Sempou E , Kostiuk V , Zhu J , Cecilia Guerra M , Tyan L , Hwang W , Camacho-Aguilar E , Caplan MJ , Zenisek D , Warmflash A , Owens NDL , Khokha MK .

R01EY021195 U.S. Department of Health & Human Services | National Institutes of Health (NIH), R01GM126122 U.S. Department of Health & Human Services | National Institutes of Health (NIH), R01HD081379 U.S. Department of Health & Human Services | National Institutes of Health (NIH), MCB-1553228 National Science Foundation (NSF), C-2021 Welch Foundation

             Wnt signaling pathway
             endodermal cell differentiation

Xtr Wt + barium (Fig. 1c,k)
Xtr Wt + barium (Fig. 1g,l)
Xtr Wt + cacna1c CRISPR + kcnh6 MO (Fig. 5a_c3)
Xtr Wt + cacna1c CRISPR + kcnh6 MO (Fig. 5b_c3, d)
Xtr Wt + ergtoxin (Fig. 3a)
Xtr Wt + Hsa.KCNH6 mRNA
Xtr Wt + K+ (20mM high) (Fig. 1b,f)
Xtr Wt + K+ (20mM high) (Fig.1d,k)
Xtr Wt + K+ (20mM high) (Fig. 1h,l)
Xtr Wt + K+ (20mM high) (Fig.6 e)
Xtr Wt + K+ (20mM high) (Fig. 7. a_c4,d, b_c4,e, c_c4,f)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig.1e)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. 1i)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. 3a)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. 4a,b, e, f, k, l, m, n)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. 5 a_c2,d, b_c2,e, c_c2,f)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. S1. b c d e)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. S1 i r1_c2, j)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. S1 i r2_c2, j)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. S1 k c1_r1, l)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. S4 l,m)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. S4. l m)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. S5 r_2,c_2)
Xtr Wt + kcnh6 CRISPR (400pg) (Fig. S5 r_2 c_3)
Xtr Wt + kcnh6 CRISPR (400pg) (Supp Fig.5 r_2 c_1)
Xtr Wt + kcnh6 CRISPR (400pg) (Suppl. Fig. 6 b r1_c3, r2_c3)
Xtr Wt + kcnh6 CRISPR + choline (Fig. 3a)
Xtr Wt + kcnh6 MO (Fig. 3b,c, d, f)
Xtr Wt + kcnh6 MO (Fig. 4b,f)
Xtr Wt + kcnh6 MO (Fig. 5a_c2, c)
Xtr Wt + kcnh6 MO (Fig. 5b_c2, d)
Xtr Wt + kcnh6 MO (Fig. S3 a)
Xtr Wt + kcnh6 MO (Fig. S4. a_c1,b c_c1,d)

	Fig. 1: Membrane potential is important for gastrulation and LR patterning. a GHK equation for Vm; R = gas constant, T = temperature, F = Faraday’s constant, p = permeability for each ion, [X]o = ion concentration outside of cell, [X]i = ion concentration inside the cell. b–i Effects of depolarizing treatments (barium chloride and high K+) and kcnh6 depletion on gastrulation (b–e; stage 15–17 embryos; arrowheads indicate incomplete blastopore closure) and organ situs (f–i; stage 45 tadpoles; ventral views; arrowheads indicate normal (D) and inverse (L) heart looping). j Different stages of barium chloride application (color key in j for bar graphs k and l; green = cleavage stages (stages 0–6 or 0–8); red = gastrulation (stages 8–12); blue = LRO signaling (stages 12–19); orange = early organogenesis (stages 19–30); gray = cleavage through LRO signaling (stages 0–19). Xenopus illustrations © Natalya Zahn (2022) from Xenbase (www.xenbase.org RRID:SCR_003280). k, l Percentages of embryos with incomplete blastopore closure at stage 15 (k) and abnormal organ situs at stage 45 (l) after treatment with barium or high K+ at different stages (see j for color code); p-values are in k: (Ba2+ gastrula vs untreated) = 2.28e–015, (high K+ gastrula vs untreated) = 1.36e–006; in l: (Ba2+ gastrula vs untreated) = 5.85e–007, (high K+ gastrula vs untreated) = 2.08e–008, (Ba2+/LRO vs untreated) = 8.51e–003, and (Ba2+ 0–19 vs untreated) = 1.2e–006. All graphs depict mean ± SEM and report total embryo numbers (N) collected over 3 independent experiments for high K+, and over 2 independent experiments for the 47 h barium time course. Key for asterisks: *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001, ns nonsignificant with p > 0.05. Source data are provided as a Source Data file.
