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Fig. 4 Effects of DAPT treatment and loss of asymmetric Vangl2 localization.
(A) Phalloidin staining (red) of a wild-type cldnB:lynGFP larva demonstrates the normal orientation of hair bundles in an anteroposteriorally polarized neuromast derived from the first primordium. This and the subsequent micrographs show apical views of neuromasts from cldnB:lynGFP larvae; the subjacent diagrams indicate the orientations of the hair cells. (B) The numerous hair cells in a neuromast of similar age treated with 100μM DAPT display a strong bias toward an anterior orientation. (C) After exposure to 100μM DAPT, the hair bundles in a neuromast derived from the second primordium show a striking dorsal bias. (D) A neuromast derived from the first primordium in a neurogenin1 larva displays an anterior bias after exposure to only 50μM DAPT. (E) Under control conditions, the production of hair cells (red) and supporting cells (blue) is relatively constant during an observation period beginning at 3 dpf. (F) In wild-type larvae a burst of hair-cell production begins 17–22 hr after the addition of 100μM DAPT; the differentiation of supporting cells is meanwhile suppressed. Note that imaging commenced 7 hr after exposure to 100μM DAPT. (G) In a neuromast of a myo6b:RFP-Vangl2;cldnB:lynGFP larva, Vangl2 (red) occurs in hair cells at the level of the apical actin belt surrounding the cuticular plate. An immature hair cell is labeled with an arrowhead. The top left inset shows the apices of the hair cells in the green channel. The lower inset in this and the subsequent illustrations schematizes the orientations of hair bundles. (H) Overexpression of Vangl2 eliminates the specific enrichment of the protein at the posterior apex and causes hair-cell misorientation. The mature hair cell with the strongest expression, which extends down the cell′s lateral borders (arrows), has a centrally placed kinocilium (arrowhead; dot in the inset). (I) Another neuromast with high levels of Vangl2 expression (red) displays a bias toward anterior orientations. (J) A neuromast from a cldnB:lynGFP;tri larva treated with 50μM DAPT and stained with phalloidin (red) contains hair cells with an anterior bias. The scale bars represent 10μm in all panels.