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• W2923081206 abstract "•p190 is a context-dependent gate for Netrin-DCC signaling•Distinct temporal activities of p190 emerge from a modular domain organization•p190 is a cell intrinsic signal-detection filter•Suppression of incongruous directional signals prevents miswiring The rich functional diversity of the nervous system is founded in the specific connectivity of the underlying neural circuitry. Neurons are often preprogrammed to respond to multiple axon guidance signals because they use sequential guideposts along their pathways, but this necessitates a strict spatiotemporal regulation of intracellular signaling to ensure the cues are detected in the correct order. We performed a mouse mutagenesis screen and identified the Rho GTPase antagonist p190RhoGAP as a critical regulator of motor axon guidance. Rather than acting as a compulsory signal relay, p190RhoGAP uses a non-conventional GAP-independent mode to transiently suppress attraction to Netrin-1 while motor axons exit the spinal cord. Once in the periphery, a subset of axons requires p190RhoGAP-mediated inhibition of Rho signaling to target specific muscles. Thus, the multifunctional activity of p190RhoGAP emerges from its modular design. Our findings reveal a cell-intrinsic gate that filters conflicting signals, establishing temporal windows of signal detection. The rich functional diversity of the nervous system is founded in the specific connectivity of the underlying neural circuitry. Neurons are often preprogrammed to respond to multiple axon guidance signals because they use sequential guideposts along their pathways, but this necessitates a strict spatiotemporal regulation of intracellular signaling to ensure the cues are detected in the correct order. We performed a mouse mutagenesis screen and identified the Rho GTPase antagonist p190RhoGAP as a critical regulator of motor axon guidance. Rather than acting as a compulsory signal relay, p190RhoGAP uses a non-conventional GAP-independent mode to transiently suppress attraction to Netrin-1 while motor axons exit the spinal cord. Once in the periphery, a subset of axons requires p190RhoGAP-mediated inhibition of Rho signaling to target specific muscles. Thus, the multifunctional activity of p190RhoGAP emerges from its modular design. Our findings reveal a cell-intrinsic gate that filters conflicting signals, establishing temporal windows of signal detection. Cellular communication is controlled by signaling processes that are inherently context dependent and temporally regulated. An extreme example of signaling complexity is manifested in the way axon growth is directed. During embryonic development, neurons build intricate connectivity networks through combinatorial, synergistic, and sequential activation of a conserved but remarkably limited set of ligand-receptor systems (Bonanomi et al., 2012Bonanomi D. Chivatakarn O. Bai G. Abdesselem H. Lettieri K. Marquardt T. Pierchala B.A. Pfaff S.L. Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals.Cell. 2012; 148: 568-582Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, Dudanova and Klein, 2013Dudanova I. Klein R. Integration of guidance cues: parallel signaling and crosstalk.Trends Neurosci. 2013; 36: 295-304Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, Kolodkin and Tessier-Lavigne, 2011Kolodkin A.L. Tessier-Lavigne M. Mechanisms and molecules of neuronal wiring: a primer.Cold Spring Harb. Perspect. Biol. 2011; (Published online June 1, 2011)https://doi.org/10.1101/cshperspect.a001727Crossref PubMed Scopus (434) Google Scholar, Morales and Kania, 2017Morales D. Kania A. Cooperation and crosstalk in axon guidance cue integration: Additivity, synergy, and fine-tuning in combinatorial signaling.Dev. Neurobiol. 2017; 77: 891-904Crossref PubMed Scopus (30) Google Scholar). Although elegant, this design strategy risks introducing signaling noise due to the temporal and spatial overlap of competing guidance signals. Nevertheless, the intrinsic filters that sort potentially conflicting instructions within neurons are poorly understood. Axon trajectories are not simply defined by the complement of guidance systems within each neuronal population; rather, they also depend on tight control of receptor deployment through differential splicing, localized synthesis, trafficking, and proteolytic processing that enable neurons to adjust their sensitivity to directional cues at intermediate steps along their projection pathways (Bai and Pfaff, 2011Bai G. Pfaff S.L. Protease regulation: the Yin and Yang of neural development and disease.Neuron. 