Otic specification, early otic vesicle patterning, and hindbrain phenotype in Cdh11 knockdown embryos. Control morpholino oligonucleotide-injected (A, D) and cdh11 antisense morpholino oligonucleotide-injected 24-hpf embryos (B,C and E,F) were labeled by in situ hybridization with cldna (A-C) and dlx3b (D-F) cRNA probes. Normal expression for cldna (which begins during otic specification and continues in all otic vesicle epithelial cells) was seen in both control and Cdh11 knockdown embryos, although the otic vesicles were smaller in Cdh11 knockdown embryos. Normal expression for dlx3b (which begins during otic specification, continues in the otic vesicle, then resolves into a dorsal pattern in the otic vesicle) was seen in both control and Cdh11 knockdown embryos. Again, otic vesicles were smaller in Cdh11 knockdown embryos. Scale bar in A = 50 μm. In A-F, rostral is left, and dorsal is up. Hindbrain markers were examined in control morpholino oligonucleotide-injected embryos (G, I) and cdh11 antisense morpholino oligonucleotide-injected embryos (H, J, K) using for krox20 in situ hybridization (G, H) or neurofilament immunofluorescence (I-K). In situ hybridization-labeled embryos at 18 hpf were imaged in flat mount using transmitted light DIC microscopy (G, H) using Nikon diaphot 200, 40x DIC 0.55 NA objective. Immunofluorescence of embryos at 48 hpf was imaged in flat mount (I-K) using multiphoton microscopy (BioRad MRC1024, 60x W 1.2 NA objective, 800 nm illumination). Projection images were made using Voxx Software in alpha blending mode. In severe Cdh11 knockdown embryos, lateral longitudinal fasciculus (llf) and medial longitudinal fasciculus (mlf) are severely reduced or absent. However, there is a small labelled fiber tract that might be part of the lateral longitudinal fasciculus, and Mauthner cells (M) axons still contribute to the medial longitudinal fasciculus in severely affected Cdh11 knockdown embryos. Scale bar in J = 50 μm for H-K. In G-K, dorsal images are viewed with rostral at left. Representative images are shown. For all panels, rostral is left, and dorsal is facing the viewer.
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