Bilateral hyperpolarized cell clusters appear in the anterior neural field before formation of eye primordia inXenopus

To document real-time changes in V_mem during eye development, we used the voltage-sensitive dye CC2-DMPE (Wolff et al., 2003) to detect hyperpolarization in the Xenopus embryo in vivo. A physiological screen for bioelectric patterns during craniofacial development (Vandenberg et al., 2011) revealed that, starting at stage 17/18, normal embryos exhibit two small areas in the anterior neural field that are bilaterally positioned and hyperpolarized relative to their neighbors (Fig. 1A, red arrowheads). Whole-cell electrophysiological recordings of V_mem from these areas and from the neighboring tissues on the flank of the same embryo (Fig. 1B) revealed that the CC2-DMPE-demarcated domains were hyperpolarized relative to neighboring tissues by ∼10 mV (also confirming the ability of the CC2-DMPE dye to detect cells with hyperpolarized V_mem). Other regions of the embryo also exhibit interesting distributions of V_mem; these are described elsewhere (Vandenberg et al., 2011) and will be investigated in subsequent work.

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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 V_mem 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 V_mem 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).

V_mem collapses upon cell death and thus cannot be imaged simultaneously with in situ hybridization. We imaged the hyperpolarized V_mem signal using CC2-DMPE at stage 17/18 and immediately fixed the embryos for in situ hybridization analysis of the EFTFs Otx2, Rx1 and Pax6 (Fig. 1C; data not shown). The relatively broad anterior expression of EFTFs appeared to overlap with the hyperpolarized cells (Fig. 1C; data not shown).

To overcome the incompatibility of in situ hybridization with in vivo physiological imaging and to more precisely characterize the timing and co-localization of the observed hyperpolarized cell cluster in relation to the formation of eye primordia, we verified the overlap by labeling the CC2-DMPE-demarcated hyperpolarized cells using a photoconvertible protein (Wacker et al., 2007). Fertilized eggs were injected with EosFP (a green fluorescent protein) mRNA and, at stage 18, as small an area as possible centered on the hyperpolarized cells was photoconverted. Observation of the resulting red signal at stage 28 confirmed that the hyperpolarized cells indeed contribute to eye formation (supplementary material Fig. S1A-C). Agarose sectioning of the embryo through the eye showed that the photoconverted cells contribute to both the lens and the retinal layers of the eye (supplementary material Fig. S1D-F). The hyperpolarized area pattern remains bilateral and largely unchanged through anterior development (stage 22; Fig. 1C; supplementary material Table S1). By contrast, the EFTFs undergo a bilateral regionalization by stage 22, as expected (Fig. 1C) (Viczian et al., 2009; Zuber et al., 2003). The distinct bioelectrical regionalization of the anterior neural field suggests that the bilateral hyperpolarized areas might be involved in the processes by which dynamic expression of the EFTFs regulates the formation of eyes.

Local perturbation of V_mem causes disruption of normal eye development

To determine whether hyperpolarization is functionally required for eye development, we performed a loss-of-function experiment by depolarizing V_mem of the relevant cells. We used the constitutively conductive EXP1 cation channel (Beg and Jorgensen, 2003) and the glycine-gated chloride channel (GlyR) (Davies et al., 2003), which can be permanently activated by the specific GlyR opener Ivermectin (IVM) (Shan et al., 2001). The use of two different molecular reagents (channels that share no sequence or structural homology and have in common only their effect on membrane potential) specifically tested the role of V_mem per se, independent of possible other functions of a particular channel protein or ion species.

