Grb2-associated binder-1 is required for extrafusal and intrafusal muscle fiber development.

The neuregulin-1 (NRG1) signaling pathway plays an important role in the development of the peripheral neuromuscular system, including in muscle spindle and postnatal myelination. We previously showed that NRG1 on the axonal membrane regulates peripheral nerve myelination through Grb2-associated binder 1 (Gab1), a scaffolding mediator of receptor tyrosine kinase signaling. Here, we determined the role of Gab1 in the development of muscles and the muscle spindle using muscle-specific conditional Gab1 knockout mice. The mutant mice showed general retardation in muscular growth and hypotrophy of extrafusal muscle fibers. In addition, the muscle-specific Gab1 knockout mutant exhibited significant underdevelopment of muscle spindles, which are normally regulated by NRG1, and abnormal proprioceptive behavior. Furthermore, the selective knockdown of Gab1 in C2C12 muscle cells reduced NRG1-induced expression of Egr3, a critical transcription factor for muscle spindle development. However, Gab2 knockout mice did not show any defects in the development of muscles or muscle spindles. Our findings suggest that Gab1 is an essential signaling molecule in mediating axonal NRG1 signaling for the development of both extrafusal and intrafusal muscle fibers.

Differential regulation of AChR clustering in the polar and equatorial region of murine muscle spindles.

Intrafusal fibers of muscle spindles are innervated in the central region by afferent sensory axons and at both polar regions by efferent γ-motoneurons. We previously demonstrated that both neuron-muscle contact sites contain cholinergic synapse-like specialisation, including aggregates of the nicotinic acetylcholine receptor (AChR). In this study we tested the hypothesis that agrin and its receptor complex (consisting of LRP4 and the tyrosine kinase MuSK) are involved in the aggregation of AChRs in muscle spindles, similar to their role at the neuromuscular junction. We show that agrin, MuSK and LRP4 are concentrated at the contact site between the intrafusal fibers and the sensory- and γ-motoneuron, respectively, and that they are expressed in the cell bodies of proprioceptive neurons in dorsal root ganglia. Moreover, agrin and LRP4, but not MuSK, are expressed in γ-motoneuron cell bodies in the ventral horn of the spinal cord. In agrin- and in MuSK-deficient mice, AChR aggregates are absent from the polar regions. In contrast, the subcellular concentration of AChRs in the central region where the sensory neuron contacts the intrafusal muscle fiber is apparently unaffected. Skeletal muscle-specific expression of miniagrin in agrin(-/-) mice in vivo is sufficient to restore the formation of γ-motoneuron endplates. These results show that agrin and MuSK are major determinants during the formation of γ-motoneuron endplates but appear dispensable for the aggregation of AChRs at the central region. Our results therefore suggest different molecular mechanisms for AChR clustering within two domains of intrafusal fibers.

Bace1 and Neuregulin-1 cooperate to control formation and maintenance of muscle spindles.

The protease ß-secretase 1 (Bace1) was identified through its critical role in production of amyloid-ß peptides (Aß), the major component of amyloid plaques in Alzheimer's disease. Bace1 is considered a promising target for the treatment of this pathology, but processes additional substrates, among them Neuregulin-1 (Nrg1). Our biochemical analysis indicates that Bace1 processes the Ig-containing ß1 Nrg1 (IgNrg1ß1) isoform. We find that a graded reduction in IgNrg1 signal strength in vivo results in increasingly severe deficits in formation and maturation of muscle spindles, a proprioceptive organ critical for muscle coordination. Further, we show that Bace1 is required for formation and maturation of the muscle spindle. Finally, pharmacological inhibition and conditional mutagenesis in adult animals demonstrate that Bace1 and Nrg1 are essential to sustain muscle spindles and to maintain motor coordination. Our results assign to Bace1 a role in the control of coordinated movement through its regulation of muscle spindle physiology, and implicate IgNrg1-dependent processing as a molecular mechanism.

γ-Aminobutyric acid-, glycine-, and glutamate-immunopositive boutons on mesencephalic trigeminal neurons that innervate jaw-closing muscle spindles in the rat: ultrastructure and development.

