Key Points

ift140-deficient zebrafish exhibited phenotypes observed in mammals, including skeletal defects and kidney cysts.Beyond ciliogenesis, ift140 also regulated cell polarity and intracellular microtubule stability, potentially contributing to cystogenesis.Microhomology-mediated end joining–induced mosaic ift140 crispants enabled evaluation of cystogenesis in adult fish and F0-based screening for modifiers of kidney cysts.

Background

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.

Methods

We generated zebrafish mutants of IFT140, 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 ift140 crispants, generated through efficient microhomology-mediated end joining–based genome editing, to compare phenotypes with mutants and conduct a pilot genetic modifier screen.

Results

ift140 mutants developed kidney cysts and bone defects similar to those seen in mammalian models. ift140 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 ift140-associated cystogenesis. The ability to detect ift140-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 mtor and ulk1a. We further found that inhibition of mtor and ulk1a reversed both cilia-related and non–cilia-related abnormalities in the kidney.

Conclusions

By establishing a zebrafish model of ift140-associated cystic kidney disease, we recapitulated ift140’s ciliary role and uncovered a nonciliary function in kidney cystogenesis. Importantly, we demonstrated the feasibility of using ift140 mosaic crispants to evaluate cystogenesis in adult fish and to perform F0-based screening for identifying genetic modifiers of kidney cysts.