	Fig. 2: kcnh6 expression analysis for kcnh6 during Xenopus development. a–h kcnh6 transcripts were detected by a full-length antisense probe via WMISH. Embryos and tissues displayed are in the following orientations: a animal pole to the top, b animal pole view c bisected with animal pole to the top, d animal pole view, e vegetal view and dorsal to the top, f gastrocoel roof plate with anterior to the top and vegetal view, g, h lateral view with anterior to the left and dorsal to the top; Ecto prospective ectoderm, Endo prospective endoderm, DMZ dorsal marginal zone (mesoderm), PM paraxial mesoderm, som somites, GRP gastrocoel roof plate. Representative images from N = 60 embryos (per developmental stage) over 3 independent experiments. i kcnh6 transcripts19 (blue) and Vm16 (black) plotted during early Xenopus development. Blue dots represent mean transcript levels by temporal resolution RNA-seq, while blue shaded region marks Gaussian process 95% confidence interval of the data; n = 2 biological replicate time courses in original study. Membrane potential is presented as mean ± SD from at least n = 14 embryos in original study.
	Fig. 3: Membrane potential is important for gastrulation and regulates calcium levels at gastrulation onset. a Percentages of embryos with abnormal gastrulation after depletion of kcnh6 (MO, CRISPR) or Kcnh channel blockade with Ergtoxin, and rescue of kcnh6 depletion with medium conditions that hyperpolarize the Vm (low K+, val valinomycin, sodium substitution with choline; treatments performed stages 8–12). Above: examples of embryos scored for the graph; posterior views (dorsal to the top) of stage 15 embryos after successful (control) or unsuccessful (kcnh6 CRISPR = CR) gastrulation; arrowhead points to blastopore closure. Graph reports mean ± SEM; total embryo numbers (N) in the graph are from 3 independent experiments (except for Ergtoxin: 2 independent experiments with devitellinized embryos); p-values are (kcnh6MO vs Control MO) = 8.66e–010, (kcnh6MO+mRNA vs kcnh6MO) = 2.59e-004, (kcnh6CRex4 vs Control) = 7.19e-018, (kcnh6CRex3 vs Control) = 5.79e-022, (kcnh6CR+low K+ vs kcnh6CR) = 1.64e-005, (kcnh6CR+val vs kcnh6CR+DMSO) = 1.22e-002, (kcnh6CR+choline vs kcnh6CR) = 2.34e-006, (ErgTx vs Control) = 6.57e-010; two-sided Fisher’s exact test. b Representative intracellular recording in the prospective ectoderm of a control stage 10 embryo; Vm is measured relative to the medium (baseline); the dip in membrane potential indicates the electrode breaking into the cell. c The Vm as measured by intercellular recordings in the prospective ectoderm of stage 10 Control MO and kcnh6 MO-injected embryos; graph reports mean ± SEM; p-value (kcnh6MO vs Control MO) is 1.07e-006 (unpaired two-tailed student’s t-test); each data point represents one cell; data from 10 cells/5 embryos/3 independent experiments. d Live animal pole images of GCaMP6/mCherry at stage 10. e Quantification of GCaMP6 fluorescence intensity normalized to mCherry in mCherry+ cells; graph shows mean ± SEM; data points represent single cells; data from N cells (in graph)/10 embryos/3 independent experiments; p = 1.14e-006; unpaired two-tailed student’s t-test. f Maximum area undergoing simultaneous Ca2+ transients within a 20 s time lapse recording as a percentage of total animal pole area; the animal poles of 13 Control MO and 15 Kcnh6 MO embryos were recorded over 3 independent experiments; p = 1.04e-003; unpaired two-tailed student’s t-test. Key for asterisks: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns nonsignificant with p > 0.05. Source data are provided as a Source Data file.