2011; 72: 9-21Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, Jung et al., 2012Jung H. Yoon B.C. Holt C.E. Axonal mRNA localization and local protein synthesis in nervous system assembly, maintenance and repair.Nat. Rev. Neurosci. 2012; 13: 308-324Crossref PubMed Scopus (3) Google Scholar, O’Donnell et al., 2009O’Donnell M. Chance R.K. Bashaw G.J. Axon growth and guidance: receptor regulation and signal transduction.Annu. Rev. Neurosci. 2009; 32: 383-412Crossref PubMed Scopus (256) Google Scholar). Despite these multiple layers of control, growing axons are often equipped ahead of time with receptors that will be used at later steps of navigation, presumably to ensure timely activation of guidance responses (Dickson and Zou, 2010Dickson B.J. Zou Y. Navigating intermediate targets: the nervous system midline.Cold Spring Harb. Perspect. Biol. 2010; 2: a002055Crossref PubMed Scopus (82) Google Scholar, Keleman et al., 2002Keleman K. Rajagopalan S. Cleppien D. Teis D. Paiha K. Huber L.A. Technau G.M. Dickson B.J. Comm sorts robo to control axon guidance at the Drosophila midline.Cell. 2002; 110: 415-427Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). The risk associated with this preparedness is accidental or premature detection of contradictory instructions from opposing guidance signals, leading to axon targeting errors. While many control mechanisms that operate at the receptor level have been documented, the degree to which intracellular components contribute to correct detection of guidance cues at specific times and places during pathfinding is less clear. A few signaling regulators preferentially associated with individual guidance receptors have been identified (Bashaw and Klein, 2010Bashaw G.J. Klein R. Signaling from axon guidance receptors.Cold Spring Harb. Perspect. Biol. 2010; 2: a001941Crossref PubMed Scopus (181) Google Scholar, O’Donnell et al., 2009O’Donnell M. Chance R.K. Bashaw G.J. Axon growth and guidance: receptor regulation and signal transduction.Annu. Rev. Neurosci. 2009; 32: 383-412Crossref PubMed Scopus (256) Google Scholar); yet, for the most part the downstream nodes of axon guidance pathways appear to be relatively unspecialized compared to cell-surface receptors and ligands. Thus, a common view is that neuron-type-specific connectivity is primarily determined by the selective expression of receptor systems that impinge on a somewhat “generic” obligatory but non-instructive signal transduction machinery, shared among many cell types for the control of basic cell motility and chemotaxis. However, because there is remarkable conservation among the intrinsic components of signaling pathways it has been challenging to distinguish their roles as core effectors versus instructive regulators. We conducted a mouse ENU (N-ethyl-N-nitrosourea) mutagenesis screen designed to identify instructive determinants of axon guidance in spinal motor neurons (Lewcock et al., 2007Lewcock J.W. Genoud N. Lettieri K. Pfaff S.L. The ubiquitin ligase Phr1 regulates axon outgrowth through modulation of microtubule dynamics.Neuron. 2007; 56: 604-620Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The cell bodies of motor neurons reside in the ventral spinal cord, while their axons exit the CNS to connect with target muscles (Bonanomi, 2019Bonanomi D. Axon pathfinding for locomotion.Semin. Cell Dev. Biol. 2019; 85: 26-35Crossref PubMed Scopus (10) Google Scholar, Jessell, 2000Jessell T.M. Neuronal specification in the spinal cord: inductive signals and transcriptional codes.Nat. Rev. Genet. 