Formation of cholinergic synapse-like specializations at developing murine muscle spindles.

Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibers, called intrafusal fibers, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. We show that AChRs are concentrated at the γ-motoneuron endplate as well as in the equatorial region where they colocalize with the sensory nerve ending. In addition to the AChRs, the contact site between sensory nerve ending and intrafusal muscle fiber contains a high concentration of choline acetyltransferase, vesicular acetylcholine transporter and the AChR-associated protein rapsyn. Moreover, bassoon, a component of the presynaptic cytomatrix involved in synaptic vesicle exocytosis, is present in γ-motoneuron endplates but also in the sensory nerve terminal. Finally, we demonstrate that during postnatal development of the γ-motoneuron endplate, the AChR subunit stoichiometry changes from the γ-subunit-containing fetal AChRs to the ε-subunit-containing adult AChRs, similar and approximately in parallel to the postnatal subunit maturation at the neuromuscular junction. In contrast, despite the onset of ε-subunit expression during postnatal development the γ-subunit remains detectable in the equatorial region by subunit-specific antibodies as well as by analysis of muscle spindles from mice with genetically-labeled AChR γ-subunits. These results demonstrate an unusual maturation of the AChR subunit composition at the annulospiral endings and suggest that in addition to the recently described glutamatergic secretory system, the sensory nerve terminals are also specialized for cholinergic synaptic transmission, synaptic vesicle storage and exocytosis.

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.

Expression of kin of irregular chiasm-like 3/mKirre in proprioceptive neurons of the dorsal root ganglia and its interaction with nephrin in muscle spindles.

Kin of irregular chiasm-like 3 (Kirrel3), a mammalian homolog of the kirre gene of Drosophila melanogaster, belongs to the immunoglobulin superfamily. Previously, we have reported that Kirrel3 is expressed in the developing and adult central nervous system. In the present study we investigated the expression of Kirrel3 in the mouse dorsal root ganglia (DRG) and their projection targets. In the adult DRGs, Kirrel3 mRNA was detected in 21.5 +/- 2.3% of total DRG neurons and the expression was mainly prevalent in the medium- and large-sized neurons. In addition, Kirrel3 mRNA predominantly colocalized with tyrosine kinase receptor (Trk) C-immunoreactivity. In the developing DRGs, Kirrel3 mRNA was first detected in a few cells at embryonic day (E) 11.5, gradually increased, and reached the adult level at E17.5. During the development, Kirrel3 was expressed in most TrkC-positive DRG neurons. The expression of Kirrel3 was observed in TrkC-positive nerve fibers around neurotrophin 3 (NT3)-positive intrafusal muscle fibers of muscle spindles at E17.5. However, Kirrel3 was not expressed in TrkC-positive nerve fibers projecting to the spinal cord throughout development. Furthermore, nephrin was expressed in the NT3-positive intrafusal muscle fibers and was in close apposition with Kirrel3-immunoreactivity. Coimmunoprecipitation assay revealed that nephrin interacted with Kirrel3 in the developing muscles. These results suggest that Kirrel3 might play a role in the axonal pathfinding, cell recognition, and synapse formation of DRG neurons on appropriate target cells, including the targeting of proprioceptive neurons on muscle spindles through the interaction with nephrin.

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.

Proprioceptive sensory neuropathy in mice with a mutation in the cytoplasmic Dynein heavy chain 1 gene.

Mice heterozygous for the radiation-induced Sprawling (Swl) mutation display an early-onset sensory neuropathy with muscle spindle deficiency. The lack of an H reflex despite normal motor nerve function in the hindlimbs of these mutants strongly suggests defective proprioception. Immunohistochemical analyses reveal that proprioceptive sensory neurons are severely compromised in the lumbar dorsal root ganglia of newborn Swl/+ mice, whereas motor neuron numbers remain unaltered even in aged animals. We have used positional cloning to identify a nine base-pair deletion in the cytoplasmic dynein heavy chain 1 gene (Dync1h1) in this mutant. Furthermore, we demonstrate that Loa/+ mice, which have previously been shown to carry a missense point mutation in Dync1h1 that results in late-onset motor neuron loss, also present with a severe, early-onset proprioceptive sensory neuropathy. Interestingly, in contrast to the Loa mutation, the Swl mutation does not delay disease progression in a motor neuron disease mouse model overexpressing a human mutant superoxide dismutase (SOD1(G93A)) transgene. Together, we provide in vivo evidence that distinct mutations in cytoplasmic dynein can either result in a pure sensory neuropathy or in a sensory neuropathy with motor neuron involvement.

