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Proc Natl Acad Sci U S A. Jun 25, 2002; 99(13): 8713–8718.
Published online Jun 11, 2002. doi:  10.1073/pnas.122571799
PMCID: PMC124364
From the Cover
Developmental Biology

Bone patterning is altered in the regenerating zebrafish caudal fin after ectopic expression of sonic hedgehog and bmp2b or exposure to cyclopamine

Abstract

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.

Keywords: shh‖bmp‖chordin‖fin regeneration‖in vivo transfection

The 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 (26).

The dermal skeleton of the zebrafish fin comprises mineralized lepidotrichia (fin rays) and more distal collagenous actinotrichia (Fig. (Fig.11A). 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 epithelial/mesenchymal interface and begin to secrete the matrix that will form the new dermal bone.

Figure 1
(A) Schematic representation of the skeleton of a ray in the zebrafish fin. Dermal bone forms two segmented, concave and opposing hemirays. The diagram illustrates the position of amputations, relative to the bifurcation of the lepidotrichia; “low ...

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-β 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 epidermis, and ptc1 and bmp2b transcripts are also found in the bone matrix-secreting scleroblasts within the mesenchyme, beneath the epithelial cells (3). By 1–2 days after initiation of expression, an imminent bifurcation is signaled in all except the outermost (lateral) rays (which never bifurcate) by the duplication of the single domain of shh and ptc1 expression into two domains (Fig. (Fig.11B).

The reexpression of shh and bmp2b in the region of cell proliferation and new bone deposition, along with their changing expression domains before ray bifurcation events, implicates them in the formation and patterning of new dermal bone and possibly cell proliferation during fin regeneration. In developmental systems that depend on epithelial–mesenchymal interactions, shh and bone morphogenetic proteins, such as bmp2 and bmp4, are often coexpressed or expressed in adjacent cell populations (11). In a few of these developing organs, such as the feather bud (12), the gut (13), and the genital tubercle (14), the bmp factors have acted as downstream targets of shh signaling. However, in other cases, such as the tooth (15), which, like the fin ray, is a dermoskeletal structure, misexpression of shh does not seem to affect expression of bmp factors suggesting that, in these organs, shh and bmp signaling are acting in parallel and independent pathways.

To test shh function and the relationship between shh and bmp2b signaling in dermal bone formation directly, we established an in vivo transfection method to express shh and bmp2b ectopically in localized regions of regenerating fin and investigated the effects of impaired shh-signaling by using cyclopamine, a Veratrum californicum alkaloid known to block shh signal transduction (1821).

Materials and Methods

Animals.

Zebrafish were purchased at a local pet store and maintained at 28.5°C by standard methods (22).

Fin Amputation.

Adult zebrafish were anesthetized in 0.6 mM Tricaine and their caudal fins amputated either one segment below (proximal to) the level of the first ray bifurcation (low cut; see Fig. Fig.11A) or in the range four to six segments above (distal to) the first bifurcation (high cut; see Fig. Fig.11A).

DNA Injections.

DNA injections were performed at 2 days after a high cut. Tissue separating the blastema of regenerating branches of rays was microinjected with 0.2–0.6 nl of 100 ng/μl circular plasmid DNA. Coinjection of shh and chordin plasmids was performed with 250 ng/μl of each DNA construct. For ectopic expression of shh and bmp2b, DNA was injected in each ray of the ventral lobe. Dorsal rays were injected with a control plasmid encoding the enhanced green fluorescent protein gene (pCMV5EGFP).

To analyze the effects of shh or bmp2b injection on bone deposition, fins were allowed to regenerate for 1 week, fixed in 4% paraformaldehyde in PBS for 2 h at room temperature. High-cut fins were stained with 0.01% Alcian blue in 70% ethanol and 30% acetic acid, rinsed in PBS, stained in 0.1% Sirius red in saturated picric acid for 1 h, washed in 0.01 N HCl, and rinsed in distilled water. Selected rays were cut into 16-μm sections by standard cryosectioning procedures (22). To analyze the effects of DNA-construct injections on endogenous gene expression, fins were allowed to regenerate for 2 days after injection, cut into 16-μm sections, and processed for in situ hybridization.

