Breathing is a basic motor behavior essential to all terrestrial vertebrates. The frequency and amplitude of respiratory contractions are driven by neural networks residing in the brainstem that coordinate the activation of dedicated sets of spinal motor neurons. Respiratory rhythm generation occurs primarily in the Pre-Bötzinger complex and can be modified by other brain stem nuclei in response to stimuli such as pH changes 1
. This rhythm is transmitted via descending pathways to motor nuclei that directly drive the activity of inspiratory and expiratory muscles. Despite the complexity of the networks that regulate respiratory rhythms, contraction of the diaphragm is controlled by a single input supplied by motor neurons within the PMC. Phrenic nerve lesions or spinal cord injuries at or above the fourth cervical segment (C4) result in diaphragm paralysis and respiratory failure, underscoring the vital role of PMC neurons within the respiratory system.
Motor neurons within the PMC are generated in the cervical spinal cord where they form a single clustered population spanning ~3 segments 2
. Most PMC axons exit the spinal cord at the C4 level, initially projecting along a medioventral path before converging with other cervical axons at the brachial plexus. Following their separation from limb-innervating axons, PMC axons extend ventrally through the thoracic cavity towards the primordial diaphragm. Upon reaching their target, phrenic axons defasciculate from the main nerve and split into multiple finer branches, prior to forming synapses across the muscle length 3
. Although PMC neurons have a central role in respiration, and their columnar organization has been recognized for over 100 years 4, 5
, surprisingly little is known about their developmental origins.
All motor neuron subtype identities emerge from the intersection of transcription factor-based programs acting along the dorsoventral and rostrocaudal axes of the spinal cord 6
. Motor neurons as a class are produced as an outcome of signaling pathways acting along the dorsoventral axis that specify features common to all subtypes, such as exit of axons from the spinal cord and neurotransmitter phenotype 7
. These signaling pathways generate motor neurons that initially express a common set of transcription factors (Hb9, Isl1/2, and Lhx3) which distinguish them from other neuronal classes 8–10
. While mutation of transcription factors required for core motor neuron programs results in phrenic nerve loss, largely due to conversion to interneuron fates 10
, no selective determinants of PMC identity have been described.
Given their discrete position within the spinal cord, the specification of PMC neurons could involve the same programs contributing to motor neuron diversity along the rostrocaudal axis. Members of the Hox
gene family are critical in generating segmentally-restricted motor neuron subtypes at limb and thoracic levels 11
. At limb levels, the diversification of lateral motor column (LMC) neurons employs a network of ~20 Hox
, while thoracic level motor neuron fates are determined by the single Hoxc9
. All Hox
gene activities in spinal motor neurons are thought to require the transcription factor FoxP1, as limb-level and thoracic Hox-dependent subtypes are lost in Foxp1
mutants 14, 15
. PMC neurons are however not depleted in Foxp1
mutants, but instead appear to increase in number 15
. These observations raise the question of whether PMC neurons are specified through mechanisms independent of Hox activities, or whether certain Hox proteins contribute to motor neuron specification independent of Foxp1
We show here that Hoxa5 and Hoxc5 have critical roles in phrenic motor neuron development. PMC neurons are defined through a broader network of Hox factors that constrain their position and number. Selective deletion of Hox5 genes from motor neurons leads to an extinction of PMC molecular determinants, cell body disorganization, and the progressive loss of PMC numbers. Hox5 genes are also essential for a diaphragm-specific pattern of intramuscular branching, independent of their roles in cell survival. Temporal analysis of Hox5 function in motor neurons indicates that survival and intramuscular branching programs are distinct from those controlling columnar organization. These results define a specific transcriptional program for PMC neurons and indicate Hox activities are required throughout motor neuron ontogeny.