Fig. S6

Ihha is not required for bone growth, initial differentiation, or patterning but does support bone re-calcification during fin regeneration. (A) Quantitative RT-PCR analysis showing relative expression levels of shha, shhb, ihha, and ihhb in 96 hpa wild-type and ihha^-/- regenerates. Normalized and relative levels of the indicated transcripts represent the means of three fins. Error bars are one standard deviation. (B-E) Von Kossa stained 96 hpa fin sections from ihha^-/- and wild-type clutchmate fish. Calcified bone is stained black and black arrows denote the extent of calcified bone in the regenerate. Dashed boxes in B and C indicate zoom panels shown in D and E, respectively. (F) Quantified re-calcification of ihha^-/- and wild-type clutchmate fish at 96 hpa using von Kossa-stained sections. The calcified fraction represents the length of the calcified region (gray bars) divided by the total regenerate length. At least four sections were measured to generate a mean value for each individual fish (plotted on the graph). Statistically significant differences are indicated (p < 0.05, two-tailed Student’s t-tests). (G-J) Brightfield images showing Alizarin red-stained ihha^-/- (n =8) and wild-type (n = 6) fins collected at 8 dpa (G, I) and six weeks later (H, J). (K-N) Whole mount Alizarin red staining of unamputated adult ihha^-/- (n = 3) and wild-type (n = 3) clutchmates. L and N show zooms of panels K and M, respectively. (O, P) Fin sections collected 72 hpa from wild-type and ihha^-/- fish stained with Runx2 (red), sp7 (green) antibodies and, by click chemistry, EdU (white, 6 hour pulse). Hoechst-stained nuclei are blue. (Q) Quantification of osteoblast subtypes and EdU incorporation at 72 hpa. Bars show the mean percentile representation of each osteoblast subtype on comparable sections (n = 10 rays (wild-type) and n = 11 rays (ihha^-/-) compiled from > 5 fish). Error bars are one standard deviation. The proportion of each cell population that incorporated EdU is indicated by the extent of gray shading relative to the bar’s height. For all panels, amputation planes are indicated with a dashed yellow line. Scale bars: 50 μm.