In Situ Hybridization.

In situ hybridization on whole-mount fins and cryosections was performed as described (2, 23). The shh digoxigenin-labeled riboprobe corresponded to a 1,440-bp C-terminal fragment that excludes a region conserved between different hedgehog genes (24). The ptc1 probe consisted of a 1,150-bp cDNA fragment encoding amino acids 400–780 (25).

Construct Subcloning.

pCMV5EGFP, the enhanced GFP gene (EGFP) was excised from plasmid pEGFP-C1 (CLONTECH) and ligated into the multiple cloning site of pCMV5 (CMV, cytomegalovirus) (Department of Molecular and General Biochemistry, University of Texas, Southwestern Medical Center). pCMV5shh-NH2, a 632-bp, N-terminal fragment of the shh cDNA (26), was obtained by PCR by using the following primers: forward, 5′-ATTAAGCTTGCGGCAAAATGCGG-3′; reverse, 5′-GAAGATCTCCCCCAGATTTCGCAGC-3′. It was cloned into the pCMV5 vector at the HindIII and BglII sites. Position 632 of the shh sequence corresponds to the cleavage site of the shh precursor. pCMV5bmp2b, a modified version of pCMV5 vector, was made to create convenient restriction sites (EcoRI and KpnI sites) to place the bmp2b cDNA under the control of the CMV promoter. pCMVchordin (kindly provided by M. Halpern, Carnegie Institute of Washington, Baltimore), a 2.5-kb fragment encoding the zebrafish chordin cDNA (27), was inserted into the pCS2+ expression vector containing the CMV promoter.

Cyclopamine Preparation and Treatment.

Water-soluble complexes of the alkaloids cyclopamine and solanidine were prepared in PBS and evaporated under nitrogen. Complexes consisted of 10 mg of cyclopamine, 5 mg of citric acid, and 125 mg of cyclodextrin or 10 mg of solanidine, 5 mg of citric acid, and 250 mg of cyclodextrin. Stocks of 10 mM alkaloid complex were made with distilled water and stored in the dark at 4°C.

Zebrafish were placed in beakers containing water with 1, 5, or 10 μM alkaloid (cyclopamine or the control, solanidine) for 2 days after a low-cut amputation. Fish were maintained in the alkaloid solution for 5 days in the dark at 28.5°C with a solution change on the third day. Each day, the fish were anesthetized and the length of the regenerated fin measured (same rays each day). After treatment, fins were recut and fixed in 4% paraformaldehyde/PBS, then either stained for histological examination by using Alcian blue and Sirius red or processed for in situ hybridization.

BrdUrd Incorporation.

Proliferating cells were labeled with BrdUrd according to ref. 7, with some modifications (8), except that after incubation with a mouse anti-BrdUrd monoclonal antibody (Sigma), BrdUrd incorporation was visualized with an ABC alkaline-phosphatase detection kit (Serotec).

Results

Ectopic Expression of shh and bmp2b Disrupts the Normal Patterning of Regenerating Rays.

To investigate the role of shh and bmp2b in bone deposition and patterning, we developed a method of local gene transfection (based on gene-therapy studies, e.g., refs. 28 and 29), to express ectopically shh and bmp2b in specific regions of the regenerating fin.

Pilot experiments showed that 10–60 mesenchymal cells could be transfected with plasmid DNA containing the EGFP reporter gene, placed under the control of the CMV promoter, when injected at concentrations between 100 and 250 ng/μl into the tissue separating the blastemas of regenerating branching rays (Fig. (Fig.11C). The normal morphology of rays 24 h after injection (assessed with the light microscope) indicated that the procedure did not interrupt normal regenerative patterning. GFP fluorescence continued for at least 1 week after injection. Transfection efficiency was low in unamputated rays, indicating that this technique is suited to either traumatized tissue and/or undifferentiated, highly proliferative tissue.

