Pai VP et al. (2017), HCN4 ion channel function is required for early...

XB-ART-53933

Biol Open 2017 Oct 15;610:1445-1457. doi: 10.1242/bio.025957.

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HCN4 ion channel function is required for early events that regulate anatomical left-right patterning in a nodal and lefty asymmetric gene expression-independent manner.

Pai VP , Willocq V , Pitcairn EJ , Lemire JM , Paré JF , Shi NQ , McLaughlin KA , Levin M .

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Laterality is a basic characteristic of all life forms, from single cell organisms to complex plants and animals. For many metazoans, consistent left-right asymmetric patterning is essential for the correct anatomy of internal organs, such as the heart, gut, and brain; disruption of left-right asymmetry patterning leads to an important class of birth defects in human patients. Laterality functions across multiple scales, where early embryonic, subcellular and chiral cytoskeletal events are coupled with asymmetric amplification mechanisms and gene regulatory networks leading to asymmetric physical forces that ultimately result in distinct left and right anatomical organ patterning. Recent studies have suggested the existence of multiple parallel pathways regulating organ asymmetry. Here, we show that an isoform of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of ion channels (hyperpolarization-activated cyclic nucleotide-gated channel 4, HCN4) is important for correct left-right patterning. HCN4 channels are present very early in Xenopus embryos. Blocking HCN channels (Ih currents) with pharmacological inhibitors leads to errors in organ situs. This effect is only seen when HCN4 channels are blocked early (pre-stage 10) and not by a later block (post-stage 10). Injections of HCN4-DN (dominant-negative) mRNA induce left-right defects only when injected in both blastomeres no later than the 2-cell stage. Analysis of key asymmetric genes' expression showed that the sidedness of Nodal, Lefty, and Pitx2 expression is largely unchanged by HCN4 blockade, despite the randomization of subsequent organ situs, although the area of Pitx2 expression was significantly reduced. Together these data identify a novel, developmental role for HCN4 channels and reveal a new Nodal-Lefty-Pitx2 asymmetric gene expression-independent mechanism upstream of organ positioning during embryonic left-right patterning.

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Species referenced: Xenopus
Genes referenced: hcn2 hcn4 lefty nodal nodal1 pitx2
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	Figure 1: HCN4 channel inhibitor, ZD7288 affects left-right organ laterality only upon early exposure of embryos (St1-10). (A) Quantification of stage 45 tadpoles for left-right organ (Heart, Gut and Gallbladder) laterality with or without exposure to 100μM ZD7288 at 22ºC at the indicated stages. A significantly high incidence of heterotaxia was observed in embryos exposed to ZD7288 between stages 1-10 in comparison to controls. Embryos exposed to ZD7288 late (St 10-40) did not show any significant increase in the incidence of left-right organ misplacement. The experiment was conducted in triplicates and data was pooled to run a χ2 analysis, ***p<0.001. (B) Representative images of stage 45 tadpoles: (i) Control tadpole showing rightward coiling gut as indicated by the red dotted lines and red arrow, rightward coiling heart as indicated by green dotted lines and green arrow, and leftward placed gallbladder as indicated by yellow dotted line and yellow arrow, (ii) tadpoles from embryos exposed to ZD7288 (100μM – St 1-10) showing inversion of gut coiling as indicated by the red dotted lines and red arrow, inversion of the heart as indicated by green dotted line and green arrow, and inversion of gallbladder placement as indicated by yellow dotted lines and yellow arrow, (iii) tadpoles from embryos exposed to ZD7288 (100μM – St10-40) showing normal gut coiling as indicated by the red dotted lines and red arrow, normal heart as indicated by green dotted line and green arrow, and normal gallbladder placement as indicated by yellow dotted lines and yellow arrow. Scale bar = 0.25 mm (C) Pie chart showing the incidence of various left-right phenotypes seen in the tadpoles from embryos exposed to ZD7288 (100μM) between stages 1-10.
