NT-3 Is Necessary for Proper Innervation of Motor Neurons
TrkA/TrkC-positive fibers in the spinal cord could be detected at E15 (Figure 1E). TrkA-positive fibers were restricted to and terminated in the dorsolateral spinal cord, whereas TrkC-positive fibers entered the cord dorsomedially, and descended into the ventral horns in WT (Ozaki and Snider 1997) and Bax KO embryos. There was no detectable TrkC expression in NT-3 KO spinal cord, indicating complete absence of proprioceptive fibers. In double KO spinal cord, TrkC-positive fibers entered the dorsal spinal cord and descended medially in a manner similar to that seen in WT or Bax KO cases. However, it was not possible to follow TrkC immunolabeled fibers all the way to their terminal zones in any of the cases. Next we examined the central projections of DRG axons with the lipophilic tracer DiI at P0. In WT and Bax KO pups, proprioceptive afferents entered the dorsal spinal cord and followed a medial course towards the ventral horn. They then turned laterally towards motor neurons in the lateral motor column, where they branched and terminated (Figure 2A). DiI labeling was confined to dorsal spinal cord in NT-3 KOs (Figure 2A), as reported earlier, consistent with a complete absence of proprioceptive innervation (Ernfors et al. 1994; Tessarollo et al. 1994). In double KOs, proprioceptive afferents initially followed a trajectory similar to that of WT counterparts, but most of them failed to project all the way to the ventral cord and into the lateral motor column. Instead, they arborized near the ventral midline; some crossed the midline and extended into the contralateral ventral cord (Figure 2A and 2C). In order to distinguish between a role for NT-3 in initiation of motor neuron innervation and a role for maintenance, we repeated the DiI labeling at E17. Innervation patterns of E17 spinal cords (Figure 3) were similar to those at P0 (Figure 2). Dorsal horns of all genotypes were filled with DiI-labeled fibers corresponding to nerve growth factor–dependent nociceptive axons. In WT and Bax KO embryos, proprioceptive fibers extended towards ventral horn motor neurons (Figure 3A and 3B), whereas the ventral horns of the NT-3 KO embryos were devoid of innervation (Figure 3C). In the Bax/NT-3 double KOs, DiI-labeled fibers entered the ventral spinal cord, but extended towards the midline instead of the ventral horn (Figure 3D), in a pattern similar to that observed at P0. Our data point to a complete absence of proprioceptive innervation of the ventral horn of the Bax/NT-3 null spinal cord throughout the developmental stages investigated. As the sensory axons never reach motor neuron dendrites in the ventral horn (Figure S1), the stretch reflex arc circuit is not established. The failure to initiate contact between sensory axons and motor neurons in the absence of NT-3 suggests a requirement for NT-3 for proper axon targeting in addition to a role in sensory axon maintenance.
Figure 2  Axonal Projections in the Spinal Cord after DiI Labeling of DRG at P0
(A) Rescued DRG proprioceptive neurons fail to properly innervate motor neurons in double KOs. Instead, some axons are directed towards the ventral midline; they cross the midline and branch.
(B) Schematic drawing of the monosynaptic reflex arc as it normally develops. Small black dots represent NT-3 released centrally by the motor neurons and peripherally by the muscle spindles.
(C) High-power magnification of the inset in (A). Arrow points to the midline, and arrowheads point to synaptic bouton-like structures.
Scale bar: 1 mm (A), 400 μm (C).
Figure 3  Sensory Axons Labeled with DiI through the DRG at E17
(A) DiI-labeled fibers in WT spinal cord. Notice proprioceptive axons extending towards the motor neurons located in the ventral horn of the spinal cord in cross section.
(B) Bax null spinal cord.
(C) NT-3 null spinal cord. Stained fibers are restricted to the nociceptive axons in the dorsal horn, as evidenced by the complete absence of labeling in the ventral spinal cord.
(D) Bax/NT-3 double null spinal cord. Although fibers extend into the ventral spinal cord, they never grow towards the motor neurons, but are directed towards the midline instead.
Scale bar: 100 μm.