Pitcairn E et al. (2017), Coordinating heart morphogenesis: A novel role ...

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Commun Integr Biol 2017 May 10;103:e1309488. doi: 10.1080/19420889.2017.1309488.

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Coordinating heart morphogenesis: A novel role for hyperpolarization-activated cyclic nucleotide-gated (HCN) channels during cardiogenesis in Xenopus laevis.

Pitcairn E , Harris H , Epiney J , Pai VP , Lemire JM , Ye B , Shi NQ , Levin M , McLaughlin KA .

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Hyperpolarization-activated cyclic-nucleotide gated channel (HCN) proteins are important regulators of both neuronal and cardiac excitability. Among the 4 HCN isoforms, HCN4 is known as a pacemaker channel, because it helps control the periodicity of contractions in vertebrate hearts. Although the physiological role of HCN4 channel has been studied in adult mammalian hearts, an earlier role during embryogenesis has not been clearly established. Here, we probe the embryonic roles of HCN4 channels, providing the first characterization of the expression profile of any of the HCN isoforms during Xenopus laevis development and investigate the consequences of altering HCN4 function on embryonic pattern formation. We demonstrate that both overexpression of HCN4 and injection of dominant-negative HCN4 mRNA during early embryogenesis results in improper expression of key patterning genes and severely malformed hearts. Our results suggest that HCN4 serves to coordinate morphogenetic control factors that provide positional information during heart morphogenesis in Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 hcn2 hcn4 lefty1 nkx2-5 nodal nodal1 otx2 pitx2 shh tbx18 tbxt tnnt2
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	Figure 1. HCN4 expression during embryogenesis (A) In situ hybridization analysis reveals HCN4 is expressed in the developing head and along the dorsal midline at the completion of neurulation embryos (NF stage 21 lateral and frontal views). At early tailbud stages, HCN4 expression is observed primarily in the craniofacial region, in 2 stripes along the neural tube, and in the developing somites. This expression pattern is maintained as development progresses (NF stage 25, lateral and anterior/ventral views). At NF stage 26, HCN4 mRNA begins to be expressed on the left side of the developing heart field (arrow; lateral and ventral views). Expression becomes bilateral beginning at NF stage 27/28 (arrows; lateral and ventral views) but remains more highly expressed on the left side of the body. Scale bars = 200Î¼m. (B) As cardiogenesis progresses during tadpole stages (NF stages 29â€“39, lateral and ventral views, arrows denote heart), HCN4 expression is diffusely present in the developing myocardium (outlined in yellow dashed lines/circles), but the highest expression is in the septum transversum where other proepicardial genes are expressed (Tbx18, NF stage 34). Scale bars = 200Î¼m. (C,D) Histological sections confirm HCN4 expression in the cardiac mesoderm at tailbud stages, and in the myocardium and proepicardial region in tadpole stages. HCN4 expression can also be observed in the notochord and somites. Red lines in schematic diagrams denote the plane of section. Sections = 10Î¼m, scale bars = 100Î¼m. (E) Immunohistochemistry shows HCN4 protein is expressed at the completion of cardiac looping (NF stage 35) and is expressed throughout the entire ventricle of mature hearts (NF stage 46). Another important cardiac isoform, HCN2, is also found throughout the ventricle of mature hearts, similar to what has been described in mammalian systems. Transverse sections = 15Â Âµm, scale bars = 100Â Âµm. Schematic images are modified from Xenbase.20
	Figure 2. HCN4 dominant-negative effect test in HEK293 cells. (A) Sequence alignment of mouse HCN channels and other K+ channel examples that have a signature cation selectivity âGYGâ motif at the channel pore region. (B) IHCN currents were elicited by a stepwise hyperpolarization to â120Â mV from a holding potential of â40Â mV; and then normalized to membrane capacitance. Representative IHCN4 current traces recorded in HEK293 cells expressing HCN4-WT, HCN4-DN(AAA) mutant, both HCN4-DN(AAA)/HCN4-WT, or a vector control are shown. Transfection with HCN4-WT produced robust IHCN4 currents, whereas transfection with HCN4-DN(AAA) did not produce any current greater than the vector control. When HCN4-WT and HCN4-DN(AAA) were co-transfected, IHCN4 was abolished. (C) Summary data for the peak IHCN4 currents recorded from HEK293 cells expressing WT-HCN4, HCN4-AAA mutant, HCN4-AAA/WT-HCN4 or vector control, confirming the ability of HCN4-DN(AAA) to act as a dominant-negative construct. n = 4â5, *p < 0.05.
