Involvement of the Sonic Hedgehog, patched 1 and bmp2 genes in patterning of the zebrafish dermal fin rays

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1 Development 125, (1998) Printed in Great Britain The Company of Biologists Limited 1998 DEV Involvement of the Sonic Hedgehog, patched 1 and bmp2 genes in patterning of the zebrafish dermal fin rays Lynda Laforest 1,2, Christopher W. Brown 1,2, Germán Poleo 1,2, Jacqueline Géraudie 3, Masazumi Tada 4, Marc Ekker 1,2 and Marie-Andrée Akimenko 1,2, * 1 Loeb Institute for Medical Research, Ottawa Civic Hospital, and 2 Department of Cellular and Molecular Medicine, University of Ottawa, 725 Parkdale Avenue, Ottawa, Ontario, K1Y 4E9, Canada 3 Laboratoire de Biologie du Développement, Université Paris 7, Denis Diderot, Case 7077, 2 place Jussieu, Paris Cedex 05, France 4 Department of Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-Ku, Sapporo 060, Japan *Author for correspondence ( akimenko@civich.ottawa.on.ca) Accepted 13 August; published on WWW 30 September 1998 SUMMARY The signaling molecule encoded by Sonic hedgehog (shh) participates in the patterning of several embryonic structures including limbs. During early fin development in zebrafish, a subset of cells in the posterior margin of pectoral fin buds express shh. We have shown that regulation of shh in pectoral fin buds is consistent with a role in mediating the activity of a structure analogous to the zone of polarizing activity (ZPA) (Akimenko and Ekker (1995) Dev. Biol. 170, ). During growth of the bony rays of both paired and unpaired fins, and during fin regeneration, there does not seem to be a region equivalent to the ZPA and one would predict that shh would play a different role, if any, during these processes specific to fish fins. We have examined the expression of shh in the developing fins of 4-week old larvae and in regenerating fins of adults. A subset of cells in the basal layer of the epidermis in close proximity to the newly formed dermal bone structures of the fin rays, the lepidotrichia, express shh, and ptc1 which is thought to encode the receptor of the SHH signal. The expression domain of ptc1 is broader than that of shh and adjacent blastemal cells releasing the dermal bone matrix also express ptc1. Further observations indicate that the bmp2 gene, in addition to being expressed in the same cells of the basal layer of the epidermis as shh, is also expressed in a subset of the ptc1-expressing cells of the blastema. Amputations of caudal fins immediately after the first branching point of the lepidotrichia, and global administration of all-trans-retinoic acid, two procedures known to cause fusion of adjacent rays, result in a transient decrease in the expression of shh, ptc1 and bmp2. The effects of retinoic acid on shh expression occur within minutes after the onset of treatment suggesting direct regulation of shh by retinoic acid. These observations suggest a role for shh, ptc1 and bmp2 in patterning of the dermoskeleton of developing and regenerating teleost fins. Key words: blastema, Danio rerio, developmental patterning, hedgehog, Patched, Bmp, limb regeneration INTRODUCTION Analysis of regenerative capabilities in many teleosts has shown that they have the ability to regenerate their fins after injury. This is restricted to the fin ray structures that enclose the dermal skeleton, since amputation close to the body does not allow fin regeneration (Goss, 1969). The skeletal elements of the fin rays, the lepidotrichia, have a dermal origin since, and in contrast to endochondral bones of the girdles or of the limb segments in tetrapods, lepidotrichial ontogenesis occurs by direct mineralization of a collagenous matrix without passage through a cartilaginous precursor (Géraudie and Landis, 1982; Landis and Géraudie, 1990). The lepidotrichia consist of pairs of segmented concave hemirays facing each other (Fig. 1) and occupy a subepidermal position (Becerra et al., 1983). Most of the lepidotrichia form a few bifurcations or forks along the proximodistal axis of the fin. The space between two hemirays is filled with connective tissue and contains the nerves and blood vessels. Each ray ends distally with a row of rigid but unmineralized elastoidin fibrils named actinotrichia (Ryder, 1885) which internally line each hemiray (Fig. 1; Chandross and Bear, 1979). During fin regeneration, fin rays can be considered as independent regeneration units separated from each other by soft tissue (Géraudie and Singer, 1992). Within the first day after amputation, epidermal cells rapidly cover the wound. A blastema of undifferentiated cells develops at the level of each bony ray stump and proliferates beneath the wound epidermis (Géraudie et al., 1998). As blastemal cells leave this area, they progressively differentiate to give rise to all cell types involved

2 4176 L. Laforest and others in the regeneration of connective, nervous, vascular and skeletal tissues. The differentiation program leading to bone regeneration has received most attention at the histological and cytological levels. Bone regeneration starts with the differentiation of blastemal cells in contact with the basement membrane into scleroblasts which synthesize and release the lepidotrichial matrix in the subepidermal space (Becerra et al., 1996; Géraudie and Singer, 1992; Marí-Beffa et al., 1996; Santamaría et al., 1992). During development, signaling between cells is essential to determine their relative positions within the embryo. Regeneration also involves precise cell communications to restore the missing part of the injured organ. In order to explore the possible function of signaling proteins during fin regeneration, we analyzed the expression of genes involved in the SHH signaling pathway. Members of the Hedgehog (HH) family of secreted proteins control a number of inductive interactions, the