchyme in 32% of the hoxd13a-overexpressing fins when compared with wild-type controls equally treated with the hormone (n = 50, Figure 2 B). Later on, at 60 and 90 hpf, the expression of this gene became largely similar to the controls in the distal border of the endoskeletal disc. However, it showed increased expression levels in a significant number of fins evaluated (45% at 60 hpf and 46% in embryos at 90 hpf, n = 20 per stage). As for the hoxd13a analyses, we then selected injected embryos with visibly affected fins (n = 8) at 120 hpf to further characterize changes in hoxa13b expression ( Figure 2 B). In wild-type embryos, this gene was expressed in the distal part of the endoskeletal disc that typically ends close to the marginal blood vessel at that stage ( Figures 2 B and 1 A). Identical expression pattern was observed in control embryos injected with hoxa4a-GR and treated with Dex ( Figure S3 ). However, in fins affected by hoxd13a overexpression, hoxa13b appeared to be expressed further distally beyond that vessel ( Figure 2 B).

Genes such as hoxa13, Pea3, and Cyp26 are known to be involved in the proximal-distal subdivision of the developing limbs (). Their expression leads to establishment of a distal cell identity domain, where formation of the autopod takes place in tetrapods. In order to investigate changes toward a distal limb fate in the region affected by the overexpression of hoxd13a, we characterized the expression of these genes in overexpressing fins and controls.

Next, we evaluated how increased hoxd13a expression may have affected the molecular identity of the ectodermal and endoskeletal components of the fins throughout development. We started by characterizing the expression dynamics of hoxd13a itself in embryos injected with hoxd13a-GR versus control wild-type embryos equally treated with Dex. We found that, in 42% of the fins 48 hr after the injection, hoxd13a expression was spread throughout most fin mesenchyme (n = 50, Figure 2 A). At 60 hpf hoxd13a expression levels progressively decayed ( Figure 2 A), showing a similar distribution in control and injected fins, although with slightly higher levels in the latter fins (72%, n = 50). However, by 90 hpf hoxd13a acquired a distinct expression domain in the area of apparent finfold reduction (72%, n = 50). We then selected embryos at 120 hpf with visible finfold reduction (n = 8) and found that they all have fins with anteriorly expanded hoxd13a expression when compared with the controls ( Figure 2 A). Although the molecular cause of this expression is not known, it might be due to an autoregulation process triggered by the ectopic Hoxd13a protein. Interestingly, the distinct hoxd13a expression domain was coincident with the distal area where extra chondrogenesis took place in the affected fins (compare Figures 2 A and 1 D–1F).

(E) Gene expression changes caused by hoxd13a overexpression controlled by hsp70 and col2a1a promoters. Transgenic fins show distally expanded hoxa13b and cyclin d1 expression 3 to 4 dpf and reduced and1 expression (arrowheads) when compared with controls (arrows).

(D) No differences of and1 expression are observed between injected and control fins at 48 hpf. However, between 60 and 120 hpf, injected fins present a progressive reduction of and1 expression in the finfold.

(A–C) hoxd13a (A), hoxa13b (B), and cyclin d1 (C) expression is located throughout most fin mesenchyme after injection (48 hpf). At 60 hpf, these genes show slightly higher levels in the injected fins. At 90 hpf, their expression domains start to be expanded distally. This expansion becomes very clear at 120 hpf.

Each panel shows expression dynamics of a gene from 48 to 120 hpf, in which wild-type controls (WT + Dex) and Dex-treated hoxd13a-GR injected fins are on left or right columns, respectively. Stages of development are indicated in each row at left side of the figure. Arrows point to the WT expression and arrowheads indicate expression induced by hoxd13a-GR injections.

Given the lethality associated with the phenotypes generated with these transgenic approaches ( Figure S2 ) and the need to obtain a considerable number of embryos with temporally controlled manipulations, further analyses were carried out using the hoxd13-GR overexpression system.

