A Novel Role for Embigin to Promote Sprouting of Motor Nerve Terminals at the Neuromuscular Junction

Abstract

Adult skeletal muscle accepts ectopic innervation by foreign motor axons only after section of its own nerve, suggesting that the formation of new neuromuscular junctions is promoted by muscle denervation. With the aim to identify new proteins involved in neuromuscular junction formation we performed an mRNA differential display on innervated versus denervated adult rat muscles. We identified transcripts encoding embigin, a transmembrane protein of the immunoglobulin superfamily (IgSF) class of cell adhesion molecules to be strongly regulated by the state of innervation. In innervated muscle it is preferentially localized to neuromuscular junctions. Forced overexpression in innervated muscle of a full-length embigin transgene, but not of an embigin fragment lacking the intracellular domain, promotes nerve terminal sprouting and the formation of additional acetylcholine receptor clusters at synaptic sites without affecting terminal Schwann cell number or morphology, and it delays the retraction of terminal sprouts following re-innervation of denervated endplates. Conversely, knockdown of embigin by RNA interference in wild-type muscle accelerates terminal sprout retraction, both by itself and synergistically with deletion of neural cell adhesion molecule. These findings indicate that embigin enhances neural cell adhesion molecule-dependent neuromuscular adhesion and thereby modulates neuromuscular junction formation and plasticity. Adult skeletal muscle accepts ectopic innervation by foreign motor axons only after section of its own nerve, suggesting that the formation of new neuromuscular junctions is promoted by muscle denervation. With the aim to identify new proteins involved in neuromuscular junction formation we performed an mRNA differential display on innervated versus denervated adult rat muscles. We identified transcripts encoding embigin, a transmembrane protein of the immunoglobulin superfamily (IgSF) class of cell adhesion molecules to be strongly regulated by the state of innervation. In innervated muscle it is preferentially localized to neuromuscular junctions. Forced overexpression in innervated muscle of a full-length embigin transgene, but not of an embigin fragment lacking the intracellular domain, promotes nerve terminal sprouting and the formation of additional acetylcholine receptor clusters at synaptic sites without affecting terminal Schwann cell number or morphology, and it delays the retraction of terminal sprouts following re-innervation of denervated endplates. Conversely, knockdown of embigin by RNA interference in wild-type muscle accelerates terminal sprout retraction, both by itself and synergistically with deletion of neural cell adhesion molecule. These findings indicate that embigin enhances neural cell adhesion molecule-dependent neuromuscular adhesion and thereby modulates neuromuscular junction formation and plasticity. At the developing neuromuscular junction the heparan sulfate proteoglycan agrin from motor nerves interacts with the receptor tyrosine kinase MuSK (muscle specific kinase) in the muscle fiber to orchestrate the differentiation of both presynaptic nerve terminals (1Kim N. Burden S.J. Nat. Neurosci. 2008; 11: 19-27Crossref PubMed Scopus (163) Google Scholar) and the postsynaptic apparatus (2Bezakova G. Ruegg M.A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 295-308Crossref PubMed Scopus (271) Google Scholar). In adult muscle, sectioning an adult muscle's own nerve is an essential condition for transplanted foreign nerves to form new ectopic endplates in its extrasynaptic regions. This model system recapitulates essential aspects of embryonic NMJ formation (3Lømo T. J. Neurocytol. 2003; 32: 835-848Crossref PubMed Scopus (43) Google Scholar). In light of these findings, muscle denervation may promote ectopic synapse formation by one or a combination of the following mechanisms: 1) by up-regulating the extrasynaptic expression of MuSK or of signaling components downstream of MuSK, thus increasing the responsiveness of the fibers to agrin (4Bowen D.C. Park J.S. Bodine S. Stark J.L. Valenzuela D.M. Stitt T.N. Yancopoulos G.D. Lindsay R.M. Glass D.J. DiStefano P.S. Dev. Biol. 1998; 199: 309-319Crossref PubMed Scopus (72) Google Scholar), 2) by inducing the secretion of factors from muscle that may stimulate the secretion of agrin from motor neurites, and 3) by promoting neuron-muscle fiber adhesion, which may promote stable agrin-MuSK interaction required for synaptic differentiation (5Meier T. Marangi P.A. Moll J. Hauser D.M. Brenner H.R. Ruegg M.A. Eur. J. Neurosci. 