Pipeline for segmentation and analysis of support cell orientation, and description of the atoh1a CRISPR experiments. a. Pipeline for the segmentation of Phalloidin stainings of neuromasts 3 hours after hair cell ablation. Each Phalloidin image is sharpened and segmented. After segmentation, only the support cells are kept and used for fitting the Ellipse that determines the cell’s long axis. For further information, see Materials and Methods. b. Phalloidin staining of 5 dpf gpc4 sibling or mutants after removal of hair cells and quantification of support cell orientation with respect to the horizontal, showing support cell disorganization only in primll-derived neuromasts of gpc4 mutants (Uniform distribution p-val gpc4 siblings priml= 1.43 x 10^-12, gpc4 siblings primll= 1.30 x 10^-14; gpc4 mutants priml= 1.84 x 10^-14, gpc4 mutants primll= 0.80). c. Agarose gels showing the efficiency of CRlSPR targeting the single exon of atoh1a in CRlSPants (upper part) and Cas9-only injections (lower part). d. DASPEl staining of uninjected and atoh1a CRlSPR injected fish, showing that atoh1a CRlSPants do not possess hair cells. Below are shown higher magnification pictures of the neuromasts, where sqet20 labels the mantle cells and sqet4 and DASPEl label the hair cells. e. Double β-ll-Spectrin and ZO-1 labeling of the uninjected MZwnt11 (wnt11f1) siblings for Figure 5q, r. Scale bar in a, b and e equals 5 µm, d equals 20µm
|