To investigate shh signaling in ray bifurcation, shh and bmp2b constructs were misexpressed between two sister ray branches, where shh and bmp2b are not normally expressed. Rays were amputated at a “high-cut” level (Fig. (Fig.11A) where normal transient fusion of the lepidotrichial branches rarely occurs (3). Fins were injected 2 days after amputation (dpa), with plasmids containing either the amino-terminal coding portion of shh [N-shh; the portion responsible for its biological activity (24, 26)], the bmp2b gene (10), or the control EGFP reporter.

When N-shh or bmp2b gene constructs were transfected between the blastemas of the two sister rays, an increase in the incidence of fusion was observed, most noticeable at four to five segments above the origin of bifurcation. At this level, 51% of rays injected with N-shh (n = 159/300) were transiently fused, compared with 15% of fins injected with EGFP (n = 48/320), and 40% of rays injected with bmp2b (n = 18/45) showed fusions compared with 13% (n = 4/30) of EGFP controls.

Chordin Can Antagonize the Formation of Bone Fusion Induced by shh Ectopic Expression.

Chordin, a secreted protein that directly binds to bmp factors and prevents BMP–BMP-receptor interactions (30) was coinjected with N-shh after a high cut. Coinjection resulted in fewer (3.4%; n = 1/29) transient fusions of the rays at four to five segments above the origin of bifurcation compared with N-shh injected alone (51%; see above). Coinjection also reduced the occurrence of natural ray fusion at 1–2.5 segments above the origin of bifurcation from 100% to 52% of injected fins (n = 10/19). The inhibition of bone fusion by chordin indicates that ectopic bone formation requires a bmp factor. Furthermore, our observation that both natural and shh-induced fusions are inhibited suggests that ectopic shh expression leading to bone fusion is mediated through activation of bmp signaling.

Dermal Bone Is Deposited in the Interray Region During Fusion.

To determine where the additional dermal bone matrix was deposited after both N-shh (Fig. (Fig.11F) and bmp2b injection (Fig. (Fig.11H), control (Fig. (Fig.11D) and injected (Fig. (Fig.11 F and H) rays (at least four per construct) were stained with Alcian blue and cut into horizontal sections in the region of the fusion (Fig. (Fig.11 E, G, and I). In each of the fused fins examined, ectopic matrix was found only in the space between the basal layer of the epithelium and the mesenchymal cells of the interray (Fig. (Fig.11 G and I). No dermal bone deposits were found in the mesenchymal interior of the lepidotrichia where exogenous N-shh and bmp2b were expressed by transfected cells, which suggests that only cells competent to form dermal bone [i.e., scleroblasts adjacent to the basement membrane (7, 31)] were able to do so in response to ectopic gene expression.

Ectopic Expression of shh but Not bmp2b Alters the Patterns of ptc1 Expression.

In the developing limb bud, shh signaling is thought to be regulated by feedback loops with other proteins such as fibroblast growth factors (32, 33). We observed that chordin, an inhibitor of bmp signaling, can antagonize the formation of bone fusion induced by shh, suggesting an interaction between shh and bmp signaling. To investigate a possible reciprocal interaction between bmp2b and shh during bone formation, we examined whether ptc1 expression was affected by the ectopic expression of N-shh and bmp2b. Two days after injection of N-shh, ptc1 expression was induced in the mesenchymal cells and in cells of the basal epithelial layer located between the two ray branches (Fig. (Fig.22D), where ptc1 and also shh and bmp2b are normally not expressed (Fig. (Fig.22 AC). In contrast, 2 days after bmp2b injection, no change occurred in ptc1 expression compared with noninjected fins (Fig. (Fig.22E). This result suggests that the shh signaling pathway is not activated in response to ectopic expression of bmp2b.

Figure 2
Transverse sections of regenerating fins showing the normal expression patterns of shh (A), ptc1 (B) in two adjacent rays; bmp2b (C) in one ray at 4 dpa; ptc1 expression 2 days after injection of N-shh (D) or bmp2b (E) constructs. Note the ectopic expression ...

Cyclopamine Inhibits Fin Regeneration and Alters shh and ptc1 Gene Expression.

