Shh promotes direct interactions between epidermal cells and osteoblast progenitors to shape regenerated zebrafish bone

Abstract

Zebrafish innately regenerate amputated fins by mechanisms that expand and precisely position injury-induced progenitor cells to re-form tissue of the original size and pattern. For example, cell signaling networks direct osteoblast progenitors (pObs) to rebuild thin cylindrical bony rays with a stereotypical branched morphology. Hedgehog/Smoothened (Hh/Smo) signaling has been variably proposed to stimulate overall fin regenerative outgrowth or promote ray branching. Using a photoconvertible patched2 reporter, we resolve active Hh/Smo output to a narrow distal regenerate zone comprising pObs and adjacent motile basal epidermal cells. This Hh/Smo activity is driven by epidermal Sonic hedgehog a (Shha) rather than Ob-derived Indian hedgehog a (Ihha), which nevertheless functions atypically to support bone maturation. Using BMS-833923, a uniquely effective Smo inhibitor, and high-resolution imaging, we show that Shha/Smo is functionally dedicated to ray branching during fin regeneration. Hh/Smo activation enables transiently divided clusters of Shha-expressing epidermis to escort pObs into similarly split groups. This co-movement likely depends on epidermal cellular protrusions that directly contact pObs only where an otherwise occluding basement membrane remains incompletely assembled. Progressively separated pObs pools then continue regenerating independently to collectively re-form a now branched skeletal structure.

Keywords: BMS-833923; Basal epidermis; Basement membrane; Bone patterning; Calcification; Caudal fins; Cyclopamine; Hedgehog signaling; Indian hedgehog; Osteoblasts; Ray branching; Regeneration; Smoothened inhibitor; Sonic hedgehog; Zebrafish.

Fig. 1.

ptch2:Kaede expression and photoconversion reveals transient Hedgehog/Smoothened signaling restricted to distal osteoblast progenitors and basal epidermis during fin regeneration. (A,B) Whole-mount images showing Kaede expression (green) in a 96 hpa fin from a ptch2:Kaede fish. (B) A high-magnification image of the boxed region in A. White brackets mark the split domains of Kaede at the distal end of the regenerate. Green arrows indicate Kaede⁺ cells at newly re-forming joints. (C-F) Antibody-stained longitudinal fin sections from a 72 hpa ptch2:Kaede fish. Individual Runx2 (C) and Kaede (D) channels are shown in gray scale. Overlay images with Runx2 in magenta and Kaede in green are shown in E and F (a magnification of the boxed region in E). Nuclei are in blue. Yellow arrows indicate Kaede⁺ basal epidermis; white arrows mark Kaede⁺/Runx2⁺ Obs; magenta arrows indicate proximal Runx2⁺ Obs lacking Kaede. The dotted magenta line indicates the boundary between Kaede⁺ basal epidermis and pObs. (G) ptch2:Kaede photo-conversion experiment overview. (H-L) Whole-mount bright-field (H,I) and fluorescent (J-L) images of the regenerating caudal fin from a ptch2:Kaede photo-conversion experiment fish. (J) The fin is shown at 5 dpa, immediately after photoconverting Kaede protein in a distal field (tissue marked by the magenta dashed line). (K,L) The same fin 24 h post-conversion (6 dpa) imaged for Kaede expression. The dashed box in K marks the enlarged region in L. Unconverted and new Kaede is green; photo-converted Kaede is magenta. The green arrow marks cells in a forming joint that expressed Kaede within the previous 24 h. The white bracket indicates the narrow distal domain of new Kaede production since photoconversion. The magenta arrow indicates epidermis near the tip of the fin that had displaced distally while retaining photo-converted Kaede. Dashed yellow lines in A,E,J,K show amputation planes. Scale bars: 50 µm in E; 10 µm in F; 500 µm in J,K; 250 µm in L.

Fig. 2.

shha is briefly transcribed by distal migrating epidermal cells, whereas ihha is restricted to re-differentiating progenitor osteoblasts. (A) qRT-PCR analysis of the relative expression levels of shha, shhb, ihha, ihhb, runx2a and sp7 in 96 hpa fin tissue. The relative levels of the indicated transcripts are means of four fins normalized to rpl8 expression. Error bars represent 1 s.d. (B-E) An immunostained fin section from a 72 hpa shha:GFP fish showing GFP (white), Runx2 (red) and sp7 (green) expression. Nuclei are in blue. E is a high-magnification view of the dashed box in D. (F-O) Fin sections from a 72 hpa fish stained by RNA in situ hybridization for shha (F-J) or ihha (K-O) transcripts (blue) and with Runx2 (red) and sp7 (green) antibodies. Single channels are shown in gray scale (F-I,K-N). Nuclei are gray in the overlay images (I,J,N,O). (J,O) Enlarged regions marked in I,N, respectively. The white bracket indicates the extent of epidermal cells expressing shha relative to pObs. Yellow arrows show Runx2⁺/sp7⁺ Obs that express ihha; magenta arrows mark distal Runx2⁺ pObs that lack ihha mRNA. The dashed yellow lines indicate amputation sites. Scale bars: 25 μm in J,O; 50 µm in E; 50 μm in B-D,F-N.