In standard 0.1× MMR medium, these channels work to depolarize cells (supplementary material Fig. S2A) (Beg and Jorgensen, 2003; Blackiston et al., 2011; Davies et al., 2003). Embryos were injected with GlyR or EXP1 mRNA in the dorsal two cells of the four-cell embryo [the blastomeres from which eyes derive (Moody, 1987)] (supplementary material Fig. S2Ai) and were stained with CC2-DMPE dye at stage 19 (supplementary material Fig. S2Aii). GlyR-injected embryos were treated with the channel opener IVM from stage 17 to 23 to enable the efflux of chloride that depolarizes GlyR-expressing cells. In contrast to uninjected embryos, which show distinct bilaterally hyperpolarized cell clusters (supplementary material Fig. S2Aiii, green arrowheads), GlyR and EXP1 misexpression strongly reduced or disrupted the hyperpolarization pattern in the injected embryos (supplementary material Fig. S2Aiv). This confirmed that our reagents produce the expected loss-of-function effect on the hyperpolarization pattern. We saw no signs of general toxicity or ill-health from ion channel mRNAs, which were titrated to the lowest levels needed to produce eye phenotypes: dorsoanterior index, midline patterning, overall growth rate and proportion, and behavior were all normal, ruling out simple toxicity as a cause of eye malformation. Consistent with existing evidence of V_mem changes being associated with specific tissue outcomes (Blackiston et al., 2011; Levin et al., 2002; Levin, 2009; Sundelacruz et al., 2009; Tseng et al., 2010), we occasionally observed additional interesting patterning phenotypes involving other organs, which will be characterized in subsequent studies.

To determine whether the observed hyperpolarization is required for normal eye development, GlyR or EXP1 mRNA was injected into the dorsal blastomeres at the four-cell stage and the embryos scored for eye development at stage 42 (Fig. 2A). Uninjected, lacZ-injected, GlyR-injected and IVM-treated embryos served as controls. Control embryos had intact, fully closed, well-formed eyes (Fig. 2B). By contrast, expression of GlyR plus IVM treatment, or expression of EXP1 mRNA, caused a high incidence of disrupted endogenous eye formation (48% and 46%, respectively; Fig. 2A). Phenotypes included incomplete eye formation (supplementary material Fig. S2Bi), small (supplementary material Fig. S2Bii) or absent (Fig. 2B) eyes, fusion to the brain (supplementary material Fig. S2Biii) and pigmented optic nerves (supplementary material Fig. S2Biv).

Although bioelectric events can exert effects at long range (Blackiston et al., 2011; Morokuma et al., 2008), the position of the bilateral hyperpolarized regions suggested a local role. We examined the effects of inducing depolarization elsewhere in the embryo. Injection of GlyR mRNA plus IVM treatment or of EXP1 mRNA into the two ventral blastomeres (which make a minor contribution, if any, to eye fate lineages) caused no significant effect on eye development (Fig. 2A). To allow a direct comparison of the injected and uninjected sides of the same embryo and to further test the effective range of influence of hyperpolarization, only one side (one dorsal cell at the four-cell stage) of the embryo was targeted by depolarizing channel mRNA (GlyR plus IVM, Fig. 2B; EXP1, supplementary material Fig. S2Biii) while leaving the contralateral side as an internal control. The mRNA was mixed with a lineage tracer (lacZ mRNA); eye defects occurred only in regions that had been targeted by the depolarizing mRNA, whereas normal eye development occurred on the uninjected region of the same embryo (Fig. 2B; supplementary material Fig. S2Biii). Taken together, these results demonstrate that the bilateral hyperpolarized cell clusters in the anterior neural field are an important, specific component of normal eye development and that this signal works in a local fashion.

To confirm the role of specific V_mem levels in eye induction, we took advantage of the ability to control the V_mem of GlyR-expressing cells (plus IVM) by modulating the extracellular chloride content [Cl^-]_ex. The depolarizing effect of GlyR misexpression can be progressively reduced by increasing [Cl^-]_ex toward the intracellular chloride concentration (Blackiston et al., 2011; Davies et al., 2003), at which point chloride efflux (and depolarization) will cease. After GlyR mRNA injection into the dorsal two cells at the four-cell stage, we incubated the embryos (with IVM) at different levels of [Cl^-]_ex (stage 17-23). A progressive decrease in the incidence of the malformed eye phenotype (from 48% to 21% at stage 42) was seen as [Cl^-]_ex was increased toward isomolar (60 mM) levels, which abolished the normal outward gradient for these negative charges (Fig. 2C, red box). The ability to rescue the phenotype and control eye patterning by modulating [Cl^-]_ex, in a dose-responsive manner in accordance with the predictions of the Goldman equation, points to the importance of specific V_mem levels in eye development. We could not achieve 100% penetrant rescue, as the levels of [Cl^-]_ex could not be raised to higher values without perturbing other voltage-sensitive systems during patterning that might act as confounding factors (Blackiston et al., 2011; Levin et al., 2002; Levin, 2009; Sundelacruz et al., 2009).

