Pai VP and Levin M (2022), HCN2 channel-induced rescue of brain, eye, hear...

XB-ART-59136

Wound Repair Regen 2022 Nov 01;306:681-706. doi: 10.1111/wrr.13032.

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HCN2 channel-induced rescue of brain, eye, heart and gut teratogenesis caused by nicotine, ethanol and aberrant notch signalling.

Pai VP , Levin M .

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Organogenesis is a complex process that can be disrupted by embryonic exposure to teratogens or mutation-induced alterations in signalling pathways, both of which result in organ mispatterning. Building on prior work in Xenopus laevis that showed that increased HCN2 ion channel activity rescues nicotine-induced brain and eye morphogenesis, we demonstrate much broader HCN2-based rescue of organ patterning defects. Induced HCN2 expression in both local or distant tissues can rescue CNS (brain and eye) as well as non-CNS (heart and gut) organ defects induced by three different teratogenic conditions: nicotine exposure, ethanol exposure or aberrant Notch protein. Rescue can also be induced by small-molecule HCN2 channel activators, even with delayed treatment initiation. Our results suggest that HCN2 (likely mediated by bioelectric signals) can be an effective regulator of organogenesis from all three germ layers (ectoderm, mesoderm and endoderm) and reveal non-cell-autonomous influences on organ formation that work at a considerable distance during embryonic development. These results suggest molecular bioelectric strategies for repair that could be explored in the future for regenerative medicine.

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Species referenced: Xenopus laevis
Genes referenced: hcn2 notch1

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	Figure 1. Both local and distant expression of HCN2 channels rescue nicotine-induced non-CNS organ defects in Xenopus embryos. (A) Schematic of embryonic bioelectric pattern, its disruption, and restoration. Our previous studies20-23 show membrane voltage difference between regions of ectoderm (neural plate and surrounding regions) are crucial for proper eye and brain morphology. Embryonic exposure to chemical (nicotine and ethanol) and genetic/biochemical (Notch disruption) erases this crucial membrane voltage difference leading eye and brain morphology defects. Predictive computational modelling suggested HCN2 as most promising channel whose activation could bring the bioelectric pattern back to normal. HCN2 channel expression either locally or at distance or exposure to HCN2 opening drugs in nicotine-exposed embryos is sufficient to restore the membrane voltage difference and rescue eye and brain morphology defects. (B–F) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or nicotine-treated (stage 10–35) tadpoles with or without microinjection with Hcn2-WT mRNA either in both blastomeres at two-cell stage or dorsal or ventral two blastomeres at four-cell stage as indicated. Cyan arrow indicates intact rightward gut coils, orange dashed lines indicate intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (G) Quantification of stage 45 tadpole non-CNS organ morphology defects under indicated conditions. Percentage of tadpoles with non-CNS morphology defects for each experimental group are: controls – 5%, nicotine – 50%, nicotine + HCN2-WT 2/2 – 11%, nicotine + HCN2-WT 2/4 dorsal – 17% and nicotine + HCN2 2/4 ventral – 14%. Data are mean + SD, ***p < 0.001 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each data point is an independent experiment with N > 50 embryos. (H,I) Representative images of stage 45 tadpoles. β-galactosidase expression assessed using X-Gal (blue) in bleached tadpoles co-injected with Hcn2-WT and β-galactosidase mRNA. In dorsal blastomere injections, β-galactosidase was observed mainly in the nostrils, eye, brain and spinal cord (cyan arrowheads). In ventral blastomere injections, β-galactosidase was absent from eye, brain and spinal cord (magenta arrowheads) and was mainly present in the brachial arches, gut, heart and muscles (cyan arrowheads).
