Fig. 4: Adult pectoral girdle phenotype in Hh-gli signaling knockout fish.

a–c, d–f, g–i Volumetric renderings of the skeleton, which was segmented manually from µCT scans of three-months old adult fish. a–c wildtype, d–f) gli3^14ins/14ins, g–i gli3^14ins/14ins; shha^30%KO fish. a, d, g the whole-body 3D reconstruction, (b, c, e, f, h, i) the enlarged images of the scapulocoracoid and mesocoracoid (b, e, h); lateral view, (c, f, i); posterior view). In contrast to the thin and long dorsal cleithrum with the postbranchial lamina in gli3^+/+ fish (a), the dorsal cleithrum was reduced in either the left or right side of gli3^14ins/14ins fish (n = 28/159) (d) and both sides of gli3^14ins/14ins; shha^30%KO fish (3/3) (g). In the scapula of gli3^+/+ fish, the glenoid is anteroposteriorly wide and flat (b, c), but it is concave and posterolaterally opened, and delineates the proximal surface of basal radials of the pectoral fin in gli3^14ins/14ins and gli3^14ins/14ins; shha^30%KO fish (e, f, h, i) (n = 5/6 for each genotype). The supraglenoid buttress is more developed in gli3^14ins/14ins and gli3^14ins/14ins; shha^30%KO fish compared to wildtype fish (b, e, h). j Quantitative analysis of the cleithrum height, scapula width, mesocoracoid width, and glenoid depth. The length and width of each bone were measured and standardized by the dorsoventral length of the skull (see Methods). The unit is aribitrary unit [a.u.]. All raw measurements are summarized in Supplementary Data 3. While the cleithrum is dorsoventrally shorter in the severely affected pectoral girdles of gli3^14ins/14ins and gli3^14ins/14ins; shha^30%KO fish than those of gli3^+/+ fish, the scapula and mesocoracoid are larger in gli3^14ins/14ins and gli3^14ins/14ins; shha^30%KO fish. The glenoid is more concave in gli3^14ins/14ins and gli3^14ins/14ins; shha^30%KO fish than wildtype fish. ANNOVA test and following Tukey-Kramer Post Hoc test were conducted to detect statistically significant differences of bone size among wildtype and knockout fish. **; absolute mean value > Q critical value in Tukey-Kramer Post Hoc Test. 5.07 and 5.83 > 3.73 in the cleithrum, 5.10 and 6.16 > 3.73 in the scapula, and 6.07 > 3.88 in the mesocoracoid, and 6.05 > 3.88 in the glenoid). Maximum, minimum, median, and average values are shown in the box and whisker plots. Two or three biologically independent fish samples were analyzed in each genotype. Both left and right pectoral girdle measurements are plotted. See all replicates and measurements in Supplementary Data 3. c coracoid, cl cleithrum, g glenoid, ms mesocoracoid, pl postbranchial lamina, sb supraglenoid buttress, and scl supracleithrum. The scale bar indicates 2.5 mm.