Fig. 3.

Ihha promotes the efficient calcification of regenerated bone by non-canonical Hedgehog signaling. (A-F) Whole-mount Kaede fluorescence images of 5 dpa caudal fins from ptch2:Kaede (A-C) and ihha^−/−;ptch2:Kaede (D-F) fish 24 h after photoconversion. Photoconverted pre-existing Kaede is magenta; Kaede produced after photoconversion is green. Magenta arrows indicate extreme distal epidermal tissue exclusively expressing converted Kaede. The white brackets indicate the domain of newly expressed Kaede protein. (G,H) Whole-mount images showing GFP-expressing osteoblasts in fins from sp7:EGFP and ihha^−/−;sp7:EGFP fish at 11 dpa. Yellow arrows mark newly formed joints. Red lines and arrows denote points of ray bifurcation. (I,J) Bright-field images of Alizarin Red-stained sp7:EGFP(I) and ihha^−/−;sp7:EGFP (J) fins at 11 dpa. Black and red brackets show the total length of the regenerate and the extent of mineralization from the site of amputation, respectively. Yellow dashed lines in all panels show amputation positions. (K) Quantification of the relative extent of calcified regenerated bone in sp7:EGFP versus ihha^−/−;sp7:EGFP fish at 5, 8 and 11 dpa. Means and data points representing individual fish are shown. Significant differences between control and ihha-null fish (P<0.05) were determined by two-tailed Student's t-tests. Scale bars: 500 μm.

Fig. 4.

BMS-833923, a newly identified potent zebrafish Smoothened inhibitor, shows that Hedgehog/Smo signaling does not impact fin regenerative outgrowth. (A-L′) Whole-mount fluorescence images of 96 hpa fins from ptch2:Kaede fish 18 h after Kaede photoconversion and 5 h after EdU injection. Regenerating fish were treated for 24 h with DMSO (A-D′), cyclopamine (E-H′) or BMS-833923 (I-L′). Newly produced Kaede and converted Kaede are green and magenta, respectively, in overlay images (C,G,K). Magenta arrows indicate cells with converted Kaede. White brackets mark the domain of new Kaede expression in the 18 h since photoconversion. (D,H,L) Fins from the same fish shown for Kaede fluorescence processed to reveal EdU-incorporating nuclei in green. (D′,H′,L′) Confocal stack images showing EdU incorporation at the distal aspect of single re-forming rays. The timeline at the top of the figure details the experimental design. Scale bars: 500 μm.

Fig. 5.

BMS-833923 demonstrates that Hedgehog/Smoothened signaling is dedicated to bony ray branching during the outgrowth phase of fin regeneration. (A-J) Whole-mount images of fins from the same two sp7:EGFP fish acquired prior to amputation and at 12 and 30 days post-amputation (dpa). The fish are treated with either control DMSO or BMS-833923 at 48 and 72 hpa. (A-C,F-H) Fluorescence images showing osteoblast GFP expression in green. (D,I) Rotterman contrast images. Periodic high-contrast patches along the rays are joints. Red lines and arrows mark ray bifurcation points. (E,J) Bright-field images of Alizarin Red-stained fins collected from the same fish to visualize mineralized bone. Amputation planes are indicated with dashed yellow lines. (K,L) Wide-field whole-mount GFP fluorescence and bright-field overlay images of 5 dpa fins from shha:GFP fish following injections with DMSO or BMS-833923 at 48 and 72 hpa. Green brackets indicate the extent of epidermal shha:GFP expression along the proximal-distal axis. Student's t-test shows the 142% mean increase in the length of the shha:GFP domain is significant [n=5 control and 10 BMS-833923-treated fish (60 scored rays), P<0.0001]. Scale bars: 2 mm in A,F; 1 mm in B-E,G-J; 500 µm in K,L.

Fig. 6.