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Fig. 2.

Local perturbation of V_mem 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 V_mem 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).

Perturbation of V_mem during early development disrupts expression patterns of eye development markers

The bilateral hyperpolarization of cell clusters precedes the bilateral regionalization of the EFTFs (Rx1 and Pax6) (Fig. 1) and this V_mem signal is important for proper eye development (Fig. 2). But where does the hyperpolarization V_mem signal function within the hierarchy of known signals, such as Otx2, Rx1 and Pax6? We analyzed the expression of these genes in V_mem-perturbed embryos. Embryos were injected in the dorsal two cells at the four-cell stage with depolarizing mRNAs (GlyR or EXP1) and fixed at stage 22. Otx2, Rx1 and Pax6 mRNA expression was detected using antisense mRNA probes (Fig. 2D). Otx2 is a marker for anterior neural plate (Acampora et al., 1995) and Pax6 and Rx1 are highly expressed in the eye field and in certain regions of the central nervous system, including the hypothalamus and the pineal gland (Bailey et al., 2004; Gehring and Ikeo, 1999). The expression pattern of Otx2 remained unchanged by depolarization (Fig. 2D, green arrowheads), suggesting that overall anterior neural/brain development [which provides crucial signals for eye induction (Lupo et al., 2002)] is not affected by the depolarization. This was confirmed by the observation that there was no change in the robust midline expression of sonic hedgehog (Shh) in treated embryos (supplementary material Fig. S2D), nor in the non-eye neural expression of Pax6 (Fig. 2D, black arrowheads). By contrast, expression of the EFTFs Rx1 and Pax6 within the eye domains was significantly reduced (in area, not expression intensity) or mispatterned in embryos in which the V_mem of hyperpolarized cell clusters had been artificially depolarized (Fig. 2D, yellow arrowheads). These results suggest that this bioelectrical signal functions to regulate the bilateral patterning of Pax6 and Rx1, confirming the functional importance of V_mem for the normal events of eye induction.

Feedback loops and cross-talk between biophysical and genetic pathways are common in patterning systems (Huang and Ingber, 2006; Ingber, 2006; Levin, 2006; Levin, 2009). We asked whether EFTFs could in turn affect the V_mem of the hyperpolarized cell clusters. Microinjection of mRNA encoding a dominant-negative mutant of Pax6 (DNPax6) resulted in embryos that lack eyes or have small eyes (Chow et al., 1999) (supplementary material Fig. S2C). We incubated the uninjected and DNPax6-injected embryos in CC2-DMPE dye to monitor the hyperpolarization signal. Interestingly, reduction in Pax6 activity caused a loss of the hyperpolarization signal in the anterior neural field (Fig. 2E), suggesting the presence of a positive-feedback loop between the hyperpolarization signal and Pax6 expression in the presumptive eye field cell cluster.

Perturbation of V_mem is sufficient to induce ectopic eyes outside the anterior neural field

Having shown that a specific bioelectrical state is necessary for normal eye development, we next asked whether modulation of V_mem outside of the normal eye field could be sufficient to induce ectopic eyes. The V_mem of cells is determined by the combined activity of numerous endogenously expressed channels and pumps. Extensive expression profiling and quantitative physiological modeling would be required for a comprehensive understanding of the natively expressed conductances that give rise to distinct V_mem signals within the Xenopus face. Our interest was in probing a range of non-eye regions for their capacity to form eyes and in developing a gain-of-function technology that might be useful in regenerative applications without requiring a complete physiomic analysis. Hence, we targeted a widely expressed ion translocator.

The ATP-sensitive potassium channel (K_ATP) is a biomedically important ion channel that is involved in linking V_mem to cellular functions in many systems (Nichols, 2006). All four subunits of the K_ATP channel (Kir6.1, Kir6.2, SUR1 and SUR2) were found to be expressed in the head region (including the eye fields) and along the dorsal trunk regions of the embryo from early development (stage 15/16) to tail bud stages (data not shown). We used two well-characterized dominant-negative constructs targeting Kir6.1: either the Kir6.1 pore mutant (DNKir6.1p) or the endoplasmic reticulum (ER)-retention mutant (DNKir6.1-ER) (Ficker et al., 2000; Schwappach et al., 2000).

