ift140 -Deficient Zebrafish as a Model for Kidney Cystogenesis and an F0-Based Screen for Genetic Modifiers of Kidney Cysts.

Abstract

Genetic modifiers are believed to play an important role in the onset and severity of polycystic kidney disease, but identifying these modifiers has been challenging due to the lack of effective methodologies. We generated zebrafish mutants of <i>IFT140</i>, a skeletal ciliopathy gene and newly identified autosomal dominant polycystic kidney disease gene, to examine skeletal development and kidney cyst formation in larval and juvenile mutants. In addition, we used <i>ift140</i> crispants, generated through efficient microhomology-mediated end joining–based genome editing, to compare phenotypes with mutants and conduct a pilot genetic modifier screen. <i>ift140</i> mutants developed kidney cysts and bone defects similar to those seen in mammalian models. <i>ift140</i> crispants recapitulated mutant phenotypes while bypassing the early lethality of the mutants, enabling the analysis of kidney cyst formation in adult fish. In addition to cilia defects, we identified nonciliary phenotypes, including disrupted cell polarity and aberrant cytoplasmic microtubule stabilization in kidney epithelial cells, as potential contributors to <i>ift140</i>-associated cystogenesis. The ability to detect <i>ift140</i>-associated kidney cysts with ease allowed us to develop an F0-based genetic screen to identify potential protective modifiers. A pilot screen of 16 genes previously implicated in dysregulated signaling pathways in autosomal dominant polycystic kidney disease revealed both known and novel modifiers, including <i>mtor</i> and <i>ulk1a</i>. We further found that inhibition of <i>mtor</i> and <i>ulk1a</i> reversed both cilia-related and non–cilia-related abnormalities in the kidney. By establishing a zebrafish model of <i>ift140</i>-associated cystic kidney disease, we recapitulated <i>ift140</i>’s ciliary role and uncovered a nonciliary function in kidney cystogenesis. Importantly, we demonstrated the feasibility of using <i>ift140</i> mosaic crispants to evaluate cystogenesis in adult fish and to perform F0-based screening for identifying genetic modifiers of kidney cysts.