Endogenous Gradients of Resting Potential Instructively Pattern Embryonic Neural Tissue via Notch Signaling and Regulation of Proliferation

Local perturbation of V_mem disrupts endogenous brain development. A, CC2-DMPE:DiBAC staining of stage 16 Xenopus control (uninjected) and Kv1.5, GlyR (+IVM), or only GlyR-microinjected (two dorsal cells at four-cell stage) embryos. Control embryos (n = 28; Ai) and only GlyR (n = 34; Aiv)-injected embryos showed characteristic hyperpolarization within the forming neural tube (blue arrow) but the Kv1.5-injected embryo (Aii) shows widespread hyperpolarization [n = 24, 21 embryos (87.5%) show changed V_mem] and the GlyR (+IVM, in standard saline) embryo (Aiii) shows widespread depolarization [n = 27, 18 embryos (66.7%) show changed V_mem]. Av, Fluorescence intensity measurements of CC2-DMPE:DiBAC-stained embryos. One-way ANOVA analysis pixel distance 100, 250, and 400, shows significant difference of CC2-DMPE:DiBAC signal in the Kv1.5 and GlyR + IVM embryos compared with control embryos. Data are represented as mean ± SEM; ***p < 0.001 and **p < 0.01. B, Quantification of tadpoles with brain phenotypes upon microinjections of Kv1.5 or GlyR (+IVM ± 70 mm Cl⁻) ion channel mRNA in the two dorsal cells (red arrows) of the four-cell Xenopus embryo. A high incidence of malformed brain is observed in dorsal injections compared with uninjected controls. A χ² analysis showed that the too-hyperpolarized and the too-depolarized embryos were significantly different from the rightly polarized controls. ***p < 0.001. C, Four-cell Xenopus embryos were injected (red arrow indicates injected cell). Ci, Stage 45 uninjected control tadpoles show well formed anterior neural tissue with red arrowheads indicating nostrils, orange arrowheads indicating forebrain/olfactory bulbs, yellow arrowheads indicating mid brain, and green arrowheads indicating hindbrain. Cii, Stage 45 tadpole injected with hyperpolarizing channel Kv1.5 in both the dorsal cells at the four-cell stage. The green arrowheads indicate the hindbrain, which remains largely unaffected. Blue arrowheads indicate severely malformed midbrain and forebrain. Eyes were also found to be malformed or absent. Ciii, Stage 45 tadpole that had been injected with hyperpolarizing Kv1.5 in only one dorsal cell of four-cell embryo with the other side as contralateral control. The uninjected side of the embryo shows unaffected nostrils, forebrain, midbrain, and hindbrain (arrowhead colors as in Ci). The injected side of the embryo shows misformed nostrils and forebrain/olfactory bulb (blue arrowheads) but unaffected midbrain and hindbrain. D, Stage 45 PNTub::GFP transgenic tadpole shows GFP fluorescence in neural tissue (arrowhead colors as in Ci). Gut autofluorescence is also seen as indicated. Di, The uninjected control tadpole shows intact nostrils, forebrain/olfactory bulbs, midbrain, and hindbrain. Dii, Stage 45 tadpoles injected with Kv1.5 ion channel mRNA in one dorsal cell at the four-cell stage, with the contralateral side of the brain as control (as in Ciii). Blue arrowheads indicate the malformed nostrils and forebrain/olfactory bulbs regions in the injected tadpole (n = 100). Black spots are melanocytes.