	Figure 2. Non-local exposure to lamotrigine and gabapentin rescues nicotine-induced non-CNS organ defects in Xenopus embryos. (A–D) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or nicotine-treated (stage 10–35) tadpoles with or without treatment with lamotrigine (stage 10–35) or gabapentin (stage 10–35). Cyan arrow indicates intact rightward gut coils, orange dashed line indicates intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (E) Quantification of stage 45 tadpole non-CNS organ morphology defects under the indicated conditions. Percentage of tadpoles with non-CNS organ defects for each experimental group are: controls – 5%, nicotine – 48%, nicotine + lamotrigine (stage 10–35) – 15%, nicotine + gabapentin (stage 10–35) – 16%, nicotine + lamotrigine (stage 25–35) – 12% and nicotine + gabapentin (stage 25–35) – 13%. Data are mean + SD, *p < 0.001, p < 0.01 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 3. Both local and distant expression of HCN2 channels rescue ethanol-induced eye and brain morphology defects in Xenopus embryos. (A–E) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or ethanol-treated (stage 9–40) tadpoles with or without microinjection with Hcn2-WT mRNA either in both blastomeres at two-cell stage or dorsal or ventral two blastomeres at four-cell stage as indicated. Blue arrowheads indicate intact nostrils, orange brackets indicate intact forebrain (FB), yellow brackets indicate intact midbrain (MB), cyan brackets indicate intact hindbrain (HB), intact eyes (e) and magenta arrowheads indicate severe eye and brain morphology defects. (F) Quantification of stage 45 tadpole eye and brain morphology defects under indicated conditions. Percentage of tadpoles with eye and brain defects for each experimental group are: controls – 6%, ethanol – 56%, ethanol + HCN2-WT 2/2 – 24%, ethanol + HCN2-WT 2/4 dorsal – 24% and ethanol + HCN2 2/4 ventral – 70%. Data are mean + SD, *p < 0.001, p < 0.01, p < 0.05 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. (G) Comparison of severity indices (SI), a measure of severity of phenotypes (eye and brain) within a population, for indicated conditions. Mean SI are: ethanol – 181 and ethanol + HCN2 2/4 ventral – 239. p < 0.05 (unpaired t-test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 4. Non-local exposure to lamotrigine and gabapentin rescues ethanol-induced eye and brain morphology defects in Xenopus embryos. (A–D) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or ethanol-treated (stage 9–40) tadpoles with or without treatment with lamotrigine (stage 9–40) or gabapentin (stage 9–40). Blue arrowheads indicate intact nostrils, orange brackets indicate intact forebrain (FB), yellow brackets indicate intact midbrain (MB), cyan brackets indicate intact hindbrain (HB) and magenta arrowheads indicate severe eye and brain morphology defects. (E) Quantification of stage 45 tadpole eye and brain morphology defects under the indicated conditions. Percentage of tadpoles with eye and brain defects for each experimental group are: controls – 6%, ethanol (state 9–40) – 57%, ethanol + lamotrigine (stage 9–40) – 31%, ethanol + gabapentin (stage 9–40) – 30%, ethanol + lamotrigine (stage 23–40) – 21% and ethanol + gabapentin (stage 23–40) – 26%. Data are mean + SD, **p < 0.0001, p < 0.001, p < 0.01, p < 0.05 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 5. Only distant expression of HCN2 channels rescue ethanol-induced non-CNS organ defects in Xenopus embryos. (A–E) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or ethanol-treated (stage 9–40) tadpoles with or without microinjection with Hcn2-WT mRNA either in both blastomeres at two-cell stage or dorsal or ventral two blastomeres at four-cell stage as indicated. Cyan arrow indicates intact rightward gut coils, orange dashed lines indicate intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (F) Quantification of stage 45 tadpole non-CNS organ morphology defects under indicated conditions. Percentage of tadpoles with non-CNS morphology defects for each experimental group are: controls – 6%, ethanol – 67%, ethanol + HCN2-WT 2/2 – 38%, ethanol + HCN2-WT 2/4 dorsal – 31% and ethanol + HCN2 2/4 ventral – 66%. Data are mean + SD, *p < 0.001, p < 0.01, p < 0.05 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos per treatment group per experiment. (G) Comparison of severity indices (SI), a measure of severity of phenotypes (heart and gut) within a population, for indicated conditions. Mean SI are: ethanol – 152 and ethanol + HCN2 2/4 ventral – 246. *p < 0.01 (unpaired t-test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 6. Non-local exposure to lamotrigine and gabapentin rescues ethanol-induced non-CNS organ defects in Xenopus embryos. (A–D) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or ethanol-treated (stage 9–40) tadpoles with or without treatment with lamotrigine (stage 9–40) or gabapentin (stage 9–40). Cyan arrow indicates intact rightward gut coils, orange dashed line indicates intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (E) Quantification of stage 45 tadpole non-CNS organ morphology defects under the indicated conditions. Percentage of tadpoles with non-CNS organ defects for each experimental group are: controls – 6%, ethanol – 65%, ethanol + lamotrigine (stage 9–40) – 29%, ethanol + gabapentin (stage 9–40) – 30%, ethanol + lamotrigine (stage 23–40) – 25% and ethanol + gabapentin (stage 23–40) – 22%. Data are mean + SD, **p < 0.0001, p < 0.001, p < 0.01, p < 0.05 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 7. Only overall uniform expression of HCN2 channels rescue overactive Notch (ICD)-induced eye and brain morphology defects in Xenopus embryos. (A–E) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or overactive Notch (ICD) mRNA microinjected (both blastomeres at two-cell stage) tadpoles with or without microinjection with Hcn2-WT mRNA either in both blastomeres at two-cell stage or dorsal or ventral two blastomeres at four-cell stage as indicated. Blue arrowheads indicate intact nostrils, orange brackets indicate intact forebrain (FB), yellow brackets indicate intact midbrain (MB), cyan brackets indicate intact hindbrain (HB), intact eyes (e) and magenta arrowheads indicate severe eye and/or brain morphology defects. (F) Quantification of stage 45 tadpole eye and brain morphology defects under indicated conditions. Percentage of tadpoles with eye and brain defects for each experimental group are: controls – 3%, ICD – 74%, ICD + HCN2-WT 2/2 – 38%, ICD + HCN2-WT 2/4 dorsal – 85% and ICD + HCN2 2/4 ventral – 76%. Data are mean + SD, **p < 0.0001, p < 0.001, p < 0.01 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. (G) Comparison of severity indices (SI), a measure of severity of phenotypes (eye and brain) within a population, for indicated conditions. Mean SI are: ICD – 133, ICD + HCN2-WT 2/4 dorsal – 228 and ICD + HCN2 2/4 ventral – 220. p < 0.05 (one-way ANOVA with Tukey's post-hoc test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 8. Non-local exposure to lamotrigine and gabapentin fails to rescue overactive Notch (ICD)-induced eye and brain morphology defects in Xenopus embryos. (A–D) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or overactive Notch (ICD) mRNA microinjected (dorsal two blastomeres at four-cell stage) tadpoles with or without treatment with lamotrigine (stage 11–40) or gabapentin (stage 11–40). Blue arrowheads indicate intact nostrils, orange brackets indicate intact forebrain (FB), yellow brackets indicate intact midbrain (MB), cyan brackets indicate intact hindbrain (HB) and magenta arrowheads indicate severe eye and brain morphology defects. (E) Quantification of stage 45 tadpole eye and brain morphology defects under the indicated conditions. Percentage of tadpoles with eye and brain defects for each experimental group are: controls – 7%, ICD – 70%, ICD + lamotrigine (stage 11–40) – 74%, ICD + gabapentin (stage 11–40) – 72%, ICD + lamotrigine (stage 22–40) – 71% and ICD + gabapentin (stage 22–40) – 75%. Data are mean + SD, **p < 0.0001 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. (F) Comparison of severity indices (SI), a measure of severity of phenotypes (eye and brain) within a population, for indicated conditions. Mean SI are: ICD – 142, ICD + lamotrigine – 245 and ICD + gabapentin – 252. p < 0.01 (one-way ANOVA with Tukey's post-hoc test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 9. Only overall uniform expression of HCN2 channels rescue overactive Notch (ICD)-induced non-CNS organ defects in Xenopus embryos. (A–E) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or overactive Notch (ICD) mRNA microinjected (both blastomeres at two-cell stage) tadpoles with or without microinjection with Hcn2-WT mRNA either in both blastomeres at two-cell stage or dorsal or ventral two blastomeres at four-cell stage as indicated. Cyan arrow indicates intact rightward gut coils, orange dashed lines indicate intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (F) Quantification of stage 45 tadpole non-CNS organ morphology defects under indicated conditions. Percentage of tadpoles with non-CNS morphology defects for each experimental group are: controls – 6%, ICD – 75%, ICD + HCN2-WT 2/2–34%, ICD + HCN2-WT 2/4 dorsal – 83% and ICD + HCN2 2/4 ventral – 70%. Data are mean + SD, **p < 0.0001, p < 0.001, p < 0.01 and n.s.