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Figure 7. Knockdown of Wnt5a or Ror2 phenocopies xPAPC depletion. Knockdown of Wnt5a (A, A', C, C', E, E') or Ror2 (B, B', D, D', F, F'), respectively, via antisense morpholino injection at 16-cell stage in each of the two adjacent ventral and dorsal cells in the animal pole. (A, A', B, B', C, C', D, D') ISH at stage 28 for (A, A', B, B') xPAPC or (C, C', D, D') Tbx2. Either Wnt5a or Ror2 depletion showed a reduction in xPAPC expression (A', B') as well as deformations of the otocyst at the injected side (A', B', C', D'). (E, E', F, F') Immunostaining of Wnt5a (E, E') or Ror2 (F, F') depleted otic epithelia. Mo was injected as described in (A) and ISH for Tbx2 was performed at stage 26. Embryos were then sliced and immunostained for different epithelial markers like (E, E') fibronectin or (F, F') C-Cadherin in red. The nuclei were stained in blue for DAPI. As in xPAPC depleted embryos the injected otocysts showed epithelial disorganization indicated through inaccurate nuclei alignment and displaced adhesion proteins (E', F'). (G, H) Angle measurements of DAPI stained nuclei in immunostained sections reveal a disorganization of the epithelial structure of the otocyst. While in uninjected otocysts DAPI cells are orientated in angles between 0 and 50ostly, the cells on the injected sides (G: Wnt5a Mo; H: Ror2 Mo) shows angles between 0 and 170 (I) Knockdown of Wnt5a is rescued by Ror2 RNA coinjection in a dose-dependent manner. Injections were carried out at 16-cell stage as described in (A). The embryos were examined at stage 26. Mo, antisense morpholino; n, number of embryos. Scale bar (A', B') 500 μm, (E', F') 20 μm. Arrow points to the deformed otocyst. * = p < 0,05. |
