Segregation of axial motor and sensory pathways via heterotypic trans-axonal signaling

Abstract

Execution of motor behaviors relies on circuitries effectively integrating immediate sensory feedback to efferent pathways controlling muscle activity. It remains unclear how, during neuromuscular circuit assembly, sensory and motor projections become incorporated into tightly coordinated, yet functionally separate pathways. We report that, within axial nerves, establishment of discrete afferent and efferent pathways depends on coordinate signaling between coextending sensory and motor projections. These heterotypic axon-axon interactions require motor axonal EphA3/EphA4 receptor tyrosine kinases activated by cognate sensory axonal ephrin-A ligands. Genetic elimination of trans-axonal ephrin-A --> EphA signaling in mice triggers drastic motor-sensory miswiring, culminating in functional efferents within proximal afferent pathways. Effective assembly of a key circuit underlying motor behaviors thus critically depends on trans-axonal signaling interactions resolving motor and sensory projections into discrete pathways.

Fig. 1

Spontaneous delineation of axial motor and sensory projections during neuromuscular circuit assembly. (A) 120-μm lumbar (L5 to L6) transversal E12.5 section at the DR-VR junction. eGFP, motor axons; anti-βGal, sensory axons (red); MN, motor neurons. (B and C) 120-μm nerve cross sections. Levels, dashed lines in (A). (C) MMCm projections through sensory fascicles. (D and E) 120-μm lumbar E10.0. Sequential advance: nonaxial motor axons, bottom arrowheads; sensory axons, top arrowheads. Asterisks denote emerging MMCm axons. (E) E10.0 dorsal ramus, emerging sensory; MMCm growth cone, top and bottom arrowheads. (F) E12.5 dorsal ramus, segregated sensory-motor pattern; TrkA, tropomyosin-related kinase A. (A to F) Scale bars, 20 μm. (G) Sensory-motor interaction assay (see SOM). MN and DRG explants. (H to J) Motor (green) and sensory (red) axons segregate in vitro. The dashed-line box indicates the enlarged area in (I) and (J); Tujl, βIII-tubulin. (I and J) Axon interface of (H). (K and L) Absence of homotypic axon segregation. (H) Scale bar, 100 μm. (I to L) Scale bar, 100 μm.

Fig. 2

Loss of proximal motor-sensory segregation upon eliminating MMCm-expressed EphA receptors. (A to I) 60-to-120–μm L3 to L5 nerve cross sections: complementary distribution of EphA4 (anti-EphA4), ephrin-As (EphA3-Fc) in motor (eGFP), and sensory axons (TrkA). Scale bar, 25 μm. (J to M) 60-μm thoracic transversal. Scale bars, 100 μm. (K and L) No motor projections in DRG (Isl1/2: sensory nuclei). (L and M) MMCm axons misproject into DRG [arrowhead in (L)]. Dashed line indicates the spinal cord. (N and O) 180-μm transversal. (N) Control motor-sensory trajectories. (O) MMCm axons invade proximal sensory pathways and DRG (arrowheads). Solid lines indicate the DR-VR junction; asterisks denote the dorsal ramus; and dashed lines indicate motor and sensory somas. Scale bar, 50 μm. (P and Q) 120-μm T10 nerve cross. Dotted lines indicate line measurements in (R) and (S). (P) Control motor-sensory trajectories. (Q) Ectopic MMCm axons associate with sensory fascicles (arrowheads). Scale bar, 20 μm. (R and S) Intensity profiles of line measurements indicated in (P) and (Q).

Fig. 3

Axial motor axons within sensory pathways retain functional coupling with spinal locomotor circuits. (A to C) E18.5 DR recordings (top). Genotype-matched VR traces (bottom). Locomotor activity evoked by 10 μM N-methyl D,L-aspartate (NMA) and 20 μM serotonin [5-hydroxytryptamine (5-HT)]. (A) No rhythmic activity in WT DR, rhythmic activity in VR. (B) Some DR activity. (C) Strong rhythmic DR activity. (D and E) Quantitative summary: motor-sensory misrouting (see SOM and fig. S5, A to C). EphA3^+/−;EphA4^−/− (n = 6 spinal cords), EphA3^−/−;EphA4^+/− (n = 8), EphA3^−/−;EphA4^−/− (n = 6). Error bars indicate SD. (F) Autocorrelation analysis. Significance: WT, EphA3/EphA4-deficient VRs (P < 0.001, each); WT DR (P > 0.05), EphA3/EphA4-deficient DR (P < 0.001). WT (n = 6), EphA3/EphA4-deficient (n = 6). Asterisks denote significance; error bars indicate SD.

Fig. 4

Motor axonal EphAs impose sensitivity toward sensory-expressed ephrin-As. (A) EphA3/EphA4 deficiency abolishes ephrin-A–induced MMCm growth-cone collapse in vitro. IgG, immunoglobulin G. (B to E) Example of control-Fc (B and C) and ephrin-A1–Fc (D and E) stimulation. Typical Hb9::eGFP⁺ MMCm growth cones (B and C); ephrin-A1–Fc triggers collapse to filopodialike thread, F-actin⁺TujI⁺ swelling. Scale bar, 10 μm. (F to K) Autonomous EphA3/EphA4 requirement in MMCm axons for repulsion by sensory axons. Scale bars, 100 μm. (F and G) EphA3/EphA4-deficient MMCm axons extensively cross WT sensory axons and (H) invade WT DRGs (dashed outline). (I and J) EphA3/EphA4 null sensory axons repel WT MMCm axons. (K) No WT motor axon invasion of EphA3/EphA4 null DRG. (L) Quantitative summary. Mean number of invasion events per DRG (left); stacked column diagram represents percent invaded DRGs per genotype (right). Explant combinations are indicated. Relative distance is between motor-sensory explants.