best characterized members being the Drosophila Hedgehog and the vertebrate Sonic Hedgehog (SHH) proteins. In vertebrates, transcripts of the shh gene are expressed in tissues associated with polarizing activity through cell-cell communication, including the notochord, the floor plate and the zone of polarizing activity (ZPA) of the limb bud (Echelard et al., 1993; Krauss et al., 1993; Riddle et al., 1993; Roelink et al., 1994). Ectopic expression of SHH mimics the effects of an ectopic ZPA, a region in the posterior part of the limb bud which organizes the anterior-posterior axis of the limb (Riddle et al., 1993). We had previously reported that in developing pectoral fin buds of zebrafish, shh is regulated in a manner consistent with a role in mediating the action of a ZPA (Akimenko and Ekker, 1995). In Drosophila, one of the secondary signals produced in response to HH activity in the wing imaginal disk is decapentaplegic (DPP). In vertebrates, misexpression of SHH at the anterior margin of the limb bud results in the induction of the ectopic expression of bmp2 (Laufer et al., 1994), a member of the transforming growth factor β (TGF-β) family which exhibits a high degree of sequence similarity with the Drosophila DPP. HH induces transcription of target genes such as dpp by relieving the repressive activity of the gene patched, ptc (Capdevila et al., 1994; Hidalgo and Ingham, 1990; Ingham et al., 1991). Ptc, which encodes a transmembrane protein (Hooper and Scott, 1989; Nakano et al., 1989) is itself a target gene of hh. Vertebrate homologs of the Drosophila ptc gene have been isolated (Concordet et al., 1996; Goodrich et al., 1996; Hahn et al., 1996; Johnson et al., 1996; Marigo et al., 1996a) and are highly expressed in cells close to those that express SHH (Bellusci et al., 1997; Goodrich et al., 1996; Marigo et al., 1996a; Marigo and Tabin, 1996; Platt et al., 1997). SHH can bind to PTC in cell culture and in frog oocytes (Marigo et al., 1996b; Stone et al., 1996) suggesting that PTC is the receptor for the SHH signal. Here we show that, during fin regeneration, a subset of cells of the basal layer of the epidermis, in close proximity with the newly formed bone structures express shh. These patterns resemble those seen during the initial growth of the bony rays that occurs around the fourth week of development of zebrafish fry, but clearly differ from shh expression in pectoral fin buds. The zebrafish ptc1 (Concordet et al., 1996) and bmp2 (Nikaido et al., 1996) genes are reinduced during fin regeneration with patterns which suggest a coordinated expression with shh. Finally, treatments with retinoic acid, previously reported to cause fusion of lepidotrichia, result in the transient loss of shh, ptc1 and bmp2 expression. These results are discussed in the light of a possible role of shh, ptc1 and bmp2 in bone patterning. MATERIALS AND METHODS Animals Zebrafish were obtained from the Oregon AB line. Adults and embryos were maintained at 28.5 C using standard methods (Westerfield, 1995). Fin amputation Adult zebrafish of at least 10 weeks of age were anesthetized by immersion in water containing 0.17 mg/ml tricaine (ethyl-maminobenzoate; Westerfield, 1995). Caudal fins of adult fish were amputated, using a scalpel, proximal to the first branch point of the lepidotrichia. Fish were returned to their tanks. At various times afterwards, the fins were excised, and fixed in a phosphate-buffered saline solution (PBS) containing 4% paraformaldehyde (PFA, Westerfield, 1995). Retinoic acid treatment All-trans-RA was purchased from Sigma and a 0.1 M stock solution was made in dimethyl sulfoxide (DMSO). For treatment of adult fish, RA was dissolved in 0.5% DMSO at a final concentration of 10 6 M. This concentration has been shown to be the most effective in producing defects in fin regenerates (Géraudie et al., 1994; White et al., 1994). After treatment, adult fish were washed extensively with system water. Controls were immersed in 0.5% DMSO for the same periods of time. Gene expression analysis In situ hybridization on whole-mount fins was performed as previously described (Akimenko et al., 1995). The shh probe consisted of antisense RNA corresponding to a 1440 bp fragment that excludes the region conserved between different hedgehog genes (Krauss et al., 1993). The shh cdna, also named vhh-1, was generously provided by V. Korzh. The ptc1 probe consisted of a 1150 bp fragment of the cdna sequence encoding for the 400 to 780 amino acid sequence of the open reading frame (Concordet et al., 1996). The bmp2 probe consisted of antisense RNA corresponding to the full length cdna, 1448 nucleotides (Nikaido et al., 1996). The bmp2 probe did not give strong hybridization signals using our standard hybridization protocol. The colorimetric detection was modified to enhance the hybridization signal by adding polyvinyl alcohol (PVA) as follows and according to (Barth and Ivarie, 1994): 5% PVA was dissolved in equilibration buffer (100 mm Tris-HCl, ph 9.5, 100 mm NaCl, 50 mm MgCl 2) in a boiling water bath. Prior to colorimetric detection, fins were washed in equilibration buffer + 1 mm levamisol + 0.1% Tween-20. BCIP and NBT were added to equilibration buffer + PVA. Color developed in darkness for up to 16 hours. To stop the reaction, fins were rinsed in PBS containing 0.1% Tween-20 (PBST). Fins to be sectioned were post-fixed with 4% PFA after in situ hybridization and embedded in agar (Westerfield, 1995). Cryostat sections were 16 µm thick. Alcian blue and Alizarin stainings Fins were fixed in 4% PFA and washed in PBS. They were stained for 6 hours at room temperature with a filtered 0.1% Alcian blue solution in 30% acetic acid, 70% ethanol, dehydrated in a waterethanol series and kept in 100% ethanol overnight at 4 C to fix the Alcian blue in the fin rays and de-stain surrounding soft tissues. Fins were rehydrated in a water-ethanol series, macerated in a fresh

