Comprendre le rôle de la dynéine et de la matrice extracellulaire au cours du développement néocortical humain à l'aide d'organoïdes cérébraux

Cortical development in mammals begins with the differentiation of neuroepithelial cells into apical radial glial (aRG) cells, a primary population of neural stem cells (NSCs). These cells drive the early stages of brain development through cycles of proliferation and the generation of post-mitotic neurons that migrate radially to form the layered structure of the cerebral cortex. In humans, cortical expansion and complexity are further supported by a second NSC type—basal radial glial (bRG) cells. Derived from aRGs, bRGs share neurogenic and gliogenic potential but localize to more basal regions of the developing cortex and lack apical attachments. bRG cells are thought to be a key evolutionary feature of primates and may give rise to a range of neural lineages, including inhibitory neurons and oligodendrocytes. Despite advances in single-cell and lineage-tracing technologies, the molecular mechanisms guiding cell fate decisions and the generation of this cellular diversity remain incompletely understood. My PhD research investigated both intrinsic (genetic) and extrinsic (environmental) mechanisms that regulate human cortical development, with a particular focus on how disruptions in these pathways contribute to neurodevelopmental disorders known as Malformations of Cortical Development (MCDs). These disorders often arise from impairments in NSC behavior, neuronal migration, or tissue organization, resulting in conditions such as microcephaly or lissencephaly. In the first part of my thesis, I studied genetic disorders caused by mutations in microtubule-associated proteins and molecular motors. A key focus was cytoplasmic dynein, a motor protein complex critical for mitosis, intracellular transport, and neuronal positioning. Mutations in the dynein heavy chain gene DYNC1H1 cause a range of neurological conditions, collectively termed dyneinopathies, including both MCDs and Spinal Muscular Atrophy with Lower Extremity Predominance (SMALED). These disorders differ in their phenotypic outcomes—MCDs involve cortical defects, while SMALED primarily affects the spinal cord—suggesting mutation-specific, context-dependent functions of dynein. To explore this, I used patient-derived induced pluripotent stem cells (iPSCs) to generate cerebral organoids, 3D in vitro models of early human brain development. I analyzed dynein-dependent processes in organoids carrying different DYNC1H1 mutations. Organoids with MCD-associated mutations exhibited reduced growth, mitotic spindle defects, and delayed NSC division, leading to increased apoptosis and reduced cell numbers—findings consistent with the microcephaly phenotype seen in patients. In contrast, SMALED-mutant organoids showed normal growth and mitotic behavior, reflecting the absence of cortical abnormalities. These results support a model where specific DYNC1H1 mutations disrupt NSC proliferation and genome integrity, resulting in lineage-specific developmental failure. In the second part of my work, I investigated how extrinsic cues, particularly the extracellular matrix (ECM), influence cortical development. The ECM provides structural and biochemical support for NSC proliferation, differentiation, and migration. I focused on fibronectin (FN1), a major ECM glycoprotein implicated in brain development. To assess its role, I created an FN1 knockout iPSC line and used it to generate cerebral organoids. Preliminary data showed impaired organoid growth in the absence of fibronectin, suggesting its critical role in maintaining progenitor function and tissue architecture. Overall, my research highlights the importance of both intrinsic mutations in key genes like DYNC1H1 and extrinsic factors like fibronectin in shaping human cortical development. These findings enhance our understanding of the cellular and molecular mechanisms underlying MCDs and may inform future strategies for modeling and potentially correcting neurodevelopmental disorders.