Combinatorial ShcA docking interactions support diversity in tissue morphogenesis.

Changes in protein-protein interactions may allow polypeptides to perform unexpected regulatory functions. Mammalian ShcA docking proteins have amino-terminal phosphotyrosine (pTyr) binding (PTB) and carboxyl-terminal Src homology 2 (SH2) domains, which recognize specific pTyr sites on activated receptors, and a central region with two phosphorylated tyrosine-X-asparagine (pYXN) motifs (where X represents any amino acid) that each bind the growth factor receptor-bound protein 2 (Grb2) adaptor. Phylogenetic analysis indicates that ShcA may signal through both pYXN-dependent and -independent pathways. We show that, in mice, cardiomyocyte-expressed ShcA directs mid-gestational heart development by a PTB-dependent mechanism that does not require the pYXN motifs. In contrast, the pYXN motifs are required with PTB and SH2 domains in the same ShcA molecule for the formation of muscle spindles, skeletal muscle sensory organs that regulate motor behavior. Thus, combinatorial differences in ShcA docking interactions may yield multiple signaling mechanisms to support diversity in tissue morphogenesis.

Transcriptional regulation of myotube fate specification and intrafusal muscle fiber morphogenesis.

Vertebrate muscle spindle stretch receptors are important for limb position sensation (proprioception) and stretch reflexes. The structurally complex stretch receptor arises from a single myotube, which is transformed into multiple intrafusal muscle fibers by sensory axon-dependent signal transduction that alters gene expression in the contacted myotubes. The sensory-derived signal transduction pathways that specify the fate of myotubes are very poorly understood. The zinc finger transcription factor, early growth response gene 3 (Egr3), is selectively expressed in sensory axon-contacted myotubes, and it is required for normal intrafusal muscle fiber differentiation and spindle development. Here, we show that overexpression of Egr3 in primary myotubes in vitro leads to the expression of a particular repertoire of genes, some of which we demonstrate are also regulated by Egr3 in developing intrafusal muscle fibers within spindles. Thus, our results identify a network of genes that are regulated by Egr3 and are involved in intrafusal muscle fiber differentiation. Moreover, we show that Egr3 mediates myotube fate specification that is induced by sensory innervation because skeletal myotubes that express Egr3 independent of other sensory axon regulation are transformed into muscle fibers with structural and molecular similarities to intrafusal muscle fibers. Hence, Egr3 is a target gene that is regulated by sensory innervation and that mediates gene expression involved in myotube fate specification and intrafusal muscle fiber morphogenesis.

Muscle spindles in the levator palpebrae superioris muscle of human foetuses.

Studies were performed on the levator palpebrae superioris muscle, isolated from foetuses aged 17 to 30 weeks. The number of muscle spindles varied from 2 in the 17th week to 7 in the 30th week. The length of the muscle spindles ranged from 20 to 500 microm, and the diameter varied from 10 to 70 microm. These observations show that the number of muscle spindles in levator palpebrae superioris muscles is significantly lesser than that in the extraocular muscles.

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.

Development of the monosynaptic stretch reflex circuit.

Significant advances have been made during the past few years in our understanding of how the spinal monosynaptic reflex develops. Transcription factors in the Neurogenin, Runt, ETS, and LIM families control sequential steps of the specification of various subtypes of dorsal root ganglia sensory neurons. The initiation of muscle spindle differentiation requires neuregulin 1, derived from Ia afferent sensory neurons, and signaling through ErbB receptors in intrafusal muscle fibers. Several retrograde signals from the periphery are important for the establishment of late connectivity in the reflex circuit. Finally, neurotrophin 3 released from muscle spindles regulates the strength of sensory-motor connections within the spinal cord postnatally.

A role for neuregulin1 signaling in muscle spindle differentiation.

The maturation of synaptic structures depends on inductive interactions between axons and their prospective targets. One example of such an interaction is the influence of proprioceptive sensory axons on the differentiation of muscle spindles. We have monitored the expression of three transcription factors, Egr3, Pea3, and Erm, that delineate early muscle spindle development in an assay of muscle spindle-inducing signals. We provide genetic evidence that Neuregulin1 (Nrg1) is required for proprioceptive afferent-evoked induction of muscle spindle differentiation in the mouse. Ig-Nrg1 isoforms are preferentially expressed by proprioceptive sensory neurons and are sufficient to induce muscle spindle differentiation in vivo, whereas CRD-Nrg1 isoforms are broadly expressed in sensory and motor neurons but are not required for muscle spindle induction.