2000; 1: 20-29Crossref PubMed Scopus (1638) Google Scholar). From our forward genetic mouse screen, we isolated the Cassin mutant, in which separate classes of motor neurons make guidance errors at multiple choice-points for axon turning. The Cassin mutation was mapped to the Arhgap35 gene, which encodes the GTPase-activating protein p190RhoGAP, a negative regulator of RhoA family GTPases required for tissue morphogenesis with evolutionarily conserved roles in axon branching and fasciculation (Billuart et al., 2001Billuart P. Winter C.G. Maresh A. Zhao X. Luo L. Regulating axon branch stability: the role of p190 RhoGAP in repressing a retraction signaling pathway.Cell. 2001; 107: 195-207Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Brouns et al., 2000Brouns M.R. Matheson S.F. Hu K.Q. Delalle I. Caviness V.S. Silver J. Bronson R.T. Settleman J. The adhesion signaling molecule p190 RhoGAP is required for morphogenetic processes in neural development.Development. 2000; 127: 4891-4903Crossref PubMed Google Scholar, Brouns et al., 2001Brouns M.R. Matheson S.F. Settleman J. p190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation.Nat. Cell Biol. 2001; 3: 361-367Crossref PubMed Scopus (200) Google Scholar, Jeong et al., 2012Jeong S. Juhaszova K. Kolodkin A.L. The control of semaphorin-1a-mediated reverse signaling by opposing pebble and RhoGAPp190 functions in Drosophila.Neuron. 2012; 76: 721-734Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We show that p190RhoGAP prevents inappropriate activation of Netrin-DCC signaling when motor axons navigate out of the spinal cord. Despite the classification of p190 as a RhoGAP, gating of Netrin-DCC signaling was independent of the control of Rho GTPases. Our findings identify an intracellular signaling regulator that imposes a context-dependent switch upon the timing of guidance receptor activation to ensure axons ignore incongruous directional signals that would divert their normal path causing miswiring of neural circuits. In order to identify recessive genes involved in the development of spinal motor neurons, we conducted a forward genetic screen in which mice expressing a motor neuron-specific farnesylated-GFP reporter (ISLMN::fGFP) were mutagenized with ENU, and the offspring were intercrossed to generate homozygous mutants (Lewcock et al., 2007Lewcock J.W. Genoud N. Lettieri K. Pfaff S.L. The ubiquitin ligase Phr1 regulates axon outgrowth through modulation of microtubule dynamics.Neuron. 2007; 56: 604-620Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). fGFP-labeled motor projections were visualized in whole embryo flat mounts, which were used to screen for defects in motor axon development (Figure 1A). To increase the probability of unmasking regulatory molecular switches of guidance pathways rather than structural or permissive components, we focused on mutants in which motor axons did not simply display abnormal morphogenesis (e.g., arrested growth, impaired branching) but were diverted from their normal trajectories. With these criteria, we identified a mutant line, named Cassin after the Italian mountaineering explorer, which displayed severe motor axon projection errors characterized by ectopic axon bundles at the pial surface (i.e., the boundary between the CNS and periphery) (Figures 1B, 1C, and S1A–S1F). Rather than leaving the spinal cord through the ventral root exit points, Cassin mutants formed aberrant motor fascicles that extended dorsally in close apposition to the limiting membrane of the spinal cord (Figures 1E–1H). Defects in motor axon exiting were highly penetrant, observed at every spinal cord level, and resulted in the corresponding depletion and disorganization of the ventral roots (Figures S1B, S1C, and S1F). In some instances, ectopic bundles grew in the interstitial space between the sensory dorsal root ganglia and the outer surface of the spinal cord, which is covered by meningeal membranes, but unlike EphA3/A4 mutants were never observed invading the sensory ganglia (Gallarda et al., 2008Gallarda B.W. Bonanomi D. Müller D. Brown A. Alaynick W.A. Andrews S.E. Lemke G. Pfaff S.L. Marquardt T. Segregation of axial motor and sensory pathways via heterotypic trans-axonal signaling.Science. 