3 shh, ptc1, bmp2 in zebrafish fin regeneration 4177 solution of 0.5% trypsin in 2% sodium borate for 10 minutes at 37 C, and extensively rinsed with frequent water changes. Bones were stained by placing the fins in a fresh solution of 0.1% Alizarin red S in 0.5% KOH for 4-5 hours at room temperature. Fins were cleared by transfer in glycerol through a graded series of 0.5% KOH-glycerol solutions and kept in 100% glycerol at 4 C. RESULTS Cell-specific induction of shh during fin regeneration Shh transcripts are expressed during embryonic development of the pectoral fin buds in a position analogous to that of the ZPA of tetrapod limbs. A role for a ZPA in development or regeneration of fin rays is uncertain, questioning the role of shh in these processes. Thirty hours post-amputation of caudal fins, a few dispersed cells express shh at the distal end of each ray (not shown). At 2 days, a strong expression of shh is detected in cells located at the level of amputation (Fig. 2A). Beginning at 4 days, shh transcripts are localized to two groups of cells on each side of the second to seventeenth fin rays (the caudal fin is composed of 18 major fin rays). Shh-expressing cells thus form a total of four groups per ray (Fig. 2B,D). Rays 1 and 18 have only one group of shh-expressing cells on each side (Fig. 2B). Sections of hybridized fins revealed that shh expression, 2 days post-amputation, is restricted to a subset of cells of the basal layer of the epidermis, in a region immediately distal of the site of amputation (Fig. 2E). As new bone is forming, expression of shh is always observed in cells of the basal epidermal layer (5-10 cells wide and cells long) immediately adjacent to the lepidotrichial material being released in the subepidermal space (Fig. 2F,G). Transverse sections reveal that when a unique group of shh-expressing cells is present, it is always centered in the fin ray, but when two groups of shh-expressing cells are observed, they are located on the lateral sides of fin ray (Fig. 2H,I). This pattern of expression, observed throughout the regeneration process, Lepidotrichia Fin Ray Actinotrichia Bifurcation Segment Hemisegments Fig. 1. Dermal skeleton of a fin ray. The lepidotrichia are composed of two tile-like hemirays made of a series of segments or hemisegments. The lepidotrichia form a few dichotomous branches along the proximodistal axis of the fin. Each lepidotrichia ends distally with a row of rigid elastoidin fibrils lining the inner surface of the hemirays. suggests that shh participates in the process of dermal bone formation. Expression of shh in two groups of cells on one side of the major rays 2-17 is observed shortly before formation of a bifurcation (Fig. 2B). After the bifurcation took place, only one group of expressing cells is observed, transiently, in the center of each newly formed branch (Fig. 2C,H). Shh expression in two subsets of cells on each side of the rays precedes any detectable morphological changes associated with the formation of a bifurcation (Fig. 2I). Therefore, the dynamics of shh expression suggests a role in the induction of bone bifurcation. We observed induction of shh in regenerating pectoral, dorsal and anal fins with patterns and time course that are similar to those observed in the caudal fins (data not shown), suggesting similar roles for shh in the regeneration of both paired and unpaired fins. Therefore, this situation contrasts with early fin development when only developing paired fin buds express shh. Shh expression in regenerating fins recapitulates expression during larval development Shh expression in pectoral fin buds of zebrafish larvae diminishes after 80 hours and is no longer detectable at 4 days post-fertilization. The growth of the fin ray structures initiates around the fourth week of life. Patterns of shh expression during development of lepidotrichia of larval fins resemble those observed during fin regeneration (Fig. 3B,D-F). Fin sections (not shown) confirm that shh expression is restricted to cells of the basal layer of the epidermis, as observed during regeneration. Shh is re-expressed in the developing fins in a sequential manner, which correlates with the order of formation of the lepidotrichia starting with the caudal fin (Fig. 3A,B), followed successively by the anal (Fig. 3C,D), dorsal (Fig. 3E), pectoral (Fig. 3F) and, finally, the pelvic fins (Fig. 3G). At the onset of pelvic fin buds outgrowth, shh expression is first confined to the cells at the posterior margin of the bud in a pattern similar to that observed during the development of the pectoral fin buds (Sordino et al., 1995). In contrast to pectoral fins, the pelvic fin lepidotrichia start to develop shortly after the onset of formation of the fin buds. As the lepidotrichia are forming, cells at the posterior margin of the pelvic bud no longer express shh but transcripts are found in each ray with patterns similar to that of other fins (Fig. 3G). Bifurcations form relatively late after the initiation of fin ray development. Consequently, we never observed bifurcating fin rays at the larval stages analyzed and shh transcripts remained confined to one domain on each side of the rays. This observation reinforces the hypothesis that shh expression in two groups of cells precedes the formation of a bifurcation. Ptc1 could mediate shh activity in the developing and regenerating fins A faint expression of ptc1 is first detected at 40 hours post amputation at the level of the stump bone (not shown). Later, ptc1 hybridization signal is located at a similar proximodistal position along the fin ray as shh (Fig. 4A,B). However, while shh expression is observed in one or two discrete groups of cells (Fig. 2), ptc1 transcripts occupy the entire width of the fin ray. The limits of the ptc1 expression domain are diffuse. Proximodistal axis sections show that ptc1, like shh, is expressed in the basal layer of the epidermis (Fig. 4C), 48