Local perturbation of V_mem signal disrupts endogenous forebrain marker expression during neural development. A, Control (Ctrl; uninjected) embryos at the indicated stage (Ai, Aiii, Av, Avii, Aix) and embryos microinjected (Inj) with Kv1.5 mRNA (Aii, Aiv, Avi, Aviii, Ax) in the right dorsal cell at the four-cell stage. In situ hybridization for emx (Ai, Aii [n = 7 of 10], Avii, Aviii [n = 10 of 14]), bf1 (Aiii, Aiv [n = 6 of 9], Aix, Ax [n = 6 of 9]), and otx2 (Av, Avi [n = 9 of 13]) show a significantly decreased or missing expression (red arrowheads) of emx, bf1, and otx2 only on the injected side of the embryos, while expression on the uninjected (Uninj) side is intact (green arrowheads). Quantification of the in situ signal (Axi–xiii) as ratio of the signal intensity for uninjected verses its contralateral injected side for emx (Axi), bf1 (Axii), and otx2 (Axiii) show a significant change in the in situ signal for these probes upon Kv1.5 mRNA injection. The data are represented as mean ± SEM (n > 8 for each). The data are analyzed via t test. ***p < 0.001. B, Control (untreated) embryos (Bi, Bv, Bix), embryos treated with LiCl (0.2 m for 10 min at 32-cell stage; Bii, Bvi, Bx), UV light (75 s; Biii, Bvii, Bxi) and microinjected with Kv1.5 mRNA in the two dorsal cells at four-cell stage (Biv, Bviii, Bxii). Phase contrast images (Bi–iv) of embryos at stage 30 show that LiCl treatment (Bii) dorsalizes the embryos with a majority of dorsal tissue and lack of ventral tissue development, while UV treatment (Biii) ventralizes the embryos with increased ventral tissue specification and lack of dorsal tissue development as compared with control embryos (Bi), which show correctly balanced development of dorsal and ventral tissues. Kv1.5-injected embryos (Biv) appear normal similar to control embryos with balanced development of dorsal and ventral tissues. In situ hybridization for dorsalization markers chordin (Bv–viii) and cerberus (Bix–xii) show normal expression (green arrows) in control embryos (Bv, Bix), a significantly increased expression (yellow arrows) in LiCl-treated embryos (Bvi [n = 9 of 10], Bx [n = 9 of 9]), a significantly decreased expression (red arrow) in UV-treated embryos (Bvii [n = 14 of 15], Bxi [n = 11 of 11]), and relatively unchanged expression (green arrows) in Kv1.5-injected embryos (Bviii [n = 20 of 20], Bxii [n = 18 of 18]) compared with control embryos.

The V_mem signal is transduced via Ca²⁺ and GJ. A, Schematic of the logic behind the suppression screen to test known candidate mechanisms for transduction of V_mem change to malformed brain tissue phenotype in tadpoles: GJ, serotonin signaling, and calcium influx. B, Quantification of tadpoles with malformed brain phenotype in control (GFP-injected) and Kv1.5-microinjected (dorsal two blastomeres at four-cell stage) embryos with or without the indicated inhibitors. A high incidence of malformed brain phenotype is seen in Kv1.5-microinjected embryos and this effect of Kv1.5 is prevented by verapamil (a blocker of voltage-gated calcium channels, stages 10–30), Lindane (a blocker of GJC among cells, stages 10–30), and H7 (a chimeric dominant-negative connexin that blocks GJ) but not by fluoxetine (Flx) or sertraline (chemical blockers of the serotonin transporter, stages 10–30) or dominant-negative SERT (DN-SERT) mutant (molecular blocker of serotonin transport). A one-way ANOVA (n = 3 experiments) analysis with post hoc test showed significant variance among the groups, with Kv1.5 significantly different from control, Kv1.5 + verapamil, Kv1.5 + Lindane, and Kv1.5 + H7. ***p < 0.001, **p < 0.01.