	Fig. 4: Membrane potential affects early gastrula patterning and pluripotency. a–j WMISH for germ-layer markers in early gastrula embryos (stage 10; a, c: vegetal views with dorsal to the top; e, g, and i: lateral view of bisected embryos, dorsal to right). Markers are for paraxial mesoderm (myoD), superficial dorsal mesoderm (foxj1), ectoderm (ectodermin = ecto), endoderm (vegT, mixer). Graphs (b, d) and (f–j) depict percentages of embryos with absent or strongly reduced expression of these markers; presented are mean ± SEM; total embryo numbers (N) are from 3 independent experiments; p-values (vs Control) are in b: (CR vs Control) = 1.61e-006, (MO vs Control) = 4.99e-003, (Ba2+ vs Control) = 4.45e-003, (high K+ vs Control) = 7.56e-004, and in f: (CR vs Control) = 6.81e-013, (MO vs Control) = 2.7e-013, (Ba2+ vs Control) = 1.58e-009, (high K+ vs Control) = 6.61e-012; ns (nonsignificant) for p > 0.05; two-sided Fisher’s exact test. k–n WMISH for Xenopus pluripotency genes pou5f3.3, pou5f3.1, ventx1.2, and sox2 at stages 9 and 10 (animal pole views). Graphs (k–n) depict mean percentages of embryos with present (+/blue) or absent (−/orange) gene expression; total embryo numbers (N) are from 3 independent experiments; p-values (vs Control) are in k: p = 2.01e-008, in l: p = 1.52e-010, in m: p = 6.73e-018 and in n: p = 1.85e-006; ns (nonsignificant) for p > 0.05. Key for asterisks in all graphs: *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, ns nonsignificant. o Differentiation potential of animal caps excised at stage 8 or 12 and treated with no activin (differentiation into epidermis marked by cytokeratin), low activin (differentiation into mesoderm marked by tbxt) and high activin (differentiation into endoderm marked by sox17β). In each image, the numbers on the bottom right report caps with the indicated phenotype vs total number of caps analyzed over 2 independent experiments. Source data are provided as a Source Data file.
	Fig. 5: The role of VGCCs in germlayer differentiation. a WMISH for ectodermin; lateral views with the animal pole to the top; asterisk marks the animal pole with loss of ectodermin expression; embryos with absent expression are quantified in c; CR CRISPR, Nfd nifedipine. b WMISH for myf5: vegetal views with dorsal to the top; arrowhead marks loss of expression; embryos with abnormal expression are quantified in d. c, d Percentages of embryos with abnormal ectodermin (c) and myf5 (d) expression. Graphs depict mean ± SEM; p-values are in c: (kcnh6MO+DMSO vs DMSO) = 2.88e-006, (kcnh6MO+Nfd vs kcnh6MO+DMSO) = 4.59e-002, (kcnh6MO+cacna1cCR vs kcnh6MO) = 1.53e-002, (kcnh6MO+cacna1gCR vs kcnh6MO) > 9.99e-001 (ns), (kcnh6CR+DMSO vs DMSO) = 3.50e-007, (kcnh6CR+Nfd vs kcnh6CR+DMSO) = 1.20e-002; in d: p-values are (kcnh6MO+DMSO vs DMSO) = 4.07e-015, (kcnh6MO+Nfd vs kcnh6MO+DMSO) = 8.92e-005, (kcnh6MO+cacna1cCR vs kcnh6MO) = 4.78e-005, (kcnh6MO+cacna1gCR vs kcnh6MO) = 2.24e-001 (ns), (kcnh6CR+DMSO) = 2.04e-014, (kcnh6CR+Nfd) = 1.45e-003; two-sided Fisher’s exact test; total embryo numbers (N) in the graphs are from at least 2 independent experiments; Key for asterisks: *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, ns nonsignificant for p > 0.05. Source data are provided as a Source Data file.