Unlike other primary sensory neurons, the neurons in the mesencephalic trigeminal nucleus (Vmes) receive most of their synaptic input onto their somata. Detailed description of the synaptic boutons onto Vmes neurons is crucial for understanding the synaptic input onto these neurons and their role in the motor control of masticatory muscles. For this, we investigated the distribution of γ-aminobutyric acid (GABA)-, glycine-, and glutamate-immunopositive (+) boutons on Vmes neurons and their ultrastructural parameters that relate to transmitter release: Vmes neurons that innervate masseteric muscle spindles were identified by labeling with horseradish peroxidase injected into the muscle, and immunogold staining and quantitative ultrastructural analysis of synapses onto these neurons were performed in adult rats and during postnatal development. The bouton volume, mitochondrial volume, and active zone area of the boutons contacting labeled somata (axosomatic synapses) were similar to those of boutons forming axoaxonic synapses with Vmes neurons but smaller than those of boutons forming axodendritic or axosomatic synapses with most other neurons. GABA+ , glycine+ , and glutamate+ boutons constituted a large majority (83%) of all boutons on labeled somata. A considerable fraction of boutons (28%) was glycine(+) , and all glycine+ boutons were also GABA+ . Bouton size remained unchanged during postnatal development. These findings suggest that the excitability of Vmes neurons is determined to a great extent by GABA, glycine, and glutamate and that the relatively lower synaptic strength of axosomatic synapses may reflect the role of the Vmes neurons in modulating orofacial motor function.

Muscle spindle composition and distribution in human young masseter and biceps brachii muscles reveal early growth and maturation.

Significant changes in extrafusal fiber type composition take place in the human masseter muscle from young age, 3-7 years, to adulthood, in parallel with jaw-face skeleton growth, changes of dentitions and improvement of jaw functions. As motor and sensory control systems of muscles are interlinked, also the intrafusal fiber population, that is, muscle spindles, should undergo age-related changes in fiber type appearance. To test this hypothesis, we examined muscle spindles in the young masseter muscle and compared the result with previous data on adult masseter spindles. Also muscle spindles in the young biceps brachii muscle were examined. The result showed that muscle spindle composition and distribution were alike in young and adult masseter. As for the adult masseter, young masseter contained exceptionally large muscle spindles, and with the highest spindle density and most complex spindles found in the deep masseter portion. Hence, contrary to our hypothesis, masseter spindles do not undergo major morphological changes between young age and adulthood. Also in the biceps, young spindles were alike adult spindles. Taken together, the results showed that human masseter and biceps muscle spindles are morphologically mature already at young age. We conclude that muscle spindles in the human young masseter and biceps precede the extrafusal fiber population in growth and maturation. This in turn suggests early reflex control and proprioceptive demands in learning and maturation of jaw motor skills. Similarly, well-developed muscle spindles in young biceps reflect early need of reflex control in learning and performing arm motor behavior.

[Influence of a liquid diet on the jaw-closing muscle spindles in growing rats].

Recently, it has been shown that prolonged feeding of a liquid diet after being weaned impedes the functional development and leads to immature mastication in growing rats. Since the jaw muscle spindles play an important role in the control of the jaw movement during the normal masticatory function, in this study we investigated the effects of prolonged feeding of a liquid diet after being weaned on the functional development of the jaw-closing muscle spindles in growing rats. Soon after weaning, 40 female Wistar rats were divided into two equal groups. The control group was fed a solid diet and the experimental group was fed a liquid diet. At 5, 7, 9 and 11 weeks, the rats were anesthetized and the response of the masseter muscle spindles to ramp-and-hold jaw stretches were recorded from the mesencephalic trigeminal nucleus. In the experimental groups, both the dynamic and the static indices were significantly lower than those of the control groups at the age of 5, 7, 9 and 11 weeks old. There was no significant change within the same group during the experimental period in both indices. These results suggest that the long-term masticatory functional change due to feeding of a liquid diet may impede the maturation of the functional properties of the jaw-closing muscle spindles, leading to immature mastication in growing rats.

α-Dystroglycan is essential for the induction of Egr3, a transcription factor important in muscle spindle formation.

