Ciliary assembly, maintenance and disassembly are complex processes required for establishing cilia as dynamic and unique signalling organelles. In WP1, ESR1 studied a key intraflagellar transport (IFT) subunit and determined a novel high-resolution crystal structure of its C-terminal domain, providing novel insights into the structure and function of the ciliogenic IFT machinery. ESR2 has investigated the effect of several different ciliopathy proteins on PCARE-induced ciliary tip expansions. Furthermore, ESR2 has determined the impact of patient mutations in the retinal ciliopathy protein INPP5E on the morphology of primary cilia in patient-derived fibroblasts. ESR3 identified protein networks to understand the molecular mechanisms of cilium disassembly. Affinity purification experiments and quantitative mass spectrometry analyses revealed several modulations in protein complex interactions during cilium disassembly. ESR4 has analysed the consequences of specific PROM1 alternative splicing on photoreceptor sensory cilia using a retinal organoid system, and was able to correlate these data to the in vivo situation using embryonic and foetal retinal samples, thus proving the concept that retinal organoids can be used to study alternative splicing.
Characterising novel ciliary signalling concepts in development and homeostasis was the focus of WP2. ESR5 studied the molecular mechanisms balancing the output of ciliary TGFβ/BMP signalling, and has scrutinized a key module in this process using patient cells, stem cell models and siRNA approaches in order to evaluate its role in signalling in health and disease. ESR6 studied the interplay between primary cilia and metabolic rewiring in Polycystic Kidney Disease, and acquired skills in evaluating cilium morphology under diverse stress conditions, and the use of cilium gene ablation models (cells and mice) to distinguish the specific response of ciliary processes to cellular stress. ESR7 has provided novel insights into the role of DLG1 and KIF13B in regulating the ciliary length and signalling capacity in kidney epithelial cells. To assess this by proximity proteomics, she successfully carried out a secondment in Nijmegen (with P1) to acquire expertise in this technique and generate relevant datasets that were subsequently validated, leading to new molecular insight relevant for cilia-related kidney disease. ESR8 used selected JBTS patient-derived hiPSCs, as well as isogenic CRIPSR/Cas9-edited lines for neuronal network measurements using microelectrode arrays (MEAs). She generated a developmental network MEA fingerprint that functionally describes the properties of the neural signalling circuits. Complemented with superresolution imaging of JBTS-associated markers and measurements of cilium formation/stability in organoids, this unveiled critical information on the defects in neuronal development ESR9 evaluated ciliary pathways and ontologies following a computational approach. In close collaboration with ESR13, she has devised a socioaffinity-like metric, which was applied to PubMed to extract disease/gene associations and (more importantly) to rank them according to the weight/uniqueness of evidence.
Determining the biomedical consequences of disrupted ciliary signalling in ciliopathies was the main focus of WP3. ESR10 zoomed in on the pathophysiological processes in Nephronophthisis (NPH) using a combination of CRISPR-based genome editing in epithelial cells and urinary epithelial cells from patients, and deployed multi-omics analyses to determine common and specific signalling pathways altered in patients. She validated the in vitro results in iPSC-derived kidney organoids and animal models (mouse). ESR11 was focusing on therapeutic approaches for renal ciliopathies to characterize the effects of new molecules and their mechanism of action. Two specific compounds, including a prostaglandin-derived molecule, were shown to have a broad positive effect on ciliogenesis in URECs of patients with mutations in NPHP genes. ESR12 has generated zebrafish CRISPR /Cas9 knock-out lines of CHD candidate genes and examined specific cardiac developmental processes in detail. He also identified and delineated novel gene networks in cardiogenesis via functional analyses in zebrafish, cell models and organoids. The goal of ESR13 was to provide an online system for the study of affinity proteomics data analysis to study edgetic effects resulting from mutations in ciliopathy genes. The "socioaffinity" metric to query single-wash affinity proteomics data, as well as datasets when data for both wild-type and mutation(s) are available, was improved. ESR14 used zebrafish to model ciliopathy disease, and generated a novel tool for addressing the link between cilia and heart regeneration. She used this novel automated platform to identify a ciliary druggable target whose modulation leads to an enhanced cardiac regeneration.
Network wide training (WP4) was provided for the fellows in the form of six complimentary skills courses and four research training courses (all compulsory for the ESRs). The Training Programme was regularly discussed and reviewed by the training committee (TPDC) with input from the fellows. These SCilS training events ran according to schedule and we have completed 100% of the planned training.