1998; 10: 3141-3152Crossref PubMed Scopus (26) Google Scholar). Adhesion could be mediated by agrin itself, anchored in the basel lamina and interacting with the neural cell adhesion molecule (NCAM) 2The abbreviations used are: NCAM, neural cell adhesion molecule; CAM, cell adhesion molecule; NMJ, neuromuscular junction; AChR, acetylcholine receptor; RT, reverse transcription; qRT, quantitative RT; RL8, ribosomal protein L8; MCK, muscle creatine kinase; MuSK, muscle specific kinase; wt, wild type; RNAi, RNA interference; shRNA, short hairpin RNA; GFP, green fluorescent protein. in the surface of motor neurons (6Storms S.D. Anvekar V.M. Adams L.D. Murray B.A. Exp. Cell Res. 1996; 223: 385-394Crossref PubMed Scopus (25) Google Scholar, 7Burg M.A. Halfter W. Cole G.J. J. Neurosci. Res. 1995; 41: 49-64Crossref PubMed Scopus (60) Google Scholar, 8Cotman S.L. Halfter W. Cole G.J. Exp. Cell Res. 1999; 249: 54-64Crossref PubMed Scopus (74) Google Scholar), or by up-regulation of adhesion molecules in muscle and/or motor neurites. Cell adhesion molecules (CAMs) such as NCAM and N-cadherin have been implicated in motor neuron sprouting. NCAM-induced sprouting is thought to be induced via homophilic binding between NCAMs in the neural and the muscle surfaces, that in turn induces growth promoting mechanisms in the nerve process (9Walsh F.S. Doherty P. Annu. Rev. Cell Dev. Biol. 1997; 13: 425-456Crossref PubMed Scopus (404) Google Scholar, 10Walsh F.S. Hobbs C. Wells D.J. Slater C.R. Fazeli S. Mol. Cell Neurosci. 2000; 15: 244-261Crossref PubMed Scopus (37) Google Scholar, 11Niethammer P. Delling M. Sytnyk V. Dityatev A. Fukami K. Schachner M. J. Cell Biol. 2002; 157: 521-532Crossref PubMed Scopus (239) Google Scholar). Interestingly, both NCAM and N-cadherin are expressed in the surface of developing muscle with a spatiotemporal pattern that is consistent with a role in NMJ formation (12Sanes J.R. Schachner M. Covault J. J. Cell Biol. 1986; 102: 420-431Crossref PubMed Scopus (242) Google Scholar): expression is along the entire myotube surface; once NMJ formation has begun, it is reduced to persist only at the NMJ. Upon denervation it is up-regulated in parallel with MuSK, AChRs, and the increased susceptibility of the muscle to innervation. Comparable evidence supports the hypothesis that tenascin-C, a glycoprotein component of extracellular matrix, is involved in neuromuscular interactions related to those of NCAMs (12Sanes J.R. Schachner M. Covault J. J. Cell Biol. 1986; 102: 420-431Crossref PubMed Scopus (242) Google Scholar, 13Gatchalian C.L. Schachner M. Sanes J.R. J. Cell Biol. 1989; 108: 1873-1890Crossref PubMed Scopus (142) Google Scholar, 14Wehrle-Haller B. Chiquet M. J. Cell Sci. 1993; 106: 597-610Crossref PubMed Google Scholar). However, it has been difficult to demonstrate the importance of these molecules in vertebrate NMJ formation, because their ablation did not critically alter synapse formation (15Moscoso L.M. Cremer H. Sanes J.R. J. Neurosci. 1998; 18: 1465-1477Crossref PubMed Google Scholar) or was embryonically lethal (16Radice G.L. Rayburn H. Matsunami H. Knudsen K.A. Takeichi M. Hynes R.O. Dev. Biol. 1997; 181: 64-78Crossref PubMed Scopus (617) Google Scholar). Combined these findings thus suggest that CAMs up-regulated by denervation are dispensable for early ectopic NMJ formation, that several nerve-muscle adhesion systems may be redundant, and/or that some may not have been identified. Here we show that motor neurones constitutively overexpressing agrin and transplanted onto muscles constitutively overexpressing MuSK do not form ectopic synapses, unless the muscle's own nerve is sectioned, indicating that in addition to agrin/MuSK other denervation-induced interactions are required. Given that NCAM and tenascin-C expression in muscle are up-regulated by denervation (12Sanes J.R. Schachner M. Covault J. J. Cell Biol. 1986; 102: 420-431Crossref PubMed Scopus (242) Google Scholar, 13Gatchalian C.L. Schachner M. Sanes J.R. J. Cell Biol. 1989; 108: 1873-1890Crossref PubMed Scopus (142) Google Scholar), yet their deletion does not seriously affect NMJ formation (15Moscoso L.M. Cremer H. Sanes J.R. J. Neurosci. 