Shha-driven Smoothened signaling directs progenitor osteoblasts to migrate in step with transiently split basal epidermal clusters at the onset of ray branching. (A-F) Runx2 and EGFP immunostaining (red and white, respectively) and EdU incorporation (2 h treatment, green) on transverse 96 hpa fin sections from individual shha:GFP fish treated at 48 and 72 hpa with DMSO (A,C,E) or BMS-833923 (B,D,F). Nuclei are blue. (A,B) Far distal sections beyond the distal-most pObs. (C,D) Sections from positions where shha-expressing epidermal cells have split into two clusters on each side of the fin. (E,F) Further proximal sections where shha is first induced by distal migrating epidermal cells. Yellow arrows indicate Runx2⁺/EdU⁺ cells located more than one cell layer from shha:GFP-expressing basal epidermis. Red arrows indicate Runx2⁺ pObs two or more cell layers distant from shha:GFP-positive epidermal cells. The magenta bracket in D highlights Runx2⁺ pObs that span the junction between split shha:GFP domains in BMS-833923-treated fish. (G) Quantification of pOb proliferation and the relative position of pObs to epidermal cells in regenerating fins from the above and similar DMSO versus BMS-833923 treated shha:GFP fish. Only images at ‘split’ positions are scored. Left plots: the fraction of EdU incorporating Runx2⁺ pObs. Middle plots: the fraction of pObs not directly adjacent to GFP⁺ epidermal cells. Right plots: the fraction of pObs aligned with split clusters of shha:GFP-expressing epidermis. Each data point represents a scored independent section (11 individual rays from five DMSO-treated fish and 23 rays from seven BMS-833923-treated fish). Two-tailed Student's t-tests were used to determine statistically significant differences (P<0.05) between the means of control versus small-molecule-treated samples. Scale bars: 50 μm.

Fig. 7.

shha:GFP-expressing basal epidermal cells extend cellular protrusions through incompletely assembled basement membrane to contact Runx2⁺ progenitor osteoblasts. (A-P) GFP (white), laminin (magenta) and Runx2 (green) antibody stained fin sections from 96 hpa shha:GFP fish. All images are 1 airy unit (∼1 µm) single optical confocal sections, except D, which is a structured illumination microscopy (SIM) image representing an ∼100 nm section. (A-D) Longitudinal section showing the distal regenerate where the shha-expressing basal epidermis is split into two clusters and Hh/Smo signaling is activate in both pObs and basal epidermis. (C) An overlay showing Runx2 staining together with A and B. (D) A high-magnification SIM image of the boxed area in C. (E-G) A proximal field from the same longitudinal section shown in A-D. (H-P) Transverse sections representing three positions along the proximal-distal axis from a single regenerating ray. (H-J) An extreme distal section beyond the distal extent of Runx2⁺ pOb pools. (K-M) Section from a position where shha:GFP-expressing basal epidermis is split into two clusters on each side of the regenerating fin. (N-P) A further proximal section where shha expression has initiated in epidermal cells but prior to their division into split clusters. The dashed boxes in I, L and O mark regions shown at higher magnification in J, M and P, respectively. White arrows indicate cellular protrusions from shha:GFP⁺ cells that contact or enshroud Runx2⁺ osteoblasts. White asterisks mark gaps in the basal lamina. Magenta arrows show a continuous laminin-containing basement membrane that physically separates the basal epidermis from Runx2⁺ pObs. Scale bars: 5 µm in D,J,M,P; 50 µm in C,G,I,L,O.

Fig. 8.

Model showing how cell movements, Hh/Smo pathway dynamics and direct cell-to-cell interactions between neighboring basal epidermal cells and progenitor osteoblasts induce ray bifurcation during fin regeneration. Basal epidermal cells generated proximal to the caudal fin amputation site continuously advance distally along a mature basal lamina. During this effective migration, groups of basal epidermal cells overlying the regenerating blastema transiently upregulate shha expression (cells outlined in blue) and then split into two clusters on each side of the ray. Shha drives active Hh/Smo signaling marked by ptch2 expression (orange cells) in a narrow distal zone of shha-expressing epidermal cells and adjacent progenitor osteoblasts (pObs). As fin outgrowth proceeds, some pObs escape self-renewal signals, initiate re-differentiation and progressively extend reforming bone. Fully differentiated Obs secrete new bone matrix, enabled by the earlier non-canonical activity of Ob-expressed Ihha (cells outlined in dark blue). The interface between Hh/Smo-responsive Obs and basal epidermis is the active site of basal lamina (red) assembly associated with fin regenerative outgrowth. Coinciding with the epidermal ‘branchpoint’, this incompletely assembled basement membrane enables shha-expressing basal epidermal cells to extend cellular protrusions that contact neighboring pObs. This direct binding activates Smo-dependent signaling (including further ptch2 expression) and progressively escorts pObs into physically separated pools. The newly divided pools of Runx2⁺ pObs then continue regenerating independently to produce a now bifurcated ray.