Pai VP , Pietak A , Willocq V , Ye B , Shi NQ , Levin M .

R01 AR055993 NIAMS NIH HHS , R01 AR061988 NIAMS NIH HHS

                anencephaly
                spina bifida
                Alzheimer's disease
                Parkinson's disease

Xla Wt + Nicotine (Fig.1.b,e,f)
Xla Wt + Nicotine (Fig.5.b,c)
Xla Wt + Nicotine (Fig.5.d,f)
Xla Wt + Nicotine (Fig.8.b,d)
Xla Wt + Nicotine (Fig.8.f,h)

	Figure 1. Nicotine induces brain morphology defects in Xenopus embryos. Representative images of stage 45 tadpoles: a) control tadpole showing nostrils (blue arrowhead), forebrain (FB) indicated by the orange bracket, midbrain (MB) indicated by the yellow bracket, and hind brain (HB) indicated by the cyan bracket, b and c) tadpoles from embryos exposed to nicotine (0.1 mg/mL – stage 10–35) showing severe brain morphology defects as indicated by magenta arrowheads. Cyan brackets indicate presence of hindbrain (HB). d) Quantification of stage 45 tadpoles for major brain morphology phenotypes in absence or presence of nicotine exposure (0.1 mg/mL – stage 10–35). A significantly high incidence of malformed brain was observed in embryos exposed to nicotine in comparison to controls. Three independent experiments (n = 3) were conducted with N > 50 embryos per treatment group for each of those experiments collected from multiple animals across independent clutches. Data were analyzed with t-test and graphed as mean ± SD; **p < 0.01
	Figure 2. BETSE model of endogenous resting potential gradients that instructively pattern embryonic neural tissue. a The BETSE model of neurula stage Xenopus embryo featured three different cell groups (referred to as tissue profiles) as indicated by color and labels. The neural tube profile was made to have 5× higher leak membrane permeability to K+ ions and 15× lower membrane permeability to Cl− ions, compared to the other two profiles, leading to hyperpolarized resting Vmem in neural tube cells as has been experimentally observed28, 48. The model is assumed to be the outer layer of cells which face 0.1 × MMR. See supplemental document for detailed description of model. b The regulatory network describing the main components of the model. All cells of all simulations expressed equal levels of Na/K-ATPase ion pumps, inward-rectifying K+ channels (Kir), and nAChR cation channels. Some simulations included HCN2 channels equally expressed on all cells, and/or nicotine introduced via environmental exposure
	Figure 3. BETSE model predicts that nicotine suppresses neural tube hyperpolarization and HCN2 recovers neural tube hyperpolarization in presence of nicotine. a–e) BETSE model of a stage 15 Xenopus embryo where cells are assumed to be facing 0.1 × MMR. a) Control model exhibits a characteristic Vmem pattern featuring relative hyperpolarization in the neural tube area and depolarization everywhere else. This Vmem pattern is actually seen in the Xenopus embryos with voltage reporter dyes28. b) Nicotine treatment of the control model is predicted to preferentially depolarize the neural tube area to suppress the Vmem gradient. c) Model with ion translocator (IT) expression in all cells, acting in a context-specific manner showing strongly enhanced neural tube Vmem pattern. d) Adding the ion translocator (IT) to the nicotine treated model shows the Vmem pattern significantly retained in comparison to the only nicotine treated model of b. e) Line graph showing the information retrieved along the white dotted lines depicted in (a–d), which allows for easy comparison between models. f) The effect of the HCN2 channel on the Vmem of a series of cells with different membrane leak permeabilities (Pmem). The ability of homogeneously expressed HCN2 channels to act in a context specific manner is predicted to stem from the channels’s opening at hyperpolarized Vmem with a hyperpolarizing effect upon opening. This selectively hyperpolarizes cells while leaving relatively more depolarized cell’s Vmem unchanged. This further emphasizes that HCN2 channels amplify the endogenous Vmem pattern of the neural tube and maintain the Vmem pattern against nicotine depolarization exactly like IT in (c, d and e)
	Figure 4. Validation of the model prediction of nicotine effects and HCN2-induced recovery of membrane voltage prepatterns. Representative CC2-DMPE:DiBAC4(3) images of stage ~15 Xenopus embryos: a) untreated controls, b) nicotine-exposed (0.1 mg/mL – stage 10–35), c) Hcn2-WT mRNA microinjected (0.75 ng/injection) in both blastomeres at 2-cell stage, and d) nicotine-exposed (0.1 mg/mL – stage 10–35) and Hcn2-WT mRNA microinjected (0.75 ng/injection) in both blastomeres at 2-cell stage. Control embryos show the characteristic hyperpolarization (solid yellow arrow) as previously reported28. Nicotine-treated embryos show reduced signal (depolarized) within the neural tube (hollow yellow arrow). Hcn2-WT mRNA microinjection show enhanced signal (hyperpolarized) within the neural tube (magenta arrows), in presence or absence of nicotine exposure. e) Quantification of CC2-DMPE:DiBAC4(3) images of stage ~15 Xenopus embryos along the red dotted line as indicated in the inset illustration, along with electrophysiology based membrane voltage approximations (as previously reported in refs.27,28). The fluorescence