We investigated the effects of shh signaling down-regulation on bone patterning by exposing regenerating fins to cyclopamine, a steroidal alkaloid that blocks hedgehog (hh) signal transduction (1821). Caudal fins were amputated at a low-cut level (Fig. (Fig.11A), then treated with 1, 5, or 10 μM alkaloid, starting at 2 dpa, when shh expression is already established (3). The morphology and length of the regenerated portion was assessed daily. As a control, an equal number of fish were treated at the same dosage with solanidine, a steroidal alkaloid of similar structure, but without known effects on hh signaling. Fin growth in the presence of solanidine is similar to the growth in untreated fins (data not shown).

Treatments with 10 μM (Fig. (Fig.33A) and 5 μM (Fig. (Fig.33B) cyclopamine significantly inhibited fin outgrowth by days 2 (P < 0.05) and 4 (P < 0.05), respectively. By day 5, both doses caused an accumulation of melanocytes in the distal blastema (Figs. (Figs.33D and and44F), where newly deposited bone seemed thicker and terminated abruptly (Fig. (Fig.33 D and F) compared with controls (Fig. (Fig.33 C and E). Also, at 10 μM, the actinotrichia found in the distal ray (Fig. (Fig.33E) were absent (Fig. (Fig.33F). Inhibition of fin outgrowth correlated with significant reductions in the number of regenerated segments (P < 0.001; Table Table1).1). However, the length of each segment did not seem to be affected (not shown). Unlike solanidine-treated fins, rays exposed to cyclopamine did not bifurcate.

Figure 3
(A, B) Growth curves of fins treated with 10 μM (A) or 5 μM (B) solanidine (black bars) or cyclopamine (diagonal hatched bars). The ordinate indicates the length of the fins in millimeters and the abscissa the day of treatment, where day ...
Figure 4
Gene expression in regenerating caudal fins treated with 5 μM solanidine (A, C, E) or cyclopamine (B, D, F). After 1 day of cyclopamine, ptc1 expression, normally expressed in a single domain (A), is absent (B) and shh, normally expressed in two ...
Table 1
Mean number of segments ± SEM in alkaloid-treated fins after 5 days of treatment

We examined the expression patterns of shh and ptc1 after 1, 3, and 5 days of treatment with 5 μM alkaloid (n = 4 per group). ptc1 expression, usually present in the distal regenerate (Fig. (Fig.44A), was absent at all time points in cyclopamine-treated fins (Fig. (Fig.44B). In contrast, shh, which in controls is expressed in two domains in each blastema (Fig. (Fig.44 C and E), was expressed in one expanded domain at 1 day (Fig. (Fig.44D) and a smaller diffused domain at 3 (Fig. (Fig.44F) and 5 (not shown) days. Exposure to 1 μM cyclopamine (n = 14) had no obvious effects on morphology or shh/ptc1 gene expression (not shown).

Cyclopamine Affects Cell Proliferation and the Morphology of the Blastema/Epithelial Interface.

Cessation of fin outgrowth and altered expression of shh and ptc1 suggest that cyclopamine can affect blastema cells, which are the sites of cell proliferation, scleroblast differentiation, and bone matrix secretion. It is likely that cyclopamine somehow affects the competence of the blastema to extend distally. To determine whether this effect was caused by decreased proliferation of cells contributing to the regenerate, we examined BrdUrd incorporation after 1 and 3 days of treatment with 10 μM cyclopamine or solanidine (n = 4 per group), beginning at 2 dpa. At 24 and 72 h after the onset of treatment, fish were injected with BrdUrd and fins harvested 7 h later (8).

One day after treatment onset, no significant difference existed in the amount of cell proliferation between solanidine (Fig. (Fig.55A) and cyclopamine (Fig. (Fig.55B) groups (P > 0.05; Table Table2).2). BrdUrd-positive cells were visible in the epithelial layer along the length of the fin (arrows) and in the mesenchyme of the blastema (Fig. (Fig.55 A and B, arrowheads). By day 3, cyclopamine treatment resulted in a significant reduction in cell proliferation in the regenerate, both in the blastema (P < 0.005; Table Table2,2, Fig. Fig.55 C and D) and epithelial cells (P < 0.005; Table Table2,2, Fig. Fig.55 C and D, arrows), and significantly more BrdUrd-positive epithelial cells in the stump (P < 0.005; Table Table2).2). Although it is possible that the absence of shh signaling affects epithelial cell in addition to blastema cell proliferation, it seems that it may rather affect the distribution of epithelial cells because these cells are known to migrate from the lateral side of the stump to the regenerate (8). Epithelial cell migration may be impaired in cyclopamine-treated fish as a result of regenerate growth arrest.

