Fig. 8

Spatially Distributed Gene Expression Combined with Negative Feedback Maintains Regenerative Osteogenesis

(A) Quantitative RT-PCR analysis of sp7 and dkk genes in primary fin osteoblasts after 24 hr of 300 nM BMPRi (red bars and error bars) relative to control DMSO-treated cells (variation shown with black error bars). For each primer pair, the normalized average fold change in transcript levels upon BMPRi treatment is plotted on a log₂ scale with DMSO samples averaged to log₂0 = 1. Each group includes three independent cultures. Error bars are one SD. Statistically significant differences are indicated with an asterisk (p < 0.05, two-tailed Student’s t tests). A representative example of three independent experiments is shown.

(B) Relative gene expression of osteogenic factors and dkk genes at 96 hpa following DMSO (black error bars) or BMPRi exposure (red bars, 5 μM from 48 to 96 hpa). Mean normalized levels of the indicated transcripts from four fins per treatment group are shown on a log₂ scale. Error bars represent one SD, and asterisks mark differentially expressed genes (p < 0.05, two-tailed Student’s t tests).

(C–H) Antibody staining for Runx2 (red) and β-catenin (green) on sectioned fins from control (C–E, two heat treatments between 48 and 72 hpa) and Tg(hsp70l:dkk1b-GFP) fish (F–H, two heat treatments between 48 and 72 hpa) harvested 72 hpa. The yellow arrow indicates Runx2⁺ cells with nuclear β-catenin.

(I–L) Immunostaining of a 72 hpa fin cryosection with Runx2 (I, white) and sp7 (J, green) antibodies and simultaneous wnt5a mRNA in situ hybridization (K, red), overlaid in (L). The red arrow shows wnt5a expression in distal mesenchymal cells.

(M–P) A fin cryosection from a 72 hpa Tg(sp7:EGFP) fish showing antibody staining for Runx2 (M, white) and EGFP (N, green) and bmp2b mRNA in situ hybridization (O, red), overlaid in (P). The white arrow points to Runx2⁺/bmp2b cells, and yellow arrows indicate overlapping expression of sp7:EGFP and bmp2b. Hoechst-stained nuclei are in blue in overlay panels. Scale bars represent 50 μm.

(Q) A signaling network model for osteogenesis during fin regeneration. Wnt acts distally to maintain a pool of Runx2⁺ osteoblast progenitor cells, whereas Bmp2b-initiated signaling in progenitor-derived cells both promotes sp7-associated differentiation and constrains Wnt activity by inducing Dkk1b.