Bulk RNAseq from cerebellar organoids

Joubert syndrome (JBTS) is a rare neurodevelopmental ciliopathy characterized by cerebellar vermis hypo-dysplasia. Despite significant advances in mouse models, the pathogenetic mechanisms linking ciliary gene dysfunction to JBTS neurodevelopmental defects are still poorly understood. In this study we generated cerebellar organoids from human induced pluripotent stem cells (hiPSCs) carrying CRISPR/Cas9 null mutations or patient-derived variants in the JBTS causal gene RPGRIP1L, which encodes a scaffold protein crucial for building functional cilia. Using immunolabelling and transcriptomics, we demonstrated the expression of characteristic markers for Granule cell progenitors and the Purkinje cell lineage in isogenic WT organoids. In contrast, cerebellar organoids derived from all RPGRIP1L-deficient lines exhibit a common severe reduction of Purkinje lineage markers, along with decreased neurogenesis and increased progenitor proliferation, coupled to an extended overactivation of the FGF pathway. Most importantly, restoring normal levels of FGF signaling in RPGRIP1L-deficient organoids rescued the proliferative/neurogenic balance. Our results uncover for the first time in a JBTS human model of cerebellar development, an early FGF deregulation that hampers correct neuronal differentiation and could account for cerebellar features observed in JBTS patients.