Notch ICD-induced brain mispatterning can be rescued by V_mem modulation. A, Quantification of tadpoles with malformed brain phenotype in control (Ctrl; uninjected) embryos and embryos microinjected with a constitutively active notch ICD mRNA (in the dorsal two blastomeres at four-cell stage) alone or in combination with Kv1.5 or Bir10 or dominant-negative Kir6.1p mRNA. As expected (see text), misexpression of Notch ICD alone results in a malformed brain phenotype. Remarkably, hyperpolarizing mRNAs Kv1.5 and Bir10 significantly rescue this effect, while depolarizing channel mRNA (DNKir6.1p) increases the number of tadpoles with malformed brains. A χ² analysis showed that the Notch ICD group is significantly different from the other (Ctrl, Notch ICD + Kv1.5, Notch ICD + Bir10, and Notch ICD + DNKir6.1p) groups. ***p < 0.001. B, Four-cell Xenopus embryos were microinjected with the indicated constructs in two dorsal cells. Bi, Stage 45 control (uninjected) embryos show well formed anterior neural tissue including nostrils, forebrain/olfactory bulbs, midbrain, and hindbrain (arrowhead colors as in Fig. 2Ci). Bii, Stage 45 tadpoles injected with constitutively active notch ICD showed severely malformed neural patterning with the forebrain almost absent and a severely mispatterned midbrain (blue arrowheads). Biii, Stage 45 tadpole injected with both Notch ICD and hyperpolarizing Bir10 ion channel showed significant restoration of neural patterning with intact nostrils and distinct forebrain, midbrain, and hindbrain (arrowhead colors as in Fig. 2Ci). Biv, Stage 45 tadpole injected with both Notch ICD and depolarizing dominant-negative DNKir6.1p channel mRNA shows severely malformed neural tissues and eye patterning with nostrils and forebrain/olfactory bulbs absent and mispatterned midbrain (blue arrowheads). C, Imaging of resting potentials during brain development reveals that Notch ICD misexpression perturbs the normal hyperpolarization patterns required for brain patterning. Fluorescent voltage-sensitive dye (CC2-DMPE:DiBAC) staining of stage 16 Xenopus control (uninjected) embryos (Ci [n = 12]) and embryos microinjected with Notch ICD in two dorsal cells at four-cell stage (Cii [n = 10]). Control embryos show the expected (Fig. 1) hyperpolarization in the forming neural tube as well as in the lateral region of the embryo (solid orange arrowheads), while the Notch ICD-injected embryos are depolarized and fail to show the characteristic hyperpolarization signal (empty orange arrowheads) and develop major neural tissue and eye-patterning defects if grown to stage 45 (see B). D, Fluorescence intensity measurements of CC2-DMPE:DiBAC-stained embryos. One-way ANOVA analysis shows significant difference of CC2-DMPE:DiBAC signal in the Notch ICD-injected embryos (n = 10) compared with control (uninjected) embryos (n = 12). Data are represented as mean ± SEM; ***p < 0.001 and *p < 0.05.

V_mem modulation potentiates ability of reprogramming factors to induce ectopic neural tissues. A, Quantification of tadpoles with ectopic neural tissue in control embryos (uninjected; Uninj) and embryos microinjected with POU + HB4 mRNA in both cells at the two-cell stage. Coinjection with the hyperpolarizing Kv1.5 channels caused a significant increase in the number of tadpoles with ectopic neural tissue compared with POU + HB4-only controls. A χ² analysis showed that the POU + HB4-only controls are significantly different from POU + HB4 + Kv1.5. **p < 0.01. B, Stage 45 PNTub::GFP transgenic tadpoles (Bi–viii). Bi, Biv, and Bvi are bright-field images. Bii, Biii, Bv, Bvii, and Bviii are GFP fluorescence. Control (uninjected) embryos (Bi–iii) show well patterned nostrils, forebrain/olfactory bulbs, midbrain, hindbrain (arrowhead colors same as Fig. 2Ci), and spinal cord (Biii; white arrowheads). Tadpoles injected with POU + HB4 + hyperpolarizing Kv1.5 (Biv–viii) showed brain tissue that was highly expanded anteriorly (Biv; yellow arrows), noticeable amounts of ectopic neural tissue in the head unattached to the brain (Bv; yellow arrows), and noticeable ectopic neural tissue in the tail (Bvi—viii; yellow arrows) away from the spinal cord (white arrowhead).