2008; 320: 233-236Crossref PubMed Scopus (79) Google Scholar). Because motor axons were rerouted onto an ectopic path in Cassin embryos, we reasoned that the ENU mutation might have affected pathfinding rather than a more general aspect of axon growth. Defects in motor projection were not accompanied by a change in total motor neuron number or cell body positioning (Figures 1E–1H and S1H–S1J). Cassin mutants were born at the expected Mendelian ratio but died soon after birth with cyanosis and respiratory failure. Although a recessive mutation, occasionally heterozygous Cassin mutants had small ectopic motor axon bundles, and a minority of homozygous mutant embryos (<2%) displayed neural tube closure defects and exencephaly (Figures S1E, S1G, and 2N ). To visualize the aberrant intraspinal motor axons at higher resolution, we imaged spinal cord “open-book” preparations (Figure S1K). While in wild-type embryos ISLMN::fGFP exclusively labeled the bilateral columns of motor neuron cell bodies, in Cassin mutants numerous ectopic GFP+ axons bundles were found throughout the intermediate region of the spinal cord (Figures 1I–1J). These intraspinal fascicles were formed by axons that converged within each spinal segment (Figures 1K, S1N, and S1O). Although the majority of misprojecting axons remained inside the spinal cord, some were found in the surrounding meninges (Figures S1L and S1M). Thus, a unique hallmark of motor neurons—their ability to project axons through the basal boundary of the neuroepithelium and into the periphery—is severely affected in Cassin mutants (Figure 1L). In addition, Cassin mutants displayed discrete defects in distal projection of subsets of motor neurons. Specifically, thoracic intercostal nerves formed by hypaxial motor neurons were defasciculated (Figures S1P–S1R), and the hindlimb-innervating peroneal nerve displayed an aberrant proximal branch resulting in thinning or ablation of the main nerve tract (Figures S1S–S1U). Taken together, the Cassin mutation affects both proximal (axon exiting) and distal (muscle targeting) pathfinding decisions but does not appear to disrupt axon growth per se. To identify the Cassin mutation with single-base pair resolution, we performed deep sequencing of mRNAs extracted from the spinal cord and surrounding mesenchyme of mutant embryos and control littermates. SNP-discovery identified an A > T transversion in the first exon of the Arhgap35 gene that encodes the Rho GTPase-activating protein p190RhoGAP (hereafter p190). The novel SNP causes the amino acid substitution I602N, which alters a highly conserved residue and is predicted as “deleterious” by in silico tools (see STAR Methods) (Figures 1M–1O). All affected embryos, but none of the phenotypically normal littermates, were homozygous for the mutation. Compared to wild-type embryos, p190 protein levels were strongly reduced in Cassin (p190I602N/I602N) mutants (Figure 1P). When equal amounts of p190 were immunoprecipitated from mutant and control embryonic spinal cords using limiting antibody, p190I602N had lower stoichiometry of tyrosine-phosphorylation and impaired association with the binding partner p120RasGAP—both are indications of altered signaling competence (Figure 1Q) (Bouton et al., 1991Bouton A.H. Kanner S.B. Vines R.R. Wang H.C. Gibbs J.B. Parsons J.T. Transformation by pp60src or stimulation of cells with epidermal growth factor induces the stable association of tyrosine-phosphorylated cellular proteins with GTPase-activating protein.Mol. Cell. Biol. 1991; 11: 945-953Crossref PubMed Scopus (61) Google Scholar, Settleman et al., 1992bSettleman J. Narasimhan V. Foster L.C. Weinberg R.A. Molecular cloning of cDNAs encoding the GAP-associated protein p190: implications for a signaling pathway from ras to the nucleus.Cell. 1992; 69: 539-549Abstract Full Text PDF PubMed Scopus (262) Google Scholar). p190 inhibits RhoA by promoting GTP hydrolysis and contributes to a substantial fraction of Rho-GAP activity in the cell (Settleman et al., 1992aSettleman J. Albright C.F. Foster L.C. Weinberg R.A. Association between GTPase activators for Rho and Ras families.Nature. 1992; 359: 153-154Crossref PubMed Scopus (252) Google Scholar, Vincent and Settleman, 1999Vincent S. Settleman J. Inhibition of RhoGAP activity is sufficient for the induction of Rho-mediated actin reorganization.Eur. J. Cell Biol. 1999; 78: 539-548Crossref PubMed Scopus (68) Google Scholar). As expected, higher amounts of GTP-bound (active) RhoA were pulled down from the spinal cord of p190I602N/I602N embryos (Figure 1R). These results indicate that the Cassin mutation severely affects p190 protein levels and activity. To confirm that the motor axon guidance defects observed in Cassin mutants were due to p190 loss of function, we crossed the ISLMN::fGFP reporter into p190 knockout mice (Brouns et al., 2000Brouns M.R. Matheson S.F. Hu K.Q. Delalle I. Caviness V.S. Silver J. Bronson R.T. Settleman J. The adhesion signaling molecule p190 RhoGAP is required for morphogenetic processes in neural development.Development. 