Ephexin1 Is Required for Eph-Mediated Limb Trajectory of Spinal Motor Axons

Abstract

The precise assembly of a functional nervous system relies on the guided migration of axonal growth cones, which is made possible by signals transmitted to the cytoskeleton by cell surface-expressed guidance receptors. We investigated the function of ephexin1, a Rho guanine nucleotide exchange factor, as an essential growth-cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using mRNA detection, we first show that ephexin1 is expressed in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Ephexin1 loss of function and gain of function using electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of ephexin1 in motor axon guidance. In addition, ephexin1 loss in mice of either sex led to LMC axon trajectory selection errors. We also show that ephexin1 knockdown attenuates the growth preference of LMC neurites against ephrins and Eph receptor-mediated retargeting of LMC axons, suggesting that ephexin1 is required in Eph-mediated LMC motor axon guidance. Finally, both ephexin1 knockdown and ectopic expression of nonphosphorylatable ephexin1 mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating ephexin1 as an Src target in Eph signal relay in this context. In summary, our findings demonstrate that ephexin1 is essential for motor axon guidance and suggest an important role in relaying ephrin:Eph signals that mediate motor axon trajectory selection. The proper development of functioning neural circuits requires precise nerve connections among neurons or between neurons and their muscle targets. The Eph tyrosine kinase receptors expressed in neurons are important in many contexts during neural-circuit formation, such as axon outgrowth, axon guidance, and synaptic formation, and have been suggested to be involved in neurodegenerative disorders, including amyotrophic lateral sclerosis and Alzheimer's disease. To dissect the mechanism of Eph signal relay, we studied ephexin1 gain of function and loss of function and found ephexin1 essential for the development of limb nerves toward their muscle targets, concluding that it functions as an intermediary to relay Eph signaling in this context. Our work could thus shed new light on the molecular mechanisms controlling neuromuscular connectivity during embryonic development.

Nck2 is essential for limb trajectory selection by spinal motor axons

“…The differential expression of Nck2 in medial LMC pathfinding implies its differential role in LMC subpopulations. Previous studies also indicate the requirement of other key molecules, including Src family kinases, a Rac GTPase activating protein (GAP), α2-chimaerin, and a Rho guanine nucleotide exchange factor (GEF), ephexin1, for the fidelity of axon trajectory from LMC subpopulations (Chang et al, 2018;Kao et al, 2009Kao et al, , 2015. Among these intermediaries, our and other observations suggest the interaction of Nck and α2-chimaerin in several neuronal types (Wegmeyer et al, 2007;Fawcett et al, 2007).…”

“…Also, ephrin:Eph signaling is sufficient to redirect LMC axons (Kania and Jessell, 2003;Luria et al, 2008). To investigate the dependence of Eph signaling on Nck2 function, we tested the response of LMC axons to stripes of ephrin-A or -B proteins in the context of Nck2 loss-or gain-of-function (Kao and Kania, 2011;Chang et al, 2018). HH st. 25/26 LMC explants were dissected and placed onto carpets of two alternating stripes: those containing a mixture of ephrin-Fc and a Cy3 secondary antibody and those containing Fc protein only.…”

“…To investigate the dependence of Eph signaling on Nck2 function, we tested the response of LMC axons to stripes of ephrin-A or -B proteins in the context of Nck2 loss-or gain-of-function (Kao and Kania, 2011;Chang et al, 2018). Stripe preference was scored as the proportion of GFP or EphA4 signal found over the different stripes after overnight explant culture (Chang et al, 2018;Kao et al, 2015). Medial LMC axons were identified by e[Isl1]::GFP electroporation, whereas lateral LMC axons were identified by their EphA4 expression.…”

“…Previous studies also indicate the requirement of other key molecules, including Src family kinases, a Rac GTPase activating protein (GAP), α2-chimaerin, and a Rho guanine nucleotide exchange factor (GEF), ephexin1, for the fidelity of axon trajectory from LMC subpopulations (Chang et al, 2018;Kao et al, 2009Kao et al, , 2015. Previous studies also indicate the requirement of other key molecules, including Src family kinases, a Rac GTPase activating protein (GAP), α2-chimaerin, and a Rho guanine nucleotide exchange factor (GEF), ephexin1, for the fidelity of axon trajectory from LMC subpopulations (Chang et al, 2018;Kao et al, 2009Kao et al, , 2015.…”

“…Medial LMC axons were identified by e[Isl1]::GFP electroporation, whereas lateral LMC axons were identified by their EphA4 expression. Stripe preference was scored as the proportion of GFP or EphA4 signal found over the different stripes after overnight explant culture (Chang et al, 2018;Kao et al, 2015). For statistical analysis, we compared the proportions of neurite growth preference in ephrin (or Fc) stripes between different experimental conditions.…”