Identification of novel downstream effector molecules of the homeodomain transcription factor, Nkx2.9, that regulate spinal accessory motor neuron development
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A distinguishing feature of motor neurons (MNs) is that their axons project out of the central nervous system (CNS) and into the periphery, where they ultimately connect with muscle targets, which control movement and autonomic function, including heart rate, respiration, and digestion. Despite the clear importance of understanding this key phase of motor axon pathfinding, the molecular mechanisms that underlie motor axon exit in mammals remain largely unknown. Whereas the majority of spinal MN axons leave the CNS through ventral exit points, spinal accessory motor neurons (SACMNs) project dorsally directed axons toward and through lateral exit points (LEP) and assemble into a readily identifiable spinal accessory nerve (SAN), which innervates neck and back muscles. Given that SACMN and their axons selectively express an immunoglobulin (Ig) domain-containing cell surface protein, BEN, and that SACMN axons are the sole MN axons that leave the CNS through a discrete exit point, these particular motor axons represent an ideal model system for investigating the mechanisms underlying motor axon exit. Moreover, we previously showed that SACMN axons likely fail to exit the mouse spinal cord in the absence of Nkx2.9, a homeodomain transcription factor (TF). Although there is increasing evidence that TFs, including Nkx2.9, control axon guidance in a variety of invertebrate and vertebrate systems, the identities of the corresponding downstream effector genes are largely unknown across species. To identify novel downstream targets of Nkx2.9 that regulate SACMN axon exit and/or pathfinding, I performed candidate-based and microarray screens designed to identify genes that are differentially expressed in Nkv2.9 deficient versus wild type mice.;In Chapter I, I describe the first genetic pathway that regulates motor axon exit from the mammalian spinal cord. Specifically, I show that Nkx2.9 is selectively required for the exit of SACMN axons and identify the Robo2 guidance receptor as a likely downstream effector of Nkx2.9. Consistent with short-range interactions between Robo2 and its Slit ligands regulating SACMN axon exit, SACMN axons express Robo2 as they exit the spinal cord and Slits are present at the LEP through which these axons emerge from the spinal cord. In support of this model, SACMN axons fail to leave the CNS in mice lacking Robo2 or Slits. I also provide evidence that Robo-Slit interactions are required for the guidance of longitudinally projecting SACMN axons. Collectively, my studies indicate that Nkx2.9 controls SACMN axon exit from the spinal cord via regulation of Robo-Slit signaling.;In Chapter 2, I identify the transmembrane neurite outgrowth inhibitor, Slitrk2, as an additional novel and putative downstream effector of Nkx2.9. Slitrk2 belongs to the Slitrk family (Slitrkl-6) of structurally related proteins that have significant homology to secreted Slit ligands and trk neurotrophin receptors. Consistent with the hypothesis that Slitrk2 is a gene target of Nkx2.9, I show that Slitrk2 levels are significantly up-regulated in Nkx2.9 null mice and that the size of the SAN appears selectively increased in the absence of Slitrk2. Collectively, these findings suggest that Slitrk2 selectively regulates the number of SACMN axons that exit the spinal cord and/or the fasciculation of longitudinally projecting SACMN axons within the SAN. Taken together, these observations provide novel insights into the molecular mechanisms underlying MN axon exit and/or the formation of motor nerves by linking Nkx2.9 to the regulation of the Slitrk2 cell surface protein. Notably, my findings provide the first evidence supporting an in vivo role for Slitrk2 in MN development.