1998; 18: 1465-1477Crossref PubMed Google Scholar), we searched for new factors affecting neuromuscular interactions by screening mRNAs differentially expressed by innervated and chronically denervated muscle. We identified the IgS molecule embigin (17Huang R.P. Ozawa M. Kadomatsu K. Muramatsu T. Differentiation. 1990; 45: 76-83Crossref PubMed Scopus (25) Google Scholar) in the muscle surface to be strongly regulated by electrical muscle activity and to exhibit functional properties consistent with a role in the outgrowth of motor neurons and the formation of NMJs. Rat soleus muscles of 7-week-old Wistar rats (weight of ∼200 g) and mouse gastrocnemius muscles of 6-week-old C57/BL6 mice were denervated by cutting or crushing the sciatic nerve just proximally to its branching into tibial and fibular nerves. Ectopic endplates were induced in mice as by (17Huang R.P. Ozawa M. Kadomatsu K. Muramatsu T. Differentiation. 1990; 45: 76-83Crossref PubMed Scopus (25) Google Scholar). and mice were with at the and muscles were for In some denervated rat soleus muscles were chronically via as A. K. H. T. J. 1998; PubMed Scopus Google Scholar). muscles were from and were by the of RNA were into and with of in and at were by on the apparatus RNA was to the the mRNA differential RNA from of and rat soleus muscles was with to mRNA differential We performed the mRNA differential display to the for mRNA differential display with the following were performed in the of in a of were on and to at were with and into and systems was performed on both on an were identified to the and were to the We used as for full-length of mouse embigin We a fragment of the mouse ribosomal protein into and was with and the was used as for were by with and on were on at RNA was with and with an the for quantitative was with for used in were on the in were to a of an on the without formation were used for were performed on an system the mRNA expression of is to ribosomal protein Rat soleus and mouse gastrocnemius muscles were into of and and were by on the apparatus were at for at and were and at of proteins was by on an of full-length embigin protein were with a M. C. J. Biol. PubMed Scopus Google Scholar). was the intracellular of embigin, we used to expression and of embigin in and in mouse was used to for protein were with Schwann were with In some nerve terminals were with and with were from full-length embigin was from denervated gastrocnemius RNA with the and was on a and into full-length mouse embigin was of the with and into with was with and and into the the with and J. P. S. G. H. Ruegg M.A. PubMed Scopus Google Scholar). form of embigin lacking the intracellular was by from the the and was in the between and This of of the intracellular of embigin, were to into the cell the were with with a and on a mouse used mice G. M. C. M. Sanes J.R. 2000; PubMed Scopus Google Burden S.J. 2002; PubMed Scopus Google Scholar), C. S. M. G. M. S. P. Sanes J.R. B. Ruegg M.A. J. Neurosci. PubMed Scopus Google Scholar), and H. A. M. G. J. S. P. S. K. W. PubMed Scopus Google Scholar). or were at in of in the of K. was for at the was at for at on of were with in a of in the of and of and reverse and mice were for a fragment with and for of for for and for for mice were for a fragment with and for of for for and for for mice were for a fragment with and for of for for and for for mice were for a fragment with and for of for for and for for embigin were the interference RNA of the the and were and in the from was used for both and to have a of between the RNA encoding were and Forced of by and of MuSK by for NMJ in the of agrin to postsynaptic differentiation in the extrasynaptic of muscle G. T. M. V. B. Brenner H.R. Sci. S. A. 1997; PubMed Scopus Google Scholar), an condition for synapse formation to MuSK expression in extrasynaptic fiber (4Bowen D.C. Park J.S. Bodine S. Stark J.L. Valenzuela D.M. Stitt T.N. Yancopoulos G.D. Lindsay R.M. Glass D.J. DiStefano P.S. Dev. Biol. 1998; 199: 309-319Crossref PubMed Scopus (72) Google Scholar), we ectopic endplates in soleus muscle of mice for agrin in motor neurones C. S. M. G. M. S. P. Sanes J.R. B. Ruegg M.A. J. Neurosci. PubMed Scopus Google Scholar) and MuSK in muscle Burden S.J. 2002; PubMed Scopus Google Scholar), without the to section the muscle's own have been to their from after fibular nerve onto the of the soleus muscle in agrin/MuSK the soleus muscles was for in to foreign nerve was in indicating that not agrin and