The role of Poorly Characterized Disease-related Proteins in Cortical Development

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PROJECT SUMMARY Many rare genetic diseases include a range of neurological phenotypes. Malformations of Cortical Development (MCDs) includes a group of syndromes that share defective cortical lamination phenotypes. However, little is known regarding axonal and dendritic morphology of neurons in affected MCDs cortices. In particular, axonal morphologies of excitatory projection neurons are remarkable because these neurons connect neocortex with multiple regions throughout the brain, and this requires precise regulation of interstitial axon branching. However, molecular mechanisms that regulate interstitial axon branching are poorly understood. We hypothesize that among many rare disease-related genes encoding Understudied Proteins Associated with Rare Diseases (UPARDs) listed in this funding opportunity announcement (FOA), there are a significant number of genes encoding UPARDs that are directly involved in the regulation of cortical neuron axon and dendrite morphology. We have previously developed an approach for studying complete cortical neuronal morphology at the single cell level which at the same time can be combined with targeted genetic or epigenetic perturbations. In this project, we will perform a genetic screen of 105 genes encoding UPARDs listed in this FOA which are expressed at high levels in the mouse cerebral cortex. We identified these genes by analysis of our scRNAseq dataset generated from postnatal day 13 (P13) upper layer cortical excitatory neurons and other publicly available RNAseq or in situ hybridization databases. We will generate a library of sgRNAs targeting these genes and clone them into vectors carrying the Cas9 enzyme. We will use in utero electroporation at embryonic day 15.5 (E15.5) to target layer 2/3 callosal projection neurons (CPNs) and downregulate expression of selected genes to study the morphology of these neurons at postnatal day 14 (P14). We will focus on changes in the density and distribution of interstitial axon branches in individual cortical layers and on the orientation and branching of apical and basal dendrites. The major outcome of this screen will be the identification of novel proteins that regulate cortical excitatory neuron morphology. These results will serve as a foundation for further analyses of these understudied proteins, allowing for the identification of novel signaling pathways that modulate neuronal morphology and for a better understanding of the pathogenesis of rare diseases connected with dysfunction of these proteins.