A sustained Hox program delineates brainstem neurons essential for breathing

Respiratory neurons in the brainstem must diversify and acquire unique properties during development to enable breathing at birth. Dbx1-expressing progenitors give rise to functionally and molecularly distinct excitatory respiratory populations, including rhythm-generating pre-Bötzinger complex (preBötC) neurons and phrenic motor neuron (MN)-projecting rostral Ventral Respiratory Group (rVRG) neurons. These neurons are organized rostrocaudally within the ventral respiratory column (VRC) but, despite their critical functions in breathing, the mechanisms that control their organization and diversification are not well understood. Here, we generate a novel genetic tool to label brainstem neurons within the VRC. We find that rVRG neurons selectively express Hox5 genes through postnatal stages. Selective deletion of all Hox5 paralogs from Dbx1-derived neurons leads to respiratory dysfunction, perinatal death, and changes in the pattern and amplitude of phrenic MN firing. We show that Hox5 inactivation leads to a caudal expansion of putative preBötC neurons, likely at the expense of the rVRG. Collectively, our findings indicate that Hox5 proteins are required for the delineation and functional specialization of excitatory brainstem neurons essential for breathing.