MuSK In in and wild-type soleus muscles did in to foreign nerve the tibial nerve the soleus innervation been to These demonstrate that synapse formation be induced by overexpression of agrin by motor neurons and of MuSK by muscle of transplanted nerves in the agrin/MuSK could not have been to expression because both and muscles extrasynaptic clusters in adult muscle Burden S.J. 2002; PubMed Scopus Google Scholar, C. S. M. G. M. S. P. Sanes J.R. B. Ruegg M.A. J. Neurosci. PubMed Scopus Google Scholar). ectopic synapse formation additional factors up-regulated by molecules promoting neuromuscular of mRNAs from versus as a by identify encoding proteins on the state of we from innervated versus adult rat soleus muscles by mRNA differential display P. PubMed Scopus Google Scholar). of the with a in expression by at These to of expression of was increased after denervation and was reduced for identified a strongly up-regulated of as a fragment of embigin mRNA embigin is a transmembrane glycoprotein with a protein component of of a the form of extracellular a transmembrane and a short to a of molecules by and in addition to embigin, and is the of with but in neural and C.R. P. M. J. Neurosci. PubMed Scopus Google Scholar). of with embigin at the is only in the and is in their transmembrane of the a suggesting that with other transmembrane molecules to at some of their the other embigin a of in the of that were to be essential for NMJ formation and in S. C. S.J. A. J. Cell Biol. PubMed Scopus Google Scholar). embigin does not cell by homophilic interactions Ozawa M. Kadomatsu K. Muramatsu T. Dev. Biol. 1993; PubMed Scopus (37) Google Scholar), suggesting of for the Given its expression in the early the role of embigin is thought to be in early embryonic (17Huang R.P. Ozawa M. Kadomatsu K. Muramatsu T. Differentiation. 1990; 45: 76-83Crossref PubMed Scopus (25) Google Scholar, Kadomatsu K. K. Muramatsu T. Dev. 1998; PubMed Scopus Google Scholar). of embigin mRNA in muscle by denervation was by and of mRNA from denervated mouse gastrocnemius muscle up-regulation of transcripts of and by an in the of the increased in the expression pattern of embigin mRNA denervation was by quantitative in both rat soleus and mouse gastrocnemius muscles. In the embigin mRNA expression was increased after denervation to a after of denervated soleus muscle via at reduced embigin mRNA to by by electrical muscle activity In the the expression at the an in mRNA by was on mouse muscles at the protein Interestingly, the in embigin mRNA expression following denervation the of extrasynaptic expression of involved in synapse formation (4Bowen D.C. Park J.S. Bodine S. Stark J.L. Valenzuela D.M. Stitt T.N. Yancopoulos G.D. Lindsay R.M. Glass D.J. DiStefano P.S. Dev. Biol. 1998; 199: 309-319Crossref PubMed Scopus (72) Google Scholar, V. Brenner H.R. B. J. Cell Biol. PubMed Scopus Google as as the formation of ectopic by transplanted foreign nerves after the soleus nerve is T. Slater C.R. J. PubMed Scopus Google Scholar) consistent with the that it may be related to ectopic synapse we the expression of embigin in developing muscle. which are expressed at in in adult muscle V. B. M. B. 1989; PubMed Scopus Google Scholar), embigin mRNA in muscle at was only in the adult that it was localized to the endplates at of and at the synapse adult by for a of embigin at the NMJ in of mice overexpressing embigin the of the muscle creatine kinase were and on NMJ were In soleus muscles of embigin protein expression were with protein in wild-type muscles after denervation overexpression the of the NMJ. motor axons and their terminal embigin were with a fluorescent protein in the cell of or nerve terminals were by were with nerve terminals in adult muscles of the of embigin mRNA and terminal sprouts were terminal the of clusters and in were not in muscles. but not of the were with clusters suggesting that the sprouts induced the clusters In one 2) the of terminal sprouting was with that in wild-type only sprouts into that were not by In sprouts were in of endplates in the in of endplates sprouting in the embigin was not to soleus muscles but was in and in one muscles it was increased and with in the of sprouts was from those endplates only sprouts did not additional postsynaptic clusters in Given that do the of endplates sprouts as in is an only of the of embigin overexpression in innervated muscle. These that embigin expression in innervated muscles