PROJECT SUMMARY Breathing is essential for terrestrial life. In mammals, phrenic motor neurons (MNs) form a single motor column (Phrenic Motor Column or PMC) and innervate the diaphragm in a stereotypical manner to control its contraction. Alterations in the development of phrenic MNs are likely a cause of respiratory dysfunction, as observed in sleep apneas and sudden infant death syndrome (SIDS). Despite the essential role of phrenic MNs, the molecular determinants that establish phrenic MN identity are not fully understood. Our previous studies have shown that Hox5 (Hoxa5 and Hoxc5) transcription factors (TFs) are critical for the specification of PMC, but the transcriptional and regulatory mechanisms that control PMC specification are yet to be elucidated. Deciphering how Hox5 proteins can selectively control their targets is fundamental in order to understand phrenic MN development. Cooperative binding of Hox proteins with select cofactors influences their target site selectivity. We have identified additional TFs that interact with Hoxa5. In this proposal I will investigate the function of Hoxa5 protein and its interaction with other cofactors in determining PMC identity. In Aim 1 I will determine how Hoxa5 specifically regulates its target effectors during the specification of phrenic MNs. In Aim 2 I will map the interaction between Hoxa5 and other cofactors and evaluate if this interaction is essential for the activation of putative phrenic enhancers and promoters. In Aim 3 I will define Hox5-dependent phrenic MN diversity. I have developed an integrative methodology encompassing genetic models, cell and molecular biology techniques, and high throughput sequencing in order to address these questions in vivo. Elucidating the molecular mechanisms underlying transcriptional regulation of phrenic motor neuron identity will allow us to identify potential therapeutic targets and bring us closer to the development of effective treatments for respiratory dysfunction.