	Figure 2: Early injection of HCN4-DN affects left-right organ laterality in Xenopus laevis. (A) Quantification of stage 45 tadpoles for left-right organ (Heart, Gut and Gallbladder) laterality with or without microinjecting HCN4-DN mRNA apically (~0.5-1 ng/injection/blastomere) in both blastomeres at two-cell stage as indicated in the illustrations. A significantly high incidence of heterotaxia was observed in only when HCN4-DN mRNA was injected in both blastomeres at 2-cell stage, in comparison to controls. The experiment was conducted in triplicates and data was pooled to run a χ2 analysis, ***p<0.001. (B) Representative ventral images of stage 45 tadpoles: (i) Control tadpole showing rightward coiling gut as indicated by the red dotted lines and red arrow, rightward coiling heart as indicated by green dotted lines and green arrow, and leftward placed gallbladder as indicated by yellow dotted line and yellow arrow, (ii) HCN4-DN mRNA injected (both blastomeres at 2-cell stage) tadpoles showing inversion of gut coiling as indicated by the red dotted lines and red arrow, inversion of the heart as indicated by green dotted line and green arrow, and inversion of gallbladder placement as indicated by yellow dotted lines and yellow arrow, (iii) HCN4- DN mRNA injected (both blastomeres at 2-cell stage) tadpoles showing bilateral gut coiling as indicated by the red dotted lines and red arrow, with normal heart (green dotted lines and green arrow) and gallbladder (yellow dotted line and yellow arrow). Scale bar = 0.25 mm (C) Pie chart showing the incidence of various left-right phenotypes seen in the HCN4-DN mRNA injected (in both blastomeres at 2-cell stage) tadpoles.
	Figure 3: Xenopus laevis embryos express endogenous HCN4 channel during early development. Immunofluorescence analysis of whole Xenopus embryos for HCN4 channel protein at indicated stages of development. (i-vi) no primary antibody controls (vii – xii) HCN4 immunofluorescence. (i, iii, v, vii, ix, xi) Bright field images of immunofluorescent embryos. (ii, iv, vi, viii, x, xii) Fluorescence images of immunofluorescence embryos. Xenopus embryos at the indicated stage of development showed a prominent HCN4 channel protein (n=15).
	Figure 4: Localization of the asymmetric gene Xnr-1 (nodal) is not affected by HCN4- DN and ZD7288. (A) Representative images of approximately stage 21 embryos assayed for Xnr-1 (nodal) expression by in situ hybridization and quantification of area of nodal expression. Red dotted line is midline and L representing left-side, R representing right-side, H representing head and T representing tail of embryos. Scale bar = 0.25 mm. (i) is no probe (negative) untreated control, (ii) Control embryos with Xnr-1 signal – red arrow, (iii) ZD7288 treated (from stage 1-10) embryos with Xnr-1 signal – red arrow, (iv) HCN4-DN mRNA injected (in both blastomeres at 2-cell stage) embryos with Xnr-1 signal – red arrows, and (v) Quantification of area of Nodal expression in embryos showed no significant change in the area of Nodal expression in HCN4-DN mRNA injected and ZD7288 treated embryos. N>10, Data was analyzed by one-way ANOVA, n.s. – Non-significant. (B) Representative images of approximately stage 23 embryos assayed for Lefty expression by in situ hybridization and quantification of area of lefty expression. Red dotted line is midline and L representing left-side, R representing right-side, H representing head and T representing tail of embryos. Scale bar = 0.25 mm. (i) is no probe (negative) untreated control, (ii) Control embryos with Lefty signal – yellow arrow, (iii) ZD7288 treated (from stage 1-10) embryos with Lefty signal – yellow arrow, (iv) HCN4-DN mRNA injected (in both blastomeres at 2-cell stage) embryos with Lefty signal – yellow arrows, and (v) Quantification of area of Lefty expression in embryos showed no significant change in the area of Lefty expression in HCN4-DN mRNA injected and ZD7288 treated embryos. N=10, Data was analyzed by one-way ANOVA, n.s. – Non-significant.
