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. 2020 Dec 30;9:e60432. doi: 10.7554/eLife.60432

Figure 5. Spatiotemporal reporter activity of emx2 in wild-type (WT) and emx2 mutants.

(A) In an emx2:Gfp zebrafish, Gfp-positive hair cells (HCs) are located in the anterior region of an A-P neuromast (top) and in the dorsal region of a D-V (bottom) neuromast. Phalloidin staining shows the hair bundle orientation (yellow arrows). (B) Time-lapse imaging of nascent HC pairs undergoing Roll or No Roll movement in emx2:Gfp; myo6b:dtTomato neuromasts. Left panel (Roll): A tdTomato-positive, nascent HC pair undergoing roll movement shows detectable Gfp expression in the posterior HC as it rolls into the anterior position (white arrow, 170 min into the Rock and Roll process). Right panel (No Roll): A HC pair that did not roll and Gfp appears in the anterior HC by 156 min into the Rock phase (white arrow). (C–E) The distribution of Gfp-positive HCs in an emx2gfp/- (loss of function [LOF]) neuromast, showing a merged image (C) of DAPI, GFP (D) and phalloidin staining (E). Outline of HCs are dotted. Gfp-positive HCs are located in the anterior region but hair bundles are pointing in P→A direction (yellow arrows in e’). (F–H) The distribution of Gfp-positive HCs in an emx2 gain of function (GOF);emx2Gfp/+ neuromast, showing a merged image (F) of DAPI, GFP (G), mCherry, and phalloidin staining (H). Among the total six pairs of GOF HCs analyzed, only two are Gfp-positive (G), one anterior- and one posterior (asterisk)-located. All hair bundles are in A→P direction (yellow arrows in h’).

Figure 5.

Figure 5—figure supplement 1. Generation of the emx2:Gfp transgenic line.

Figure 5—figure supplement 1.

(A) Schematic of the emx2 genomic structure and its replacement with donor DNA using CRISPR. Red arrowhead indicates the target position for the guide RNA. The reporter eGfp and a polyA sequence in the targeting vector are flanked by left and right homologous arm (LHA and RHA) of the emx2 allele as indicated with dotted line. The targeting position for the guide RNA is not included in either the LHA or RHA. (B) Hybridization signals of emx2 transcripts in the brain (b), otic vesicles (ov), and pharyngeal arches (pa). (C) Gfp-positive regions in emx2:Gfp reporter larvae are similar to emx2 positive regions indicated by in situ hybridization.