	Fig. 6: High-resolution temporal RNA-seq identifies mTOR and Ca2+ Gene Regulatory Network. a, b Summary of activated gene clusters by a heatmap and b cluster average. Data is Gaussian process median for each gene normalized by maximal value, shaded region in b is ±1 SD for each cluster. UC untreated control; High K+ (=depolarizing conditions). c Bubble plot for selection of gene set enrichment terms, calculated with Enrichr, see Methods for definition of terms, and Supplementary Data 1 for full set of enrichments. Bubble size reflects Enrichr Combined score and color indicates −log10 FDR. d Bubble plot enrichment of TF motifs in 500 bp upstream of cluster promoters, see also Supplementary Fig. 7g. Bubble size reflects fold change over background and color is −log10 Hypergeometric right tail p-value for enrichment. e Expression of exemplar genes in control and high K+. Central line and shaded region are transformed Gaussian process median and 95% CI. Circle in top right hand corner gives cluster number. UC (=untreated control), High K+ (=depolarizing conditions). Data analysis performed from N = 13 samples/each 10 embryos over one biological replicate time course.
	Fig. 7: Vm polarization limits mTOR which promotes pluripotency. a–c WMISH for pluripotency markers pou5f3.3, ventx1.2, and differentiation marker ectodermin in stage 10 embryos, depolarized by kcnh6 depletion (kcnh6CR) or high K+ (hiK), and treated with vehicle (DMSO) or rapamycin (Rapa) as indicated; views are a animal pole; b, c lateral view with dorsal to the right; AP animal pole, VMZ ventral marginal zone. d–f Quantification of stage 10 embryos with present (+) or absent (−) expression of markers pou5f3.3 (d), ventx1.2 (e), and ectodemin (f) in the animal pole area. p-values in d are: (kcnh6CR+Rapa vs kcnh6CR+DMSO) = 3.34e-087 and (hiK+Rapa vs hiK+DMSO) = 2.89e-047, in e: (kcnh6CR+Rapa vs kcnh6CR+DMSO) = 1.60e-110 and (hiK+Rapa vs hiK+DMSO) = 1.93e-050, and in f: (kcnh6CR+Rapa vs kcnh6CR+DMSO) = 3.38e-040 and (hiK+Rapa vs hiK+DMSO) = 4.89e-02; two-sided Fisher’s exact test; total embryo numbers (N) in the graphs were collected over 3 independent experiments for pou5f3.3 and ventx1.2 and over 4 independent experiments for ectodermin; key for asterisks: ****p ≤ 0.0001. Source data are provided as a Source Data file.
	Fig. 8: Potassium channels affect pluripotency in hESCs. [a-d] a Images showing untreated hESCs grown in mTeSR1 media or cells treated with 1 mM Barium or 25 nM Ergtoxin and immunostained for pluripotency factors. Scale bar = 100 μm. b Quantification of the results in a; AU arbitrary units. p-values are Oct4: (control vs Ba) = 0.03, (control vs Erg) = 4.5e-04; Sox2 (control vs Ba) = 0.06, (control vs Erg) = 4.4e-04. c Images showing untreated hESCs grown in MEF-CM media or cells treated with 100 nM rapamycin with or without 25 nM Ergtoxin. Scale bar = 100 μm. d Quantification of the results in c; AU arbitrary units. p-values are Nanog: (control vs Rapa) = 4.5e-04, (control vs Rapa+Erg) = 4.3e-04; Oct4: (control vs Rapa) = 0.002, (control vs Rapa+Erg) = 0.002; Sox2 (control vs Rapa) = 7.7e-04, (control vs Rapa+Erg) = 7.6e-04. Graphs (b) and (d) present mean ± SEM; key for asterisks: *p ≤ 0.05, **p ≤ 0.01; ***p ≤ 0.001; ns nonsignificant for p < 0.05; data were derived from 400 cells/3 independent replicates over at least 2 independent experiments. Source data are provided as a Source Data file. e Model for the onset of embryonic differentiation depicting classical biochemical signaling (right) that is complemented by regulation via membrane potential (left). In the electrophysiological pathway, potassium channels set the membrane potential, which limits activation of voltage-gated calcium channels and suppresses intracellular Ca2+ levels. Both pathways result in changes in gene expression, mediated by intracellular signal transducers (right), or by factors that require calcium (left). While biochemical pathways are essential to induce expression of differentiation factors, the electrophysiological pathway affects cell fate indirectly by controlling the timing of downregulation of pluripotency genes. Adapted from “Transporters”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates.