Figure 5
BrdUrd-incorporation in fins treated with cyclopamine or solanidine. After 1 day of treatment (initiated at 2 dpa) with either solanidine (A) or cyclopamine (B), proliferating cells were identified in the epidermis (solid arrows) and mesenchyme (arrowheads) ...
Table 2
Mean number of BrdUrd-positive cells ± SEM in alkaloid-treated fins after 1 and 3 days of treatment

Discussion

Ectopic Bone Deposition after N-shh and bmp2b Ectopic Expression.

By using in vivo transfection of DNA, we were able to target expression of N-shh and bmp2b in the amputated fin, between the branches of already bifurcated rays where endogenous shh and bmp2b are not usually expressed. Injection of reporter-gene DNA in the blastema of amputated rays had no adverse effects on regeneration. Ectopic expression of N-shh and bmp2b resulted in additional bone deposition in the interray normally devoid of bone. However, excess bone was found only between the basal layer of the epidermis and the mesenchyme where the bone matrix-secreting scleroblasts are found (7, 31). This effect depended on the distance between the amputation site (injection location) and origin of bifurcation, being maximal four to five segments above the origin of bifurcation and diminishing when DNA was injected further away. This finding suggests that N-shh and bmp2b can stimulate the secretion of bone matrix, possibly by influencing the differentiation of bone-secreting scleroblasts, but only in mesenchymal regions competent to do so. No bone was found in the vicinity of N-shh-expressing cells located in the mesenchymal interior of the lepidotrichia, where scleroblasts and their precursors are not usually found. shh expression in epithelial cells is known to play a role in interactions with adjacent mesenchymal tissue in other systems. For example, shh expression in the chick feather-bud epithelium is thought to induce mesenchymal condensations that form the feather placode (34), and shh expression in the skin is required for the morphogenesis of the hair follicle (17). shh is also important in the differentiation of mesenchymal cells in the developing lung (16) and in the normal growth and morphogenesis of the tooth, but it does not seem to be required for the differentiation of ameloblasts and odontoblasts (15). It is not yet clear, however, whether the ectopic secretion of bone matrix described here is due to the differentiation of new scleroblasts from existing precursor cells in the interray region or to the stimulation of bone secretion by existing scleroblasts.

We showed that coinjection of chordin, an inhibitor of bmp signaling, can antagonize the formation of ectopic bone by shh, suggesting an interaction between the shh and bmp pathways in fin dermal bone formation and that a bmp factor acts as a downstream target of shh. Conversely, we observed that, in contrast to shh, ectopic expression of bmp2 does not affect endogenous ptc1 expression, a target of shh signaling, questioning existence of a positive feedback mechanism between the bmp2 and shh pathways in dermal bone formation.

Inhibition of Fin Outgrowth by Cyclopamine.

Exposure of regenerating fins to cyclopamine initially reduced, then inhibited fin outgrowth and resulted in fewer or no actinotrichia and a distal accumulation of pigment cells. These effects were accompanied by a reduction in cell proliferation within the blastema and a diminution in blastema size.

Inhibition of the hh signaling pathway by cyclopamine is consistent with an interference of blastema maintenance, including proliferation and differentiation of specialized cell types within the mesenchyme, possibly including the bone-secreting scleroblasts and/or the cells synthesizing the actinotrichia, which are thought to help new tissue migrate distally (35, 36). Although cyclopamine may affect signaling by all hedgehog-related proteins, the effects we observed are likely mediated by shh, rather than other hedgehog-related genes such as tiggy-winkle hedgehog (37) and echidna hedgehog (38), because these family members are not expressed in the regenerating fin during the course of these experiments (unpublished observations). We cannot exclude the possibility that as-yet undescribed hedgehog-like genes are expressed in the regenerating fin that may be affected by cyclopamine treatment.