4 4178 L. Laforest and others Fig. 2. Induction of shh during regeneration of the caudal fin. shh expression was determined (A,E) 2 days; (B,D,F-I) 4 days, (C) 12 days after amputation. Two fin rays are shown in A and D, three fin rays in B and branches of three fin rays are shown in C. Shh is confined to one or two subsets of cells in individual fin rays on each side of the fin (B- D). Shh is expressed in only one subset of cells as soon as a bifurcation is formed (C) or in the lateral-most fin rays of a caudal fin which never form branches (marked with an asterisk in B). (D) The distal part of the fin is slightly tilted; the open triangles and the filled triangles indicate the two subsets of shh-expressing cells on each side of the ray. (E-G) Proximodistal and (H,I) transverse sections of regenerating caudal fins, 2 days (E) and 4 days (F-I) after amputation. The planes of sections are shown in B. (G) Higher magnification of a section comparable to that shown in F with shh-expressing cells delimited by brackets. The scleroblasts can be distinguished from the rest of the blastemal cells by their location and by their more condensed appearance. The region containing shh-expressing cells seems to overlap the limit where lepidotrichial material can be observed in the epidermal-blastemal interface. (H) shh is expressed in a unique broad domain when no bifurcation is forming. (I) shh expression in two subsets of cells on one side of the fin ray precedes the formation of a bifurcation of the lepidotrichia. The large arrows in A, B and E indicate the level of amputation. P (in B) indicates the proximal direction; b, blastema; l, lepidotrichia; e, epidermis; s, scleroblasts. Scale bars, A-D, 80 µm; E,F,H,I, 30 µm; G, 4 µm. Fig. 3. Shh and ptc1 expression in the developing fins of zebrafish larvae. (A,B) Caudal fins of 5.5 mm larvae. (C,D) Anal fins of 6.5 mm larvae. (E) dorsal fin, (F) pectoral fin and (G) pelvic fin of 7.2 mm larva. (A) Alcian blue staining of the cartilage. The lepidotrichia stained in light blue are already well developed in the caudal fin fold. (B) Caudal part of a similar larva to the one in A showing shh expression in each developing lepidotrichia. Note that whole-mount in situ hybridization procedure causes a shrinkage of the tissues. (C) Alcian blue staining of the cartilage of the anal fin. Some elements of the endoskeleton as well as the exoskeletal lepidotrichia are not yet apparent. (D) In a comparable fin to that in C, shh transcripts are already found in the fin fold in subset of cells where the lepidotrichia are forming. Shh is also strongly expressed in the analia-genitalia region. (E-G) Shh expression subsequently appears in dorsal (E), pectoral (F), and pelvic (G) fin rays. The waving of the dorsal fin (E) shows the two groups of cells, one per side, expressing shh. Ptc1 expression in a caudal fin (H) and a pectoral fin (I) of a 7 mm larva. The caudal fin is already heavily pigmented. Arrows in B,D-F and in H,I indicate shh and ptc1 expression, respectively. Anterior is to the left; h, cartilage condensation of hypural bones; l, lepidotrichia; n, notochord; ff, fin fold; pr, proximal radial of the endoskeleton of an anal fin. Scale bars, A, 200 µm; B, 100 µm; C,D,E,G,H,I, 160 µm; F, 80 µm hours after amputation. Later, ptc1 expression still overlaps that of shh (Fig. 4D) but occasionally extends more proximally. In addition, ptc1 transcripts are also found in adjacent mesenchymal cells which border the lateral sides of the blastema (Fig. 4D). Transverse sections (not shown) indicate that ptc1 is expressed across the width of the ray in both

5 shh, ptc1, bmp2 in zebrafish fin regeneration 4179 epidermal and mesenchymal cells. The ptc1-expressing mesenchymal cells are more compact than those of the blastema core. These cells are scleroblasts which release the lepidotrichial material beneath the subepidermal basement membrane during regeneration (Marí-Beffa et al., 1996; Santamaría et al., 1996). Their differentiation progresses from the distal to the proximal part of the regenerate and the ptc1- expressing cells correspond to newly formed scleroblasts. Although this pattern of expression remains unchanged 4 days post amputation in 71% of the fins analyzed (10/14), in 29% of the fins, ptc1 transcripts are only found in the basal layer of the epidermis (Fig. 4E). Similarly, 6 days after amputation, ptc1 transcripts are strictly found in the basal layer of the epidermis in approximately 50% of the fins (not shown). We consistently observe stronger hybridization signals in fins where ptc1 is uniquely expressed in the basal epidermal cells, compared to fins where ptc1 transcripts are found both in the mesenchymal and epidermal compartments. A similar coordinated expression of shh and ptc1 takes place in developing fin rays of larvae (Fig. 3H,I). Coordinate expression of bmp2, shh and ptc1 Bmp2, a member of the TGF-β family of genes, is induced in many tissues in response to SHH. To investigate the possible interactions between shh, ptc1 and bmp genes, we examined the expression of bmp2 during fin regeneration. Four days after amputation, the distribution of bmp2 mrna is very similar to that of shh (Fig. 4F). However, bmp2 expression is detected before shh expression, 24 hours after amputation, in cells of the basal layer of the epidermis whose distal limit corresponds to the stump bone. At that stage, the basal layer of the wound epidermis is not yet organized. At 30 hours, bmp2 transcripts are found in a few cells of the basal layer of the epidermis just below the level of amputation as well as in basal cells covering the wound (Fig. 4G). At this time, bmp2 is also expressed in the newly forming blastemal cells (not shown). Starting 40 hours