	Fig. 8: Potassium channels affect pluripotency in hESCs. e Model for the onset of embryonic differentiation depicting classical biochemical signaling (right) that is complemented by regulation via membrane potential (left). In the electrophysiological pathway, potassium channels set the membrane potential, which limits activation of voltage-gated calcium channels and suppresses intracellular Ca2+ levels. Both pathways result in changes in gene expression, mediated by intracellular signal transducers (right), or by factors that require calcium (left). While biochemical pathways are essential to induce expression of differentiation factors, the electrophysiological pathway affects cell fate indirectly by controlling the timing of downregulation of pluripotency genes. Adapted from “Transporters”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates.
	Supplementary Figure 1. kcnh6 depletion or Ergtoxin induces LR patterning defects. a-e) Examples of organ situs in stage 47 X. tropicalis tadpoles (ventral view with anterior to the top). Arrowheads indicate normal (D) and abnormal (L and A; A=outflow tract is midline) cardiac looping; the heart and gall bladder are minimally pseudo-colored in pink and green respectively for visualization; asterisks indicate the location of the liver whenever discernible. f-h) Percentages of stage 47 tadpoles with situs defects upon injection of CRISPRs targeting independently two sites in different exons of the kcnh6 locus, translation blocking MO, or after treatment with Ergtoxin. Graphs report mean ±SEM. p-values for (f) are (kcnh6CRex3 vs Co)=6.02e-008, (kcnh6CRex4 vs Co)=4.47e-006, (kcnh6MO vs ControlMO)=4.57e-005, (ErgTx vs Co)=1.08e-007; p-values for (g) are (kcnh6CRex3 vs Co)=5.64e-012, (kcnh6CRex4 vs Co)=8.39e-007, (kcnh6MO vs ControlMO)=6.06e-046, (ErgTx vs Co)=8.14e-012; p-values for (h) are (kcnh6CRex3 vs Co)=3.08e-024, (kcnh6CRex4 vs Co)=2.17e-010, (kcnh6MO vs ControlMO)=5.70e-025, (ErgTx vs Co)=1.54e-015; two-sided Fisher’s exact test. Total tadpole numbers (N) in graphs were collected over at least 3 independent experiments; Ergtoxin data is from 2 independent experiments; Tadpoles with multiple defects were counted once. i-j) Detection of dand5 expression via WMISH in stage 16 (pre-ciliary flow) and stage 19 (post flow) embryos viewed ventrally (i; A= anterior, P= posterior; R= right, L= left); (j) indicates percentages of embryos with absent dand5 expression. k-l) Detection of pitx2c expression in stage 28 embryos via WMISH; embryos in (k) are lateral views with dorsal to the top and either the left (L) or right (R) side visible; (l) indicates percentages of embryos with abnormal (absent or bilateral) pitx2c expression. Graphs (j) and (l) report mean ±SEM. Total tadpole numbers (N) in graphs were collected over at least 2 independent experiments; p-values in (j) are (kcnh6CRex3 vs Co, pre-flow)=8.22e-008, (kcnh6MO vs Co, pre-flow)=1.50e-008, and (kcnh6CRex3 vs Co, post-flow)=8.34e-005, (kcnh6MO vs Co, post-flow)=2.68e-007; p-values in (l) are (kcnh6CRex3 vs Co)=2.33e-003, (kcnh6MO vs Co)=1.55e-010, (MO+mRNA vs MO)=6.75e-004; two-sided Fisher’s exact test. Key for asterisks: *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****=p≤0.0001; ns= non-significant for p>0.05. Source data are provided as a Source Data file.
