Pai VP et al. (2012), Transmembrane voltage potential controls embryo...

XB-ART-44558

Development 2012 Jan 01;1392:313-23. doi: 10.1242/dev.073759.

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Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis.

Pai VP , Aw S , Shomrat T , Lemire JM , Levin M .

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Uncovering the molecular mechanisms of eye development is crucial for understanding the embryonic morphogenesis of complex structures, as well as for the establishment of novel biomedical approaches to address birth defects and injuries of the visual system. Here, we characterize change in transmembrane voltage potential (V(mem)) as a novel biophysical signal for eye induction in Xenopus laevis. During normal embryogenesis, a striking hyperpolarization demarcates a specific cluster of cells in the anterior neural field. Depolarizing the dorsal lineages in which these cells reside results in malformed eyes. Manipulating V(mem) of non-eye cells induces well-formed ectopic eyes that are morphologically and histologically similar to endogenous eyes. Remarkably, such ectopic eyes can be induced far outside the anterior neural field. A Ca(2+) channel-dependent pathway transduces the V(mem) signal and regulates patterning of eye field transcription factors. These data reveal a new, instructive role for membrane voltage during embryogenesis and demonstrate that V(mem) is a crucial upstream signal in eye development. Learning to control bioelectric initiators of organogenesis offers significant insight into birth defects that affect the eye and might have significant implications for regenerative approaches to ocular diseases.