post-amputation, bmp2 transcripts are found immediately distal to the level of amputation (Fig. 4H). Unlike shh, bmp2 is also expressed in two groups of mesenchymal cells lining the blastema, adjacent to the bmp2-expressing epithelial cells (Fig. 4H). Starting at day 4, bmp2 transcripts are detected either in the basal epidermal cells and the scleroblasts (15/21 fins) or in the basal epidermal cells only (Fig. 4I,J). Two days later, each pattern of expression is found in 50% of the fins. Thus, this aspect of bmp2 expression Fig. 4. Expression of ptc1 and bmp2 in the regenerating caudal fin. Expression of ptc1 (A-E) was determined at 2 days (A,C) and 4 days (B,D,E) after amputation. (C-E) Proximodistal sections. (C) Two days after amputation, ptc1 is expressed in the basal epithelial layer of the wound epidermis just distal to the stump. (D,E) Four days after amputation, ptc1 is expressed at the same proximodistal level as shh but in 71% of the fins analyzed, it is not only expressed in the basal epithelial layer but also in the adjacent scleroblasts (D); in the remaining 29%, ptc1 is only expressed in a subset of cells of the basal epithelial layer (E). Expression of bmp2 (F-K) was determined at 4 days (F,I-K), 30 hours (G) and 2 days (H) after amputation. G-K are proximodistal sections. (H) Two days after amputation, bmp2 is expressed in cells of the basal epidermal layer just distal to the stump bone on each side of the fin and in adjacent scleroblasts. (I,J) At 4 days after amputation, bmp2 like ptc1 is expressed in 71% of the fins in the basal epithelial cells as well as in the scleroblasts (I); in the remaining 29%, bmp2 transcripts are only found in the epidermal compartment (J). (K) Another site of bmp2 at 4 days is a group of cells invading the space between the newly forming lepidotrichia and the epidermis as indicated by the single arrowhead. The double arrowhead in K indicates the distal domain of bmp2 expression. Arrows indicate the level of amputation. b, blastema; l, lepidotrichia; e, epidermis; s, scleroblasts. Scale bars, A,B, 80 µm; C,D,E,H,K, 30 µm; F, 65 µm; G,I,J, 25 µm.

6 4180 L. Laforest and others resembles that of ptc1 and occurs with similar proportions. The alternative patterns of expression of bmp2 and ptc1 may be linked to the formation of segment boundaries of the lepidotrichial matrix as discussed below. Starting 3 days post-amputation, bmp2 transcripts are also found in a group of compact cells located at the level of the amputation between the basal layer of the epidermis and the developing bone (Fig. 4K). A transient change in shh expression precedes fusion of bony ray segments Amputations of the caudal fin immediately after a branching point of the lepidotrichia will result in the fusion of the lepidotrichiap branches. To test the proposed role for shh in bone formation during fin regeneration, we cut a zebrafish caudal fin either within the first or second segment immediately after the first branching point ( short cut ), a procedure that should result in fusion of the lepidotrichia, or at the end of the third lepidotrichia segments ( long cut ), a procedure that does not lead to fusion (Fig. 5A,B). Three days after a short cut, shh expression is weaker and forms more diffuse patterns (Fig. 5C) compared to rays with a long cut (compare Figs. 2 and 6G). This is only observed in fin rays undergoing a fusion of the two newly formed branches. Later, when the fused rays are regenerating as a unique fin ray, shh expression is resolved into two groups of shh-expressing cells, announcing a bifurcation (Fig. 5E). Expression of shh, ptc1 and bmp2 in regenerating fins is down-regulated following treatment with retinoic acid Treatments with retinoic acid (RA) during fin regeneration can result in multiple fusions of adjacent lepidotrichia (Géraudie et al., 1994; Géraudie et al., 1995; White et al., 1994). Therefore, it was interesting to investigate the effects of RA on the expression of genes involved in the shh pathway. Caudal fins were amputated and left to regenerate for an initial 1 day period. The fish were then treated with 10 6 M all-trans RA. Twenty-four hours later, the fish were returned to normal tank water and shh expression was determined at several time points thereafter. It was previously reported that, while the growth of the regenerate is inhibited during RA administration, it quickly resumes shortly after the end of treatment (Ferretti and Géraudie, 1995). Regenerates of treated and control fish are similar in size, 3 days after treatment, although RA has induced a narrowing of the tissues which shows as a characteristic curving of the fin (Fig. 6A,B). A loss of shh expression is observed in regenerating caudal fins of RA-treated fish, when determined 1 or 3 days after the end of treatment (Fig. 6A,B). At these times, shh transcripts are normally expressed at high levels in regenerating fin rays of non-treated fish (Fig. 2) or of controls treated with DMSO (not shown). A faint shh re-activation can be detected in a small percentage of fins, 3 days after the end of RA treatment (i.e. 5 days after amputation; data not shown). This re-activation is progressive and occurs first in the dorsal- and ventral-most rays (Fig. 6C). To correlate the effects of RA on shh gene expression with a possible effect on bone formation, we repeated the above RA treatments and, 3 days or 5 days later, caudal fins were cut into two pieces. Shh expression was determined in one half of the fin while the other half was stained with Alizarin red and Alcian blue. Although lepidotrichia and actinotrichia are dermal elements which do not differentiate through cartilage formation, the lepidotrichial matrix contains glycosaminoglycans which can be efficiently stained using Alcian blue. Three days after the end of the treatment, Alcian blue staining, distal to the stump bone, covers