PROJECT SUMMARY/ABSTRACT Normal brain function requires proper establishment of cerebral cortical layering during development. A central aspect of development of the cerebral cortex layers is the migration of newly-born neurons to their specific destination in one of the six cortical layers. Neuronal migration involves orchestrated changes to the cytoskeleton driven in part by dynamic changes to microtubules with the aid of microtubule associating proteins (MAPs). Doublecortin (DCX) is a MAP that is highly expressed in immature, migrating neurons. DCX dysfunction has been linked to lissencephaly, a malformation characterized by a lack of gyri in the cortex, and subcortical band heterotropia (SBH) or a "double cortex". However, the role of DCX in neuronal migration is not understood. Studies in mouse models have only investigated the effects of a complete knockout (KO) of Dcx, which resulted in a minor hippocampal lamination phenotype but no phenotype in the cortex as seen in humans. This example has been cited to support the claim that mice may be poor models to study complex disorders of human cortical development and disease, due to species differences. However, documented disease-causing human DCX mutations involve missense mutation in one of DCX's microtubule binding domains, which does not remove DCX function entirely. In this regard, my preliminary data indicate that Dcx protein with a human missense mutation is robustly expressed. I thus propose to determine whether the lack of a cortical phenotype in mice is due to species differences or due to compensatory mechanisms after complete Dcx ablation. Specifically, I will introduce a human disease-causing DCX mutation (T203R) into the endogenous mouse Dcx locus. This will enable direct analysis of how a patient-specific mutation affects the function of doublecortin as a MAP during neuronal migration in the cortex. Conversely, I will completely ablate DCX or introduce the DCX T203R mutation into the endogenous DCX locus in human induced pluripotent stem cells (hiPSCs), to directly address the limited experimental studies performed on a human genetic background, and to complement the proposed studies of endogenous Dcx in the mouse. I will use these hiPSC lines to generate neurons and cerebral organoids, in order to define the extent to which complete DCX ablation or the human DCX T203R mutation causes impaired neuronal migration in a human experimental context. The proposed studies are important because the impact of complete DCX inactivation versus patient missense mutations has not been evaluated in isogenic human lines or in a three-dimensional human tissue context. Together, this project is expected to yield fundamental insights into a key organizational event in cortical development, as well as to establish approaches to improve studies of mutations associated with human developmental disorders more broadly.