trio-a depletion affects neurogenesis in zebrafish, to model microcephalic phenotype in haploinsufficient patients

TRIO is a key regulator protein in multiple processes involved in neurodevelopment that require actin cytoskeletal reorganization, such as migration and axonal pathfinding. Patients with mutations that alter the activity of the GEF1 domain of the protein present an aberrant activation of its downstream effector, the Rho GTPase RAC1, displaying a broad range of neurodevelopmental disorders including microcephaly and intellectual disability. By reproducing the TRIO haploinsufficiency condition found in patients by means of morpholino knockdown in zebrafish, we demonstrated that it is possible to study the disease pathoethiology starting from its onset during embryonic development. trioa morphants displayed the typical morphological abnormalities of neural crest cell migration impairment, on top of a reduced head area, developmental delay, and impaired axonal migration. The histological and molecular analysis of proliferation markers showed a delayed depletion of the neural progenitor cell pool, paired with a decreased expression of terminally differentiated neuronal markers. TRIO knockdown of HeLa cells showed an increased disorganization of F-actin filaments, but no defects in cytokinesis. However, cell cycle analysis showed a delay in cell division timing, indicating that TRIO may also be involved in cell cycle regulation. Contextually, the study of patient derived cortical organoids corroborated the results found in vivo as organoids exhibited substantial morphometric alterations in terms of size and shape. Altogether these results demonstrated that TRIO may play a role in neural progenitor cell proliferation and division conserved from teleosts to mamamls.