mtor Haploinsufficiency Ameliorates Renal Cysts and Cilia Abnormality in Adult Zebrafish tmem67 Mutants

Significance Statement Zebrafish embryos are well recognized for their value in studying polycystic kidney disease, but they cannot fully recapitulate the pathogenesis of the disease. The establishment of tmem67 e3/e3 mutants provides the first adult zebrafish model for cystogenesis. The model exhibits progressive renal cysts and enables the discernment of compensational effects from pathogenic events. Development of a tissue-clearing, whole-mount imaging protocol allows the analysis of renal cysts and cilia abnormality at a single-nephron resolution. Evidence also indicates potential benefit of mammalian target of rapamycin inhibition in tmem67 -based cystogenesis. Combining powerful genetics and phenotyping tools, this novel adult zebrafish model shall facilitate mechanistic studies and therapeutic development for cystic diseases. Background Although zebrafish embryos have been used to study ciliogenesis and model polycystic kidney disease (PKD), adult zebrafish remain unexplored. Methods Transcription activator-like effector nucleases (TALEN) technology was used to generate mutant for tmem67 , the homolog of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Classic 2D and optical-clearing 3D imaging of an isolated adult zebrafish kidney were used to examine cystic and ciliary phenotypes. A hypomorphic mtor strain or rapamycin was used to inhibit mTOR activity. Results Adult tmem67 zebrafish developed progressive mesonephric cysts that share conserved features of mammalian cystogenesis, including a switch of cyst origin with age and an increase in proliferation of cyst-lining epithelial cells. The mutants had shorter and fewer distal single cilia and greater numbers of multiciliated cells (MCCs). Absence of a single cilium preceded cystogenesis, and expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of renal cysts in young adult zebrafish, suggesting a primary defect and an adaptive action, respectively. Finally, the mutants exhibited hyperactive mTOR signaling. mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants. Conclusions Adult zebrafish tmem67 mutants offer a new vertebrate model for renal cystic diseases, in which cilia morphology can be analyzed at a single-nephron resolution and mTOR inhibition proves to be a candidate therapeutic strategy.