to those in wild after denervation the formation of of embigin overexpression to terminal sprouts at innervated endplates was on the of its intracellular in mouse overexpressing or of mRNA encoding a embigin lacking of of the intracellular of the the of endplates terminal sprouts was as in muscles that the mRNA was expressed at that of the full-length embigin in the and that both were expressed at in cell and the of sprouts in the mice was not to in expression or of and of the terminal sprouting additional clusters as the number of clusters at an be with endplates in muscles a number of soleus Interestingly, a but was in the mice overexpressing consistent with the that the of additional is promoted in by additional sprouts by neuromuscular adhesion that in turn in a of agrin on a fiber with of surface MuSK, or by sprouts of in a of agrin on a fiber with of surface At early of vertebrate muscle fibers are innervated by several motor neurons a of the motor axons for the innervation of muscle fibers in a process as synapse J.R. Annu. Rev. Neurosci. 1999; PubMed Scopus Google Scholar). of factors between nerve the and muscle fibers and thus promoting synapse has been to embigin is expressed at at and delays retraction of terminal sprouts in we synapse was in the mice by the of sites in and wild-type at and in the of the synapse could be not of of by of nerve terminals is a of neuromuscular PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). clusters following denervation. the is sprouts to Annu. Rev. Neurosci. 4: PubMed Scopus Google Scholar). embigin expression retraction of terminal sprouts by axons denervated we denervated wild-type and embigin overexpressing soleus muscles by crushing the sciatic nerves at of the and the of endplates with sprouts with a of embigin on terminal the of endplates with sprouts at was in in in wild-type in was in muscles after the nerve the of embigin mRNA expression was to not indicating that the in sprout retraction in the mice was by the embigin expression We by the number of terminal sprouts after nerve in denervated muscle in which embigin expression been by RNA interference In a of hairpin RNA were by for their to embigin protein in induced to embigin by of an expression used for knockdown of N-cadherin M. J. Cell Sci. PubMed Scopus Google Scholar) as a for of embigin was these were into denervated soleus muscle but in At in be to the that at of fibers in a muscle are by the used the of embigin knockdown on sprout retraction, the sciatic nerve of wild-type mice was and the encoding the was into the soleus muscle only on one with a to the of sprouts was in in the fibers with and it was in in fibers not with In innervated muscles with an expression for the of endplates with terminal sprouts in was as in muscle indicating that did not affect sprouting. knockdown of embigin sprout retraction These the that the of sprouting is with the of embigin by in the of adult muscle expression of NCAMs is regulated in parallel with it is localized to the neuromuscular up-regulated and re-innervation F.S. J. 4: PubMed Scopus Google Scholar, J. Sanes J.R. J. Cell Biol. 1986; 102: PubMed Scopus Google Scholar). both embigin and NCAM F.S. Hobbs C. Wells D.J. Slater C.R. Fazeli S. Mol. Cell Neurosci. 2000; 15: 244-261Crossref PubMed Scopus (37) Google Scholar) promote terminal sprouting we the sprout re-innervation in NCAM mice in which embigin was by in with L.M. Cremer H. Sanes J.R. J. Neurosci. 1998; 18: 1465-1477Crossref PubMed Google Scholar, the of NCAM did not affect retraction of sprouts re-innervation with wild the knockdown of embigin in sprout retraction with that in muscles or in muscles embigin knockdown of with with to sprout retraction re-innervation the of embigin knockdown was that that of NCAM the of Schwann muscle denervation terminal Schwann both in those endplates that been denervated and those innervated 1995; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar, J. 1999; PubMed Scopus Google Scholar). These Schwann cell terminal sprouts from innervated endplates to denervated sites T. T. 2003; 18: Google Scholar). for a of Schwann in their was in and by with an the protein In endplates both in and soleus muscle, Schwann were with motor nerve were in of endplates in in and in of endplates in in soleus muscle of wild-type in with a T. T. 2003; 18: Google Scholar). we were to evidence of an of Schwann in the of sprouting of motor nerve We have the