Investigating the Post-Transcriptional Regulation of Nodal Inhibitor dand5 during Left-Right Patterning in Zebrafish

Abstract

Most vertebrates appear bilaterally symmetric; however, their visceral organs are left-right (L- R) asymmetric in structure and position. Improper L-R asymmetry leads to a spectrum of congenital disorders, including congenital heart disease. Thus, understanding the molecular and cellular mechanisms that determine L-R axis development during early embryogenesis is critical for understanding how defects in this process contribute to structural birth defects. Cilia-mediated fluid flow directing L-R symmetry breaking in the left right organizor (LRO) establishes the L-R axis, resulting in the L-R asymmetric expression of Nodal inhibitor dand5. However, the exact mechanism by which dand5 is regulated in response to flow remains unknown. Previous studies have demonstrated that the 3’ untranslated region (UTR) is essential for driving the asymmetric expression of dand5 in the zebrafish LRO, Kupffer’s Vesicle (KV). My work aims to identify the regulatory domains within the dand5 3’-UTR that are sufficient and/or necessary for regulating its asymmetric expression. Utilizing Tol2 mediated transgenesis and expression of a GFP reporter construct, I identified a 112-nucleotide region of the 3’-UTR to be sufficient for L-R asymmetric accumulation of dand5 RNA. To identify specific regulatory sequence elements that are necessary for dand5 regulation, ~100-200 bp domains within the dand5 3’- UTR were systematically deleted using CRISPR/Cas9-mediated genome editing. Scoring of heart looping in F0 crispants and successive generations at 48 hpf demonstrates increased L-R heart looping defects compared to un-injected embryos for multiple regions of the 3’-UTR. By contributing to the identification of regulatory domains in the dand5 UTR, these studies bring us one step closer to elucidating the regulation of dand5 and, subsequently, the molecular mechanisms downstream of fluid flow that establish dand5 asymmetry during L-R organogenesis. Ultimately, these findings will provide critical insight into the molecular mechanisms that underlie major laterality defects, such as congenital heart disease.