a reduced surface compared to controls (Fig. 6D); this surface is narrower and has a triangular shape not observed in controls. Five days after the end of treatment, although the lengths of the regenerates of RA-treated and untreated fins are similar, the lepidotrichial matrix in treated fins does not extend as far as the matrix in untreated fins and its distal limit looks more diffuse compared to controls (Fig. 6E,F). At both times, shh-expressing cells mark the distal part of the lateral limits of the surface stained with Alcian blue, suggesting a correlation between the progression of shh re-expression and the lepidotrichial matrix deposition. To determine how rapid are the effects of RA on shh expression, we amputated caudal fins, let them regenerate for 4 days, began the treatment with RA but fixed fins at various time points after the onset of treatment. A decrease in shh expression was observed after only 30 minutes of RA treatment (not shown) and transcript levels are greatly reduced after 1 hour (Fig. 6G,H). By 4 hours, shh transcripts are no longer detectable (Fig. 6I). The same transient disappearance of shh expression was observed in larvae treated for 2 hours with RA (not shown). Following RA treatment, bmp2 expression disappears as fast as shh whereas ptc1 expression disappears with a slight delay (Fig. 6J-L). This delay could be attributable to the persistence of SHH protein molecules even though shh transcription is rapidly down-regulated. DISCUSSION During zebrafish fin regeneration, shh and two genes, ptc1 and bmp2, known to be part of the SHH signaling pathway in multiple systems, are up-regulated in a coordinated manner suggesting a role for the products of these genes in epithelial mesenchymal interactions that lead to synthesis and patterning of the bone. Shh is exclusively expressed in cells of the basal layer of the epithelial compartment located at the level where scleroblast cells of the blastema start to secrete the lepidotrichial matrix in the subepithelial space. Ptc1 and bmp2 transcripts are detected in the same cells as shh but also in the scleroblasts. Shh and ptc are expressed in overlapping or adjacent domains in many systems, such as the posterior mesenchymal cells of the mouse and chick limb buds and of the zebrafish pectoral fin buds, the neural tube, the developing gut, whisker and hair follicles, the lungs, and the developing mouse retina (Bellusci et al., 1996, 1997; Concordet et al., 1996; Goodrich et al., 1996; Marigo et al., 1996a; Jensen and Wallace, 1997). Furthermore, when ectopically expressed, shh can up-regulate the expression of ptc in the neural tube, limb buds and lungs (Bellusci et al., 1997; Goodrich et al., 1996; Marigo et al., 1996a). Expression of ptc1 in epithelial cells overlapping the shh domain and in surrounding mesenchymal cells suggests that SHH signaling is mediated through the interaction with PTC1. This relationship is further supported by bmp2 expression analysis. The

7 shh, ptc1, bmp2 in zebrafish fin regeneration 4181 Drosophila HH and the vertebrate SHH proteins appear to regulate the expression of decapentaplegic (dpp), and the related bmp2 and bmp4 genes of vertebrates, respectively (Basler and Struhl, 1994; Bitgood and McMahon, 1995; Laufer et al., 1994; Roberts et al., 1995). For example, bmp2, whose domain overlaps that of shh and ptc in the posterior margin of the chick limb bud, is up-regulated after ectopic expression of shh in anterior mesenchymal cells of the bud (Laufer et al., 1994). During tooth development, shh and bmp2 transcripts are both located in the enamel knot which is proposed to function as an organizing center (Vaahtokari et al., 1996). During fin regeneration, bmp2 transcripts are detected prior to shh or ptc1 transcripts, suggesting that bmp2 expression is not induced by SHH signaling, although we cannot exclude the possibility that very low level of shh expression, undetectable by in situ hybridization, may be sufficient to induce early expression of bmp2. Later, bmp2 transcripts co-localize with shh transcripts in the epithelial compartment but are also found in scleroblasts facing shh-expressing cells. The SHH signal may regulate the expression of bmp2 both in basal epidermal cells and in the facing blastemal cells. It is tempting to propose that bmp2 regulation by shh may be transduced via SHH binding to PTC1. However, the ptc1 expression domain is broader than that of bmp2 in both the epithelial and mesenchymal compartments: transcripts of ptc1 but not of bmp2 are found in the center of the fin rays. This suggests that, if regulated through the SHH/PTC1 signaling pathway, bmp2 expression might be inhibited in the center of the fin ray through a yet unknown mechanism. It is also possible that secretion of SHH from the two lateral groups of cells might be enough to regulate ptc1 but not bmp2 expression in more centrally located cells. Throughout fin regeneration, the shh domain of expression always remains adjacent to the newest scleroblasts of the blastema. While we do not know how the distal limit of this domain of expression is regulated, it is possible that the proximal limit of shh domain of expression may be determined by the thickness of the lepidotrichial matrix already deposited in the subepidermal space. In other systems, maintenance of shh expression depends on signals which are themselves regulated by SHH to form a positive feedback loop. For example, Fgf4 expression in the posterior part of the AER of chick limb buds allows the maintenance of shh expression in the ZPA through a positive feedback loop (Laufer et al., 1994; Niswander et al., 1994). In regenerating fins, it is possible that the cumulating lepidotrichial matrix may represent a physical barrier that impedes the transfer of the signal provided by the scleroblasts to maintain shh expression in the basal cells of the epidermis. The absence of SHH signal from the epidermal cells proximally located may, in turn, downregulate the expression of targets, such as ptc1 and bmp2, in the mesenchymal compartment (Fig. 7). Function of the shh signaling pathway The position of the shh domain of expression suggests that it may be involved in the induction of scleroblast differentiation, or in the signaling to the newly differentiated scleroblasts to initiate lepidotrichial matrix synthesis and/or release. Shh, ptc1 and bmp2 may not play a role in cell proliferation. BrdU incorporation experiments show that the rate of proliferation in the parts of the regenerate where these genes are expressed is not higher than in surrounding areas (G. P., C. B. and M.