	Figure 5: Pitx2 expression is affected by HCN4-DN and ZD7288. (A) Representative images of approximately stage 28 embryos assayed for Pitx2 expression by in situ hybridization. Left orientation of the embryo is indicated at the bottom of the image. Red line indicates the anterior-posterior spread of the Pitx2 expression and yellow dotted line indicates the area of the Pitx2 expression (i) is no probe (negative) untreated control (ii) Control embryos with Pitx2 signal – yellow arrow. (iii) embryos injected with HCN4-DN mRNA in both blastomeres at 2-cell stage with Pitx2 expression - yellow arrow, (iv) ZD7288 treated (100μM stage1-10) embryo with Pitx2 expression - yellow arrow. Scale bar = 0.25 mm. (B) Quantification of anterior-posterior spread of Pitx2 expression (as indicated by red lines in A) in embryos showed a significant reduction in the spread of Pitx2 expression in HCN4- DN mRNA injected and ZD7288 treated embryos. N=20, Data was analyzed by oneway ANOVA. *p<0.001. (C) Quantification of area of Pitx2 expression (as indicated by yellow dotted lines in A) in embryos showed a significant reduction in the area of Pitx2 expression in HCN4-DN mRNA injected and ZD7288 treated embryos. N>25 Data was analyzed by one-way ANOVA. p<0.001, *p<0.01.
	Figure 6: Model for HCN4 function in establishing laterality. The developmental timeline along the left illustrates early cleavage stages to post-gastrulation asymmetric gene expression of the Nodal-Lefty-Pitx2 cascade leading to final organ situs. Previously established important laterality events are outlined adjacent to the developmental time line and portray very early laterality events of cytoskeletal rearrangement and physiological amplification, as well as later events such as gastrulation-stage ciliary flows, all funnel into the canonical Nodal- Lefty-Pitx2 gene regulatory network to bring about invariant asymmetric organ situs. HCN4 action is required during early clevage stages and can largely bypass the Nodal- Lefty asymmetric gene expression cascade to affect organ situs. HCN4 mediated Nodal-Lefty asymmetric gene expression-independent effect could be due to directly acting on downstream factors of the Nodal-Lefty pathway or through a non-canonical pathway. Players in this HCN4 mediated laterality patterning remain to be discovered.
	Figure S1: HCN4 channel inhibitor, ivabradine affects left-right organ laterality only upon early exposure of embryos (st. 1-10). (A) Quantification of stage 45 tadpoles for left-right organ (Heart, Gut and Gallbladder) laterality with or without exposure to 400μM ivabradine at 22 ºC at the indicated stages. A significantly high incidence of heterotaxia was observed in embryos exposed to ivabradine between stages 1 -10 in comparison to controls. Embryos exposed to ivabradine late (st. 10-40) did not show any significa nt increase in the incidence of leftright organ misplacement. The experiment was conducted in triplicates and data was pooled to run a χ2 analysis, ***p<0.001.
	Figure S2: HCN4-DN mRNA depolarizes membrane voltage in Xenopus embryos in vivo. Fluorescence intensity measurements of CC2 -DMPE:DiBAC stained embryos extrapolated to estimate approximate membrane voltage values using electrophysiology measurements. N=7 for each experimental group. Neural measurements (red square) of controls are significantly hyperpolarized versus non-neural measurements (blue squares). Embryos injected with HCN4-DN mRNA in both blastomeres at 2 -cell stage show depolarization in the neural measurements. Data are represented as mean + S.E.M., ***p<0.001. A one -way ANOVA analysis was performed.
	Figure S3: HCN2-DN mRNA injections in both blastomeres at 2-cell stage do not result in left-right phenotypes. Quantification of stage 45 tadpoles for left- right organ (Heart, Gut and Gallbladder) laterality with or without microinjecting HCN2-DN mRNA (~0.7 ng/injection) in both blastomeres at two -cell stage as indicated in the illustrations. No significant incidence of heterotaxia was observed when HCN2-DN mRNA was injected in both blastomeres at 2 - cell stage, in comparison to controls. The experiment was conducted in triplicates and data was pooled to run a χ2 analysis.
