Investigating the Functions of Tinagl1 in Embryonic Development

Abstract

This research project was designed to explore the functions of Tubulointerstitial Nephritis Antigen-Like protein 1 (Tinagl1) in embryonic development. Prior work using morpholino knockdowns in zebrafish suggested that downregulating the tinagl1 gene has profound effects on development, resulting in defects including small eyes, body axis curvature, renal cysts, missing craniofacial cartilages, and reversed heart looping. Several of these defects could result from observed shortening and reduction in the number of motile cilia. However, morpholino knockdown techniques have fallen out of favor in zebrafish research and have been replaced by gene editing methods, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), to completely remove gene function throughout the embryo permanently. This project seeks to further test the function of tinagl1 on development through two independent and current approaches. The first approach will seek to validate or refute the prior morpholino results by knocking out tinagl1 via a 4-guide CRISPR method as it is described by Wu et al. The second approach will examine if overexpression of an engineered tinagl1 mutant mRNA can result in a dominant-negative effect that causes all the previously mentioned defects with the addition of asymmetrical craniofacial defects. These approaches will help establish Zebrafish animal models for studying functional requirements for Tinagl1 and its interactions with signaling pathways.