	Figure 3. Overexpression and injection of dominant-negative HCN4 mRNA induces aberrant heart morphology. (A) Mature NF stage 46 tadpole hearts were visualized by immunohistochemistry using a cardiac troponin-T antibody to detect mature cardiac muscle. In control tadpoles, the ventricle (V) and outflow tract (OFT) are readily visible with the atria located directly behind the ventricle (i). Altering HCN4 expression induced a variety of morphological defects including hearts that were twisted into the dorso-ventral (D-V) body axis (ii), hearts rotated in the same plane of the D-V axis (iii), unlooped hearts (iv), and double ventricle phenotypes (v). In twisted hearts (ii) the atria are no longer positioned behind the ventricle and can now be seen (At = atria). (B) Both injection conditions of the HCN4-DN(AAA) construct induced significantly higher percentages of morphological defects compared with uninjected tadpoles, whereas overexpression of HCN4 did not cause significant occurrences of malformed hearts Ï‡2 = 58.174, df = 4, * p < 0.05; n = 75 embryos per condition across 3 biological replicates.
	Figure 4. Inhibiting HCN4 function in different locations disrupts heart morphology. (A) Uninjected tadpoles have a clear chest cavity at NF stage 43/44, making the heart easily visible laterally (arrowhead, i). Hearts have normal ventricle and outflow tract morphology as seen via IHC for cardiac troponin-T antibody (ventral view, ii). The left eye is circled (white dashed line) in all images as a landmark. (B-D) HCN4-DN(AAA) injected embryos display a variety of heart defects, irrespective of the location of the lineage label membrane-bound RFP. Examples of tadpoles with mem-RFP located in the: heart (arrowhead, B, i), posterior flank (C, i), and dorsal region (D, i) of HCN4-DN(AAA) injected animals. White arrowheads denote position of heart. Ventral views of malformed heart phenotypes: symmetric midline hearts (B, ii), unlooped hearts (C, ii), and twisted hearts (D, ii). n = 18â€“31embryos per injection condition. Scale bars = 200Â Âµm.
	Figure 5. Injection of HCN4-DN(AAA) results in altered cardiac function. (A) Injecting wild-type HCN4 (HCN4-OE) into 2-cell embryos did not significantly alter heart rate. (B) Inhibiting HCN4 ion channel function (HCN4-DN[AAA]) resulted in significantly higher heart rates in all injection conditions (Welch's test, *p < 0.05). Dots represent outlier individuals who were greater or less than 1.5Â times the upper and lower quartiles respectively. n = 30 embryos per condition across 3 biological replicates.
	Figure 6. HCN4 helps coordinate the distribution of Xenopus nodal-related 1 (Xnr-1) expression. (A) During normal development, Xnr-1 is expressed exclusively in the left lateral plate mesoderm following the completion of neurulation as seen via in situ hybridization (NF stage 21, i). Both overexpression and injection of HCN4-DN(AAA) resulted in bilateral expression of Xnr-1, with the secondary Xnr-1 field ranging from a small partial field containing few cells (yellow arrow, iii) to an equivalent right-sided field (white arrow, ii). In addition to generating a second field of Xnr-1, punctate dots of expression were observed along the fused neural tube (iii, red arrow) and flanks of embryos. These ectopic dots were found on both sides of embryos regardless of the presence of endogenous Xnr-1 expression. Dorsal views, anterior at top. (B) While abnormal (bilateral, punctate, and right-sided reversed) expression was observed in all injection conditions, only HCN4-DN(AAA) injection caused a significant increase in abnormal phenotypes compared with uninjected embryos (Ï‡2 = 31.0207, df = 4, *p < 0.05). n = 73â€“84 per condition across 3 biological replicates. OE = HCN4-OE, DN = HCN4-DN(AAA). Scale bars = 200Â Î¼m.
	Figure 7. Altering normal HCN4 expression results in aberrant Lefty expression. (A) As visualized via in situ hybridization analysis, Lefty is normally expressed unilaterally in the left lateral plate mesoderm in NF stage 27 embryos (i). Altering HCN4 expression induced both full or partial bilateral and abnormal Lefty expression but did not result in a complete reversal of expression (solely right-sided). One of the most frequently observed bilateral phenotypes was a partial secondary field on the right side of embryos with the midline maintained by an absence of Lefty expression (ii). In the other commonly seen phenotype, Lefty transcripts were expressed across the anterior ventral surface of animals in an undefined shape with no visible midline gap (iii). Images are ventral views with anterior at top. (B) Overexpressing HCN4 and HCN4-DN(AAA) injection led to significant percentages of abnormal embryos compared with uninjected embryos (Ï‡2 = 19.0086, df = 4, *p < 0.05). There was no statistical difference between overexpression and dominant-negative phenotypes. n = 56â€“71 embryos per condition across 3 biological replicatesOE = HCN4-OE, DN = HCN4-DN(AAA). Scale bars = 200Â Î¼m.
