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Fig. 4
The necab2–/– larvae display grossly normal morphology and neuronal marker expression. (A) The necab2+/– and necab2–/– larvae exhibited grossly normal appearance compared to wildtype siblings. The 7 dpf larvae derived from the heterozygous necab2+/– mating were imaged blindly and followed by genotyping. Scale bar = 1 mm. (B) The necab2+/– and necab2–/– larvae exhibited normal neural cytoarchitecture compared to wildtype siblings. A total of 7 dpf larvae were derived from heterozygous necab2+/– mating in the Tg(Pnecab2:EGFP) background. Genotyping using the dissected tail was done after fixation for anti-EGFP immunofluorescence staining. Scale bar = 100 μm. The region in the dashed white box was shown at higher magnification below. Scale bar = 20 μm. (C) The necab2+/– and necab2–/– larvae exhibit grossly normal composition of neuronal types. Spatial analysis of the glutamatergic (vglut2a), GABAergic (gad1b), glycinergic (glyt2), and dopaminergic (TH) neurons by whole-mount mRNA in situ hybridization was conducted blindly in the embryos derived from in-cross of necab2+/– followed by genotyping. For each group, a total of 20 larvae were processed for in situ hybridization in order to obtain at least three homozygous larvae respectively. Scale bar = 200 μm. dpf, day post-fertilization; Tel, telencephalon; Di, diencephalon; Ce, cerebellum; re, retina; Ha, habenula; OT, optic tectum; MHB, midbrain-hindbrain boundary; HB, hindbrain; lens, crystalline lens.