Tulp3 quantitative alleles titrate requirements for viability, brain development, and kidney homeostasis but do not suppress Zfp423 mutations in mice.

Abstract

Tubby-like protein 3 (TULP3) regulates receptor trafficking in primary cilia and antagonizes SHH signaling. Tulp3 knockout mice are embryonic lethal with developmental abnormalities in multiple organs, while tissue-specific knockouts and viable missense alleles cause polycystic kidney disease. Human patients with TULP3 mutations present with variable, but often multi-organ fibrotic disease. We previously showed that mouse and human Tulp3 expression is negatively regulated by ZNF423, which is required for SHH sensitivity in some progenitor cell models. The level of TULP3 function required to prevent mutant phenotypes has not been known. Here we report a Tulp3 quantitative allelic series, designed by targeting the polypyrimidine tract 5 to the splice acceptor of a critical exon, that shows distinct dose-response effects on viability, brain overgrowth, weight gain, and cystic kidney disease. We find limited evidence for genetic interaction with Zfp423 null or hypomorphic mutations. Together, these results establish an approach to developing quantitative allelic series by exon exclusion, rank-order dose-sensitivity of Tulp3 phenotypes, and model thresholds for TULP3 function to prevent severe outcomes. Author SummaryTULP3 protein plays critical roles in regulating receptor trafficking and signaling in the primary cilium. Mutations in the TULP3 gene can cause severe, multi-organ disorders in both mice and humans, yet the amount of TULP3 activity needed to avoid these outcomes has been unclear. In this study, we used precise genome editing in mice to create a set of new Tulp3 gene variants that reduce TULP3 expression to varying degrees. This allowed us to test how much TULP3 is required for survival, normal brain and kidney development, and weight regulation. We found that as little as 5% of normal TULP3 levels is enough to avoid lethal birth defects, but still leads to obesity, mild brain overgrowth, and progressive kidney cysts preceded by reductions in cilium frequency and length in situ. The severity of these effects was related to TULP3 protein levels, highlighting a dose-dependent response. We also investigated whether reducing TULP3 levels would suppress brain abnormalities in Zfp423 mutant mice, based on prior evidence of a genetic interaction, but did not find evidence to support this effect. Our work provides a framework for understanding how varying levels of TULP3 affect various organ systems and offers a general strategy for creating quantitative genetic models of human disease.