Muscle spindle fibers are specialized stretch receptors that allow the perception and coordination of limb movement. The differentiation of these specialized structures is initiated by signals derived from the in growing Ia sensory neurons during development. While the direct molecular signaling mechanisms between sensory neurons and developing muscle at nascent spindle fibers have been well documented in past studies the roles of muscle basal lamina components on this process have not previously been described. As such, our initial experiments addressed potential roles for agrin (AGRN) and laminin (LN) in the expression of the transcription factor Egr3. Levels of Egr3 were monitored using immunoblot analysis and both basal lamina molecules proved effective in inducing Erg3 expression. Previous work had established neuregulin (NRG) as a critical signaling component in spindle fiber development so blocking experiments with NRG and ErbB inhibitors were then used to determine if LN-induced Egr3 expression was occurring as a result of NRG-ErbB signaling and not via other, novel pathway. Inhibiting signaling through this pathway did indeed reduce the expression of Egr3. Finally, we looked at alpha-dystrogylcan, a shared receptor for AGRN and LN at neuromuscular junctions. Using a alpha-dystroglycan (alpha-DG) silenced muscle cell line and an anti-alpha-DG antibody we attempted to block basal lamina/alpha-DG interactions. Again, and in both instances, Egr3 expression was significantly decreased. Taken together, analysis of the results from these experiments revealed that indeed AGRN, LN, and alpha-DG influence Egr3 levels and therefore may play an important role in spindle fiber differentiation.

A comparison of the postnatal development of muscle-spindle and periodontal-ligament neurons in the mesencephalic trigeminal nucleus of the rat.

The trigeminal mesencephalic nucleus (Vmes) is known to include primary afferent neurons of jaw muscle spindles (MS neurons) and periodontal ligament receptors (PL neurons). The aim of this study was to clarify the postnatal development of Vmes neurons by comparing MS neurons with PL neurons using horseradish peroxidase labeling. We measured somal diameter and somal shape of MS and PL neurons in rats from postnatal day (P)7 to P70. No significant changes were seen between postnatal day P7 and P70 in somal diameter or somal shape of MS neurons. Conversely, PL neurons showed a larger somal diameter at P7 than at P14, and in terms of somal profile, multipolar neurons comprised 0% at P7, but 4.8% at P14 and 16.9% at P70. These findings suggest that PL neurons develop with the eruption of teeth, taking into account the fact that tooth eruption occurs from around P14 in rats. Conversely, the lack of postnatal changes in MS neurons is due to the fact that these neurons have been active since the embryonic period, as swallowing starts in utero.

Retention of Pax3 expression in satellite cells of muscle spindles.

Intrafusal fibers within muscle spindles retain features characteristic of immaturity, unlike the larger and more numerous extrafusal fibers constituting the bulk of skeletal muscle. Satellite cells (SCs), myogenic progenitors, are detected on the surfaces of both intrafusal and extrafusal fibers, but little is known of spindle SCs. We have recently demonstrated that, like their extrafusal counterparts, SCs in muscle spindles of posthatch chickens express paired box transcription factor 7 (Pax7) protein. During vertebrate embryogenesis, myogenic progenitors express both Pax7 and Pax3 proteins. In postnatal mice, Pax3 appears in rare SC subsets, whereas Pax7 is expressed by all SCs within extrafusal fibers. Here we test the hypothesis that Pax3 protein maintains localized expression within SCs of muscle spindles. Immunohistochemical techniques were used to identify SCs by their Pax7 expression within anterior latissimus dorsi muscle excised from posthatch chickens of various ages. A greater percentage of SCs express Pax3 within intrafusal than extrafusal fibers at each age, and the proportion of SCs expressing Pax3 declines with aging. This is the first study to localize Pax3 expression in posthatch avian muscle and within SCs of muscle spindles. We suggest that Pax3-positive SCs are involved in fiber maintenance.

The neurotrophic effects of glial cell line-derived neurotrophic factor on spinal motoneurons are restricted to fusimotor subtypes.

Glial cell line-derived neurotrophic factor (GDNF) regulates multiple aspects of spinal motoneuron (MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor alpha1 (GFRalpha1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFRalpha1, or GDNF was deleted and replaced by reporter alleles. Whereas nearly all hindlimb muscles exhibited normal gross innervation, intrafusal muscle spindles displayed a significant loss of innervation in most but not all muscles at P0. Furthermore, we observed a dramatic and restricted loss of small myelinated axons in the lumbar ventral roots of adult mice in which the function of either Ret or GFRalpha1 was inactivated in MNs early in development. Finally, we demonstrated that the period during which spindle-innervating MNs require GDNF for survival is restricted to early neonatal development, because mice in which the function of Ret or GFRalpha1 was inactivated after P5 failed to exhibit denervation of muscle spindles or MN loss. Therefore, although GDNF influences several aspects of MN development, the survival-promoting effects of GDNF during programmed cell death are mostly confined to spindle-innervating MNs.