transmembrane molecule embigin to be expressed in skeletal muscle with a spatiotemporal pattern and that are consistent with its in NMJ formation and muscle re-innervation following nerve embigin is expressed in developing muscle is in the is at the NMJ from and is strongly up-regulated denervation as a of the of embigin expression is increased with a that the of expression and the formation of ectopic by transplanted foreign nerves T. Slater C.R. J. PubMed Scopus Google Scholar). overexpression in adult innervated muscle the formation of sprouts inducing additional expression affect the of sprout retraction after endplates are nerve delays and knockdown by accelerates the retraction of retraction is embigin expression is in muscle. sprout by embigin is on the on its intracellular In adult muscle embigin or are required for expression and activity of in the muscle fiber M. C. J. Biol. PubMed Scopus Google Scholar). However, denervation the up-regulation of embigin with the of mRNA and H. denervation in muscle is the for embigin expression of embigin in muscle to terminal sprouts and to sprout retraction re-innervation synergistically with NCAM are consistent with the that embigin promotes the adhesion of motor to the muscle the sprouts at of in wild-type muscle may the of outgrowth and of terminal as has been at the mouse NMJ D.J. J. Neurosci. 1989; PubMed Google Scholar). increased adhesion to the muscle fiber surface by embigin sprouts that in its thus increasing their in the muscle. increased of the sprouts the of new because stable sprouts agrin in the lamina of the muscle a condition for agrin to ectopic clusters in muscle (5Meier T. Marangi P.A. Moll J. Hauser D.M. Brenner H.R. Ruegg M.A. Eur. J. Neurosci. 1998; 10: 3141-3152Crossref PubMed Scopus (26) Google Scholar). This in increased number of clusters at an With a overexpression of MuSK the fibers to of agrin that in muscle with MuSK may for the of additional on of neuromuscular interactions embigin in muscle could promote sprouting of nerve terminals in the following by signaling Schwann and/or by increasing adhesion to the muscle surface; in such adhesion could be mediated via or interaction of embigin in the muscle surface with molecules in the embigin may with NCAM on the surface to by of NCAM has been to branching and outgrowth C.L. S. J. J. Scopus Google Scholar). an Schwann is that the number of endplates with Schwann cell in innervated muscle is to that in innervated muscle the other the of with Schwann cell both in innervated and in muscles the of in muscles sprouts consistent with the that Schwann cell may be for terminal sprouting. embigin in muscle is to promote sprouting via homophilic interactions with embigin in the nerve cell because expression of embigin in does not their to one and H. and because its extracellular short to the synaptic neuromuscular interactions embigin binding on the neural surface to a molecule with a extracellular be via because the of embigin on terminal sprouting on its intracellular embigin may terminal by adhesion mediated by other such as In muscle NCAM is expressed with a spatiotemporal pattern to that for embigin J. C. Sanes J.R. J. Cell Biol. 1986; 102: PubMed Scopus Google Scholar), and in innervated muscle it induces terminal sprouts F.S. Hobbs C. Wells D.J. Slater C.R. Fazeli S. Mol. Cell Neurosci. 2000; 15: 244-261Crossref PubMed Scopus (37) Google Scholar) by binding to its on the motor neuron (9Walsh F.S. Doherty P. Annu. Rev. Cell Dev. Biol. 1997; 13: 425-456Crossref PubMed Scopus (404) Google Scholar). retraction re-innervation is embigin is Conversely, retraction is embigin expression is by it is by embigin knockdown and of NCAM sprouts in wild-type muscle to both molecules for to after are and This that sprout retraction re-innervation is not to the of embigin and NCAM, but may on other Given that the of NCAM did not affect sprout retraction, and that the NCAM expressed in to denervation an intracellular is C.L. S. J. J. Scopus Google the of sprout retraction by and NCAM deletion that embigin may or NCAM both its and extracellular because did not interaction between and embigin in or in muscle In of the of the of embigin, its expression and denervation those of NMJ formation and of the susceptibility of the muscle to its to promote terminal sprouts inducing and its functional with NCAM, indicate that it the of CAMs involved in neuromuscular synapse formation and plasticity.