	Fig. 1. HCN4 channel inhibitor ZD7288 affects left-right organ laterality only upon early exposure of embryos (St 1-10). (A) Quantification of stage 45 tadpoles for left-right organ (heart, gut and gallbladder) laterality with or without exposure to 100 µM ZD7288 at 22°C at the indicated stages. A significantly high incidence of heterotaxia was observed in embryos exposed to ZD7288 between stages 1-10 in comparison to controls. Embryos exposed to ZD7288 late (St 10-40) did not show any significant increase in the incidence of left-right organ misplacement. The experiment was conducted in triplicates and data was pooled to run a χ2 analysis, ***P<0.001. (B) Representative images of stage 45 tadpoles: (i) Control tadpole showing rightward coiling gut as indicated by the red dotted lines and red arrow, rightward coiling heart as indicated by green dotted lines and green arrow, and leftward placed gallbladder as indicated by yellow dotted line and yellow arrow, (ii) tadpoles from embryos exposed to ZD7288 (100 µM – St 1-10) showing inversion of gut coiling as indicated by the red dotted lines and red arrow, inversion of the heart as indicated by green dotted line and green arrow, and inversion of gallbladder placement as indicated by yellow dotted lines and yellow arrow, (iii) tadpoles from embryos exposed to ZD7288 (100 µM – St 10-40) showing normal gut coiling as indicated by the red dotted lines and red arrow, normal heart as indicated by green dotted line and green arrow, and normal gallbladder placement as indicated by yellow dotted lines and yellow arrow. Scale bar: 0.25 mm. (C) Pie chart showing the incidence of various left-right phenotypes seen in the tadpoles from embryos exposed to ZD7288 (100 µM) between stages 1-10.
	Fig. 2. Early injection of HCN4-DN affects left-right organ laterality in Xenopus laevis. (A) Quantification of stage 45 tadpoles for left-right organ (heart, gut and gallbladder) laterality with or without microinjecting HCN4-DN mRNA apically (∼0.5-1 ng/injection/blastomere) in both blastomeres at 2-cell stage as indicated in the illustrations. A significantly high incidence of heterotaxia was observed in only when HCN4-DN mRNA was injected in both blastomeres at 2-cell stage, in comparison to controls. The experiment was conducted in triplicate and data was pooled to run a χ2 analysis, ***P<0.001. (B) Representative ventral images of stage 45 tadpoles: (i) Control tadpole showing rightward coiling gut as indicated by the red dotted lines and red arrow, rightward coiling heart as indicated by green dotted lines and green arrow, and leftward placed gallbladder as indicated by yellow dotted line and yellow arrow, (ii) HCN4-DN mRNA injected (both blastomeres at 2-cell stage) tadpoles showing inversion of gut coiling as indicated by the red dotted lines and red arrow, inversion of the heart as indicated by green dotted line and green arrow, and inversion of gallbladder placement as indicated by yellow dotted lines and yellow arrow, (iii) HCN4-DN mRNA injected (both blastomeres at 2-cell stage) tadpoles showing bilateral gut coiling as indicated by the red dotted lines and red arrow, with normal heart (green dotted lines and green arrow) and gallbladder (yellow dotted line and yellow arrow). Scale bar: 0.25 mm. (C) Pie chart showing the incidence of various left-right phenotypes seen in the HCN4-DN mRNA injected (in both blastomeres at 2-cell stage) tadpoles.
	Fig. 3. Xenopus laevis embryos express endogenous HCN4 channel during early development. Immunofluorescence analysis of whole Xenopus embryos for HCN4 channel protein at indicated stages of development. (i-vi) No primary antibody controls, (vii – xii) HCN4 immunofluorescence, (i, iii, v, vii, ix, xi) bright field images of immunofluorescent embryos, (ii, iv, vi, viii, x, xii) fluorescence images of immunofluorescence embryos. Xenopus embryos at the indicated stage of development showed a prominent HCN4 channel protein (n=15).