	Supplementary Figure 2. Inference of Crispr Edits (ICE) analysis in kcnh6 CRISPR stage 47 tadpoles. a,c) Average percentages of frameshift (KO= knockout), in-frame (other) or unedited kcnh6 (no edits) sequences for CRISPRs targeting exon 3 (a) or exon 4 (c). b,d) Distribution of INDEL sizes; INDELs with representation >5% of total sequences are represented in individual bars, whereas those with <5% are summed in the last bar (*). Graphs display mean ±SEM; 16 tadpoles were analyzed for each CRISPR (control, ex3 or ex4) over 2 independent experiments. e,f) Representative examples of CRISPR edits by (e) kcnh6 CR exon 3 or (f) kcnh6 CR exon 4. Sequences from two individual tadpoles are shown. The unedited control sequence is highlighted by a black box and the PAM sequence is in red; vertical dotted lines indicate the presumptive cut site. Source data are provided as a Source Data file.
	Supplementary Figure 3. Controls to Figure 1m and Vm measurements of High K+ and choline embryos a) Effects of KCNH6 mRNA expression and hyperpolarizing conditions on gastrulation in control or kcnh6 MO embryos. Percentages of embryos with incomplete blastopore closure at stage 17. Graph presents mean ±SEM; p-values are: (kcnh6mRNA vs Co)=4.22e-037, (kcnh6MO+mRNA vs mRNA)=4.00e-003, (kcnh6MO vs ControlMO)=2.87e-025, (kcnh6 MO+lowK+ vs kcnh6MO)=8.91e-005; two-sided Fisher’s exact test. Total embryo numbers (N) are from at least 2 independent experiments. Key for asterisks: *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****=p≤0.0001; ns= non-significant for p>0.05. b) Intracellular recordings in the prospective ectoderm of stage 9 embryos; Vm was measured relative to the medium. CholineCl refers to substitution of ½ Na+ with choline chloride. Centers of bars represent mean, error bars represent ±SEM. Each data point represents one cell. Normal K+ (n=16 cells/14 embryos) vs. High K+ (n=14 cells/13 embryos) p=0.04, High K+ vs. CholineCl Normal K+ (n=16 cells/12 embryos) p=0.002, High K+ vs. CholineCl High K+ (n=16 cells/12 embryos) p=0.01, one-way ANOVA with Bonferroni correction; Total embryo numbers collected over 3 independent experiments. Key for asterisks: *=p≤0.05, **=p≤0.01, ***=p≤0.005, ****=p≤0.0001. Source data are provided as a Source Data file.
	Supplementary Fig 4. Effects of kcnh6 depletion and Vm depolarization on germ layer differentiation. a-e) WMISH for mesodermal transcripts in stage 10 embryos. a) Vegetal views with dorsal to the top showing expression of paraxial mesodermal marker myf5. b) Percentages of embryos with absent or reduced (abnormal) myf5 expression; p-values are (kcnh6CR vs Co)=7.12e-005, (kcnh6MO vs Co)=4.44e-004, (Ba2+ vs Co)=3.03e-004, (highK+ vs Co)=7.38e-007; two-sided Fisher’s exact test. c) Lateral views showing tbxt expression (highK+ is a vegetal view); asterisk (*) marks absent expression in the paraxial region of the mesoderm. d) Percentages of embryos with abnormal tbxt expression (as shown in (c)); p-values are (kcnh6CR vs Co)=6.93e-003, (kcnh6MO vs Co)=6.77e-004, (Ba2+ vs Co)=4.61e-003, (highK+ vs Co)= 4.05e-003; two-sided Fisher’s exact test. (e) vegetal views with dorsal to the top showing foxj1 expression, see Main Figure 4 for quantification. f-k) WMISH for transcripts of dorsal organizer (gsc, xnr3) and ventral mesoderm (ventx2.2) marker genes; vegetal views of stage 10 embryos with dorsal to the top; (g, i, and k) show percentages of stage 10 embryos with abnormal expression of these markers (no effects are observed; non-significant, p>0.05; two-sided Fisher’s exact test). l,m) WMISH for foxI1a, expressed in the prospective ectoderm; animal views. (m) shows percentages of embryos with absent foxI1a expression; p-value (kcnh6CR vs Co)=3.09e-006; two-sided Fisher’s exact test. All graphs show mean ±SEM; total embryo numbers (N) in the graphs are from 3 independent experiments; data for foxI1a are from 2 independent experiments; key for asterisks: ***=p≤0.001, ****=p≤0.0001, ns=non-significant for p>0.05. Source data are provided as a Source Data file.