-non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. (G) Comparison of severity indices (SI), a measure of severity of phenotypes (heart and gut) within a population, for indicated conditions. Mean SI are: ICD – 119, ICD + HCN2-WT 2/4 dorsal – 215 and ICD + HCN2 2/4 ventral – 239. **p < 0.001 (one-way ANOVA with Tukey's post-hoc test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 10. Non-local exposure to lamotrigine and gabapentin fails to rescue overactive Notch (ICD)-induced non-CNS defects in Xenopus embryos. (A–D) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or overactive Notch (ICD) mRNA microinjected (dorsal two blastomeres at four-cell stage) tadpoles with or without treatment with lamotrigine (stage 11–40) or gabapentin (stage 11–40). Cyan arrow indicates intact rightward gut coils, orange dashed line indicates intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (E) Quantification of stage 45 tadpole non-CNS organ morphology defects under the indicated conditions. Percentage of tadpoles with non-CNS organ defects for each experimental group are: controls – 7%, ICD – 53%, ICD + lamotrigine (stage 11–40) – 55%, ICD + gabapentin (stage 11–40) – 56%, ICD + lamotrigine (stage 22–40) – 58% and ICD + gabapentin (stage 22–40) – 57%. Data are mean + SD, **p < 0.0001 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. (F) Comparison of severity indices (SI), a measure of severity of phenotypes (heart and gut) within a population, for indicated conditions. Mean SI are: ICD – 161, ICD + lamotrigine – 256 and ICD + gabapentin – 247. p < 0.01 (one-way ANOVA with Tukey's post-hoc test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 11. Schematic model of HCN2 repair of organ defects. (A) Stage 3 four-cell Xenopus embryo with dorsal two blastomeres being main precursors of eye and brain organogenesis and ventral two blastomeres as main precursors of non-neural/CNS organogenesis92, 143 (B) Our new data along with those of our previous studies20, 21, 23, 69 show that: nicotine-induced eye and brain or non-CNS (heart and gut) morphology defects are completely repaired by HCN2 channel expression in either overall, dorsal two or ventral two blastomeres suggesting both local and long-range mode of action. Ethanol-induced eye and brain or non-CNS (heart and gut) morphology defects are completely repaired by HCN2 channel expression in either overall or dorsal two blastomeres suggesting local action for eye and brain repair and long-range action for heart and gut repair. Notch ICD – induced eye and brain or non-CNS (heart and gut) morphology defects are completely repaired by HCN2 channel expression in overall embryo suggesting both local and long-range action together might be necessary.

	Figure 1. Both local and distant expression of HCN2 channels rescue nicotine-induced non-CNS organ defects in Xenopus embryos. (A) Schematic of embryonic bioelectric pattern, its disruption, and restoration. Our previous studies20-23 show membrane voltage difference between regions of ectoderm (neural plate and surrounding regions) are crucial for proper eye and brain morphology. Embryonic exposure to chemical (nicotine and ethanol) and genetic/biochemical (Notch disruption) erases this crucial membrane voltage difference leading eye and brain morphology defects. Predictive computational modelling suggested HCN2 as most promising channel whose activation could bring the bioelectric pattern back to normal. HCN2 channel expression either locally or at distance or exposure to HCN2 opening drugs in nicotine-exposed embryos is sufficient to restore the membrane voltage difference and rescue eye and brain morphology defects. (B–F) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or nicotine-treated (stage 10–35) tadpoles with or without microinjection with Hcn2-WT mRNA either in both blastomeres at two-cell stage or dorsal or ventral two blastomeres at four-cell stage as indicated. Cyan arrow indicates intact rightward gut coils, orange dashed lines indicate intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (G) Quantification of stage 45 tadpole non-CNS organ morphology defects under indicated conditions. Percentage of tadpoles with non-CNS morphology defects for each experimental group are: controls – 5%, nicotine – 50%, nicotine + HCN2-WT 2/2 – 11%, nicotine + HCN2-WT 2/4 dorsal – 17% and nicotine + HCN2 2/4 ventral – 14%. Data are mean + SD, ***p < 0.001 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each data point is an independent experiment with N > 50 embryos. (H,I) Representative images of stage 45 tadpoles. β-galactosidase expression assessed using X-Gal (blue) in bleached tadpoles co-injected with Hcn2-WT and β-galactosidase mRNA. In dorsal blastomere injections, β-galactosidase was observed mainly in the nostrils, eye, brain and spinal cord (cyan arrowheads). In ventral blastomere injections, β-galactosidase was absent from eye, brain and spinal cord (magenta arrowheads) and was mainly present in the brachial arches, gut, heart and muscles (cyan arrowheads).