To verify the effect of DNKir6.1p constructs on the V_mem of injected cells, we injected the mRNAs into the dorsal two cells of four-cell embryos and then imaged them using the CC2-DMPE dye. Control (uninjected) embryos showed the characteristic hyperpolarization pattern (supplementary material Fig. S3A). DNKir6.1p-injected embryos showed highly diminished, or an absence of, hyperpolarized cell clusters (supplementary material Fig. S3A), confirming the ability of DNKir6.1p to depolarize cells.

mRNAs encoding these dominant-negative K_ATP constructs were injected into all four blastomeres of four-cell embryos. The embryos were scored for eye phenotype at stage 42. Green fluorescent protein (GFP) and lacZ mRNA negative-control injections did not produce any eye-relevant phenotypes (Fig. 3A-C). By contrast, DNKir6.1p and DNKir6.1-ER injections resulted in a high incidence (45% and 35%, respectively) of eye-related phenotypes (Fig. 3A). Approximately 25% of injected embryos had defects in their endogenous eyes, as expected when dorsal cell V_mem is perturbed (Fig. 3A,D). Strikingly, ∼20% of the embryos exhibited ectopic eye tissue (only lens or only pigmented epithelium), including patches of retinal pigmented epithelium (RPE) (Fig. 3E, red arrowheads) and lens tissue (Fig. 3I,J, blue arrowheads). We also observed embryos (7.5%) with ectopic and well-formed large complete eyes (RPE, retina and lens) (Fig. 3F-H, red arrowheads). Remarkably, ectopic eyes were found not only in the head but also on the gut (Fig. 3G, red arrowhead) and other caudal areas (Fig. 3H, red arrowheads). Ectopic lens tissue, revealed by β-crystallin antibody, occurred in the head region (Fig. 3J, inset) and even the tail (Fig. 3I, blue arrowheads), with islands of ectopic lens tissue found on both the ventral and dorsal side of the embryo (Fig. 3J, blue arrowheads). Although the majority of the ectopic tissues formed in the head (supplementary material Fig. S3B), a unique aspect of this phenotype is that eye components were frequently observed in locales well outside the anterior neural field, including the distal tail (Fig. 3H-J) and the lateral plate mesoderm (Fig. 3L). This was not accompanied by any axial or anterior duplication.

Our results suggest that perturbation of V_mem is sufficient to induce an eye fate even in regions that are outside of the normal fate map of tissues competent to become eyes (Zaghloul et al., 2005). Moreover, a single mRNA encoding a bioelectric target is sufficient to kick-start the entire eye development process in distal tissues.

To ensure that the key instructive signal was indeed carried by V_mem per se, and not by an ion-specific or ion-independent (e.g. scaffolding) role of our channel constructs, we tested several different ion channels that share no sequence or structural homology but do control V_mem. A majority of these induced ectopic eyes, as predicted (supplementary material Table S2). We also asked whether the eye phenotype (ectopic eyes and disrupted endogenous eyes) induced by the depolarizing DNKir6.1p channel could be rescued by co-injection of hyperpolarizing Kv1.5 channel. Among the embryos co-injected with Kv1.5 and DNKir6.1p, only 2% had ectopic eyes and 11% had disrupted eyes, in comparison to 20% and 25%, respectively, in embryos injected with only DNKir6.1p. The rescue of phenotype by a genetically unrelated but physiologically counteractive protein demonstrates the importance of V_mem itself as a regulatory factor in the modulation of eye development.

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Fig. 3.

Dominant-negative K_ATP 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 K_ATP 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 V_mem 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.

Further, we analyzed whether a narrow V_mem range is required for eye formation. We injected embryos with mRNA encoding a sodium channel, Nav1.5, and incubated them in medium containing a range of [Na⁺]_ex, thus generating conditions ranging from hyperpolarizing to depolarizing (as predicted by the Goldman equation). Maximum ectopic eye induction was observed within a small range of hyperpolarizing [Na⁺]_ex (Fig. 3N), confirming specific V_mem values as the information-bearing signal. Taken together, these results support the hypothesis that regulation of V_mem per se is an instructive signal in initiating eye induction.

