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Fig. 1. Disruption of dorsal midline structures inhmmrmorphants. (A-C) Dorsal (d) views of the head region of Xenopus laevis embryos at stage (st.) 45, rostral to the left. Control embryo (A) and embryos injected into both dorsal animal blastomeres at the eight cell stage with hmmr morpholino (MO; B) or hmmr MO together with full length (FL) hmmr (C). Brains outlined in white, white and red brackets indicate wildtype and reduced interocular distance, respectively. Arrowhead in (B) points to pigmented optic stalk. (D-F) Dorsal views of dissected forebrains (fb), brain area indicated by yellow boxes in (A-C); brackets indicate level of fb width measurement, arrowheads point to bilateral olfactory bulbs (OB). Note decrease in OB size and fb width in (E) and restoration of both in (F). (G-I) Fluorescently labeled nuclei and F-actin with immunofluorescence detecting α-tubulin (Tuba4a) reveal morphology of control (G) and treated (H, I) brains transversally bisected at level indicated in (D-F); scale bars: 50 µm. Ventricular lumina outlined in white, yellow arrowheads indicate septum separating hemispheres; note lack of midline separation and absence of lumina in morphant indicated by white arrowheads in (H). Restoration of ventricular lumina and septal structure (I). (J, K) Quantification and statistical analysis of fb width (J) as indicated in (D-F) and fb phenotypes (K). WT: normal lumina / septum, mild: lumina / septum reduced; severe: lumina / septum absent. Number of embryos indicated in each graph. (L-N) not expression in control (L, M) and morphant (N) neurula embryos after whole-mount in situ hybridization (anterior views). Arrowheads indicate medial convergence of bilateral not-expressing cell populations. (L’-N’) Transverse sections at the forebrain level as indicated in (L-N), neural tissue outlined in white. Note failure of medial convergence in morphant embryos (N, N’). Scale bars: 100 µm. l, left; r, right; v, ventral. |