2000; 127: 4891-4903Crossref PubMed Google Scholar). Embryos with a targeted disruption of p190 exhibited motor axon guidance phenotypes similar to Cassin mutants, including failure to exit the spinal cord (Figure 1D), aberrant “bridging” of intercostal nerves (Figure S2K), and limb innervation defects (Figure S2M). As observed for Cassin mutants, p190 knockout mice died at birth, and a subset exhibited neural tube closure defects (Brouns et al., 2000Brouns M.R. Matheson S.F. Hu K.Q. Delalle I. Caviness V.S. Silver J. Bronson R.T. Settleman J. The adhesion signaling molecule p190 RhoGAP is required for morphogenetic processes in neural development.Development. 2000; 127: 4891-4903Crossref PubMed Google Scholar). Next, we crossed the Cassin and p190 knockout alleles to generate embryos heterozygous for both mutations. Compound Cassin and p190 hemizygotes displayed motor projection phenotypes virtually identical to homozygous mutants of the two alleles (Figures 2A–2G and 2N). Lack of complementation between the two recessive mutations demonstrates that the ENU mutant Cassin is a new loss-of-function allele of p190. These findings highlight the critical role of p190 for a discrete subset of motor axon guidance decisions. The specificity of these phenotypes is remarkable in light of the wholesale increase in RhoA activation observed in p190 mutants. p190 is expressed ubiquitously, with enrichment in the developing CNS (Brouns et al., 2000Brouns M.R. Matheson S.F. Hu K.Q. Delalle I. Caviness V.S. Silver J. Bronson R.T. Settleman J. The adhesion signaling molecule p190 RhoGAP is required for morphogenetic processes in neural development.Development. 2000; 127: 4891-4903Crossref PubMed Google Scholar) (Figures S2A and S2B). Despite the widespread distribution, the overall pattern of peripheral nerves comprising both sensory and motor axons appeared largely intact in mutant embryos (Figures S2C and S2D; data not shown). Thus, identification of the p190 mutant was likely only possible because of the motor neuron-specific GFP reporter employed in our screen. The broad expression of p190 raised the question of whether the motor phenotypes observed in mutant mice reflected a requirement in motor neurons or in other cells that may indirectly influence motor axon targeting. To distinguish between these possibilities, we generated a transgenic mouse line (p190-RFPLSL) that enables Cre-dependent expression of RFP-tagged p190 upon removal of a “floxable” STOP cassette (Figure 2H). Olig2MN::Cre (Dessaud et al., 2007Dessaud E. Yang L.L. Hill K. Cox B. Ulloa F. Ribeiro A. Mynett A. Novitch B.G. Briscoe J. Interpretation of the sonic hedgehog morphogen gradient by a temporal adaptation mechanism.Nature. 2007; 450: 717-720Crossref PubMed Scopus (412) Google Scholar) was used to achieve restricted expression of p190-RFP in motor neurons at levels ∼2.5-fold higher than endogenous p190 (Figures 2J, 2K, and S2E). Next, we combined p190-RFPLSL and Olig2MN::Cre with the p190 knockout allele and visualized motor projections in embryos that lack p190 in all tissues except motor neurons expressing exogenous p190-RFP (Figure 2I). Reestablishing p190 expression in motor neurons was sufficient to correct all motor axon guidance phenotypes of p190 mutants, including spinal cord exiting defects and abnormal projections of intercostal and limb nerves (Figures 2L–2N and S2F–S2N). Therefore, p190 functions intrinsically within motor neurons for proper axonal targeting. In Cassin mutants, p190 loss of function leads to an abnormal increase in active RhoA. To address whether the control of motor axon targeting by p190 was mediated by its inhibitory effect on Rho, we employed CRISPR/Cas-mediated gene targeting to generate mice harboring a point mutation in p190 that disrupts the catalytic arginine R1284 required for GAP activity (Li et al., 1997Li R. Zhang B. Zheng Y. Structural determinants