Regulation of low affinity neurotrophin receptor (p75(NTR)) by early growth response (Egr) transcriptional regulators.

The low affinity neurotrophin receptor p75(NTR) is a multifunctional receptor with important roles in neurotrophin signaling, axon outgrowth, and oligodendroglia and neuron survival. It is transcriptionally regulated with spatial and temporal precision during nervous system development, injury and regeneration. Very little is known about how p75(NTR) expression is dynamically regulated but it is likely to influence how p75(NTR) signals in particular cellular contexts. Here, we identify the early growth response (Egr) transcriptional regulators, Egr1 and Egr3, as direct modulators of p75(NTR) gene expression. Egr1 and Egr3 bind and transactivate the p75(NTR) promoter in vitro and in vivo, using distinct response elements on the p75(NTR) promoter. Consistent with these results, p75(NTR) expression is greatly diminished in muscle spindle stretch receptors and in peripheral nerve Schwann cells in Egr gene deficient mice. Taken together, the results elucidate a novel mechanism whereby Egr proteins can directly modulate p75(NTR) expression and signaling in vivo.

Distribution of slow muscle fiber of muscle spindle in postnatal rat masseter muscle.

We investigated the properties of the muscle spindle in the masseter muscle at an immunohistochemical level in rats fed for 6 weeks. Slow myosin heavy chain (MyHC) isoforms were measured and intrafusal fibers in the muscle spindle were studied to determine the relationship between the superficial and deep regions of rat masseter muscle after alternated feeding pattern. However, muscle spindles were found in both regions, mainly in the deep region of the posterior superficial region of masseter muscle. The total number of the slow fiber in the intrafusal fiber and number of muscle spindle in the deep region were high from 5 to 8 weeks old in spite of various dimensions of data such as diameter and the compositions of the intrafusal fiber. The relationship of the protein expression of slow MyHC in the two regions at 5 weeks old reversed five weeks later (10 weeks old). This period is an important stage because the mastication system in masseter muscle with muscle spindle may be changed during the alternated feeding pattern of suckling to mastication. The changes may be a marker of the feeding system and of the control by the tension receptor of muscle spindle in this stage of masseter muscle after postnatal development.

Expression of vesicular glutamate transporter 1 immunoreactivity in peripheral and central endings of trigeminal mesencephalic nucleus neurons in the rat.

The major neuronal components of the trigeminal mesencephalic nucleus (Vmes) are primary afferent neurons that convey proprioceptive information from the cranioorofacial regions. In the present study, we examined expression of vesicular glutamate transporters (VGLUTs), VGLUT1 and VGLUT2, in the primary afferent neurons of the Vmes (Vmes neurons) in neonatal and adult rats. VGLUT1 immunoreactivity was detected in the cell bodies of Vmes neurons in neonatal rats younger than 11 days old, but not in older rats. However, in situ hybridization signals for VGLUT1 mRNA were detected in both neonatal and adult rats. No VGLUT2 immunoreactivity was detected in Vmes neurons of neonatal or adult rats. VGLUT1 immunoreactivity was also seen in the peripheral sensory endings on the equatorial regions of intrafusal fibers of muscle spindles in the masseter muscles in both neonatal and adult rats. In adult rats injected with cholera toxin B subunit (CTb) into the masseter nerve, central axon terminals of Vmes neurons were identified on masseter motoneurons within the trigeminal motor nucleus (Vm) by transganglionically and retrogradely transported CTb. VGLUT1-immunopositive axon terminals in close apposition to CTb-labeled Vm motoneurons were also detected by dual-immunofluorescence histochemistry for VGLUT1/CTb. Electron microscopy after dual immunolabeling for VGLUT1/CTb by the VGLUT1/immunoperoxidase and CTb/immunogold-silver methods further revealed synaptic contact of VGLUT1- and CTb-immunopositive axon terminals upon CTb-labeled neuronal profiles within the Vm. These data indicate that VGLUT1 is expressed in both the central axon terminals and the peripheral sensory endings of Vmes neurons, although no VGLUT1 immunoreactivity was detectable in the cell bodies of Vmes neurons in adult rats.

