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Fig. 2. Local perturbation of Vmem disrupts endogenous eye development. (A) Quantification of tadpoles with eye phenotypes upon microinjection of EXP1 or GlyR (± IVM treatment) ion channel mRNA in the dorsal or ventral two cells (red arrows) of the four-cell Xenopus embryo. A high incidence of malformed eyes is observed in dorsal injections in comparison to controls or ventral injections. (B) (i) Four-cell Xenopus embryos were injected (red arrow indicates injected cell). (ii,iii) Stage 42 control (uninjected) tadpoles. (iv,v) Stage 42 tadpoles injected with GlyR mRNA plus IVM treatment. lacZ lineage tracer mRNA was co-injected with the ion channel mRNA. Yellow arrowhead indicates normal eye in the absence of β-gal (blue-green), whereas the red arrowhead indicates an absent eye on the contralateral side of the same tadpole in the presence of β-gal. (C) Quantification of tadpoles with malformed eyes at stage 42 after microinjecting GlyR plus IVM treatment in two dorsal cells at the four-cell stage and incubation in different concentrations of choline chloride (0, 20 and 60 mM). 60 mM is the isomolar concentration. A depolarization dose-dependent decrease in malformed eye incidence is observed. Values are mean ± s.e.m. (n=3). *, P<0.05; **, P<0.01; one-way ANOVA with Tukey's post test. (D) Stage 22 control (uninjected) embryos (i,iv,vii) and embryos microinjected with GlyR mRNA plus IVM treatment (ii,v,viii) or EXP1 mRNA (iii,vi,ix) in the two dorsal cells at the four-cell stage. In situ hybridization for Otx2 (i-iii), Rx1 (iv-vi) and Pax6 (vii-ix) shows a significantly disrupted expression (yellow arrowheads) of Rx1 [GlyR+IVM, 53% disrupted (n=15); EXP1, 50% disrupted (n=18)] and Pax6 [GlyR+IVM, 56% disrupted (n=9); EXP1, 57% disrupted (n=7)], whereas Otx2 expression remains unchanged (green arrowheads) [GlyR+IVM, 92% normal (n=26); EXP1, 94% normal (n=17)]. The black arrowheads indicate Pax6 expression in the forebrain and the spinal cord, which remain largely unchanged. (E) CC2-DMPE staining showing the relative Vmem of cells of the indicated region (i) in the stage 19 Xenopus embryo. Red arrowheads mark the specific bilateral cluster of hyperpolarized cells in the anterior neural field of control (uninjected) embryos (ii). Green arrowheads mark the disrupted hyperpolarization pattern in embryos microinjected with dominant-negative Pax6 in the dorsal two cells at the four-cell stage (iii); 76% (n=25) of dominant-negative Pax6-injected embryos showed a disrupted CC2-DMPE staining pattern. See also supplementary material Fig. S2. Illustrations reproduced with permission from Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). |