Postnatal regulation of limb proprioception by muscle-derived neurotrophin-3.

To investigate the effects of neurotrophin-3 (NT-3) on postnatal proprioceptive neurons and their targets, transgenic mice were generated that use the myosin light chain 1 (mlc) promoter to overexpress NT-3 in skeletal muscle. Ribonuclease protection assays revealed that NT-3 overexpression in hindlimb skeletal muscle began at embryonic day 14 (E14) and continued throughout adulthood. Overexpression of NT-3 during late embryogenesis resulted in increased numbers of large sensory and small fusimotor axons. Within a week of birth, mlc/NT-3 mice retract their limbs to the torso when lifted by the tail. Footprint analysis revealed that mlc/NT-3 mice had significant abnormalities in their gait compared with wild-types. Beam walking and rotorod analysis confirmed the poor limb control by mlc/NT-3 mice. These locomotive deficits progressively worsened with age and were likely related to the formation of morphologically abnormal muscle spindles. The most common spindle anomaly was the presence of excessive intrafusal bag fibers within individual muscle spindles. To assess the role of NT-3 in recovery from nerve injury, sciatic nerve crushes were performed in young adult mice. Two days after injury, mlc/NT-3 mice displayed significantly improved sciatic functional indexes and a significant increase in muscle spindles that remained associated with axons. The latter finding suggests that excess NT-3 in muscle may retard the degeneration of proprioceptive axons after nerve crush. Long-term survival after nerve injury in mlc/NT-3 mice did not induce further changes in spindle number or morphology. These findings demonstrate that, in addition to promoting embryonic proprioceptive neuron survival, postnatal overexpression of NT-3 in muscle leads to abnormal spindle formation and deficits in locomotive control. However, our results also show that NT-3 may be therapeutic for proprioceptive axons immediately after nerve injury by delaying axon degeneration.

Quantitative study of muscle spindles in suboccipital muscles of human foetuses.

The proprioceptive inputs from the cervical musculature play an important role in head-eye co-ordination and postural processes. Deep cervical muscles in humans are shown to have high spindle content. The density, distribution and morphology of muscle spindles were studied in superior oblique capitis, inferior oblique capitis and rectus capitis posterior major and minor three small suboccipital muscles. The muscles were obtained, post-mortem from stillborn human foetus. The spindle density was calculated as the ratio of mean spindle content to the mean wet weight of that muscle in grams. The distribution and arrangement of spindles within the muscle and their arrangement was studied. The spindle density of superior oblique muscle was found to be 190, that of inferior oblique was 242 and the rectus capitis posterior contained 98 spindles per gram of muscle. No tendon organs were seen. The serial transverse sections of inferior oblique muscle revealed muscle spindles of varying sizes, length varying between 100-650 microns and, diameter 50-250 microns. A complex parallel arrangements of group of large spindles were seen in the belly of the inferior oblique muscle, while the polar regions contain few small isolated spindles. The relevance of such high spindle receptor content in these tiny muscles is discussed.

Formation of a full complement of cranial proprioceptors requires multiple neurotrophins.

Inactivation of neurotrophin-3 (NT3) completely blocks the development of limb proprioceptive neurons and their end organs, the muscle spindles. We examined whether cranial proprioceptive neurons of the trigeminal mesencephalic nucleus (TMN) require NT3, brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT4) for their development. Complements of TMN neurons and masticatory muscle spindles were decreased by 62% in NT3 null mutants, 33% in BDNF null mutants, and 10% in NT4 null mutant mice at birth. The extent of proprioceptive deficiencies differed among different masticatory muscles, particularly in NT3 null mice. Masticatory muscles of embryonic mice heterozygous for the NT3(lacZneo) or BDNF(lacZ) reporter genes expressed both NT3 and BDNF, consistent with target-derived neurotrophin support of TMN neurons. Although more than 90% of TMN neurons expressed TrkB as well as TrkC receptor proteins by immunocytochemistry in wild-type newborns, TrkC or TrkB null mice exhibited only partial proprioceptive deficiencies similar to those present in NT3 or BDNF;NT4 null mice. Thus, in terms of the survival outcome, two main subpopulations of TMN neurons may exist during embryogenesis, one dependent on TrkC/NT3 functioning and the other utilizing TrkB/BDNF signaling. The differential dependence of TMN neurons on neurotrophins may reflect differential accessibility of the neurons to limiting amounts of NT3, BDNF, or NT4 in target tissues, especially if the tissue distribution or levels of BDNF, NT3, and NT4 were dynamically regulated both spatially and temporally.

