Fig. 5. Analysis of muscle spindle development. (A,B) EM pictures of a primary myotube being contacted by Ia afferent sensory fibers at E16 in a wild-type (A) and a mutant (B) embryo. Single myotubes (circled black) were contacted by nerve terminals (circled green) as indicated by the arrow. Schwann cells (circled yellow) accompanied the ingrowing afferent fiber. (C,D) At E17, many muscle spindles in wild-type embryos consisted of two intrafusual fibers (black) contacted by multiple nerve endings (green). Schwann cells (yellow) accompanying the spindle nerve bundle formed a rudimentary unilamelar capsule surrounding the developing spindle. In some cases two nuclei were observed at the equatorial plane of the myotube that was developing into a nuclear bag₂ fiber. In muscle in mutant mice (D), myotubes contacted by sensory nerve terminals were still frequently observed close to nerve bundles but muscle spindle differentiation appeared halted. Arrow in D indicates contact between myotube and nerve terminal. Rudimentary spindles were still formed by a single myotube contacted by unexpanded afferent nerve terminals and were not surrounded by Schwann cell processes. Innervated myotubes did not progress towards a nuclear bag phenotype and other myotubes were not recruited as intrafusal fibers. Nevertheless, synaptic contacts by sensory nerve terminals, as revealed by the lack of a basal lamina in the sypnaptic cleft still persisted. (E,F) By E18, spindles in wild-type mice were surrounded by a multi-lamellar spindle capsule, and spindle fibers were innervated by a Ia afferent (boxed contact zone enlarged in E'). Spindles in the mutant mice (F) did not differentiate further. Only one spindle fiber was present, and no capsule had formed. However, contacts between the muscle fiber and Ia-afferent was maintained (boxed area enlarged in F'). (G,H) Plastic sections through P0 muscle of wild-type and mutant mice showed that although muscle spindles in wild-type mice (G) typically consisted of three or four encapsulated multinucleated intrafusal fibers innervated by multiple nerve terminals, spindles in the mutants (H) only consisted of one muscle fiber still surrounded by nerve terminals. Some of the afferents appeared abnormally enlarged and swollen. Scale bars: 2 µm in A-F; 5 µm in G,H.