-A. A., unpublished observations). SHH signaling may be involved in patterning the fin rays by determining the lateral limits of the dermal bones. The ptc1 expression domain, which should reflect the zone under SHH influence, covers the width of the lepidotrichia but does not extend beyond the limits of the lepidotrichia. Expression of shh in epidermal cells is normally centered in the fin ray, in the absence of bifurcations such as in the lateral rays, or just after formation of a branching point. Prior to any morphological change, the single shh expression domain resolves into two separate groups of cells, located laterally. This division is soon followed by the formation of a bifurcation of the lepidotrichia. In the new branches, shh is again expressed in the median region and may provide the information necessary to establish the limits of the dermal bones. Formation of a bifurcation begins with the suppression of bone synthesis in the central region of the lepidotrichia. We observed that the bmp2 domain of expression in epidermal and blastemal cells splits into two domains in the same manner as shh prior to branch formation. If BMP2 is required for the synthesis or secretion of the lepidotrichial matrix, the transient absence of this factor in the center of the ray may be a requisite for the formation of a bone bifurcation. Expression of bmp2 and ptc1 is interrupted in scleroblasts in a number of fins at 4-6 days post-amputation. This interruption is likely temporary; i.e. the fins that do not express bmp2 and ptc1 in the scleroblasts 4 days after amputation, are probably expressing these genes 2 days later. It is possible that this alternating pattern of expression is associated with the formation of segment boundaries in the lepidotrichia. During larval development, growth of the lepidotrichia occurs by addition of new bony segments of a fixed length, rather than by progressive growth of these segments. Bone segmentation is regular and, within a fin ray, segments have very similar sizes. Segmentation of the lepidotrichia is reestablished in the regenerate; all newly formed segments within a fin ray have approximately the same size (except for the first segment formed after amputation). We are presently investigating how the cyclic expression of bmp2 and ptc1 in the scleroblasts correlates with the formation of a segment boundary. We observed that patterning of the regenerate depends on the level at which the fin is amputated. When fin amputation is made only one segment distal from a branching point, two blastema should form, one from each lepidotrichial branch. Instead, a unique blastema is established which gives rise to one large regenerate. It is possible that this patterning may result from a mis-interpretation of shh expression. Two days after amputation, shh expression is never centered in the fin ray but spreads over the entire surface of the severed bone, regardless of the level of amputation (Fig. 2). If the fin is amputated just distal from a branching point, the proximity of the two severed lepidotrichial branches leads to the induction of two very close domains of shh expression which are presumably interpreted as a unique source of SHH signal by the receiving cells. The unique diffuse shh expression domain finally divides into two domains by the fifth day and this is followed by the formation of a bone bifurcation. Together, these results suggest that the SHH/PTC1 signaling pathway is involved in patterning of the fin dermal bone during regeneration. During mouse and chick development, another member of the Hedgehog family, Indian Hedgehog (Ihh), is involved in the process of endochondral bone development (Vortkamp et

8 4182 L. Laforest and others Fig. 5. Effect of amputation level on shh expression. (A) Schematized representation of the levels of amputation with respect to the first bifurcation of the caudal fin rays. A short cut was done within 1-2 segments after the bifurcation. A long cut was made further than 2 segments distal to the bifurcation. The small insert shows the ray fusions induced by the short, but not by the long cut. (B) Representative fusions of caudal fin. Due to the uneven level of the first bifurcations across the fin, some rays underwent a short cut (s), whereas some underwent a long cut (l). Only rays with short cuts have fused branches. (C-E) Shh expression in short cut fin rays at 3 days (C), 4 days (D), and 5 days (E). The cells expressing shh form more diffuse patterns at 3 and 4 days compared to fins that had a long cut (see Fig. 2). The two groups of cells expressing shh, announcing a bifurcation, are clearly resolved at 5 days. The bracket in E indicates ray fusion. Arrows in C,D,E, indicate the level of amputation. Scale bars, 80 µm. al., 1996). Ihh transcripts are found in prehypertrophic chondrocytes. Ptc, Bmps and the transcriptional regulator Gli, both targets of Ihh are expressed in adjacent cells (Iwasaki et al., 1997). The same factors are also involved in the fracture repair of the long bone in a way that recapitulates their functions in bone development (Vortkamp et al., 1998). Of the known zebrafish hh-related