We also adopted alternative methods to overexpress hoxd13a in zebrafish fins to further confirm the results obtained with the Dex-inducible hoxd13a construct. Using the Tol2kit technology (), we generated constructs in which a hoxd13a-GFP fusion gene was placed under the control of either heat-shock protein 70 (hsp70) () or collagen (col2a1a) promoters (). These constructs allowed us to generate Tol2-mediated transient transgenic embryos () where hoxd13a levels could be modulated in time (hsp70) or space (col2a1a) by those specific promoters ( Figures 1 G and S2 ). Upon heat-shock induction (see Experimental Procedures ), at 3–4 dpf, the hsp70:hoxd13a-GFP transgenic fins showed finfold reduction in 27% of the cases ( Figure 1 G; n = 300). Similar results were found for col2a1:hoxd13-GFP transgenic fins (24%, n = 175), although more mosaic effects were observed in these fishes ( Figures 1 G and S2 ). Both of these transgenics presented distally expanded sox9a expression domains in 75% of the fins (n = 12), when compared with controls exposed to heat shock treatment ( Figure 1 H). The similarity of effects detected for each of the three overexpression systems indicated that these effects were specific to the overexpression of hoxd13.

In order to evaluate whether these changes were due to a distal expansion of the chondrogenic tissue or to an indirect consequence of the finfold reduction, we performed measurements starting from the cleithrum toward the distal end of the col2a1a expression in controls versus Dex-treated fins (see Figure 1 A). These analyses revealed a significant increase of the chondrogenic tissue (t test p < 0.03) from 148.9 ± 17 mm in control fins (n = 4) to 182.4 ± 14.6 mm (n = 4) in hoxd13a-GR overexpressing fins. Together, these results suggest that increased levels of hoxd13a induce an expansion of chondrogenesis distally in zebrafish fins.

In order to confirm the chondrogenic nature of the altered tissue in hoxd13a-overexpressing fins, we analyzed the expression profiles of two genes involved in chondrogenesis, sox9a and col2a1 (). Sox9 activity is expected to regulate both chondrogenesis and chondrocyte proliferation and Col2a1 is its known trans-activation target (). These genes were expressed mostly in the distal part of the endoskeletal disc and absent from the finfold in embryos not injected but treated with Dex at 120 hpf ( Figures 1 E and 1F). In 80% of hoxd13a-overexpressing fins with severe finfold reduction (n = 20), the expression of these genes was observed further distally, in a domain coincident with the positive alcian blue staining ( Figures 1 D–1F).

We then performed histological sections of injected and wild-type embryos treated with Dex ( Figure 1 C) and stained them with hematoxylin together with two cartilage matrix stains (periodic acid-Schiff [PAS] and Alcian blue [AB]). Analyses of these sections suggest a distal expansion of the endoskeletal territory toward the affected finfold region in hoxd13a overexpressing fins ( Figure 1 C). Whereas in control fins the proximal/distal cartilage length was in average 194.2 ± 4.6 mm (n = 3), in the injected ones this length was significantly expanded (t test p < 0.01) to 273.2 ± 16.7 mm (n = 3). In addition, whole mount alcian blue skeleton preparations also pointed to a strong finfold reduction associated with distal expansion of sulfated proteoglycans ( Figure 1 D), endemic to chondrogenic cells (). After these experiments, larvae with altered fins have problems with swimming, and consequently with feeding, and died massively before individual skeletal elements are formed, precluding the analyses of the abnormal fins at later stages.

The effect of Dex-inducible overexpression could be detected in one or both pectoral appendages presenting a variable degree of finfold reduction clearly visible 96 hpf onward. Overexpression of this gene did not seem to affect visibly other embryonic territories. Just a few treated embryos (3%–5%) had deformed bodies and were excluded from further analyses. Moreover, these phenotypes were not observed in the absence of the hormone, when Dex was added at later stages or in embryos injected with a control hoxa4-GR construct ( Figure S1 available online).