required for the interaction between Rho GTPase and the GTPase-activating domain of p190.J. Biol. Chem. 1997; 272: 32830-32835Crossref PubMed Scopus (62) Google Scholar) (Figure 3A). The p190R1284A variant lacking GAP activity was expressed at levels comparable to the wild-type protein (Figure S3A). Embryos expressing exclusively p190R1284A (p190R1284A/−) were compared to p190+/− controls (see STAR Methods). As expected, loss of GAP activity resulted in a substantial increase in GTP-bound (active) RhoA in p190R1284A embryo spinal cords (Figure S3B). Strikingly, motor axon exiting and intercostal nerves were normal in p190R1284A mutants, whereas the peroneal nerve in the hindlimb was severely affected (Figures 3B–3I). We conclude that distinct motor axon pathways display differential dependence on the GAP activity of p190. These results argue for a non-canonical function of p190 in mediating motor axon exiting from the spinal cord. p190 contains additional signaling motifs besides the GAP domain, including an N-terminal GTP-binding domain (Foster et al., 1994Foster R. Hu K.Q. Shaywitz D.A. Settleman J. p190 RhoGAP, the major RasGAP-associated protein, binds GTP directly.Mol. Cell. Biol. 1994; 14: 7173-7181Crossref PubMed Scopus (48) Google Scholar). Knockin mice containing a point mutation (S36N) that impairs GTP binding display all three distinctive phenotypes of p190 mutants (defective axon exiting, intercostal bridging, and peroneal nerve mistargeting), albeit with reduced expressivity (Figures S3C, S3D, and S3G–S3K, cf. Figures 2N and 7P). Although p190S36N transcript levels were comparable to those of the wild-type allele, p190S36N protein levels were lower (Figures S3E and S3F). Because p190S36N is a hypomorphic mutant, it remains unclear whether GTP binding is required per se for motor pathfinding. Nevertheless, these results provide evidence that motor axons are sensitive to lowering of p190 levels beyond a critical threshold. The proximal projection phenotype of p190 mutants was reminiscent of the defects observed in knockout embryos for the chemokine receptor Cxcr4 (Lieberam et al., 2005Lieberam I. Agalliu D. Nagasawa T. Ericson J. Jessell T.M. A Cxcl12-CXCR4 chemokine signaling pathway defines the initial trajectory of mammalian motor axons.Neuron. 2005; 47: 667-679Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). This similarity led us to hypothesize that p190 might mediate Cxcr4 signaling required for motor axon exiting. To study epistatic interactions, we generated an allelic series of compound p190/Cxcr4 mutants and visualized motor projections with ISLMN::fGFP (Figures 4A–4H and S4E–S4I). Cxcr4−/− embryos displayed ectopic motor axon fascicles at the spinal cord interface, which were more irregular and abundant compared to p190 knockouts, possibly reflecting guidance errors immediately past the exit points (Figures 4F, 4I, and S4A–S4C) (Lieberam et al., 2005Lieberam I. Agalliu D. Nagasawa T. Ericson J. Jessell T.M. A Cxcl12-CXCR4 chemokine signaling pathway defines the initial trajectory of mammalian motor axons.Neuron. 2005; 47: 667-679Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The number of ectopic axon bundles was markedly increased in embryos harboring a combination of p190 and Cxcr4 mutant alleles compared to single mutants (Figure 4I). Strikingly, with the removal of one or two p190 alleles from Cxcr4 knockouts (p190+/−;Cxcr4−/− or p190−/−;Cxcr4−/−) the vast majority of motor axons failed to reach the periphery (Figures 4G–4K′, S4D, and S4G–S4I). To further examine the relationship between p190 and Cxcr4 signaling, we used in vitro assays. Since the transient expression of Cxcr4 in motor neurons makes the use of primary cells impractical (Lieberam et al., 2005Lieberam I. Agalliu D. Nagasawa T. Ericson J. Jessell T.M. A Cxcl12-CXCR4 chemokine signaling pathway defines the initial trajectory of mammalian motor axons.Neuron. 