Our studies implicate shh signaling in the proliferation and/or differentiation of the specialized bone-secreting cells in the blastema during regeneration, which may be by epithelial–mesenchymal interactions involving shh/ptc1 and bmp2b, with a subset of basal epithelial cells expressing shh, and the same cells plus adjacent mesenchymal cells expressing ptc1 and bmp2b. In regenerating zebrafish fins, inhibition of fibroblast growth factor function leads to a down-regulation of epithelial shh expression and in turn to an inhibition of cell proliferation and blastema outgrowth (5). In developing limb buds a relay between fibroblast growth factors in the apical ectodermal ridge and shh in the zone of polarizing activity has been well documented (32, 33, 39, 40). Expression analysis of fibroblast growth factors after treatment with cyclopamine may help determine the importance of epithelial–mesenchymal interactions during fin regeneration.

Changes in shh and ptc1 Expression Patterns after Exposure to Cyclopamine.

We found that rays treated with cyclopamine rarely bifurcated, which may be due to the disruption of shh expression domains, which are clearly linked to the bifurcation process. A difference in gene expression was noticeable after 1 day of cyclopamine treatment where fins treated with 5 μM cyclopamine showed an expansion of shh expression along the proximal–distal axis and a loss of ptc1 expression. After 3 days of treatment, when fin outgrowth had virtually stopped, only one weak domain of shh expression existed, and still no ptc1 transcripts. The rapid up-regulation of shh expression after cyclopamine treatment suggests that a feedback mechanism is involved in the definition of shh expression domains and further supports the hypothesis that such mechanisms lead to the confinement of shh expression in the distal-most portion of the regenerate. The eventual down-regulation of shh that accompanies cessation of fin outgrowth suggests that shh signaling may be important in either the proliferation and differentiation of bone-secreting cells in the blastema or bone deposition itself. However, a direct effect on bone deposition seems unlikely because bone is still deposited during the first 3 days of cyclopamine treatment, even in the absence of the shh receptor, ptc1.

We reported that shh expression in regenerating fins correlated with the region of new bone matrix deposition (3). In addition, we observed that division of shh expression into two domains correlated with the formation of a bone bifurcation, the two domains of shh expression corresponding to the bone-forming region of the new branches. These observations suggested a role for shh in dermal bone patterning (3). However, it was not clear whether the absence of shh function was the consequence or the cause of absence of bone between branches. Here, we have presented evidence that ectopic expression of shh or bmp2b in the blastema of regenerating fins leads to excess bone deposition in regions competent to produce bone-secreting cells. Conversely, treatment of regenerating fins with cyclopamine results in a reduction in fin outgrowth and eventual cessation of cell proliferation and distal bone deposition in the blastema. These results suggest that shh signaling plays a role in the proliferation and/or differentiation of the bone-secreting cells within the blastema during fin outgrowth and is supported by the observation that treatment with cyclopamine results in an early expansion, then later reduction of shh expression once fin outgrowth has ceased. shh and bmp2b signaling may play a fundamental role in defining the region of bone deposition in the regenerating fin, possibly by influencing the differentiation of the bone-secreting scleroblasts. However, shh signaling is not required for the secretion of bone matrix by already differentiated scleroblasts.

Acknowledgments

We thank Christopher W. Brown and Reby H. W. Lee for their earlier contributions to this work, Marc Ekker for critical reading of the manuscript, and Marnie Halpern for providing the chordin-expressing construct. This work was supported by Grant MT11775 from the Canadian Institutes of Health Research (to M.A.A.). E.Q. was supported by Grant 1-FY99-572 from the March of Dimes Birth Defects Foundation to Marc Ekker. F.A. was supported by a postdoctoral fellowship from the “Fondation pour la Recherche Médicale,” France.

Abbreviations

CMV
cytomegalovirus
shh
sonic hedgehog
hh
hedgehog
bmp
bone morphogenetic protein
GFP
green fluorescent protein
EGFP
enhanced GFP
dpa
days after amputation

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

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