Myf5 expression in satellite cells and spindles in adult muscle is controlled by separate genetic elements.

The myogenic regulatory factor Myf5 is integral to the initiation and control of skeletal muscle formation. In adult muscle, Myf5 is expressed in satellite cells, stem cells of mature muscle, but not in the myonuclei that sustain the myofibre. Using the Myf5(nlacZ/+) mouse, we now show that Myf5 is also constitutively expressed in muscle spindles-stretch-sensitive mechanoreceptors, while muscle denervation induces extensive reactivation of the Myf5 gene in myonuclei. To identify the elements involved in the regulation of Myf5 in adult muscle, we analysed reporter gene expression in a transgenic bacterial artificial chromosome (BAC) deletion series of the Mrf4/Myf5 locus. A BAC carrying 140 kb upstream of the Myf5 transcription start site was sufficient to drive all aspects of Myf5 expression in adult muscle. In contrast, BACs carrying 88 and 59 kb upstream were unable to drive consistent expression in satellite cells, although expression in muscle spindles and reactivation of the locus in myonuclei were retained. Therefore, as during development, multiple enhancers are required to generate the full expression pattern of Myf5 in the adult. Together, these observations show that elements controlling adult Myf5 expression are genetically separable and possibly distinct from those that control Myf5 during development. These studies are a first step towards identifying cognate transcription factors involved in muscle stem cell regulation.

Motor, sensory and autonomic nerve terminals containing NAP-22 immunoreactivity in the rat muscle.

Neuron-enriched acidic protein having a molecular mass of 22 kDa, NAP-22, is a Ca(2+)-dependent calmodulin-binding protein and is phosphorylated with protein kinase C (PKC). This protein is localized to the biological membrane via myristoylation and found in the membrane fraction of the brain and in the synaptic vesicle fraction. Recent studies showed that NAP-22 is localized in the membrane raft domain in a cholesterol-dependent manner and suggest a role for NAP-22 in maturation and/or maintenance of nerve terminals by controlling cholesterol-dependent membrane dynamics. The present study revealed the immunohistochemical distribution of NAP-22 in the peripheral nerves in rat muscles. In all examined muscles, nerve terminals in the motor endplates showed NAP-22 immunoreactivity associated with the membranes of synaptic vesicles and nerve terminals. In the muscle spindles, annulospiral endings, which made spirals around the intrafusal muscles, showed intense NAP-22 immunoreactivity. Autonomic nerve fibers around the intramuscular blood vessels also showed the immunoreactivity for NAP-22. NAP-22 immunoreactivity in these peripheral nerves was observed from birth to adulthood (100 days after birth). Though growth-associated protein-43 (GAP-43) immunoreactivity in these nerves was observed from birth, this immunoreactivity decreased from 20 days after birth. These findings suggest that NAP-22 is distributed and regulates functions in the motor, sensory and autonomic nerve terminals in the peripheral nervous system.

Peripheral NT3 signaling is required for ETS protein expression and central patterning of proprioceptive sensory afferents.

To study the role of NT3 in directing axonal projections of proprioceptive dorsal root ganglion (DRG) neurons, NT3(-/-) mice were crossed with mice carrying a targeted deletion of the proapoptotic gene Bax. In Bax(-/-)/NT3(-/-) mice, NT3-dependent neurons survived and expressed the proprioceptive neuronal marker parvalbumin. Initial extension and collateralization of proprioceptive axons into the spinal cord occurred normally, but proprioceptive axons extended only as far as the intermediate spinal cord. This projection defect is similar to the defect in mice lacking the ETS transcription factor ER81. Few if any DRG neurons from Bax(-/-)/NT3(-/-) mice expressed ER81 protein. Expression of a NT3 transgene in muscle restored DRG ER81 expression in NT3(-/-) mice. Finally, addition of NT3 to DRG explant cultures resulted in induction of ER81 protein. Our data indicate that NT3 mediates the formation of proprioceptive afferent-motor neuron connections via regulation of ER81.

Erbb2 regulates neuromuscular synapse formation and is essential for muscle spindle development.