	Supplementary Figure 5. Effects of kcnh6 depletion on LRO patterning. Immunostaining of the gastrocoel roof plate, where the LRO is located, in stage 17 embryos for acetylated tubulin (cilia) and MyoD (paraxial mesoderm); cell morphology is highlighted with phalloidin (actin); ventral views with anterior to the top; arrowheads indicate areas of absent MyoD labelling in the paraxial margins of the LRO in kcnh6 CRISPR embryos. Representative images shown from a total of N=12 gastrocoel roof plates per condition, collected over 2 independent experiments.
	Supplementary Figure 6. Pou5f3.2 and the role of VGCCs in pluripotency. a) animal pole view; WMISH detecting pou5f3.2 expression in stage 10 control and kcnh6 CR (CRISPR) embryos, quantitated in right panel. Graph shows mean percentages of embryos with absent(-)/present(+) pou5f3.2 expression; total embryo numbers (N) in graph from 3 independent experiments; ns=non-significant for p>0.05 by two-sided Fisher’s exact test. b) lateral views with animal pole to the top; WMISH for pou5f3.3 and ventx1.2 in stage 12 embryos, quantified in (c) and (d) respectively; Nfd (nifedipine), VMZ (ventral marginal zone). c and d) Mean percentages of embryos with present/absent pou5f3.3 (c) and ventx1.2 (d) expression; p-values in (c) for pou5f3.3 are (kcnh6CR+DMSO vs Control+DMSO)=4.54e-005, (kcnh6CR+Nfd vs kcnh6CR+DMSO)=5.60e-005 and in (d) for ventx1.2 (kcnh6CR+DMSO vs Control+DMSO)= 2.14e-006, (kcnh6CR+Nfd vs kcnh6CR+DMSO)=2.87e-006; two-sided Fisher’s exact test; total embryo numbers (N) in graph are from 3 independent experiments. Key for asterisks: ****=p≤0.0001; ns= non-significant for p>0.05. Source data are provided as a Source Data file.
	Supplementary Figure 7. High Temporal Resolution RNAseq [a-e panels] a) Schematic of RNAseq collection from stage 8 to stage 12. 10 embryos were collected every 30 mins from a clutch of synchronized embryos after IVF. Xenopus illustrations © Natalya Zahn (2022) from Xenbase (www.xenbase.org RRID:SCR_003280). b) Quantification of ERCC spike-in transcripts before (top) and after (bottom) dinucleotide correction to account for GC bias for UC (=Untreated) Control and High K+ embryos. Note that true spike-in concentration is on the horizontal axis, and spikes with low GC content are underrepresented in sequencing. Corrected quantifications are used in all subsequent panels. c) Pairwise Spearman correlation between all UC and High K+ samples after filtering for genes with sufficient temporal expression d) Principal components analysis of log(TPM + 1) transformed expression for UC and HK+=High K+ samples. Sample index 1-13 labelled. e) Visualisation of total differentially expressed genes and their magnitude as 2D histogram, colour indicating the frequency of genes in each bin. Horizontal axis gives log-likelihood-ratio, with LR > 0 defined as differentially expressed. Vertical axis gives maximal divergence z-score of UIC and High K+ trajectories in transformed GP space, averaging signal and noise variance for UC and High K+.