genes, echidna hedgehog (ehh) is the most likely ortholog of Ihh (Zardoya et al., 1996). However, the restricted expression of ehh in muscle pioneers and notochord (Currie and Ingham, 1996) is not consistent with a role for ehh in fish that would be homologous to the role of Ihh in birds or mammals. We cannot rule out at this point, the existence of an additional hh-related gene in zebrafish which may be the true ortholog of Ihh. Fig. 6. Retinoic acid impairs shh and ptc1 expression in regenerating caudal fins. (A-C) Zebrafish were treated with 10 6 M all-trans-ra for a period of 1 day, starting 1 day after amputation. Shh expression was determined (A), 1 day; (B), 3 days; (C), 6 days after the end of the RA treatment. The curve in the caudal fin in A and B is due to the fusion of rays. Shh expression is abolished in treated fins at 1 or 3 days but gradually re-established at 6 days. (D-F) Alcian blue and Alizarin red stainings showing the lepidotrichial matrix and the mineralized bones of the stump, respectively, in half-fins treated with RA (D,E) or in untreated fins (F). Three days after the end of the treatment, the regenerate is developing but there is little lepidotrichial matrix deposition at the tip of each native fin rays (D); in situ hybridization on the second half of this fin shows a weak shh expression (not shown). Five days after the end of the treatment (E), the lepidotrichial matrix does not extend in the regenerate as far as in the control fin (F). Expression of shh (G-I) or of ptc1 (J-L) is abolished by short RA treatments. Zebrafish were treated with 10 6 M RA for 1 hour (H,K), or 4 hours (I,L), starting 3 days after amputation. Expression of shh and ptc1 was determined immediately before RA treatment (G,J); or immediately after the end of the treatment (H,I,K,L). Three days after amputation, shh (G) and ptc1 (J) transcripts are clearly detectable in fin rays, but markedly decreased (H,K) or undetectable (I,L) after 1 and 4 hours of RA treatment, respectively. Arrows indicate the level of amputation; dotted lines in D-F indicate the distal limit of the regenerate. Scale bars, C-L, 80 µm.

9 shh, ptc1, bmp2 in zebrafish fin regeneration 4183 A Epidermis shh ptc1 Blastema ptc1 B Epidermis shh bmp2 Blastema bmp2 Fig. 7. The SHH signaling pathway in developing and regenerating dermal skeleton of the fin rays in zebrafish. (A) SHH signaling emanating from the basal epidermal cells may activate ptc1 expression in the same cells as well as in adjacent basal epidermal cells and scleroblasts of the blastema. (B) Once expression of shh and of bmp2 is established in the basal epidermal cells and bmp2 expression is established in the adjacent scleroblasts, shh and bmp2 may coordinately regulate each other expression through cell-cell interactions. The proximal limits of the respective domains of expression may be determined by the thickness of the lepidotrichial matrix already deposited (brown triangular shape) in the interspace between the epidermal and blastemal compartments. Retinoic acid downregulates shh expression in the fin regenerate Administration of retinoic acid to fish during fin regeneration can lead to morphogenetic and teratogenic effects in the regenerate. The severity of the effects produced by RA depends on drug concentration, length of the treatment and the time at which treatment is initiated (Géraudie et al., 1994, 1995). RA treatments of at least 3 days with 10 6 M RA lead to fusion of adjacent rays. Although histological analysis has revealed the presence of blastemal cells in such regenerates, these cells do not form any structured blastema but rather spread across the stump underneath the wound epidermis (Ferretti and Géraudie, 1995; White et al., 1994). This observation, and the fact that regeneration quickly resumes after RA removal, suggests that RA does not impede the formation of these blastemal cells but rather inhibits their distal migration within the regenerate (Ferretti and Géraudie, 1995). Induction of apoptosis, essentially in the epidermal compartment, led to the proposition that loss of patterning in distinct fin rays could be due to primary defects in the epidermal compartment (Géraudie and Ferretti, 1997). Here we show that a 1-day treatment with RA inhibits shh expression and highly perturbs bone development. During this period, bone matrix deposition is either delayed or completely stopped. At the end of the treatment, bone synthesis is reinitiated but normal bone patterning requires a few days to take place, and coincides with re-activation of shh expression. Furthermore, we show that a 1 hour treatment with RA is enough to cause large decreases in shh expression while a 4 hour treatment completely abolishes shh expression. Downregulation of shh, ptc1 and bmp2 by RA is specific and unlikely to be attributable to a toxic effect of RA. First, the effects of RA are rapid. Second, the same treatments do not downregulate the expression of the apolipoprotein E gene, in the wound epidermis or msxb expression in distal blastemal cells (unpublished results). The rapid effects of RA on shh expression suggests that RA may directly regulate shh. A functional RA response element (RARE) of the DR5 type has been found in the 5 region of the zebrafish shh gene and alltrans-ra activates the shh promoter in HeLa cells (Chang et al., 1997). The RARE can be specifically bound in vitro by the retinoid receptors (RARs and RXRs). It is not yet known whether this RARE may have opposite regulatory effects (activation or downregulation) on shh expression depending, for example, on the identity of the RARs and RXRs subtypes or other factors present in various cell types. The present study revealed that shh/ptc1 expression during fin regeneration recapitulates expression of these genes during larval fin development. 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