Localization patterns of fascin 2 mRNAs and proteins in zebrafish.
RNA in situ hybridizations (A–E, G–J) were performed on larvae at 4 dpf. Fascin 2b mRNA is detected in the otocysts (A, arrowhead; C) and the eye (A, arrow). Fascin 2a mRNA is expressed in the eye (G, arrow), but not the otocyst (H). Fascin 2b sense RNA (B,E) and fascin 2a sense RNA (I,J), each labeled, were used as controls. A cryosectioned ear labeled with fascin 2b antisense probe is displayed (D). AM, PC, and asterisk indicate the anterior macula, the posterior crista, and the lumen of the otocyst, respectively (C,D,E,H,J). RT-PCR analysis (F) shows expression of fascin 2b in zebrafish hair cells. Agarose gel confirms the expected product size of the fascin 2b amplicon, left lane (+ hair-cell cDNA); no product is observed without cDNA template, right lane (no template control). Labeling using fascin 2 antiserum reveals strong fluorescent signals (green) in hair bundles of an anterior macula (K,O) and a posterior crista (L) from larvae at 4 dpf and of an adult lagena (M,N). In red, fluorophore-coupled phalloidin labels the filamentous actin of stereocilia and cuticular plates (K–O). Higher magnification of M is displayed (N). Scale bar is 2 μm. Soma is indicated by asterisk (K). An enlarged view of a hair bundle from an anterior macula (O) is shown with regions of interest (ROI) selected for the stereocilia (orange line) and the cuticular plate (blue line). Fluorescence intensity profiles of stereocilia (P), using the orange-line ROI from O, show that the fascin 2b- (green) and phalloidin-associated (red) signals are overlapping. Intensity profiles of the cuticular plate (Q), using the blue-line ROI from O, demonstrate no significant labeling with fascin 2 antiserum (green). Intensity scales are linear, but the units are arbitrary (P,Q). X-axes (P,Q) represent the lengths of the respective orange and blue lines (O).