Identity of ectopic neural tissue induced by V_mem modulation was confirmed by in situ hybridization for developing brain-tissue markers. In situ hybridization for forebrain markers emx (ii–viii) and bf1 (ix–xv) of stage 30 control embryos (i–iv [n = 7], ix–xi [n = 11]) and embryos microinjected (both cells at two-cell stage) with POU + HB4 + Kv1.5 ion channel mRNA (v–xv) [emx: n = 8 of 15; bf1: n = 6 of 12]. The areas marked by white squares in ii, v, vii, ix, xii, and xiv are expanded in iii, iv, vi, viii, x, xi, xiii, and xv, respectively. Red arrows mark ectopic expression and green arrowheads mark the endogenous normal signal; especially striking was the appearance of brain marker-positive tissues in the flank and mid-body dorsal fin (xv; red arrows).

Ectopic neural tissue induction by V_mem is local but initially not cell autonomous. A, In situ hybridization for forebrain markers emx (Ai) and bf1 (Aii; blue) of stage 30 embryos that were microinjected (both cells at two-cell stage) with POU + HB4 + Kv1.5-β-galactosidase mRNA (red). β-Galactosidase stain was developed using Magenta-Gal (red/magenta) substrate. At this stage Magenta-Gal signal and emx/bf1 in situ signal overlap with each other, although not perfectly; especially notable are regions where ectopic blue signal (yellow arrowheads; neural marker expression) is next to, but not overlapping with, the misexpressed ion channel (black arrowheads; Magenta-Gal), indicating that not only hyperpolarized cells but also their neighbors are initially driven to turn on neural markers. B, A typical stage 45 tadpole that had been microinjected with POU + HB4 + Kv1.5-EGFP shows that by this stage, ectopic neural tissues induced in the head (Bi, Bii) and tail (Biii, Biv; yellow arrows) are always positive for the EGFP signal indicating hyperpolarizing ion channel expression (this was true in 100% of embryos examined [n = 20]). Thus, by stage 45, any neighboring cells that were not hyperpolarized have turned off the aberrant neural markers, and only cells that were expressing the channel remain and form ectopic brain. Conversely, at both stages, we observed expression of the hyperpolarizing channel in some regions in which it was not sufficient to induce neural markers or ectopic brain. C, A model for de novo neural tissue induction by V_mem change was formulated to explain the local but initially not cell-autonomous misexpression of neural fate induced by hyperpolarization. This model is based on cells sharing their resting potential through GJs. Such electrical coupling of cells has been documented during neural induction and within the developing neural plate cells (Properzi et al., 2013) with the transfer of V_mem signals occurring to the surrounding cells through these GJ couplings (Blackshaw and Warner, 1976; Shi and Borgens, 1995). As cell fields become partitioned into more finely defined regions with developmental age, large areas of GJ connections are progressively shut down to isolate specific groups of cells with different fates and to insulate such isopotential groups from the long-range signals (Warner, 1985, Fig. 8C). Early pluripotent embryonic cells are connected by GJ to form isopotential cell clusters with respect to V_mem (Ci). Introduction of an ion channel in one cell results in the hyperpolarization of the V_mem of that cell (Cii). As the cell adopts a neural fate and turns on expression of neural markers (Ciii), GJs spread this hyperpolarization to coupled neighbors, causing them also to express the observed ectopic neural markers even though they were not injected with the channel (Civ). As embryonic development proceeds, the tissues become progressively more subdivided with respect to gap junctional connections (Cv). As coupling is reduced, the uninjected cells lose the temporarily hyperpolarized V_mem and then revert back to their original non-neural fate, whereas the injected cells retain their new/changed V_mem giving rise to de novo neural tissues detected at much later stages (Cvi).