The activation of hoxd13a has been described in the posterior zebrafish fin mesenchyme at 30 hr postfertilization (hpf) and, 6 hr later, was observed expanding to more distal and anterior domains (). Taking in consideration the mouse data, this second phase of expression seems to be the one that potentially would mostly affect the formation of distal structures also in fish (). We therefore added Dex at 24, 30, 32, 36, and 48 hpf to increase the levels of hoxd13a activity around this phase of hoxd13a expression. When Dex was added at 30 or 32 hpf, which is just prior to when the anterior expansion of the endogenous hoxd13a gene takes place (), thickening and reduction of the finfolds was found in 40% of the treated embryos (n > 200, Figure 1 B). The remaining embryos did not show any altered phenotypes, which might be due to insufficient amount of RNA injected and/or induction efficiency.

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages.

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages.

Several reports suggest that high levels of 5′Hox genes would be required for proper limb development (). In order to evaluate the impact of 5′hoxd gene modulation during development of fish fins, we generated a hormone-inducible construct with the zebrafish hoxd13a fused to a glucocorticoid receptor (hoxd13a-GR). The steroid hormone-inducible system allows temporal control of protein activity and has been widely use in cell culture and in vivo assays (). In the absence of hormone dexamethasone (Dex), GR fusion proteins are held in an inactive state, presumably due to complex formation with Hsp90 (). Addition of hormone causes a conformational change that dissociates Hsp90, resulting in rapid activation of the fusion protein (). This method allowed us to specifically control the time of hoxd13a overexpression by adding Dex at specific developmental times in hoxd13a-GR injected embryos.

Evidence that the hormone binding domain of steroid receptors confers hormonal control on chimeric proteins by determining their hormone-regulated binding to heat-shock protein 90.

The formation of a fish pectoral fin requires two major components: mesenchyme derived from the lateral plate mesoderm that gives rise to the endoskeleton, and ectodermic cells that form the distal finfold ( Figure 1 A). Prior to the formation of individual endoskeleton elements, the fin mesenchyme condensates and chondrifies, forming an endoskeletal disc in actinopterygians. Zebrafish larvae use this transitory structure to swim up to 17 days postfertilization (dpf) (). After the chondrification of the disc, a thin layer of cells along its distal margin retains a mesenchymal character ( Figures 1 A.1 and 1A.2). This layer then grows distally, beyond the marginal blood vessel, giving rise to de novo cartilaginous condensations later in development ( Figure 1 A.3).

(G and H) Fin alterations caused by hoxd13a overexpression controlled by hsp70 and col2a1a promoters. Stages of development are indicated in left up corner for each panel. Left, middle, and right column show wild-type (WT), hsp70:hoxd13a and col2a1a:hoxd13a transgenic fins, respectively. Ed, endoskeletal disc; Ff, finfold. Fins overexpressing hoxd13a show finfold reduction (G, arrows) and show distally expanded sox9a expression domains (H), compare arrowheads in transgenics with arrows in controls fins).

(E and F) col2a1a (E) and sox9a (F) expressions expand further distally toward the area affected by hoxd13a overexpression (arrowheads) when compared with wild-type fins equally treated with Dex (WT + Dex) at the same stage (arrows).

(B–D) Left and right columns show control wild-type (WT) and hoxd13a-GR injected fins, respectively. Embryonic stages are indicated on the upper left corners of each panel. (B) Different degrees of finfold truncation (severe and mild) are found in hoxd13a-overexpressing fins from 96 hpf onward. (C) Histological sections stained with AB, PAS, and Gill's hematoxylin (Hematox) show expansion of chondrogenic tissue distally to the Mbv in hoxd13a-overexpressing fins (arrows). Dashed yellow lines represent the length of the chondrogenic tissue measured for statistical evaluations. (D) AB skeleton preparations show staining extending distally in hoxd13a-overexpressing fins (arrowhead) when compared with the controls (arrow).

