Hcn4-deficient zebrafish embryos develop a normal left‒right axis

Background Left‒right (LR) asymmetry is a well-conserved feature of the Vertebrate body plan and is essential for the correct positioning and morphogenesis of visceral organs. While asymmetric Nodal signaling established by the LR organizer (LRO) is widely accepted as a core mechanism driving LR patterning, additional early processes that act during—or even before—the cleavage stage have been proposed to contribute to LR axis specification, including bioelectrical mechanisms mediated by ion fluxes. On the basis of studies in Xenopus laevis , the hyperpolarization-activated cyclic nucleotide-gated channel HCN4 is a candidate regulator of early LR asymmetry induction. Results To assess evolutionary conservation, we investigated whether Hcn4 contributes to LR patterning in zebrafish. We show that both zebrafish hcn4 ohnologs, namely, hcn4 and hcn4-like (hcn4l) , are maternally expressed, with specific mRNAs and proteins detectable from the earliest stages of embryonic development. Despite Hcn4s’ maternal synthesis, multiple independent approaches aimed at interfering with their function, including pharmacological and dominant-negative inhibition, as well as the genetic ablation of both genes, have failed to produce defects in cardiac LR patterning. We also performed pharmacological inhibition of Hcn4 in X. laevis without detecting, under our experimental conditions, evidence of alterations in heart or gut laterality, questioning the real role of HCN4 in the establishment of LR asymmetry. Conclusions Together, these results indicate that loss of Hcn4 function does not affect LR patterning in zebrafish and argue against a conserved role for Hcn4 as a molecular mediator of early bioelectrical contributions to Vertebrate laterality. Our findings do not exclude the existence of an ion flux-based mechanism that acts during early development but reveal that such a process is not mediated by Hcn4 function.