Investigating the Activation and Asymmetric Regulation of dand5 in Zebrafish

Abstract

Establishing the left-right (L/R) axis in the vertebrate body is a crucial step in proper development, as it governs the asymmetric positioning and formation of the organs and vasculature. The L/R axis is established in the early embryonic stages through the breaking of symmetry at the left right organizer. In the zebrafish organizer, Kupffer’s vesicle (KV), cilia generate directional fluid flow that results in the preferential right-sided expression of dand5. dand5 is a key repressor of the Nodal signaling cascade, which leads to formation of left-side morphology. However, the factors that are responsible for asymmetric dand5 expression remain elusive. Previous studies have demonstrated that the 1.2kb upstream regulatory region (referred to as the URR) and the 3’ untranslated region (UTR) are essential for driving dand5 transcription and asymmetric expression, respectively. Building on these findings, this work first aims to identify the regulatory sequences that are necessary and sufficient for dand5 transcription, using a series of Tol2 transposon-mediated URR deletion assays in the zebrafish embryo. Injections of plasmids containing URR-driven EGFP demonstrate that we have a working model for visualizing dand5 transcriptional activation. Second, this study investigates the cis-regulatory elements responsible for the post-transcriptional asymmetric regulation of dand5 expression, using CRISPR/Cas9 to generate mutants with deletions along the 3’UTR. Following deletions of specific regions within the URR and 3’UTR, dand5 expression was analyzed via immunofluorescence and in situ hybridization. The findings of this study expand our current understanding of asymmetric determination and organogenesis, and provide new insights into the molecular mechanisms that underlie major laterality defects, such as congenital heart diseases.