2005; 47: 667-679Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), we differentiated motor neurons from either wild-type or p190 mouse embryonic stem cells (ESCMN). ESCMN expressed Cxcr4 and activated signaling in response to Cxcl12 (Figure S4J; data not shown). Cxcl12 treatment enhanced motor axon outgrowth in both wild-type and p190-null ESCMN neurospheres to a comparable extent (Figures 4L–4P). In addition, chemokine signaling was normal in p190−/− ESCMN (Figure S4J), and the levels of tyrosine-phosphorylated p190 did not change after Cxcl12 stimulation (Figure S4K). Thus, Cxcl12/Cxcr4 signaling promotes motor axon growth independently of p190. Counter to our initial hypothesis, these results indicate that p190 and Cxcr4 function in parallel pathways that synergize to instruct motor axon projection from the spinal cord (Figure S4L). p190 has been linked to semaphorin signaling in other systems, and mouse mutants with impaired Sema3A and/or Sema3F activity display intercostal nerve defasciculation similar to p190 mutants (Barberis et al., 2005Barberis D. Casazza A. Sordella R. Corso S. Artigiani S. Settleman J. Comoglio P.M. Tamagnone L. p190 Rho-GTPase activating protein associates with plexins and it is required for semaphorin signalling.J. Cell Sci. 2005; 118: 4689-4700Crossref PubMed Scopus (72) Google Scholar, Huber et al., 2005Huber A.B. Kania A. Tran T.S. Gu C. De Marco Garcia N. Lieberam I. Johnson D. Jessell T.M. Ginty D.D. Kolodkin A.L. Distinct roles for secreted semaphorin signaling in spinal motor axon guidance.Neuron. 2005; 48: 949-964Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, Jeong et al., 2012Jeong S. Juhaszova K. Kolodkin A.L. The control of semaphorin-1a-mediated reverse signaling by opposing pebble and RhoGAPp190 functions in Drosophila.Neuron. 2012; 76: 721-734Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). However, we found that motor axon exiting was normal in either Sema3A or Sema3F mutant embryos. In addition, the severity of the p190−/− phenotype was not significantly altered in p190/Sema3A and p190/Sema3F double knockouts, indicating that these semaphorins are dispensable for motor axon exiting and are not implicated in the defects observed in p190 mutants (Figure S4M) In order to gain insight into the signaling pathways that require p190 to control motor axon guidance, motor neurons isolated from p190−/− embryos were gene profiled using RNA sequencing (RNA-seq). This analysis did not reveal gene expression changes that could underlie defects in motor neuron development (Figure S5A). Next, p190 was immunoprecipitated from the ventral spinal cord of e12.5 embryos, and the associated proteins were identified by tandem mass spectrometry. Bioinformatic analysis of p190-interacting proteins revealed enrichment in regulators of cell morphogenesis, cytoskeletal organization, and axon guidance (Figure S5B). Of particular interest was the identification of components of Netrin signaling, a conserved guidance pathway that influences motor neuron projection (Poliak et al., 2015Poliak S. Morales D. Croteau L.P. Krawchuk D. Palmesino E. Morton S. Cloutier J.F. Charron F. Dalva M.B. Ackerman S.L. et al.Synergistic integration of Netrin and ephrin axon guidance signals by spinal motor neurons.eLife. 2015; (Published online December 3, 2015)https://doi.org/10.7554/eLife.10841Crossref PubMed Scopus (52) Google Scholar, Serafini et al., 1994Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6.Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1161) Google Scholar). Moreover, the prominent p190 interactor p120RasGAP functions downstream of the Netrin receptor DCC in cortical neurons (Antoine-Bertrand et al., 2016Antoine-Bertrand J. Duquette P.M. Alchini R. Kennedy T.E. Fournier A.E. Lamarche-Vane N. p120RasGAP protein mediates Netrin-1 protein-induced cortical axon outgrowth and guidance.J. Biol. Chem. 2016; 291: 4589-4602Crossref PubMed Scopus (9) Google Scholar). These findings prompted us to examine whether p190 participates in Netrin-1/DCC signaling and whether this guidance system is required for motor axon exiting. DCC was detected on motor axons emerging from ventral exit points (Figures 5A and 5B" @default.
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• W2923081206 date "2019-05-01" @default.
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• W2923081206 title "p190RhoGAP Filters Competing Signals to Resolve Axon Guidance Conflicts" @default.
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• W2923081206 doi "https://doi.org/10.1016/j.neuron.2019.02.034" @default.
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