	Figure 2. Non-local exposure to lamotrigine and gabapentin rescues nicotine-induced non-CNS organ defects in Xenopus embryos. (A–D) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or nicotine-treated (stage 10–35) tadpoles with or without treatment with lamotrigine (stage 10–35) or gabapentin (stage 10–35). Cyan arrow indicates intact rightward gut coils, orange dashed line indicates intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (E) Quantification of stage 45 tadpole non-CNS organ morphology defects under the indicated conditions. Percentage of tadpoles with non-CNS organ defects for each experimental group are: controls – 5%, nicotine – 48%, nicotine + lamotrigine (stage 10–35) – 15%, nicotine + gabapentin (stage 10–35) – 16%, nicotine + lamotrigine (stage 25–35) – 12% and nicotine + gabapentin (stage 25–35) – 13%. Data are mean + SD, *p < 0.001, p < 0.01 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 3. Both local and distant expression of HCN2 channels rescue ethanol-induced eye and brain morphology defects in Xenopus embryos. (A–E) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or ethanol-treated (stage 9–40) tadpoles with or without microinjection with Hcn2-WT mRNA either in both blastomeres at two-cell stage or dorsal or ventral two blastomeres at four-cell stage as indicated. Blue arrowheads indicate intact nostrils, orange brackets indicate intact forebrain (FB), yellow brackets indicate intact midbrain (MB), cyan brackets indicate intact hindbrain (HB), intact eyes (e) and magenta arrowheads indicate severe eye and brain morphology defects. (F) Quantification of stage 45 tadpole eye and brain morphology defects under indicated conditions. Percentage of tadpoles with eye and brain defects for each experimental group are: controls – 6%, ethanol – 56%, ethanol + HCN2-WT 2/2 – 24%, ethanol + HCN2-WT 2/4 dorsal – 24% and ethanol + HCN2 2/4 ventral – 70%. Data are mean + SD, *p < 0.001, p < 0.01, p < 0.05 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. (G) Comparison of severity indices (SI), a measure of severity of phenotypes (eye and brain) within a population, for indicated conditions. Mean SI are: ethanol – 181 and ethanol + HCN2 2/4 ventral – 239. p < 0.05 (unpaired t-test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 4. Non-local exposure to lamotrigine and gabapentin rescues ethanol-induced eye and brain morphology defects in Xenopus embryos. (A–D) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or ethanol-treated (stage 9–40) tadpoles with or without treatment with lamotrigine (stage 9–40) or gabapentin (stage 9–40). Blue arrowheads indicate intact nostrils, orange brackets indicate intact forebrain (FB), yellow brackets indicate intact midbrain (MB), cyan brackets indicate intact hindbrain (HB) and magenta arrowheads indicate severe eye and brain morphology defects. (E) Quantification of stage 45 tadpole eye and brain morphology defects under the indicated conditions. Percentage of tadpoles with eye and brain defects for each experimental group are: controls – 6%, ethanol (state 9–40) – 57%, ethanol + lamotrigine (stage 9–40) – 31%, ethanol + gabapentin (stage 9–40) – 30%, ethanol + lamotrigine (stage 23–40) – 21% and ethanol + gabapentin (stage 23–40) – 26%. Data are mean + SD, **p < 0.0001, p < 0.001, p < 0.01, p < 0.05 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 5. Only distant expression of HCN2 channels rescue ethanol-induced non-CNS organ defects in Xenopus embryos. (A–E) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or ethanol-treated (stage 9–40) tadpoles with or without microinjection with Hcn2-WT mRNA either in both blastomeres at two-cell stage or dorsal or ventral two blastomeres at four-cell stage as indicated. Cyan arrow indicates intact rightward gut coils, orange dashed lines indicate intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (F) Quantification of stage 45 tadpole non-CNS organ morphology defects under indicated conditions. Percentage of tadpoles with non-CNS morphology defects for each experimental group are: controls – 6%, ethanol – 67%, ethanol + HCN2-WT 2/2 – 38%, ethanol + HCN2-WT 