(A) Schemes based on. Endoskeletal disc (Ed) in blue, distal undifferentiated mesenchyme (Dm) in light blue, marginal blood vessel (Mbv) in yellow, ectodermal finfold (Ff) in gray, cleithrum (Cle). Black arrows indicate the distal mesenchymal expansion, taking place during zebrafish fin development. Left scheme (1) shows morphology of a zebrafish pectoral fin at ∼96 hpf. Dashed line indicates the approximate place where length measurements were performed for statistical analyses. Central (2) and right (3) schemes represent longitudinal sections through fins at two stages of the mesenchymal expansion. Undifferentiated mesenchymal cells (2) are detected at the proximal vicinity of the Mbv at 4 dpf and extend further distally later (3).

Discussion

Sordino et al., 1995 Sordino P.

van der Hoeven F.

Duboule D. Hox gene expression in teleost fins and the origin of vertebrate digits. In this work, we functionally evaluate the impact that modulation of hoxd13 expression could have in promoting distal changes in zebrafish fins and discuss the implications that such process could have had in the evolution of vertebrate appendages. Using three independent methods, we found that overexpression of hoxd13a produces a fin phenotype that resembles the morphological changes expected to have happened during fin evolution (). When these appendages overexpress hoxd13a, they have a proliferative expansion of chondrogenic tissue distally that express markers characteristic of autopod development. Moreover, they present a shorter finfold with a reduced domain of and1 and fgf8a expression.

Zhang et al., 2010 Zhang J.

Wagh P.

Guay D.

Sanchez-Pulido L.

Padhi B.K.

Korzh V.

Andrade-Navarro M.A.

Akimenko M.A. Loss of fish actinotrichia proteins and the fin-to-limb transition. As with our results, a shortening of the zebrafish finfold was also detected after knockdown of and1, a gene encoding for its structural proteins (). In this situation, the truncated finfolds showed also reduced fgf8a expression and anteriorly expanded hoxd13a expression, indicating that truncation of the finfold can also cause increased hoxd13a levels. Therefore, a tight ectoderm/mesoderm crossregulation likely occurred during fin evolution, involving both modulation of 5′hoxd transcripts by specific enhancers and reduction of structural finfold proteins. Our results, however, suggest that overexpression of 5′hoxd primarily affects the fin endoskeletal compartment.

Sordino et al., 1995 Sordino P.

van der Hoeven F.

Duboule D. Hox gene expression in teleost fins and the origin of vertebrate digits. Duboule, 1994 Duboule D. Temporal colinearity and the phylotypic progression: a basis for the stability of a vertebrate Bauplan and the evolution of morphologies through heterochrony. Zákány and Duboule, 1999 Zákány J.

Duboule D. Hox genes in digit development and evolution. It has been suggested that the ectoderm/mesoderm signaling might be mechanistically interrupted by the development of the fish finfold leading to low distal proliferation (). This could explain the absence of an autopod-like structure or other further distal elements in fishes. Indeed, in tetrapod limbs the AER is never converted into a fold, and chondrogenic cells are kept proliferating distally until the formation of the autopod (). Supporting this hypothesis, our results show that finfold reduction in zebrafish following overexpression of hoxd13 is concomitant with overproliferation in fin territories expressing chondrogenic markers.

Dollé et al., 1991 Dollé P.

Izpisúa-Belmonte J.C.

Boncinelli E.

Duboule D. The Hox-4.8 gene is localized at the 5′ extremity of the Hox-4 complex and is expressed in the most posterior parts of the body during development. Nelson et al., 1996 Nelson C.E.

Morgan B.A.

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DiMambro E.

Murtaugh L.C.

Gonzales E.

Tessarollo L.

Parada L.F.

Tabin C. Analysis of Hox gene expression in the chick limb bud. Cooper et al., 2011 Cooper K.L.

Hu J.K.

ten Berge D.

Fernandez-Teran M.

Ros M.A.

Tabin C.J. Initiation of proximal-distal patterning in the vertebrate limb by signals and growth. Roselló-Díez et al., 2011 Roselló-Díez A.

