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Fig. S2. EXP1 mRNA and GlyR mRNA plus IVM disrupt the hyperpolarization pattern and induce localized endogenous eye defects. (A) CC2-DMPE staining of stage 19 Xenopus embryos. (i,ii) Four-cell and stage 19 Xenopus embryos, respectively (Bowes et al., 2010; Faber and Nieuwkoop, 1967). (iii) Control (uninjected) embryos show characteristic bilateral hyperpolarized cell clusters (green arrowheads). (iv) Seventy-five percent (20 total) of EXP1 mRNA and 54% (69 total) of GlyR mRNA plus IVM (two dorsal cells injected at the four-cell stage) embryos show weak and disrupted hyperpolarization (red and blue arrowheads). (iv) A representative image of disrupted hyperpolarization signal due to GlyR plus IVM. (B) Embryos microinjected with EXP1 mRNA or GlyR mRNA plus IVM, co-injected with lineage tracer lacZ (blue-green). Malformed eyes (red arrowheads) are seen only on the side where β-gal is expressed. (i) Incomplete eye formation, (ii) small eye, (iii) only one eye fused to brain, and (iv) pigmented optic nerves. The β-gal lineage label staining in panel i illustrates that eye defects occur in the region targeted with the ion channel construct mRNA. (C) Embryos (i-iii) microinjected with dominant-negative Pax6 mRNA in two dorsal cells at the four-cell stage. Malformed or absent eyes are indicated with red arrowheads. (D) Control (uninjected) embryo and embryo microinjected with GlyR mRNA plus IVM into the two dorsal cells at the four-cell stage, processed for in situ hybridization with a probe to sonic hedgehog (Shh) (green arrowheads) at stages 18 and 30, showing no apparent change in midline (or other) expression of Shh due to the voltage change. See also Fig. 2. Illustrations reproduced with permission from Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). |
