Motor axon exit from the mammalian spinal cord is controlled by the homeodomain protein Nkx2.9 via Robo-Slit signaling

Arlene Bravo-Ambrosio 2 Grant Mastick 1 Zaven Kaprielian () 0 2 0 Department of Pathology, Albert Einstein College of Medicine , Bronx, NY 10461 , USA 1 Department of Biology, University of Nevada , Reno, NV 89557 , USA 2 Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine , Bronx, NY 10461 , USA SUMMARY Mammalian motor circuits control voluntary movements by transmitting signals from the central nervous system (CNS) to muscle targets. To form these circuits, motor neurons (MNs) must extend their axons out of the CNS. Although exit from the CNS is an indispensable phase of motor axon pathfinding, the underlying molecular mechanisms remain obscure. Here, we present the first identification of a genetic pathway that regulates motor axon exit from the vertebrate spinal cord, utilizing spinal accessory motor neurons (SACMNs) as a model system. SACMNs are a homogeneous population of spinal MNs with axons that leave the CNS through a discrete lateral exit point (LEP) and can be visualized by the expression of the cell surface protein BEN. We show that the homeodomain transcription factor Nkx2.9 is selectively required for SACMN axon exit and identify the Robo2 guidance receptor as a likely downstream effector of Nkx2.9; loss of Nkx2.9 leads to a reduction in Robo2 mRNA and protein within SACMNs and SACMN axons fail to exit the spinal cord in Robo2-deficient mice. Consistent with short-range interactions between Robo2 and Slit ligands regulating SACMN axon exit, Robo2-expressing SACMN axons normally navigate through LEP-associated Slits as they emerge from the spinal cord, and fail to exit in Slit-deficient mice. Our studies support the view that Nkx2.9 controls SACMN axon exit from the mammalian spinal cord by regulating Robo-Slit signaling. INTRODUCTION Motor neurons (MNs) project their axons out of the central nervous system (CNS) and make stereotyped connections with peripheral muscle targets to form circuits that control movement (Bonanomi and Pfaff, 2010; Dalla Torre di Sanguinetto et al., 2008; Sharma and Peng, 2001). Motor axons grow in a directed manner to specialized exit points through which they emerge from the CNS (Bravo-Ambrosio and Kaprielian, 2011; Jacob et al., 2001; Lieberam et al., 2005; Schneider and Granato, 2003; Sharma et al., 1998; Shirasaki and Pfaff, 2002). MN subtypes can be distinguished by the positions of their exit points: ventral MNs (vMNs) and dorsal MNs (dMNs) utilize ventral and dorsal exit points, respectively (Chandrasekhar, 2004; Cordes, 2001; Dillon et al., 2005; Guthrie, 2007; Lieberam et al., 2005; Schubert and Kaprielian, 2001; Sharma et al., 1998; Snider and Palavali, 1990). Although Cxcl12-Cxcr4 signaling regulates the growth of vMN axons to their exit points in mice (Lieberam et al., 2005) and myotomal-derived diwanka (plod3 ZFIN) glycosyltransferase is required for motor axon growth into the periphery in zebrafish (Schneider and Granato, 2006), the molecular mechanisms that control motor axon exit from the vertebrate spinal cord are poorly understood. Transcription factors (TFs) control axon pathfinding by regulating the expression of cell surface molecules (Broihier et al., 2004; Garcia-Frigola et al., 2008; Labrador et al., 2005; Landgraf et al., 1999; Lee et al., 2008; Wilson et al., 2008). In Drosophila, Zfh1 (Layden et al., 2006) and Nkx6 (HGTX FlyBase) (Broihier et al., 2004) are required for the exit of vMN axons, whereas Eve (Landgraf et al., 1999) is necessary for dMN axon exit. Nkx6 and Eve are likely to mediate motor axon exit by regulating the expression of Fas3 (Broihier et al., 2004) and Unc5 (Labrador et al., 2005), respectively. In vertebrates, Lhx3/Lhx4 (Sharma et al., 1998) and Phox2b (Hirsch et al., 2007) are required for the directed growth of vMN and dMN axons, respectively, to their exit points. However, these TFs appear to control the specification of vMNs/dMNs rather than motor axon exit per se. Spinal accessory motor neurons (SACMNs) are branchiomotor dMNs that reside within the cervical spinal cord and project dorsally directed axons to and through a highly localized lateral exit point (LEP) situated midway along the dorsoventral axis of the spinal cord (Dillon et al., 2005; Hirsch et al., 2007; Lieberam et al., 2005). Upon exiting the CNS, SACMN axons execute a rostral turn and assemble into the longitudinally projecting spinal accessory nerve (SAN), which innervates particular neck and back muscles (Dillon et al., 2005; Dillon et al., 2007; Schubert and Kaprielian, 2001; Snider and Palavali, 1990). We identified the immunoglobulin (Ig) domaincontaining protein BEN (Alcam or SC1 Mouse Genome Informatics) (Dillon et al., 2005; Schubert and Kaprielian, 2001) as a selective marker of SACMN cell bodies/axons (Dillon et al., 2005; Schubert and Kaprielian, 2001). Since SACMNs are a molecularly homogenous and readily identifiable population of spinal MNs, which leave the CNS through a circumscribed exit point, they represent an ideal model system for elucidating molecular programs that control motor axon exit. Our observation that the homeodomain TF Nkx2.9 is likely to be required for SACMN axons to leave the CNS (Dillon et al., 2005) prompted us to further characterize the role of Nkx2.9 in motor axon exit. Here we show that, in mice lacking Nkx2.9, SACMN axons appropriately project to the LEP but assemble into an ectopic longitudinally projecting SAN within the spinal cord. We also identify the axon guidance receptor roundabout 2 (Robo2) (Ypsilanti et al., 2010) as a likely downstream effector of Nkx2.9 by showing that Robo2 expression in SACMNs is downregulated in Nkx2.9 null mice and that SACMN axons fail to exit the spinal cord in Robo2deficient animals. Furthermore, the Robo2 ligands Slit1-3 are present at the LEP, SACMN axons fail to exit the CNS in Slit null mice, and Slit promotes SACMN axon outgrowth in vitro. Collectively, our findings are consistent with Nkx2.9 controlling SACMN axon exit from the CNS by regulating Robo2-Slit interactions at the LEP. MATERIALS AND METHODS Mice CD-1 wild-type (WT) embryos were used for expression studies (Charles River Laboratories). Nkx2.9 mutant embryos were generated by mating Nkx2.9+/ mice and genotypes determined by PCR (Tian et al., 2006). Nkx2.9 breeding pairs were obtained from J. Locker (Albert Einstein College of Medicine). Pregnant dams were sacrificed as described (Dillon et al., 2005). The morning on which a vaginal plug was detected was considered embryonic day (E) 0.5. Robo and Slit mutant embryos were generated and genotyped as described (Andrews et al., 2008; Farmer et al., 2008; Grieshammer et al., 2004; Long et al., 2004; Lopez-Bendito et al., 2007; Lu et al., 2007; Plump et al., 2002). Fig. 1. In Nkx2.9/ embryos, SACMN axons fail to exit the CNS and assemble into an ectopic SAN within the spinal cord. (A-H)Wild-type (WT) (A,B, E10.5; E,F, E11.5) and Nkx2.9/ (C,D, E10.5; G,H, E11.5) embryos were labele (...truncated)