2/4 dorsal – 31% and ethanol + HCN2 2/4 ventral – 66%. Data are mean + SD, *p < 0.001, p < 0.01, p < 0.05 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos per treatment group per experiment. (G) Comparison of severity indices (SI), a measure of severity of phenotypes (heart and gut) within a population, for indicated conditions. Mean SI are: ethanol – 152 and ethanol + HCN2 2/4 ventral – 246. *p < 0.01 (unpaired t-test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment) [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 6. Non-local exposure to lamotrigine and gabapentin rescues ethanol-induced non-CNS organ defects in Xenopus embryos. (A–D) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or ethanol-treated (stage 9–40) tadpoles with or without treatment with lamotrigine (stage 9–40) or gabapentin (stage 9–40). Cyan arrow indicates intact rightward gut coils, orange dashed line indicates intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (E) Quantification of stage 45 tadpole non-CNS organ morphology defects under the indicated conditions. Percentage of tadpoles with non-CNS organ defects for each experimental group are: controls – 6%, ethanol – 65%, ethanol + lamotrigine (stage 9–40) – 29%, ethanol + gabapentin (stage 9–40) – 30%, ethanol + lamotrigine (stage 23–40) – 25% and ethanol + gabapentin (stage 23–40) – 22%. Data are mean + SD, **p < 0.0001, p < 0.001, p < 0.01, p < 0.05 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 7. Only overall uniform expression of HCN2 channels rescue overactive Notch (ICD)-induced eye and brain morphology defects in Xenopus embryos. (A–E) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or overactive Notch (ICD) mRNA microinjected (both blastomeres at two-cell stage) tadpoles with or without microinjection with Hcn2-WT mRNA either in both blastomeres at two-cell stage or dorsal or ventral two blastomeres at four-cell stage as indicated. Blue arrowheads indicate intact nostrils, orange brackets indicate intact forebrain (FB), yellow brackets indicate intact midbrain (MB), cyan brackets indicate intact hindbrain (HB), intact eyes (e) and magenta arrowheads indicate severe eye and/or brain morphology defects. (F) Quantification of stage 45 tadpole eye and brain morphology defects under indicated conditions. Percentage of tadpoles with eye and brain defects for each experimental group are: controls – 3%, ICD – 74%, ICD + HCN2-WT 2/2 – 38%, ICD + HCN2-WT 2/4 dorsal – 85% and ICD + HCN2 2/4 ventral – 76%. Data are mean + SD, **p < 0.0001, p < 0.001, p < 0.01 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. (G) Comparison of severity indices (SI), a measure of severity of phenotypes (eye and brain) within a population, for indicated conditions. Mean SI are: ICD – 133, ICD + HCN2-WT 2/4 dorsal – 228 and ICD + HCN2 2/4 ventral – 220. p < 0.05 (one-way ANOVA with Tukey's post-hoc test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 8. Non-local exposure to lamotrigine and gabapentin fails to rescue overactive Notch (ICD)-induced eye and brain morphology defects in Xenopus embryos. (A–D) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or overactive Notch (ICD) mRNA microinjected (dorsal two blastomeres at four-cell stage) tadpoles with or without treatment with lamotrigine (stage 11–40) or gabapentin (stage 11–40). Blue arrowheads indicate intact nostrils, orange brackets indicate intact forebrain (FB), yellow brackets indicate intact midbrain (MB), cyan brackets indicate intact hindbrain (HB) and magenta arrowheads indicate severe eye and brain morphology defects. (E) Quantification of stage 45 tadpole eye and brain morphology defects under the indicated conditions. Percentage of tadpoles with eye and brain defects for each experimental group are: controls – 7%, ICD – 70%, ICD + lamotrigine (stage 11–40) – 74%, ICD + gabapentin (stage 11–40) – 72%, ICD + lamotrigine (stage 22–40) – 71% and ICD + gabapentin (stage 22–40) – 75%. Data are mean + SD, **p < 0.0001 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. (F) Comparison of severity indices (SI), a measure of severity of phenotypes (eye and brain) within