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Abstract

ANKS3 is an Ank and SAM domain containing protein. It interacts with ciliopathy associated NPHP proteins as well as ANKS6 and the RNA binding protein BICC1 which cause polycystic kidney disease (PKD) when mutated in rodents. The ANKS3 SAM domain is capable of assembling homopolymers and forms large complexes with BICC1. This ANKS3 homopolymerisation is prevented by ANKS6-ANKS3-SAM domain binding. The PKD causing Anks6p.R823W mutation prevents ANKS6 from binding to ANKS3 which might result in increased ANKS3-SAM domain homopolymerisation. The function of ANKS3 in mammals is quite unresolved. This thesis aimed to clarify the role of Anks3 in rats and the pathophysiological role of ANKS3-SAM domain homopolymerisation in PKD. To this end, we generated novel rat models, using CRISPR/Cas9, carrying either an Anks3 knockout (Anks3KO), an ANKS3-SAM domain deletion (Anks3ΔSAM) or a missense mutation in the SAM domain (Anks3KI) which prevents ANKS3-SAM domain homopolymerisation but not ANKS3-ANKS6 binding. Furthermore, we crossed the Anks3KI rat into the Anks6p.R823W PKD rat (TGRAnks6) to study the effect of defective ANKS3-SAM domain homopolymerisation in PKD (TGRAnks6-Anks3KI/KI). We provide the following crucial results: 1. The spatial expression pattern of ANKS3 and ANKS6 in the kidney is tightly regulated during development and is disturbed by the Anks6p.R823W mutation in PKD rats. The Anks6p.R823W mutation decreases aquaporin 2 (Aqp2) expression and urinary concentration ability while the defective ANKS3 homopolymerisation in the Anks3KI/KI rats increases them, supporting a role of ANKS3 homopolymerisation in water regulation in the kidney. 2. Defective ANKS3 polymerisation in TGRAnks6-Anks3KI/KI rats, retards cyst growth and reverses most pathways altered in TGRAnks6 PKD rats, including the key signalling pathways Hippo, Wnt and cAMP as well as metabolic pathways. In addition, all DNA replication and repair pathways were upregulated in TGRAnks6-Anks3KI/KI rats vs. TGRAnks6 and vs. wildtype rats. 3. We provide evidence that the ANKS3-SAM domain, but not ANKS3-SAM domain homopolymerisation, is required for proper morphogenesis in embryos. Both, Anks3 knockout and ANKS3-SAM domain deletion, results in an embryonically lethal, ciliopathic phenotype including disturbances in organ morphogenesis and laterality defects. Cilia formation did not appear to be disturbed. Expression profiling in Anks3KO/KO embryos revealed a significant downregulation of DNA replication and repair pathways vs. wildtype embryos, indicating a significant role of ANKS3 in DNA damage response and repair, which becomes crucial during periods of high proliferative stress, including embryogenesis and PKD. Unlike Anks6, the Anks3 mutations did not cause a PKD phenotype. Altogether, in the course of this thesis we provided three novel mutated Anks3 rat models which, for the first time, allow the in vivo study of Anks3 function in mammals and will contribute to further elucidate the molecular pathways of PKD and other ciliopathies, and their interactions.