Figure 5. Fltp is required for kinocilium positioning and stereocilia bundle formation.
(A) Whole-mount LSM of an FltpZV/+ organ of Corti (OC) showing its different regions. Fltp reporter expression is restricted to all sensory HCs (IHC and OHC) of the OC (red dashed line). (B–M) Whole-mount SEM pictures of an E18.5 OC. (B and C) Fltp+/+ (WT) HCs at 50% of the OC length are perfectly polarized (B) whereas FltpZV/ZV (HOM) HCs show orientational defects (C). (D–I) Enlargements of HCs reveal perfect polarized kinocilia as well as highly ordered stereocilia in WT (D). HOM cells are misaligned and show split stereocilia bundles (E, G–I), general bundle morphology defects (E–I), as well as detached kinocilia (E). Polarity and stereocilia bundles disruption increases in HOM hair cells from 75% (K) to >75% (M) of OC length compared to WT cells (J and L). (N) Deviation of HCs from the polarity axis for WT and HOM animals significantly differs in all OC regions (<50%, 50%, 75%, >75%). Quantification of HC rotation was performed by measuring the angle from the normal tissue polarity (measured by the medial to distal alignment of the HC rows) to the middle of the stereocilia bundle. In total we examined n = 4 (219 HCs) of WT and n = 7 (426) of HOM animals. Statistical analysis uses circular statistics (****p < 0.0001). Error bars show the standard deviation. Kinocilia are marked by the red dashed lines, nuclei by DAPI, and nuclei of Fltp reporter expressing cells by GFP. Abbreviations: inner hair cell (IHC), outer hair cell (OHC1-3). Scale bars; 10 µm (B and C), 1 µm (D–I).