	Supplementary Figure 7. High Temporal Resolution RNAseq [f-j panels] f-g) K-means clustering of activated and repressed genes as (f) heatmap and (g) cluster average. Shaded region in (f) gives +/- 1 SD for each cluster as in Main Fig. 6b. h) Top 16 motif enrichments promoters (500 bp upstream of TSS) of activated clusters A1, A2, A3, A4. Heatmap gives –log10 Hypergeometric right tail p-value sorted by maximal enrichments such that motifs enriched in A1/A2 are at the top and those in A4 are at the bottom. i) Expression of calcium responsive TFs, ets1, crem, creb1, atf1, demonstrating that these genes are not transcriptionally activated over our time course. Log-likelihood ratio given with LR > 0 differentially expressed. Central line and shaded region are transformed Gaussian process median and 95% CI. j) Enrichments of public ChIP-seq peaks for CREB1, CREM and ETS in tissues and cell lines given in Human, Mouse and Rat, in proximity to genes in activated clusters A1, A2, A3, A4. Enrichments calculated with Enrichr and ChEA_2016 gene set7 which integrates data from a broad range of publicly available ChIP assays. ChEA_2016 term names given on horizontal axis describing target, Pubmed ID, cell/tissue and organism. Left panel gives size of intersection as percent of total genes in each cluster, right panel gives –log10 FDR for Fisher’s Exact test for overrepresentation in each cluster.
	Supplementary Figure 8. Blocking KCNH channels slows differentiation of hESCs while rapamycin treatment induces differentiation. a and b) Cells were grown with or without ErgToxin reagent (25 nM) for 2 (a) or 5 days (b) and then the indicated pluripotency markers were measured by qRT-PCR; mean fold-change (±SEM) over control hESCs is shown; data from 3 independent experiments. c and d) Quantification of immunofluorescence against SOX2 (c) and NANOG (d) following treatment with 50 ng/ml BMP4 with or without Ergtoxin addition; presentation is in AU=arbitrary units; mean ±SEM from 3 independent experiments; *=p≤0.05; two-tailed unpaired student’s t-test. e) Expression of pluripotency markers OCT4, SOX2 and NANOG in hESCs (relative immunofluorescence intensity, arbitrary units) upon treatment with rapamycin alone, or rapamycin and ErgToxin together, after seeding at different densities. 1000, 3000, or 7000 cells were seeded into an 18 well slide (Ibidi) and treated with rapamycin (100 nM) for 5 days. As a control, 1000 cells were seeded and grown without rapamycin treatment. Mean ±SEM from 3 independent experiments. f) Pluripotency marker expression data from (e) plotted as a function of final cell density (relative immunofluorescence intensity, arbitrary units). Numbers indicated for final density are the average number of cells in one image, whose dimensions correspond to 0.636mmx0.636mm. Pluripotency markers are reduced in the rapamycin treated cells regardless of whether the final density is higher or lower than in the control condition. Mean ±SEM from 3 independent experiments. Source data are provided as a Source Data file.
	kcnh6 (potassium channel, voltage gated eag related subfamily H, member 6) gene expressionin X. tropicalis embryo, NF stage 28, assayed via in situ hybridization, lateral view, anterior left, dorsal up.
	kcnh6 (potassium channel, voltage gated eag related subfamily H, member 6) gene expressionin X. tropicalis embryo, NF stage 22, assayed via in situ hybridization, lateral view, anterior left, dorsal up.
	kcnh6 (potassium channel, voltage gated eag related subfamily H, member 6) gene expressionin X. tropicalis embryo, NF stage 8, assayed via in situ hybridization, bissected embryo, lateral view, animal pole up.
	kcnh6 (potassium channel, voltage gated eag related subfamily H, member 6) gene expressionin X. tropicalis embryo, NF stage 6 (32-cell), assayed via in situ hybridization, animal pole up.

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