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Fig. 3 Pou3f3 is required for formation of the opercular skeleton in zebrafish. (A) Micro-computed tomography of an adult zebrafish skull, with the opercle (Op) and subopercle (Sop) bones pseudocolored pink. (B and C) Reduction or loss (*) of Op and Sop bones exposes the gills (black arrow) in adult pou3f3b mutants. In C, dissected jaw skeletons were stained with Alcian blue (cartilage) and Alizarin red (bone), and the ceratohyal and branchiostegal ray series were removed for clarity. (D) In larval mutants, the Op and supporting hyomandibula cartilage (Hm) are progressively reduced (*) with decreasing Pou3f3 dosage. (E) Loss of the Op bone in double mutants is preceded by loss of RUNX2:mCherry+ preosteoblasts in this domain (*) but not the branchiostegal ray (Br) bone domain at 3 dpf. (F) Double mutants show a reduction of sox9a expression in the Hm precartilage condensation but not the neighboring symplectic (Sy) or ceratohyal (Ch) domains. (G) Opercular outgrowth initiates but is not sustained in pou3f3a; pou3f3b mutants. Control pou3f3a+/−; pou3f3bGal4ff/+ and mutant pou3f3a−/−; pou3f3bGal4ff/− larvae carrying UAS:nlsGFP and sox10:DsRed (cartilage) transgenes were repeatedly imaged between 49 and 120 hpf. Compaction of mesenchyme around the forming opercle bone (outlined in Upper Right) was not evident in mutants. (H) Quantification of total dorsal hyoid arch area in individually tracked control and pou3f3a−/−; pou3f3bGal4ff/− siblings (repeated-measures ANOVA: genotype P = 0.0069; time: P < 0.0001; genotype × time: P < 0.0001). (I) A trend toward moderately lower rates of proliferation in double mutants at 72 hpf becomes significant at 96 hpf (unpaired t test). Horizontal lines denote the mean. (J) Representative BrdU-labeled control and mutant samples, with the quantified opercular region outlined. (K) PEM markers shha, bmp7b, and fgf24 are expressed at normal levels in pou3f3a; pou3f3b double mutants at 48 hpf (white arrows). sox10:GFPCAAX labels arch mesenchyme. (L) Unilateral transplantation of fli1a:EGFP donor neural crest cells into a pou3f3a+/−; pou3f3b−/− host rescued Op formation. Images in G, J, and L are maximum-intensity projections; single optical sections are presented in K. (Scale bars: C, 500 μm; D and L, 50 μm; E–G, J, and K, 20 μm.)