Exploring Roles of pkd2 in Left-Right Axis and Mesendoderm Patterning during Zebrafish Development

Abstract

Mutations in the gene pkd2 result in a range of severe health issues including improper organ lateralization, congenital heart defects, and Autosomal Dominant Polycystic Kidney Disease (ADPKD). While pkd2 is known to encode for cation channel polycystin-2 (PC2), the precise function of PC2 in the body is not fully understood. Studies in vertebrate left-right (L-R) patterning have proposed that PC2 acts as a mechanical sensor in the cilium where it responds to fluid flow to initiate L-R asymmetries in Nodal signing in the developing embryo. Interestingly, PC2 is expressed earlier in development as a maternally deposited gene, which suggests that PC2 may play an earlier role in regulating Nodal signaling and mesendoderm patterning. My work has sought to utilize emerging experimental tools to develop a better understanding of the functions of pkd2 in the patterning of both the L-R body axis and the mesendoderm. Utilizing new protocols for homologous recombination in zebrafish, I attempted to endogenously tag pkd2 with GFP. Knock-in of GFP was ultimately unsuccessful, however, despite the incorporation of several modifications shown to increase the efficiency of the technique. While several factors may have impacted the knock-in of GFP, these results suggest that the efficiency of HDR-mediated knock-in may be highly locus-dependent. Additionally, to explore possible functions of pkd2 in the early embryo, I utilized Cas13 protein to degrade maternally deposited and early zygotic pkd2 in early zebrafish embryos. I observed knockdown of pkd2 but did not see an effect on the expression of Nodal-activated mesendodermal patterning genes. Injection of Cas13 protein into zebrafish embryos produced significant toxicity effects, in contrast to published reports, which restricted the utility of the tool for analyses. The successes and failures identified from my adaptation of two novel protocols inform the utilization of these techniques which can be applied across experimental work in zebrafish more broadly.