ANC of esrp1/2 double mutants is populated by undifferentiated cells. Representative z-stacks of RNAscope ISH of coronal sections of esrp1/2 double CRISPR mutants and WT clutch-mate controls at 4 dpf. (A) Sections through ANC anterior to the eyes. col2a1 (red) staining depicts normal morphology of the ANC cartilage elements in WT, while a cleft is apparent in the esrp1/2−/− zebrafish, with dapi (blue)-stained cells between adjacent trabeculae (arrowheads). These col2a1– cells do not express epithelial markers krt4 (cyan) or krt5 (magenta), except around the periphery. (B) Sections posterior to those in A show col2a1– cells continuing inferior to the trabeculae in the esrp1/2 mutant zebrafish, and cells have low expression of irf6 (boxed area). (C) Zoomed image of col2a1– cells from the boxed area in B, showing irf6 expression (green). Dashed lines outline ANC cartilage elements. Scale bars: 50 μm.
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