Neuregulins and their Erbb receptors have been implicated in neuromuscular synapse formation by regulating gene expression in subsynaptic nuclei. To analyze the function of Erbb2 in this process, we have inactivated the Erbb2 gene in developing muscle fibers by Cre/Lox-mediated gene ablation. Neuromuscular synapses form in the mutant mice, but the synapses are less efficient and contain reduced levels of acetylcholine receptors. Surprisingly, the mutant mice also show proprioceptive defects caused by abnormal muscle spindle development. Sensory Ia afferent neurons establish initial contact with Erbb2-deficient myotubes. However, functional spindles never develop. Taken together, our data suggest that Erbb2 signaling regulates the formation of both neuromuscular synapses and muscle spindles.

The effect of aging on dynamic position sense at the ankle.

The present study addressed whether dynamic position sense at the ankle--or sense of position and velocity during movement--shows a similar decline as a result of aging as previously described for static position sense and movement detection threshold. Additionally, the involvement of muscle spindle afferents in the possible age-related decline was studied. To assess dynamic position sense, blindfolded subjects had to open the hand briskly when the right ankle was rotating passively through a prescribed target angle. To assess the involvement of muscle spindles, the effect of tibialis anterior vibration was studied. The results showed that aging lead to a significant increase in deviation from the target angle at hand opening as well as in variability of performance. Vibration resulted in larger undershoot errors in the elderly compared to the young adults, suggesting that the age-related decline in performance on the dynamic position sense task is not (solely) due to muscle spindle function changes. Alternatively, this degeneration might be due to altered input from other sources of proprioceptive input, such as skin receptors. The elderly subjects did show a beneficial effect of practice with the task, which may provide solid fundaments for rehabilitation.

Tetranectin in slow intra- and extrafusal chicken muscle fibers.

Tetranectin is a C-type lectin that occurs in the mammalian musculoskeletal system. In the present report we describe the first studies on an avian tetranectin. A full-length chicken tetranectin cDNA was isolated. Comparison of the deduced amino acid sequence of chicken tetranectin with mouse and human tetranectin showed an identity of 67 and 68%, respectively. Northern blot analysis demonstrated broad expression of chicken tetranectin mRNA, which was first detected on embryonic day 4. Tetranectin protein was detected in chicken serum and egg yolk. Since muscle is one of few tissues in which tetranectin protein is retained, we examined the distribution of tetranectin in various muscle types in chicken. Myofibers strongly positive for tetranectin were observed in several muscles including m. tibialis ant. and m. sartorius (from embryonic day 10 to adult). Using antibodies to fast and slow myosin heavy chains (MHC) and double immunostaining techniques, we found that tetranectin was restricted to slow (type I) muscle fibers. Similarly only slow intrafusal fibers accumulated tetranectin. The pattern of immunostaining in chickens differs markedly from that seen in mouse muscles, indicating that tetranectin performs a role in muscle that is not associated with a hitherto recognized muscle type or function.

The transcription factor Egr3 modulates sensory axon-myotube interactions during muscle spindle morphogenesis.

The Egr family of zinc-finger transcription factors, consisting of Egr1, Egr2, Egr3, and Egr4, are involved in cellular growth and differentiation. Adult Egr3-deficient mice are ataxic and lack muscle spindle proprioceptors that normally develop at the sites of Ia afferent-myotube contacts during embryogenesis. To resolve whether spindles form and then degenerate, or whether they never form in the absence of Egr3, we examined the spatiotemporal expression of Egr3 relative to spindle development. In wild type mice, Egr3 was expressed in developing myotubes shortly after they were innervated by Ia afferents and its expression was controlled by innervation because it dissipated following nerve transection. In Egr3-deficient mice, myotubes received Ia afferent innervation and assembled normally into spindles during embryogenesis. However, newborn Egr3-deficient spindles had few internal myonuclei in intrafusal fibers and thin capsules. Moreover, slow-developmental myosin heavy chain was not induced in embryonic Egr3-deficient spindles suggesting that impairments in differentiation were present before they could be detected morphologically. After birth, sensory and motor innervation withdrew from the Egr3-deficient spindles, and the spindles disassembled. In spite of the spindle disassembly and retraction of afferents from muscles, the cell bodies of proprioceptive neurons within dorsal root ganglia were retained. We conclude that Egr3 has an essential role in regulating genes required for the transformation of undifferentiated myotubes into intrafusal fibers, and hence for the phenotypic differentiation of spindles.