Topographic Specificity within Membranes of a Single Muscle DetectedIn Vitro

Abstract

Spinal motor pools project to target muscles forming distinct rostrocaudal topographic maps during development and regeneration. To define the mechanisms underlying these neuromuscular maps we studied the preferential outgrowth of embryonic spinal cord neurites on muscle membranes from different axial positions and explored the role of ephrin A ligands. We found all five ephrin As (EphAs) expressed in serratus anterior, gluteus maximus and diaphragm muscles. In the diaphragm, four of the five ephrin As are expressed as a caudal to rostral gradient. When ephrin A function is disrupted in muscle membranes by deletion of glycosyl-phosphatidylinositol anchored ephrin A ligands with phosphatidylinositol-specific phospholipase C enzyme treatment or by blocking of ephrin A ligands with EphA fusion proteins, or by genetic manipulation leading to ephrin A2/A5 mutant mice, the spinal cord neurites loose their preference for the membranes of corresponding axial position; suggesting a significant role for ephrins in topographic choices made by growing motor neurons. To closely approximate topographic choices presented to embryonic neurites in vivo, neurites within the phrenic motor pool were challenged to make outgrowth choices on membranes of their normal target, the diaphragm muscle. We observed that neurites from rostral cervical segments (C 1 and C 2 ) prefer to grow on rostral diaphragm membranes; caudal cervical neurites (C 6 -C 8 ) choose caudal diaphragm membranes; a transition of positional preference occurs at C 4 and this ability is lost in ephrin A2/A5 mutant mice. These results demonstrate for the first time topographical outgrowth of axons from within a motor pool onto a single target muscle in vitro.

Ephrin-A3 promotes and maintains slow muscle fiber identity during postnatal development and reinnervation

“…Eph/ephrin interactions direct a broad variety of cellular and intercellular processes in the context of neuronal and neuromuscular development ( Kao et al, 2012 ; Lisabeth et al, 2013 ; Laussu et al, 2014 ); the role for Eph/ephrin signaling we describe in the context of myofiber innervation thus adds to the well-known activity of these signaling molecules in motor axon repulsion ( Orioli and Klein, 1997 ), attraction ( Dudanova et al, 2012 ), fasciculation ( Luxey et al, 2013 ), dorsal/ventral patterning ( Luria et al, 2008 ), and topographic mapping ( Chadaram et al, 2007 ). The number and variety of processes mediated by this signaling pathway often results in expression of multiple Ephs and ephrins on the same cell, where they may act cooperatively in cis ( Kao and Kania, 2011 ), independently in trans ( Feng et al, 2000 ), or even oppositely in trans, with opposite effects on the cell from distinct membrane compartments ( Marquardt et al, 2005 ).…”

“…Intriguingly, another aspect of muscle identity that shows specificity of innervation is position on the rostrocaudal axis, for which both muscle and nerves possess a heritable identity ( Donoghue et al, 1992a ) that promotes interactions between cells originating at the same level. This specificity appears to be a result of differential expression of specific ephrins (a family of cell surface contact-mediated repulsive ligands) on myofibers that prevents interactions with neurons from inappropriate axial levels ( Donoghue et al, 1996 ; Feng et al, 2000 ; Chadaram et al, 2007 ).…”

Reverse Signaling by Glycosylphosphatidylinositol-LinkedManducaEphrin Requires a Src Family Kinase to Restrict Neuronal MigrationIn Vivo

“…Moreover, given our demonstration that MsEph-Fc dimers are fully capable of stimulating a reverse signaling response through GPI-linked Ephrins, the widespread use of EphA-Fc constructs to block Ephrin-A forward signaling may require re-evaluation (e.g. Brantley et al, 2002; Chadaram et al, 2007; Passante et al, 2008). …”

Eph/ephrin involvement in skeletal muscle development and regeneration

“…The contractile function of skeletal muscle requires innervation by motor neurons, whose cell bodies reside in the spinal cord and project axons into the muscle according to cell-autonomous topographic maps (Landmesser and Morris, 1975;Landmesser, 1978;Landmesser, 2001;Laskowski and Sanes, 1987;Milner et al, 1998). Involvement of forward, reverse, and autoregulatory Eph/ephrin signals (as well as the activity of other families of contact-mediated guidance proteins) in motor axon pathfinding and neuromuscular synaptogenesis is wellestablished (Chadaram et al, 2007;Dudanova et al, 2012;Franz et al, 2008;Kao and Kania, 2011;Luria et al, 2008;Luxey et al, 2013;Nguyen et al, 2002;Wang et al, 1999); expression data also suggest that Eph/ephrins may act at the neuromuscular junction itself (Lai et al, 2001). Recent work from our group, showed that ephrin-A3 expression exclusively on slow muscle fibers inhibits their innervation by fast motor neurons (which would cause them to convert to fast muscle fibers) as neuromuscular junctions are stabilized during postnatal maturation .…”