Primary cilia (PC) are ubiquitous microtubule-based organelles essential for signal transduction, and their dysfunction in humans is implicated in neurodevelopmental ciliopathies such as Joubert syndrome (JS). JS primarily affects cerebellar development, yet pathogenic mechanisms linking ciliary gene mutations to JS manifestation remain poorly understood. Consistent with the established role of cilia in SHH pathway transduction, studies in mouse ciliary gene mutants and human JS foetal samples suggest that altered Shh-dependent expansion of granule cell precursors may contribute to the cerebellar vermis hypoplasia characteristic of JS. Yet, this cannot account for the full spectrum of phenotypes observed in patients. Moreover, characterisation of PC function in different human cerebellar progenitors is still missing.

To address these questions, we have developed protocols of human induced pluripotent stem cell 3D differentiation into cerebellar organoids, focusing on two JS causal genes: RPGRIP1L and TMEM67, which encode proteins of the ciliary transition zone (TZ). The TZ is an essential protein complex at the cilium base acting as the “ciliary gate” to control ciliary content. Interestingly, mutations in TZ genes lead to overlapping phenotypes but also present distinct cellular outcomes in humans, suggesting a cell-specific requirement for certain TZ proteins.

Our initial results reveal a phenotype that precedes granule cell amplification. Specifically, RPGRIP1L loss leads to FGF pathway upregulation, progenitor pool expansion, and a consequent delay in neurogenesis. This suggests that dysregulation of Shh is not the sole pathogenic driver and highlights the broader roles of PC in cerebellar development (Brunetti et al., BioRxiv). We are currently testing how a more directed differentiation protocol incorporating FGF8, a key factor in mid-hindbrain boundary (MHB) formation (Atamian et al., 2024), modifies this phenotype. We are also characterizing TMEM67 deficient cerebellar organoids. So far, both mutants show increases in progenitor pools and altered FGF signalling, but we see striking differences in ciliary phenotype and neurogenesis depending on the TZ protein affected. By analyzing both mutants and comparing them at whole organoid and single cell level, we now wish to 1) Study the role of PCs in human cerebellar development 2) Understand the cellular and molecular causes of JS and 3) Assess the cell-type specific functions of ciliary TZ proteins.