Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily affecting girls. In its classical form, RTT is predominantly caused by mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). MeCP2 is a multifunctional regulator of gene expression which regulates transcription through diverse mechanisms such as DNA-binding, interaction with transcription factor complexes, modulation of chromatin structure and regulation of miRNAs – mechanisms that are engaged pleiotropically through different developmental stages. MeCP2 was considered to act predominantly through late development into adulthood, but recent clinical studies of RTT children point to very early signs of the disorder. The early developmental mechanisms of MeCP2 are poorly understood. We previously used RTT patient iPSCs to show that reduction of MeCP2 leads to overexpression of miRNA-199 and miRNA-214, an increase in neural progenitors, and reduction in neurogenesis and neuronal migration in cortical organoids. We now propose to analyze the migration deficits in detail, and examine the mechanisms underlying the deficits. The objective of this proposal is to develop a novel live-cell imaging platform merging 3D stem cell technologies, microfluidics and multiphoton microscopy, and combine it with state-of-the- art molecular approaches, including mass spectrometry proteomics and single cell RNA sequencing, to examine mechanisms of neuronal migration deficits associated with RTT-causing mutations in MECP2. In Aim 1, we propose to develop label-free third-harmonic generation three-photon microscopy and use it to characterize neuronal migration deficits in RTT organoids compared to isogenic controls. We will additionally develop a microfluidics-based live imaging platform where organoids can be stably imaged and neurons tracked for days. In Aim 2, we will examine the consequence of MECP2 mutations on downstream molecular pathways involved in neuronal differentiation and migration. We will examine mechanisms of anomalous overexpression of AKT in RTT organoids and neural progenitors, and use a proteomic and phospho-proteomic screen to define new proteins and pathways of neuronal migration dysregulated in RTT. We will exploit the transcriptomic profile of single cells to reveal cell types, populations and transcriptomic differences between RTT and control organoids. In Aim 3, we will use the technologies of Aim 1, and results of Aim 2, to examine the role of implicated signaling pathways in neuronal migration. We will interrogate the function of AKT and downstream signaling molecules, and that of new proteins, including modulators of cell adhesion and cytoskeleton organization identified from our screens, that are predicted as involved in migration. We will validate in vivo in mice the ability of specific pathways and focal adhesion proteins to rescue RTT neuronal migration deficits. Together, we expect that these results will advance our understanding of mechanisms involved...