Project Abstract Respiratory abnormalities are a common symptom in a wide variety of developmental and genetic disorders affecting infants and children, including sudden infant death syndrome (SIDS), Rett syndrome, and autism spectrum disorders. These abnormalities are likely due to defects in the neural circuits that control breathing, but the underlying mechanisms remain unknown. In mammals, contraction of the diaphragm muscle is critical for drawing air into the lungs during inspiration. Phrenic motor neurons (MNs) in the cervical spinal cord provide the only motor input to the diaphragm, and therefore phrenic nerve dysfunction has devastating effects on respiration. Thus, understanding the molecular mechanisms that control phrenic MN development is critical for novel treatments for the respiratory dysfunction seen in developmental disorders. We have previously established that the transcription factors Hoxa5 and Hoxc5 are required for the development of the phrenic motor column (PMC). Mice with a motor neuron-specific deletion of Hox5 genes (Hox5MN∆) exhibit perinatal lethality due to severe respiratory defects, and exhibit a loss of phrenic motor neuron identity, characterized by reduced expression of PMC specific genes, progressive motor neuron loss and disorganization, alterations in dendritic arbor orientation, and a dramatic reduction in axonal branching and synaptic contacts at the diaphragm. Restoring MN cell numbers by inhibiting cell death does not prevent axonal branching defects at the diaphragm, demonstrating that the phenotype is not due to a reduction in MN numbers. Hox5MN∆ embryos show abnormal retraction bulb axonal growth cone morphology, suggesting defects in the cytoskeleton. This proposal will focus on identifying the molecular pathways downstream of Hox5 genes that establish and maintain PMC identity by employing an integrative approach combining unbiased genetic screens, high-resolution imaging, and electrophysiological and behavioral assays. In Aim 1, I will define the role of Hox5 genes in PMC morphology via high-resolution imaging of cytoskeletal components in Hox5MN∆ growth cones. I will also perform sparse fluorescent labeling of phrenic MNs to characterize dendritic branching defects at the single cell level. Results from this analysis will guide the selection of relevant genes in Aim 2. In Aim 2, novel Hox5 target genes identified by RNA-seq will be confirmed with in situ hybridization and analyzed further. One promising target, Col25a1, will be overexpressed in MNs to attempt to rescue diaphragm innervation defects in Hox5MN∆ mice. In Aim 3, knockout mice for two previously identified Hox5 target genes, Pcdh10 and Negr1, will be examined for defects in PMC clustering, the dendritic arbor, and diaphragm innervation. In addition, embryonic phrenic nerve recordings and plethysmography will be performed. By identifying how Hox5 genes establish neuronal form, I will provide insight to a fundamental principle of ner...