Project Summary/Abstract The axial neuromuscular system plays a critical role in many essential motor behaviors in mammals, including breathing, postural stability, and integration of movement by the trunk and limbs. While the mechanisms that allow for molecular and functional diversification of limb innervating motor neurons (MN) have been relatively well characterized, less is understood about how axial motor circuits are specified during development. Further, it is not well understood whether axial MNs exist in discrete pools that innervate distinct muscle populations to achieve motor activities through activation of specific muscles. It is also not known whether the molecular identities of axial motor neurons are related to a set of functional properties in tetrapods, nor if these molecular identities dictate the connectivity patterns of spinal premotor interneurons that modulate activity of downstream muscle targets. The proposed work in this study seeks to elucidate the fate determinants that govern organization of molecularly distinct populations of motor neurons within the medial motor column (MMC). In Aim1, I will characterize the molecular subtypes and anatomical organization of MMC neurons. I will use single nuclear (sn) RNA-seq to define the molecular diversity of MMC MNs in mouse embryos. I will then use immunohistochemistry (IHC) and hybridization chain reaction RNA-FISH (HCR) to further characterize the anatomical organization of molecularly defined MNs. I will also use retrograde tracing in embryos and early postnatal mice to determine whether molecularly unique populations of MMC neurons correspond to motor pools. Aim 2 explores the role of fate determinants in axial MN diversity and muscle target specificity. I will use snRNA-seq, IHC and HCR to map the molecular diversity and organization of MMC MNs in mice with mutations in three classes of fate determinants, Mecom/Prdm16, Satb2, and Lhx3/4. Further, I will use mice that co-express the Hb9-GFP reporter to assess the pattern of axial muscle innervation. Finally, to define the epistatic relationships between these transcription factors in the MMC gene network, I will use chick neural tube electroporation to misexpress Mecom, Satb2 or Lhx3 in all MNs and examine the impact on axial MN specification. Aim 3 seeks to elucidate the role of MMC molecular identity in axial circuit assembly and function. I will use monosynaptic rabies tracing to define the distribution of spinal premotor inputs targeting MMC in control and mutant mouse models. I will also perform motorized treadmill assays while recording muscle activity from epaxial muscle, in addition to capturing gait and posture during locomotion. This work will provide an understanding of how axial motor systems are developed, their functions, the molecular identity of unique axial motor pools, and the role of fate determinants in specifying circuits that dictate the function of axial muscle. Understanding this is essential to our ...