Establishing the cell-type specificity of cilia during human cerebellar development – CiCerO

How perturbations of ubiquitous subcellular structures affect specific brain functions is a major issue in neurobiology. Primary cilia are widely distributed in the central nervous system, yet their dysfunction in humans leads to specific cerebellar defects in Joubert syndrome, a neurodevelopmental ciliopathy. The understanding of the mechanisms underlying this specificity and, more globally, of the role of primary cilia in human cerebellar development, are still very fragmentary due the lack of human models. Recently, using human induced pluripotent stem cell (iPSC)-derived neural 3D differentiation approaches, Partner 1 has uncovered species and progenitor type-specific roles of a ciliopathy protein in the human central nervous system. The CiCerO project gathers two partners with shared interest in cerebellar development and complementary expertise in PSC-derived cerebellar differentiation approaches and multi-Omics, with the aim to address the mechanisms of this cell type specificity, focusing on primary cilia structure, dynamics and function in human cerebellar development.

The project has two main objectives: 1) To dissect the function of cilia and ciliopathy proteins in human cerebellar development, 2) To understand how cell-type specific differences in ciliary structure and dynamics are established in human cerebellar lineages and influence their function. We will use human induced pluripotent stem cell-derived cerebellar 2D and 3D (organoids) differentiation into cerebellar progenitors and neurons as model systems.

In Objective 1, we will focus on two Joubert Syndrome causal genes, RPGRIP1L and TMEM67, which encode proteins of the transition zone, a region at the base of the cilium involved in controlling the composition and function of cilia. Being structurally and functionally different, these proteins are expected to contribute differently to cilia stability and composition, and consequently to cerebellar development. To test this hypothesis, we will combine immunofluorescence studies, scRNAseq, proteomic and phospho-proteomic profiling of RPGRIP1L and TMEM67 mutant cerebellar cells in order to obtain a global understanding on the role of these two proteins in human cerebellar development, in particular in early phases of lineages specification and neurogenesis. From this analysis we expect to 1) tackle the role of cilia and of distinct TZ proteins in the main cell trajectories of cerebellar progenitors 2) identify altered signaling pathways in different cerebellar progenitors and 3) Identify master regulators of specific ciliary programs within different cerebellar lineages.

In Objective 2, we will directly address the cell-type specificity of primary cilia. We aim to characterize the variations in cilia structure and content between different cerebellar lineages during the differentiation process in human cerebellar progenitors, and to correlate them to the functions of transition zone proteins in cerebellar development uncovered in Objective 1. To do so, we will apply high resolution imaging techniques to study the spatio-temporal changes in structural and molecular features of cilia in the human developing cerebellum. We will set-up a new protocol to purify, analyze and compare the protein content of cilia from cerebellar cells in different lineages and at different stages of their differentiation, in order to link the structural features of cilia to their molecular composition.

We expect the CiCerO project to have important insight on the understanding of the cell-type specific roles of cilia in human brain development, but also on our ability to understand human neurodevelopmental diseases such as Joubert syndrome and related ciliopathies.