PROJECT SUMMARY Polycystic kidney disease (PKD) is a chronic, progressive disease in which the kidneys accumulate fluid filled sacs or cysts that lead to loss of kidney function, culminating in end stage renal disease and even death. PKD affects an estimated 140,000 Americans and has an associated economic burden in excess of $7 billion annually. An estimated 95% of PKD cases are caused by mutations in PKD1 or PKD2, which encode the cilia- localized polycystin proteins, PC1 and PC2. In PKD mouse models, loss of renal primary cilia also results in cyst formation. PC1 and PC2 form a complex that localizes to the primary cilia where it inhibits a cilia- dependent cyst activation (CDCA) pathway, yet the driving force behind this CDCA pathway remains unknown. Recent mouse models indicate that the cilia-associated protein TULP3 traffics both the unknown driver(s) of the CDCA and the inhibitor of the CDCA (PC1/2) to the primary cilium. ARL13B is a ciliary GTPase, and its ciliary localization requires TULP3 in the kidney (but not other tissues examined), making it an excellent candidate as a regulator of the CDCA. In the clinic, mutations in ARL13B underlie a debilitating ciliopathy known as Joubert Syndrome, which also presents with renal cysts. Arl13b-null mice are embryonic lethal, while kidney-specific loss of Arl13b results in renal cysts and a loss of cilia, making it impossible to determine whether loss of ARL13B or loss of cilia resulted in cystogenesis. Our lab recently used CRISPR to generate a cilia-excluded Arl13b mutant mouse, V358A, which develops normally. Cilia-excluded Arl13bV358A mice exhibit renal cysts, suggesting ARL13B plays a critical ciliary role in regulating renal cystogenesis. Delineating ciliary signaling pathways in vivo is exceptionally challenging with current genetic models as loss of cilia ablates all ciliary signaling, while genetic deletion of ciliary proteins removes both ciliary and cellular pools. To address these gaps in knowledge and provide insight into how ciliary ARL13B regulates renal cystogenesis, I will use our cilia-excluded Arl13bV358A mouse model. Because the kidney provides a unique context for ciliary signaling, especially ARL13B signaling, the kidney is the only tissue in which to gain a better understanding of ARL13B’s ciliary signaling in vivo. The long-term goal of this proposal is to understand ciliary ARL13B’s regulation of kidney cystogenesis. I will test my central hypothesis that ciliary ARL13B plays a critical role in regulating kidney cystogenesis through two complementary aims. In Aim 1, I will define the ciliary and cellular contributions of ARL13B to kidney cystogenesis through extensive morphological and molecular phenotyping as well as candidate and discovery-based approaches. In Aim 2, I will determine whether ciliary ARL13B regulates the CDCA pathway. Successful completion of these aims will (1) reveal ARL13B’s ciliary function in kidney cystogenesis, (2) identify downstream molecular r...