intensity/membrane voltage pattern within the neural tube (indicated by the black dotted line in the inset illustration and corresponding lack dotted line in the graph) is significantly reduced (depolarization) in nicotine-exposed embryos in comparison to controls. Hcn2-WT mRNA microinjection significantly enhances the fluorescence intensity/membrane voltage patterns within the neural tube in comparison to controls. Nicotine-exposed embryos that are microinjected with Hcn2-WT mRNA maintain a significantly enhanced fluorescence intensity/membrane voltage pattern within the neural tube in comparison to only nicotine-treated embryos. N = 10 embryos for each treatment group at each of the indicated spatial distance in pixels were collected from multiple animals across independent clutches. Data is plotted as mean ± S.E.M. Data at black line (400 Pixels) was analyzed using one way ANOVA, *p < 0.05, **p < 0.01. (f) Quantification of peak fluorescence intensity and electrophysiology based membrane voltage approximations (as previously reported in ref. 27,28) from voltage reporter dye images (CC2-DMPE:DiBAC4(3)) of ~stage 15 Xenopus embryos within the neural tube at the intersection of the red and black dotted lines in the inset illustration in (a). Nicotine exposure significantly reduced (depolarizes) the neural tube peak intensity/membrane voltage in comparison to controls. Hcn2-WT mRNA microinjection, both in presence or absence of nicotine, significantly enhances (hyperpolarizes) the neural tube peak intensity/membrane voltage in comparison to controls. N = 10 embryos for each treatment group at each of the indicated spatial distance in pixels were collected from multiple animals across independent clutches. Data is plotted as mean ± SD and was analyzed using one way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001
	Figure 5. HCN2 channels rescue nicotine-induced brain morphology defects in Xenopus embryos. a) Representative image of stage 45 tadpole with an example of a minor brain morphology defect showing smaller forebrain and nostril on the left (magenta arrowhead), normal midbrain (MB - yellow bracket) and normal hindbrain (HB - cyan bracket). b) Quantification of stage 45 tadpoles for subtle changes in overall brain morphology with or without microinjecting Hcn2-WT (wild-type) mRNA (0.75 ng/injection) in both blastomeres at 2-cell stage as indicated in the illustrations. Hcn2-WT mRNA injections significantly suppress the minor brain defects seen in uninjected control embryos. Three independent experiments (n = 3) were conducted with N > 50 embryos per treatment group for each of those experiments, collected from multiple animals across independent clutches. Data were analyzed with t-test and graphed as mean ± SD, **p < 0.01. Representative images of stage 45 tadpoles: c) control tadpole showing nostrils (blue arrowhead), forebrain (FB) indicated by the orange bracket, midbrain (MB) indicated by the yellow bracket, and hind brain (HB) indicated by the cyan bracket, d) tadpole from embryos exposed to nicotine (0.1 mg/mL – stage 10–35) showing severe brain morphology defects as indicated by magenta arrowheads. e) tadpole from embryos exposed to nicotine (0.1 mg/mL – stages 10–35) and microinjected with Hcn2-WT mRNA (0.75 ng/injection) in both blastomeres at 2-cell stage showing intact nostrils (blue arrowheads), forebrain (FB - orange brackets), midbrain (MB - yellow brackets), and hindbrain (HB - cyan brackets). f) Quantification of stage 45 tadpoles for major brain morphology phenotypes in absence or presence of nicotine exposure (0.1 mg/mL – stage 10–35) with or without microinjection of Hcn2-WT or Hcn2-DN (dominant-negative) mRNA (0.75 ng/injection) in both blastomeres at 2-cell stage as indicated in the illustrations. A significantly high incidence of malformed brain was observed in embryos exposed to nicotine in comparison to controls. Hcn2-WT mRNA injection significantly reduced the incidence of malformed brain, while Hcn2-DN mRNA injection had no significant effect on nicotine exposure induced malformed brain. Three independent experiments (n = 3) were conducted with N > 50 embryos per treatment group for each of those experiments collected from multiple animals across independent clutches. Data were analyzed with one way ANOVA and Tukey’s post-test and graphed as mean ± SD,***p < 0.001, n.s. non-significant
	Figure 6. HCN2 channels restore the brain size relation to anterior head shape in nicotine-exposed embryos. Morphometrics canonical variate analysis of brain size relation to anterior head shape of stage 45 tadpoles. a) Graphical output, showing confidence ellipses for means, at a 0.95 probability, of shape data from controls and nicotine-exposed (0.1 mg/mL – stage 10–35) tadpoles with or without Hcn2-WT mRNA (0.75 ng/injection) microinjected in both blastomeres at 2-cell stage. Ellipses are colored to correspond with treatment as indicated. N > 26 for each group. Procrustes distances show objective alteration of brain size in relation to head morphology by nicotine exposure in relation to controls as indicated by the red-blue arrow. Hcn2-WT mRNA microinjection along with nicotine exposure moves the brain size—head shape relation closer to controls as indicated by the blue-green arrow. b) Stage 