We then performed experiments to determine whether ectopic eye induction involves: (1) the ‘migration’ of endogenous eye fate cells to ectopic locations upon perturbation of their V_mem; (2) the ‘colonization’ of ectopically injected tissues (perturbed V_mem) by attracting endogenous eye fate cells; or (3) the induction of non-eye fate cells to take on eye fate upon perturbation of their V_mem. We injected DNKir6.1p (with lacZ mRNA as a lineage label) into the dorsal and ventral blastomeres at the four-cell stage. There was no statistical difference between the incidence of ectopic eyes in dorsal (16%) versus ventral (20%) injections (data not shown). Further, whole-mount staining and sections through the ectopic eye tissue at stage 43 showed that all of the ectopic eye tissues were β-gal-positive (supplementary material Fig. S3Biii; data not shown). These results suggest that ectopic eye tissues are induced in a local manner by inducing the injected non-eye fate cells to assume eye identity and not by the migration of, or colonization by, endogenous eye-fated cells.

Bioelectrically induced eyes exhibit morphology and tissue organization similar to endogenous eyes

We next characterized in detail the ectopic eyes induced by V_mem change, to determine how closely the ectopic structures resemble normal eyes. Sectioning at stage 46 revealed that the ectopic tissues (Fig. 3K,L, red arrowheads) have a cup-shaped architecture similar to that of endogenous eyes (Fig. 3K,L, green arrows). Histological staining with Hematoxylin and Eosin showed that the ectopic cells (Fig. 3Mii) are organized in morphological layers similar to those of wild-type endogenous eyes (Fig. 3Mi).

We were able to identify and localize the key retinal cell populations in endogenous and ectopic eye tissues by immunostaining: Muller glia (Glutamine synthetase, red), amacrine cells (Islet-1, cyan), rods (XAP2, yellow) and cones (Calbindin, green) (Fig. 4A). A functional retina requires a precise arrangement of retinal cell layers. Co-immunolocalization showed that, in the ectopic eye tissue (Fig. 4B), the Muller glia, amacrine cells, rods and RPE are arranged almost identically to those in wild-type or endogenous eyes (Fig. 4B). Our histological and immunohistochemical analysis showed that bioelectric induction can give rise to most tissues present in the mature eye. Moreover, the ectopic structures formed by perturbation of V_mem contain the same tissues, in a similar arrangement, as endogenous eyes.

Formation of ectopic eye tissues by perturbation of V_mem involves ectopic induction of eye marker genes

Pax6 has been shown to induce ectopic eyes in Xenopus embryos upon overexpression (Chow et al., 1999; Zuber et al., 2003). Does V_mem modulation induce eyes through the same EFTFs that are involved in primary (endogenous) eye development? Embryos were injected with DNKir6.1p mRNA, fixed at stage 30, and expression of the anterior neural marker Otx2 and of the EFTFs Rx1 and Pax6 detected by in situ hybridization.

DNKir6.1p injections (which produce ectopic eyes) did not induce ectopic expression of Otx2; by contrast, injected embryos showed numerous foci of ectopic Pax6 and Rx1 expression well away from the head (Fig. 4C). Thus, bioelectric induction of ectopic eye tissue involves the upregulation of key EFTFs. These data also demonstrate that V_mem changes are able to induce de novo expression of EFTFs (Fig. 4C), in addition to regulating their bilateral patterning (Fig. 1, Fig. 2D).

V_mem changes are transduced by Ca²⁺ influx during ectopic eye induction

To determine how V_mem changes are transduced into effector cascades during eye development, we injected Kv1.5 mRNA into two cells of four-cell embryos to induce ectopic eye tissue and performed a simple suppression screen (Table 1), blocking a range of targets known to mediate the linkage between bioelectric and transcriptional pathways in other systems (Levin, 2007). Blockade of gap junction-mediated or serotonergic (Fukumoto et al., 2005) signaling did not prevent ectopic eye tissue induction. By contrast, blockade of voltage-gated Ca²⁺ channels (VGCCs) (100 μM Verapamil), significantly reduced the ectopic eye tissue induction by ion channel misexpression. Hence, changes of V_mem that induce ectopic eye tissues are likely to be transduced into downstream cascades by VGCC-mediated Ca²⁺ influx — a common mechanism for coupling electrical signaling to changes in cell behavior (Nakanishi and Okazawa, 2006; Nishiyama et al., 2008).