Fig. 5. Morphological analysis of snakehead and nagie oko brain ventricle mutants. (A-F) Light microscopy images of brain at 28 hpf. Dorsal views (A-C) or side views (D-F) of living, anesthetized embryos are shown, anterior to left. Ventricles are injected with Texas Red dextran in A. Relative to wild-type (A,D), brain ventricles in snakehead (B,E) and nagie oko (C,F) mutants appear to be absent: nok mutants sometimes form a small hindbrain ventricle that never enlarges. Note the characteristic refractivity of the neural tube in snk, in that neither the outline of the neural tube nor the brain folds are visible in the snk mutant (B,E). In addition, note that arrows in A and C point to the MHB constriction in wild type and nok, respectively. By light microscopy, the snk MHB constriction is not visible (even though it is in the proper location; see Q below). Bracket in D indicates hindbrain ventricle height. (G-O) Histology of snk and nok mutants. Embryos were fixed and transverse-sectioned at 22 hpf, at the level of forebrain, midbrain or hindbrain, and stained with hematoxylin and eosin. Relative to wild-type embryos (G,J,M), snk mutant embryos (H,K,N) show appropriate ventricle morphology; however, the cells appear to be adhered to one another and no lumen is present. By contrast, nok mutant embryos (I,L,O) fail to undergo any ventricle morphogenesis, and the epithelium appears disorganized. Asterisks label midbrain hinge-points in wild type (J) and snk (K), but hinge-points are absent in nok (L). (P-R) Confocal images through mid- and hindbrain ventricles of 24 hpf living embryos stained with bodipy ceramide. (P) Wild type, (Q) snk, (R) nok. Note that snk embryos (Q) assume correct ventricle morphology but fail to open the ventricles. By contrast, the brain tube in nok embryos (R) remains straight and no hinge-points form (although the MHB constriction remains). Scale bar: 50 µm. F, forebrain ventricle; H, hindbrain ventricle; M, midbrain ventricle. Asterisks: hinge-points.