ihha is required for regional Op growth through cell addition. Aligned and overlapping confocal projections of a wild-type Op (red) and an ihha mutant Op (green) from a time-lapse of sp7:EGFP-expressing larvae (see supplementary materialMovie 1) reveal early shape changes due to reduced cell addition. Images are lateral views with dorsal upwards and anterior towards the left. (A) Quantitation of peripheral osteoblast addition to the anterior and posterior halves of the Op from 3-4 dpf shows decreased anterior, but not posterior, cell addition. (B-D) Osteoblasts are added to the anteroventral region of the wild-type Op after 3 dpf (arrows) but are lacking in ihha mutants. (E-G) Threshold images derived from A-C showing separation of the Op into quadrants by a best-fit ellipse (broken line). Alignments are accompanied by graphs representing the fraction of wild-type Op that overlaps with ihha mutant Op (WT:ihha, 36 comparisons) or the fraction of wild-type Op overlapping with another wild-type Op (WT:WT, 21 comparisons) for each quadrant/time point. (E) WT:ihha overlap at 3 dpf is not significantly different from WT:WT overlap. The anteroventral edge (quadrant 1) shows a significant reduction in WT:ihha overlapping area at 3.5 dpf (F) and 4 dpf (G), but other quadrants are not significantly altered. Data are presented as mean±s.d., **P<0.01 by unpaired t-test, ne7 per genotype (A); and mean±s.e.m. ***P<0.001 by Tukey-Kramer method following one-way ANOVA, n=6 per genotype in E-G. Scale bar: 50 μm.
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