	Figure 8. Altering HCN4 expression leads to improper distribution of the homeodomain transcription factor Pitx2. (A) In situ hybridization of representative embryos displaying normal and examples of the most commonly observed Pitx2 expression defects. Uninjected embryos have asymmetric, left-sided expression (i lateral view; iv ventral view). Overexpression and injection of the dominant-negative construct resulted in a variety of molecular phenotypes including expansion or reduction of the endogenous field (ii, iii) and bilateral Pitx2 expression (v). Anterior at top of images. (B) All manipulations caused significantly higher percentages of abnormal Pitx2 expression compared with uninjected embryos (Ï‡2 = 64.71, df = 4, *p < 0.05). There was no statistical difference between overexpression and dominant-negative phenotypes. n = 54â€“86 embryos per condition across 3 biological replicates. OE = HCN4-OE, DN = HCN4-DN(AAA). Scale bars = 200Â Î¼m.
	Figure 9. BMP-4 expression is reduced and incorrectly localized in response to altering HCN4 expression. (A) At NF stage 27 BMP-4 is expressed in 2 bilateral patches around the developing heart field (i). Altering HCN4 caused a loss of definition to the fields (ii) as well as an overall reduction in the size of the fields and intensity of expression (iii). (B) BMP-4 remains bilaterally expressed during heart looping (NF stage 32) with an additional asymmetric area of expression in the myocardium (yellow arrow, i). This left-sided myocardial expression helps to direct and orient heart looping. Overexpression of HCN4 and expression of HCN4-DN(AAA) not only disrupted the lateral BMP-4 fields of expression (ii), it also caused both a randomization and loss of myocardial expression (white arrow, iii). (C) By the end of looping at NF stage 35, BMP-4 is evenly expressed in the myocardium and retains its bilateral expression in the surrounding tissues (i). Similar to stage 27 and 32, disruption of HCN4 led to both a reduction in expression as well as a loss of field definition (ii, iii). Injected embryos frequently displayed malformed hearts who did not complete looping properly (yellow vs. white arrows). All abnormal images are from HCN4-DN(AAA) injected embryos but similar phenotypes were observed with HCN4-OE embryos. (A-C) Images are ventral views with anterior at top. (D) All stages and injection conditions induced significantly higher percentages of abnormal phenotypes compared with controls (Stage 27: Ï‡2 = 32.4768, df = 4; Stage 32: Ï‡2 = 82.2366, df = 4; Stage 35: Ï‡2 = 36.0791, df = 4; All: *p < 0.05). There was no statistical difference between overexpression and dominant-negative phenotypes. n = 58â€“79 embryos per condition across 3 biological replicates. OE = HCN4-OE, DN = HCN4-DN(AAA). Scale bars = 200Â Î¼m.
	Supplemental Figure 1. HCN2 is not expressed in the developing heart during early stages of cardiogenesis. ISH for HCN2 expression on selected stages. Unlike HCN4 that is robustly expressed during early stages of cardiogenesis, HCN2 it is not present in the heart during early cardiac development. However, HCN2 is expressed in the neural tube at these stages (insert, stage 29). Images are ventral view with anterior at top.DScale bars=200 Âµm.
	Supplemental Figure 2. Altering HCN4 expression does not disrupt general axis patterning, germ layer specification, or myocardial differentiation. (A) The expression pattern of Xbra, Otx2, and Nkx2.5 in HCN4-DN(AAA) injected embryos was not different from stage-matched, sibling controls as seen by ISH. Representative images are from uninjected sibling controls and HCN4-DN(AAA) one of 2-cell (left) injected embryos. n=30, N=1 for Xbra (vegetal view) and Otx2 (anterior view, dorsal at top). n=25, N=1 for Nkx2.5 (ventral view anterior at top). dorsal lip of blastopore denoted by a yellow dashed line. (B) Shh expression is also not disrupted with injection of HCN4-OE or HCN4-DN(AAA). At gastrulation, Shh is whisker-shaped above the dorsal lip of the blastopore (black line; vegetal view). As development progresses it is found along the midline, in the notochord and somites. n=30, N=1 for stage 10.5 and stage 21 (dorsal view, anterior at top). n=50, N=1 for stage 29/30 (lateral view, anterior at top). Scale bars=200 Âµm.
	hcn4 ( hyperpolarization activated cyclic nucleotide gated potassium channel 4 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, dorsal view, anterior left.
	hcn4 ( hyperpolarization activated cyclic nucleotide gated potassium channel 4 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
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