Local and distant V_mem signals regulate the proliferation in the developing brain. A, Agarose sections of stage 30 control (uninjected) embryos (Ai) and embryos microinjected with hyperpolarizing Kv1.5 mRNA (Aii, Aiii) in the indicated blastomeres (red arrows) at four-cell stage [n > 11 for each group including controls]. Immunostaining of sections through the developing brain with H3P (Ai–iii) shows a distinct change in the H3P (orange arrowheads) staining in the developing brains of microinjected embryos with highly increased H3P staining in the ventral-injected embryos compared with uninjected controls. B, Quantification of H3P immunostaining in the agarose sections through developing brains of stage 30 control (uninjected) and Kv1.5 microinjected (red arrows indicate injected blastomeres at four-cell stage) embryos. Dorsal blastomere injections significantly decrease the H3P signal whereas ventral injections significantly increase the H3P signal. Values are mean ± SEM (n ≥ 10); *p < 0.05, **p < 0.01, and ***p < 0.001 one-way ANOVA with post-test. C, Agarose sections of Fucci-microinjected (both cells at two-cell stage) stage 30 control embryos (Ci) and embryos also microinjected with hyperpolarizing Kv1.5 mRNA (Cii, Ciii) in the indicated blastomeres (red arrows) at four-cell stage [n = 12 for each experimental group]. Fluorescence detection in sections through the developing brain (Ci–iii) shows a distinct change in the cells' fluorescence pattern (indicator of their placement in different stages of cell cycle; green, S/G₂/M; red, G₁; yellow, S) in the developing brains of microinjected embryos, with decreased numbers of green and yellow fluorescent cells (S/G₂/M phase-dividing cells) and increased red fluorescent cells (G₁ nondividing cells) in the dorsal-injected embryos (Cii), and increased numbers of yellow fluorescent cells (G₁ to S phase–cells entering division) in ventrally injected embryos (Ciii), compared with controls (Ci). D, Quantification of Fucci fluorescence (green, red, and yellow) in the agarose sections through developing brains of stage 30 controls (only Fucci-injected) and Kv1.5 (in addition to Fucci)-microinjected (red arrows indicate injected blastomeres at four-cell stage) embryos. Kv1.5 microinjection significantly changes the fluorescence pattern of cells compared with the controls. Dorsal blastomere injections significantly decrease green fluorescence and significantly increased red fluorescence whereas ventral blastomere injections significantly increase yellow fluorescence. Values are mean ± SEM (n = 12); *p < 0.05, **p < 0.01, and ***p < 0.001; two-way ANOVA with post-tests. E, Quantification of H3P immunostaining in the agarose sections through developing brains of stage 30 control (uninjected) and Kv1.5 mRNA microinjected into two ventral blastomeres at four-cell stage embryos. Injected embryos were kept with or without the indicated inhibitors. A high incidence of H3P signal is seen in the brain tissue in Kv1.5 microinjected embryos and this effect of Kv1.5 is prevented by Lindane (stages 10–30) and H7 (respective pharmacological and chimeric dominant-negative blockers of GJC among cells) but not by fluoxetine (Flx; stages 10–30) or dominant-negative SERT (DN-SERT; a chemical blocker and molecular blocker of the serotonin transporter, respectively) or verapamil (stages 10—30; a blocker of voltage-gated calcium channels). A one-way ANOVA (values are mean ± SEM; n ≥ 10) analysis with post hoc test showed significant variance among the groups, with Kv1.5 significantly different from control and Kv1.5 + Lindane and Kv1.5 + H7 (***p < 0.001). F, Model for V_mem control of neural tissue size by regulating proliferation over long range. The set of studies reported here show that there is a specific degree of hyperpolarized resting membrane potential within the developing neural tissue, with the surrounding tissue depolarized (Fi). This specific degree of hyperpolarized potential regulates the proliferation within that tissue as disrupting this specific hyperpolarization signal decreases the proliferation in the brain. Concurrently, the surrounding depolarized tissues restrict proliferation in the brain tissue over long distance thus governing the tissue size and sculpting the brain tissue (Fii). Hence, when the surrounding tissue is hyperpolarized this restriction is lost and there is increased proliferation in the brain tissue observed (Fiii). These ectopic hyperpolarized tissues not only induce ectopic brain tissues but also might regulate the proliferation within these ectopic brain tissues in conjugation with the surrounding depolarized tissue to bring about sculpting these ectopically induced brain tissues. These results suggest an important role of resting potential distribution in regulating brain tissue size and sculpting.