Proportions of slow myosin heavy chain-positive fibers in muscle spindles and adjoining extrafusal fascicles, and the positioning of spindles relative to these fascicles.

Chicken leg muscles were examined to calculate the percentages of slow myosin heavy chain (MHC)-positive fibers in spindles and in adjacent extrafusal fascicles, and to clarify how the encapsulated portions of muscle spindles are positioned relative to these fascicles. Unlike mammals, in chicken leg muscles slow-twitch MHC and slow-tonic MHC are expressed in intrafusal fibers and in extrafusal fibers, suggesting a close developmental connection between the two fiber populations. In 8-week-old muscles the proportions of slow MHC-positive extrafusal fibers that ringed muscle spindles ranged from 0-100%. In contrast, proportions of slow MHC-positive intrafusal fibers in spindles ranged from 0-57%. Similar proportions in fiber type composition between intrafusal fibers and surrounding extrafusal fibers were apparent at embryonic days 15 and 16, demonstrating early divergence of extrafusal and intrafusal fibers. Muscle spindles were rarely located within single fascicles. Instead, they were commonly placed where several fascicles converged. The frequent extrafascicular location of spindles suggests migration of intrafusal myoblasts from developing clusters of extrafusal fibers toward the interstitium, perhaps along a neurotrophic gradient established by sensory axons that are advancing in the connective tissue matrix that separates adjoining fascicles.

Morphometric study of the muscles spindles of the eye muscle in human fetuses between 24-29 weeks.

The studies were carried out on human fetuses with the crow-rump length between 230 to 270 mm (24-29 weeks). The length of recti muscles varies from 12-15 mm in 24th week to 25-28 mm in 29th week. Number of muscle spindles varies from 29-36 in 24th week to 54-59 in 29th week. The length of the muscle spindles is from 30-300 microns in 24th week to 40-450 microns in 29th week. The diameter of the muscle spindles varies from 11-36 microns in 24th week to 15-70 microns in 29th week.

Sensory ataxia and muscle spindle agenesis in mice lacking the transcription factor Egr3.

Muscle spindles are skeletal muscle sensory organs that provide axial and limb position information (proprioception) to the central nervous system. Spindles consist of encapsulated muscle fibers (intrafusal fibers) that are innervated by specialized motor and sensory axons. Although the molecular mechanisms involved in spindle ontogeny are poorly understood, the innervation of a subset of developing myotubes (type I) by peripheral sensory afferents (group Ia) is a critical event for inducing intrafusal fiber differentiation and subsequent spindle formation. The Egr family of zinc-finger transcription factors, whose members include Egr1 (NGFI-A), Egr2 (Krox-20), Egr3 and Egr4 (NGFI-C), are thought to regulate critical genetic programs involved in cellular growth and differentiation (refs 4-8, and W.G.T. et al., manuscript submitted). Mice deficient in Egr3 were generated by gene targeting and had gait ataxia, increased frequency of perinatal mortality, scoliosis, resting tremors and ptosis. Although extrafusal skeletal muscle fibers appeared normal, Egr3-deficient animals lacked muscle spindles, a finding that is consistent with their profound gait ataxia. Egr3 was highly expressed in developing muscle spindles, but not in Ia afferent neurons or their terminals during developmental periods that coincided with the induction of spindle morphogenesis by sensory afferent axons. These results indicate that type I myotubes are dependent upon Egr3-mediated transcription for proper spindle development.

Microscopic study of muscle spindles in recti muscles of human fetal eye.

Muscle spindles of recti muscles of the eyebulb appear in the 11th week of intrauterine life. At first, the spindle manifests the stage of myotube with 2-3 intrafusal fibers and is surrounded by the monolayer of internal capsule. In the course of subsequent development, the number of intrafusal fibers increases. Internal capsule begins to demonstrate two layers (14th week) and, then, external capsule develops (17th week). In the 20th week, mature spindles have been observed with a multilayered external capsule.

Number, length and localization of muscle spindles in recti muscles of the eyeball in human fetuses.

Studies were performed on recti muscles of the eyeball, isolated from human fetuses in 14th to 24th week of their development. Number of muscle spindles and their length increased with progressing age of the fetuses. Most numerous muscle spindles were present in the rectus inferior muscle. The spindles were spread along the entire length of the muscle. Somewhat more numerous spindles were present in the half of the muscle belly positioned from the side of original attachment (24th week). Spindles were localized in the peripheral part of the muscle.