DEV073759 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -
• Supplemental Table S2 -
• Supplemental Figure S1 -

  Fig. S1. The hyperpolarized cells of the anterior neural field possibly contribute to eye formation. (A) CC2-DMPE staining showing the hyperpolarized cells in the anterior neural field (red arrowhead). (B) Photoconversion from green to red florescence centered on the hyperpolarization signal using a 40× lens as per the protocol (Wacker et al., 2007). (C) At stage 28 the contribution of the photoconverted cells to eye formation was observed (yellow arrowhead). Note that the photoconverted area also contributes to some regions of the forebrain, similar to Pax6. (D) Stage 28 embryo showing plane of sectioning through the eye. (E) Confocal image of a section through a stage 28 photoconverted eye, showing labeling of the majority of the lens and retinal tissue. (F) Dark-field image of the section through the eye. See also Fig. 1. Illustrations reproduced with permission from Nieuwkoop and Faber (Nieuwkoop and Faber, 1967).

• Supplemental Figure S2 -

  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).

• Supplemental Figure S3 -

  Fig. S3. Hyperpolarization patterns and ectopic eye tissue induction. (A) CC2-DMPE staining of stage 19 embryos. (i) Illustration of stage 19 Xenopus embryo.. (ii) Control (uninjected) embryos show characteristic bilateral hyperpolarized cell clusters (red arrowheads). (iii) DNKir6.1p mRNA-injected (two dorsal cells at the four-cell stage) embryos show disrupted or lost hyperpolarization signal (57%, n=7). (B) Stage 42 tadpoles microinjected with DNKir6.1p mRNA in all four cells at the four-cell stage show (i,ii) ectopic eye tissue (red arrows) like patches of RPE with lens-like tissue having lateral pigmented connections to brain. (iii) β-gal signal in embryos co-injected with lacZ and the ion channel construct, showing that ectopic eye tissues arise in the regions in which V_mem was modulated. See also Fig. 3. Illustrations reproduced with permission from Nieuwkoop and Faber (Nieuwkoop and Faber, 1967).