45 control tadpole image illustrating the 7 landmarks considered for this analysis comparing brain size and head shape. Landmarks 2, 3, 4, and 5 show the start of forebrain, transition to midbrain, transition to hindbrain, and end of hindbrain, respectively. Landmarks 1, 6, and 7 indicate the anterior most, and lateral most points of the head shape. c) Canonical variate 1 axis legends (ball and stick diagrams) showing movement of each of the 7 landmarks in mainly anterior-posterior direction. Each ball represents the landmark as indicated by the number and the accompanying stick represents the direction and extent of movement of that particular landmark. d) Canonical variate 2 axis legends (ball and stick diagrams) showing movement of each of the 7 landmarks in mainly lateral direction. Each ball represents the landmark as indicated by the number and the accompanying stick represents the direction and extent of movement of that particular landmark
	Figure 7. HCN2 channels restore associative learning capacity in nicotine-exposed embryos. Associative learning capacity analysis for stage 45–50 tadpoles either left untreated (controls) or exposed to nicotine (0.1 mg/mL – stage 10–35) with or without microinjections of Hcn2-WT mRNA (0.75 ng/injection) in both blastomeres at 2-cell stage as indicated in the illustrations. a) The training regime used consisted of an innate preference test, a training phase, a rest period, and a learning probe. Tadpoles were placed individually in the behavior analysis robot. Motion tracking cameras under each tadpole recorded its position/behavior, and automated software executed a training cycle where animals received a shock when occupying the red half of the arena. Training, rest and testing sessions were repeated a total of six times across the trial. b) Quantification of time spent in the red color during the final testing probe for tadpoles in each treatment group. N > 20 for each experimental group. Error bars indicate ± S.E.M. Data were analyzed using one way ANOVA ***p < 0.001. c Representation of individual tadpole’s associative learning test. N > 20 for each experimental group. Following the training sessions a significant red light aversion was generated in control tadpoles. Majority of nicotine-exposed tadpoles failed to demonstrate red light aversion learning during testing. Nicotine-exposed tadpoles microinjected with Hcn2-WT mRNA showed a restored ability to learn associative red light aversion
	Figure 8. HCN2 channels restore nicotine exposure induced mispatterning of brain markers during neural development. Stage 25 embryos as illustrated with the angle of view marked by the black arrow. Control (untreated/uninjected) embryos (a, e, i, l), embryos exposed to nicotine (0.1 mg/mL – stage 10 onwards) (b, f, j, n), and nicotine-exposed embryos microinjected with Hcn2-WT mRNA (0.75 ng/injection) in both blastomeres at 2-cell stage (c, g, k, o). In situ hybridization for Otx2 (a–d), Xbf1 (e–h), Emx (i–l), and Pax6 (m–p) show that nicotine exposure leads to significantly mispatterned expression (magenta arrows) of Otx2 [46% n = 24] and Xbf1 [35% n = 23], but has little effect on Emx [17%, n = 29] and Pax6 [12%, n = 33] in comparison to controls [10%, n = 28, 4.5%, n = 22, 0%, n = 28, and 3%, n = 29, respectively]. Nicotine-exposed embryos that were also microinjected with Hcn2-WT mRNA showed largely normal expression of Otx2 [4% n = 23], Xbf1 [4% n = 26], Emx [9% n = 23], and Pax6 [6% n = 34] in comparison to controls
	Supplementary Figure 2: Dominant-negative effects of HCN2-AAA in HEK293 cells. Representative HCN current traces recorded from HEK293 cells expressing HCN2-WT (A), HCN2-AAA (B), and empty vector control (C). IHCN (HCN current) was elicited by recording protocol (D) containing stepwise hyperpolarization to -120 mV from holding potential of -40 mV and normalized to membrane capacitance. Summary data for peak IHCN from HEK293 cells expressing HCN2-WT, HCN2-AAA, and vector control is shown in (E). One way ANOVA, *p<0.05 (n=4).
	Supplementary Fig. 3: HCN2 expression levels in Hcn2-WT injected embryos. Quantification of HCN2 immunostained stage ~12 Xenopus embryos which were either left uninjected (controls) or injected with Hcn2-WT mRNA in both blastomeres at two-cell stage. Hcn2-WT injected embryos show a significantly high level of HCN2 channel expression (~3.1 times/310%). Embryos were obtained from multiple animals across independent clutches. Immunostaining was quantified using ImageJ software. Data were analyzed by t-test and plotted as mean ± SD; ***-p<0.001.
	Supplementary Fig. 4: Hcn2 Dominant-negative mRNA has no effect on brain patterning. Quantification of stage 45 tadpoles for major brain morphology phenotypes in uninjected (controls) and Hcn2-Dominant-negative (Hcn2-DN) injected (in both blastomeres at two-cell stage) embryos. No significant increase in incidence of malformed brain was observed in embryos injected with Hcn2-DN. Three independent experiments (n=3) were conducted with N>50 embryos per treatment group for each of those experiments collected from multiple animals across independent clutches. Data were analyzed with t-test and graphed as mean ± SD; n.s. = not significant.

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