	Fig. 4. Localization of the asymmetric gene Xnr-1 (nodal) is not affected by HCN4-DN and ZD7288. (A) Representative images of approximately stage 21 embryos assayed for Xnr-1 (nodal) expression by in situ hybridization and quantification of area of nodal expression. Red dotted line is midline and L representing left-side, R representing right-side, H representing head and T representing tail of embryos. Scale bar: 0.25 mm. (i) No probe (negative) untreated control, (ii) control embryos with Xnr-1 signal – red arrow, (iii) ZD7288-treated (from stage 1-10) embryos with Xnr-1 signal – red arrow, (iv) HCN4-DN mRNA injected (in both blastomeres at 2-cell stage) embryos with Xnr-1 signal – red arrows, and (v) quantification of area of Nodal expression in embryos showed no significant change in the area of Nodal expression in HCN4-DN mRNA-injected and ZD7288-treated embryos. N>10; data was analyzed by one-way ANOVA; n.s., non-significant. (B) Representative images of approximately stage 23 embryos assayed for Lefty expression by in situ hybridization and quantification of area of lefty expression. Red dotted line is midline and L representing left-side, R representing right-side, H representing head and T representing tail of embryos. Scale bar: 0.25 mm. (i) No probe (negative) untreated control, (ii) control embryos with Lefty signal – yellow arrow, (iii) ZD7288-treated (from stage 1-10) embryos with Lefty signal – yellow arrow, (iv) HCN4-DN mRNA-injected (in both blastomeres at 2-cell stage) embryos with Lefty signal – yellow arrows, and (v) quantification of area of Lefty expression in embryos showed no significant change in the area of Lefty expression in HCN4-DN mRNA-injected and ZD7288-treated embryos. N=10; data was analyzed by one-way ANOVA; n.s., non-significant.
	Fig. 5. Pitx2 expression is affected by HCN4-DN and ZD7288. (A) Representative images of approximately stage 28 embryos assayed for Pitx2 expression by in situ hybridization. Left orientation of the embryo is indicated at the bottom of the image. Red line indicates the anterior-posterior spread of the Pitx2 expression and yellow dotted line indicates the area of the Pitx2 expression. (i) No probe (negative) untreated control, (ii) control embryos with Pitx2 signal – yellow arrow, (iii) embryos injected with HCN4-DN mRNA in both blastomeres at 2-cell stage with Pitx2 expression - yellow arrow, (iv) ZD7288-treated (100 µM stage1-10) embryo with Pitx2 expression - yellow arrow. Scale bar: 0.25 mm. (B) Quantification of anterior-posterior spread of Pitx2 expression (as indicated by red lines in A) in embryos showed a significant reduction in the spread of Pitx2 expression in HCN4-DN mRNA-injected and ZD7288-treated embryos. N=20; data was analyzed by one-way ANOVA; *P<0.001. (C) Quantification of area of Pitx2 expression (as indicated by yellow dotted lines in A) in embryos showed a significant reduction in the area of Pitx2 expression in HCN4-DN mRNA-injected and ZD7288-treated embryos. N>25; data was analyzed by one-way ANOVA; P<0.001, *P<0.01.
	Fig. 6. Model for HCN4 function in establishing laterality. The developmental timeline along the left illustrates early cleavage stages to post-gastrulation asymmetric gene expression of the Nodal-Lefty-Pitx2 cascade leading to final organ situs. Previously established important laterality events are outlined adjacent to the developmental time line and portray very early laterality events of cytoskeletal rearrangement and physiological amplification, as well as later events such as gastrulation-stage ciliary flows, all funnel into the canonical Nodal-Lefty-Pitx2 gene regulatory network to bring about invariant asymmetric organ situs. HCN4 action is required during early cleavage stages and can largely bypass the Nodal-Lefty asymmetric gene expression cascade to affect organ situs. HCN4-mediated Nodal-Lefty asymmetric gene expression-independent effect could be due to directly acting on downstream factors of the Nodal-Lefty pathway or through a non-canonical pathway. Players in this HCN4-mediated laterality patterning remain to be discovered.

References [+] :

Adams, Endogenous voltage gradients as mediators of cell-cell communication: strategies for investigating bioelectrical signals during pattern formation. 2013, Pubmed