Ros M.A.

Torres M. Diffusible signals, not autonomous mechanisms, determine the main proximodistal limb subdivision. Shubin et al., 2009 Shubin N.

Tabin C.

Carroll S. Deep homology and the origins of evolutionary novelty. We also found that hoxd13a overexpression leads to a proximo/distal segregation of the endogenous gene expression pattern 3 days later, closely resembling the expression dynamics characteristic of this gene during murine limb development (). As previously mentioned, this may be explained by a positive autoregulatory mechanism triggered by the earlier hoxd13a overexpression. This autoregulatory activation likely occurred only at distal territories that are far apart from the influences of flank-derived signals that promote proximal fates in limbs (). In contrast with these distally segregated domains, the second phase of 5′Hoxd expression in actinopterygian and lungfish fins spatially overlaps with earlier phases of expression (). Therefore, our results suggest that, during fin evolution, an early increase in 5′Hoxd expression might have triggered a later, spatially segregated expression domain. If this process indeed took place, it probably occurred after lungfish radiation. Generation of this distinct domain, under the influence of the second phase of Hoxd13 transcription and further apart from the influences of proximal signals, may have been crucial for the distal endoskeletal elaboration of fins and limbs.

Kmita et al., 2005 Kmita M.

Tarchini B.

Zàkàny J.

Logan M.

Tabin C.J.

Duboule D. Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function. Kmita et al., 2005 Kmita M.

Tarchini B.

Zàkàny J.

Logan M.

Tabin C.J.

Duboule D. Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function. Sordino et al., 1995 Sordino P.

van der Hoeven F.

Duboule D. Hox gene expression in teleost fins and the origin of vertebrate digits. Shubin et al., 2009 Shubin N.

Tabin C.

Carroll S. Deep homology and the origins of evolutionary novelty. It has been increasingly clear that 5′Hoxd genes are essential for the proper formation of the mouse autopod (). However, inactivation of these genes causes limb truncation rather than homeotic transformations, which suggests that they are strongly involved in regulation of distal growth (). Their different expression levels in zebrafish pectoral and pelvic fins also pointed to a relationship with distal growth of endoskeleton elements in fishes (). If Hoxd13 influences distal fin/limb morphology by regulating late events of growth, this may then explain the variety of expression patterns described for this gene in different phylogenetic groups (). Nevertheless, our results suggest that modulation of this gene might have been a strong driving force during evolution to generate distal growth in fish fins promoting the origin of novel endoskeletal elements.

Gonzalez et al., 2007 Gonzalez F.

Duboule D.

Spitz F. Transgenic analysis of Hoxd gene regulation during digit development. In addition, we provide evidence that a tetrapod-specific 5′Hoxd enhancer (mCsC) is active during zebrafish fin development in domains that resemble the expression pattern of murine hoxd13a, one of its potential target genes. Moreover, the distal localization of its activity during later fin development shows similarities with the expression driven by the enhancer in the mouse autopod (). This points to a conservation of the upstream machinery acting on this enhancer, at least since the common ancestor of actinopterygians and sarcopterygians. The function that this machinery might have in the common ancestor, before the arising of novel regulatory elements, is a matter of speculation. However, this machinery could be required for the specification of the proximodistal axis of the ancestor's fins. Precursor sequences to CsC could have hitchhiked on the distal regulatory state to generate an upregulation of 5′Hoxd expression and thus initiated the process of distal expansion and diversification of fin structures, which likely gave a selective advantage to the bearers of these new sequences.

Within the developmental constraints imposed by a highly derived teleost fin, our results suggest that modulation of Hoxd13 results in downstream developmental changes expected to have happened during fin evolution. This, together with the evidence we provide that the upstream regulators of CsC were also present prior to tetrapod radiation, makes us favor an evolutionary scenario in which gain of extra 5′Hoxd enhancers might have allowed the developmental changes necessary for the elaboration of distal bones in fishes that evolved, ultimately, into the tetrapod hand.