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Species referenced: Xenopus laevis
Genes referenced: calb1 crybb2 gal.2 glul isl1 nav1 otx2 pax6 rax rpe shh tbx2 tfap2a
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	Fig. 1. A bilateral cluster of hyperpolarized cells appears in the anterior neural field before the formation of eye primordia. (A) CC2-DMPE staining (ii) showing the relative Vmem of cells in the indicated region (i) of a stage 18 Xenopus embryo. Arrowheads indicate the specific clusters of hyperpolarized cells in the anterior neural field. (B) Electrophysiological Vmem measurements of cells identified by CC2-DMPE staining versus their neighboring unstained cells. Readings were recorded from at least six embryos. Values are mean ± s.e.m. ***, P=0.0002. (C) CC2-DMPE staining (iii,iv) and, immediately thereafter, Rx1 in situ hybridization (v,vi) of the indicated regions (i,ii) of Xenopus embryos at stages 18 and 22. Red arrowheads mark specific clusters of hyperpolarized cells in the anterior neural field. Blue arrowheads indicate Rx1 expression in the same embryos. The bilateral hyperpolarization signal occurs before the formation of bilateral eye primordia. See also supplementary material Fig. S1, Table S1. Illustrations reproduced with permission from Nieuwkoop and Faber (Nieuwkoop and Faber, 1967).
	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).
	Fig. 3. Dominant-negative KATP channel subunits cause defects in endogenous eye development and induce a variety of ectopic eye tissues. (A) Stage 42 tadpoles showed a significantly high incidence of eye phenotypes (both malformed eyes and ectopic eye tissue) upon microinjection of dominant-negative KATP mRNAs (DNKir6.1p and DNKir6.1-ER), in comparison to control injections, in all four cells of four-cell embryos. Values are mean ± s.e.m. (n=5). , P<0.05; *, P<0.01; one-way ANOVA, Tukey's post test. (B-H) Stage 42 control tadpoles (B,C) and those microinjected with DNKir6.1p mRNA (D-H) as detailed in A. Green arrow and arrowhead mark endogenous eyes. Red arrow and arrowheads mark ectopic eye tissues. E shows multiple ectopic patches of RPE. Note whole ectopic eyes with lens (F,G) and ectopic eye tissue in tail (H). (I,J) β-crystallin immunostaining (red) in stage 42 whole tadpole (I) and lateral sections through tadpoles (J) injected with DNKir6.1p mRNA in all four cells at the four-cell stage. Blue arrowheads mark distinct ectopic lens tissue in head and tail region, with green arrowheads marking the endogenous eye lens tissue. (K,L) Transverse sections of stage 42 tadpoles microinjected with DNKir6.1p mRNA in all four cells at the four-cell stage. Green arrows marks endogenous eyes and red arrowheads mark ectopic eye tissues. Note that the ectopic eye is present on the ventral side of the tadpole in K and along lateral mesoderm in L. (M) Hematoxylin and Eosin staining of the endogenous eye (i) and ectopic eye tissue (ii) show a similar histological organization of cell layers. (N) A narrow range of membrane voltage induces ectopic eye tissue. Quantification of tadpoles with ectopic eye tissues at stage 42 upon microinjection of neonatal Nav1.5 channel mRNA in all four cells at the four-cell stage. To vary the Vmem in injected cells by control of influx of positive ions through this channel, embryos were incubated in different concentrations of sodium gluconate (10, 15, 20 and 40 mM); 20 mM is isomolar with intracellular sodium (equilibrium). Ectopic eyes were induced only in the hyperpolarizing condition created by 15 mM sodium gluconate. See also supplementary material Fig. S3, Table S2.
	Fig. 4. Ectopic eyes induced by Vmem signal are similar to endogenous eyes and exhibit ectopic expression of canonical eye development factors. (A) Confocal images of sections through endogenous (i,iii,v,vii,ix) and ectopic (ii,iv,vi,viii,x) eyes immunostained for the retinal differentiation markers Glutamine synthetase (Muller cells, red), Islet-1 (amacrine cells, cyan), XAP2 (rods, yellow) and Calbindin (cones, green) and representative phase contrast images show similar differentiated retinal cell populations in endogenous and ectopic eyes. (B) Confocal images of sections through endogenous (i) and ectopic (ii) eyes co-immunostained for retinal cell differentiation markers of rods (yellow), Muller cells (magenta) and amacrine cells (cyan) and for nuclei (blue) show a similar organization of differentiated retinal cell populations. (C) In situ hybridization of stage 30 control embryos (i,iii,v) and embryos microinjected with DNKir6.1p mRNA (ii,iv,vi) in all four cells at the four-cell stage for eye development markers Otx2 (i,ii), Pax6 (iii,iv) and Rx1 (v,vi). Red arrowheads mark ectopic expression, and green arrowheads mark endogenous expression. No ectopic Otx2 expression is observed in the injected embryos.
	Fig. 5. Model integrating the Vmem signal into eye development and depicting the effects of its change on endogenous and ectopic eye development. (A) Neural induction is crucial in the process of eye development. Bilateral hyperpolarization of a cluster of cells in the anterior neural field regulates the expression pattern of eye development markers (EFTFs) such as Rx1 and Pax6. Note that there is a positive-feedback loop between factors such as Pax6 and local hyperpolarization. (B) Eye development factor expression in relation to changes in Vmem. Normal eye development involves regulation of bilateral patterning of EFTFs by the bilateral Vmem signal (hyperpolarization). Underlying mesodermal inhibitory signal also plays an important role in this bilateral regionalization of EFTFs. Disruption of the bilateral Vmem signal results in diminished or lost EFTF signal, resulting in disrupted endogenous eye development. Induction of ectopic Vmem changes results in de novo EFTF expression and formation of ectopic eyes. (C) Hyperpolarization within a window of Vmem is important for the proper formation and development of eyes. Perturbation (depolarization, 1) of endogenous Vmem signal takes the cells outside this window and results in improper eye development. Perturbation (depolarization, 2) of Vmem of non-eye cells, such that their Vmem now lies within the window of eye formation, results in the formation of ectopic eye tissue by these cells. Perturbation (depolarization, 3 and 4) of Vmem in non-eye cells, such that their Vmem still does not fall within the eye formation window, does not result in ectopic eye tissue formation from these cells.
	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 40lens 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).
	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).
	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 Vmem was modulated. See also Fig. 3. Illustrations reproduced with permission from Nieuwkoop and Faber (Nieuwkoop and Faber, 1967).

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