NT-3 Is Required for Proper Peripheral Innervation
In order to study peripheral innervation in double KO animals, we investigated spindle development in the gastrocnemius muscle. Muscle spindles could be identified easily in P0 WT and Bax KO animals by their characteristic morphology (Figure 4A and 4B). Proprioceptive fibers labeled with neurofilament-M (NF-M) antibody formed ring-like spiral endings wrapped around intrafusal bag fibers labeled with S46 antibody, specific for slow developmental myosin heavy chain protein (Miller et al. 1985). As reported earlier, there were no muscle spindles in NT-3 KO animals (Ernfors et al. 1994). On the other hand, Bax KO animals had more spindles than WT, and spindles were in clusters similar to animals over-expressing NT-3 in the muscle (Wright et al. 1997). Although NT-3-dependent cells were rescued, no muscle spindles were detected in the limbs of double KOs (Figure 4A and 4B). Muscle spindles could be observed with TrkC antibody in WT and Bax KO animals, but not in NT-3 or double KOs (Figure 4E). In both NT-3 and double KO animals, NF-M-labeled fibers could be detected in muscles, thus the muscles of these animals were not completely devoid of nerve fibers. Since there were no TrkC-positive fibers in these muscles, we think that these NF-M-labeled fibers correspond to motor axons. Absence of muscle spindles might be due to a failure in initiation of differentiation by proprioceptive axons, or a failure of maturation and maintenance in the absence of NT-3. To distinguish between these two possibilities, we investigated muscle spindle development at E15 and E17 with S46/NF-M immunostaining as well as DiI labeling. No structures with the characteristic muscle spindle shape could be detected in any of the genotypes at E15 (Figure S2). However, numerous developing spindles could be identified at E17 in WT and Bax KOs, but not in NT-3 KO embryos (Figure 4C). Bax/NT-3 double null muscles were devoid of spindles (Figure 4C), except for one spindle-like structure observed in one leg of an embryo (Figure 4C, inset, denoted by the asterisk). DiI labeling through the DRGs at E17 yielded similar results, with muscle spindles identified in parallel sections in WT and Bax null muscles (Figure 4D), but not in NT-3 or double KO animals. Although DiI-labeled fibers could be detected in double null muscles, they never formed ring-like structures characteristic of muscle spindles. We also examined TrkA/TrkC expression at P0 in the tibial nerve, which carries sensory fibers to the gastrocnemius muscle as well as the skin of the lower leg. In NT-3 KOs, TrkA-positive axons could be seen in the tibial nerve but there were no TrkC-positive axons; in contrast, TrkA- and TrkC-labeled axons are both present in WT, Bax KO, and double KO animals (Figure 4F). These results suggest that although proprioceptive axons follow proper trajectories in distal peripheral nerves, they fail to innervate their target muscles in the absence of NT-3.
Figure 4  Muscle Spindles in Gastrocnemius Muscle and TrkA/TrkC Staining in the Tibial Nerve at P0
(A) NF-M (red) and S46 (green) immunostaining in cross section of gastrocnemius muscle at P0. There are no muscle spindles detected in double KOs.
(B) NF-M (red) and S46 (green) immunostaining in parallel sections of gastrocnemius muscle at P0.
(C) NF-M (red) and S46 (green) immunostaining in parallel sections of gastrocnemius muscle at E17. Double null muscles are mostly devoid of muscle spindles, except for one spindle-like structure detected (shown in inset, denoted by the asterisk).
(D) Muscle spindles detected by DiI labeling through the DRG. Gastrocnemius muscle is sectioned at 40 μm thickness in parallel plane to the muscle fibers.
(E) Muscle spindles detected by TrkC staining in cross section of gastrocnemius muscle at P0.
(F) TrkA (red) and TrkC (green) immunostaining in the tibial nerve cross section at P0. TrkC-positive fibers are missing in NT-3 KOs. Red-green overlap (yellow) is due to the thickness of the section and overlapping of red- and green-labeled (TrkA and TrkC) fibers present at different focal depths, rather than co-localization.
Arrows indicate muscle spindles.
Scale bar: 50 μm (A, B, D), 25 μm (C, E), 75 μm (F).