Investigating the patterning role of the hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) during cardiogenesis in Xenopus laevis.
- This thesis focuses on the embryonic role of the hyperpolarization-activated cyclic-nucleotide gated channel 4 (HCN4). HCN4 belongs to the HCN family of proteins, which is composed of 4 isoforms (HCN1-4), and regulates the spontaneous excitability of cells in the heart and nervous system. Specifically, HCN4, the main isoform in the heart, is responsible for the initial, spontaneous depolarization ... read moreof cardiac cells in the sinoatrial node (SAN). As a result, HCN4 acts as the heart’s pacemaker in adults. Interestingly, this protein is first expressed in murine models during gastrulation, well before cardiac contraction begins, suggesting that it has additional roles to regulating heart rate. In contrast to its well characterized adult role, HCN4 functions during embryogenesis are still widely unclear. Here, we investigate its embryonic roles in regard to heart development by taking advantage of the externally-developing frog, Xenopus laevis. We show that genetically altering HCN4 channel functions leads to defects in heart rate, cardiac morphology and positioning, but not differentiation. Additionally, we take advantage of chemical methods to temporally manipulate HCN4 functions and show that HCN4 is most important during cardiac morphogenesis. We also provide evidence that the defects in cardiac morphology are likely due to an overall disruption in the expression pattern of key patterning genes. Finally, we demonstrate that HCN4 function inhibition results in the improper localization of the epicardial marker Tbx18 and prevents clustering of the proepicardium, but does not impair epicardial differentiation. Together our data suggest that HCN4 is a master regulator of cell positional identity during development, but is not involved with organ differentiation. These conclusions further highlight the importance of bioelectric signaling in coordinating organ patterning during embryogenesis.read less
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