a population, for indicated conditions. Mean SI are: ICD – 142, ICD + lamotrigine – 245 and ICD + gabapentin – 252. p < 0.01 (one-way ANOVA with Tukey's post-hoc test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 9. Only overall uniform expression of HCN2 channels rescue overactive Notch (ICD)-induced non-CNS organ defects in Xenopus embryos. (A–E) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or overactive Notch (ICD) mRNA microinjected (both blastomeres at two-cell stage) tadpoles with or without microinjection with Hcn2-WT mRNA either in both blastomeres at two-cell stage or dorsal or ventral two blastomeres at four-cell stage as indicated. Cyan arrow indicates intact rightward gut coils, orange dashed lines indicate intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (F) Quantification of stage 45 tadpole non-CNS organ morphology defects under indicated conditions. Percentage of tadpoles with non-CNS morphology defects for each experimental group are: controls – 6%, ICD – 75%, ICD + HCN2-WT 2/2–34%, ICD + HCN2-WT 2/4 dorsal – 83% and ICD + HCN2 2/4 ventral – 70%. Data are mean + SD, **p < 0.0001, p < 0.001, p < 0.01 and n.s.-non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. (G) Comparison of severity indices (SI), a measure of severity of phenotypes (heart and gut) within a population, for indicated conditions. Mean SI are: ICD – 119, ICD + HCN2-WT 2/4 dorsal – 215 and ICD + HCN2 2/4 ventral – 239. **p < 0.001 (one-way ANOVA with Tukey's post-hoc test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 10. Non-local exposure to lamotrigine and gabapentin fails to rescue overactive Notch (ICD)-induced non-CNS defects in Xenopus embryos. (A–D) Representative images of stage 45 tadpoles. Control (untreated and uninjected) or overactive Notch (ICD) mRNA microinjected (dorsal two blastomeres at four-cell stage) tadpoles with or without treatment with lamotrigine (stage 11–40) or gabapentin (stage 11–40). Cyan arrow indicates intact rightward gut coils, orange dashed line indicates intact rightward coiling heart and magenta arrowheads indicate severe morphology defects in gut and heart. (E) Quantification of stage 45 tadpole non-CNS organ morphology defects under the indicated conditions. Percentage of tadpoles with non-CNS organ defects for each experimental group are: controls – 7%, ICD – 53%, ICD + lamotrigine (stage 11–40) – 55%, ICD + gabapentin (stage 11–40) – 56%, ICD + lamotrigine (stage 22–40) – 58% and ICD + gabapentin (stage 22–40) – 57%. Data are mean + SD, **p < 0.0001 and n.s., non-significant (one-way ANOVA with Tukey's post-hoc test). Each datapoint is an independent experiment with N > 50 embryos. (F) Comparison of severity indices (SI), a measure of severity of phenotypes (heart and gut) within a population, for indicated conditions. Mean SI are: ICD – 161, ICD + lamotrigine – 256 and ICD + gabapentin – 247. p < 0.01 (one-way ANOVA with Tukey's post-hoc test for n = 3 independent experiments with N > 50 embryos per treatment group per experiment). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 11. Schematic model of HCN2 repair of organ defects. (A) Stage 3 four-cell Xenopus embryo with dorsal two blastomeres being main precursors of eye and brain organogenesis and ventral two blastomeres as main precursors of non-neural/CNS organogenesis92, 143 (B) Our new data along with those of our previous studies20, 21, 23, 69 show that: nicotine-induced eye and brain or non-CNS (heart and gut) morphology defects are completely repaired by HCN2 channel expression in either overall, dorsal two or ventral two blastomeres suggesting both local and long-range mode of action. Ethanol-induced eye and brain or non-CNS (heart and gut) morphology defects are completely repaired by HCN2 channel expression in either overall or dorsal two blastomeres suggesting local action for eye and brain repair and long-range action for heart and gut repair. Notch ICD – induced eye and brain or non-CNS (heart and gut) morphology defects are completely repaired by HCN2 channel expression in overall embryo suggesting both local and long-range action together might be necessary.