PROJECT SUMMARY There is a great interest in deriving induced neuronal (iN) cells from human pluripotent stem cells (hPSCs) for central nervous system disease modeling and drug discovery. However, current differentiation methods are laborious and inefficient, thus, identifying new ways to improve neuronal differentiation is desirable. Strikingly, in my most recent publication I discovered that knockout of the axon guidance receptor Plexin-B2 in hNPCs reduced cell stiffness, increased Yes-Associated Protein 1 (YAP) phosphorylation, and accelerated neuronal differentiation. As a critical downstream mechanism, my preliminary data points to a pathway of Plexin-B2 altered mechanoregulation affecting lamin-mediated nuclear envelope architecture and localization of repressive histone marks at genomic loci containing neuron-specific genes. Based on these data, I will investigate an intrinsic ‘force-based’ method to generate iNs by manipulating Plexin-B2, Angiomotin (AMOT; a mechanosensitive component of Hippo pathway), and YAP in both 2D and 3D neuronal cultures and examine the link between Plexin-B2 and Hippo/YAP mechano-signaling in regulating lamin-mediated nuclear physical properties, force transmission to the nuclear envelope, and changes in chromatin accessibility during neural induction. To carry out this investigation, I propose the following aims: #1. Investigate the impact of Plexin-B2-altered cell mechanics on neuronal differentiation, by optimizing the differentiation method to generate iNs by both Plexin-B2 knockdown and knockout in multiple hPSC lines. I will test the robustness, stability, and functionality of iNs by evaluating transcriptional identity, neuronal lineage, maturation, and survival markers, electrophysiological properties, and neurotransmitter release. #2. Investigate a mechanochemical loop between Plexin-B2 and YAP during neuronal differentiation, by performing a comprehensive gain- and loss-of-function epistasis studies to map out the relationships between Plexin-B2, YAP, and AMOT during neural induction and evaluate their impact on enhancing neuronal differentiation, maturation, survival, electrophysiological functionality, and expression of major neuronal lineage markers. #3. Investigate lamin-mediated nuclear architecture and chromatin interactions in Plexin-YAP mechano-regulation of neuronal differentiation, by performing a comprehensive gain- and loss-of-function epistasis studies to validate lamin as the mediator of a Plexin-B2 and Hippo/YAP intrinsically-regulated mechano-axis to promote neuronal differentiation. I will then evaluate the role of Plexin-B2 / Hippo/YAP / lamins in releasing genomic loci harboring neuron-specific genes from repressive chromatin at nuclear periphery to interior regions with open chromatin. This K01 Award will provide me training in neuronal electrophysiology, transcriptomics and epigenomics, lab management and mentoring skills, as well as time and mentoring to develop and write my fir...