Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Apr 1;108(4):1527–1535. doi: 10.1083/jcb.108.4.1527

Agrin-related molecules are concentrated at acetylcholine receptor clusters in normal and aneural developing muscle

PMCID: PMC2115523  PMID: 2538482

Abstract

Agrin induces the clustering of acetylcholine receptors (AchRs) and other postsynaptic components on the surface of cultured muscle cells. Molecules closely related if not identical to agrin are highly concentrated in the synaptic basal lamina, a structure known to play a key part in orchestrating synapse regeneration. Agrin or agrin-related molecules are thus likely to play a role in directing the differentiation of the postsynaptic apparatus at the regenerating neuromuscular junction. The present studies are aimed at understanding the role of agrin at developing synapses. We have used anti-agrin monoclonal antibodies combined with alpha-bungarotoxin labeling to establish the localization and time of appearance of agrin-related molecules in muscles of the chick hindlimb. Agrinlike immunoreactivity was observed in premuscle masses from as early as stage 23. AchR clusters were first detected late in stage 25, coincident with the entry of axons into the limb. At this and all subsequent stages examined, greater than 95% of the AchR clusters colocalized with agrin- related molecules. This colocalization was also observed in unpermeabilized whole mount preparations, indicating that the agrin- related molecules were disposed on the external surface of the cells. Agrin-related molecules were also detected in regions of low AchR density on the muscle cell surface. To examine the role of innervation in the expression of agrin-related molecules, aneural limbs were generated by two methods. Examination of these limbs revealed that agrin-related molecules were expressed in the aneural muscle and they colocalized with AchR clusters. Thus, in developing muscle, agrin or a closely related molecule (a) is expressed before AchR clusters are detected; (b) is colocalized with the earliest AchR clusters formed; and (c) can be expressed in muscle and at sites of high AchR density independently of innervation. These results indicate that agrin or a related molecule is likely to play a role in synapse development and suggest that the muscle cell may be at least one source of this molecule.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anglister L., McMahan U. J. Basal lamina directs acetylcholinesterase accumulation at synaptic sites in regenerating muscle. J Cell Biol. 1985 Sep;101(3):735–743. doi: 10.1083/jcb.101.3.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barald K. F., Phillips G. D., Jay J. C., Mizukami I. F. A component in mammalian muscle synaptic basal lamina induces clustering of acetylcholine receptors. Prog Brain Res. 1987;71:397–408. doi: 10.1016/s0079-6123(08)61841-5. [DOI] [PubMed] [Google Scholar]
  3. Brown M. C., Jansen J. K., Van Essen D. Polyneuronal innervation of skeletal muscle in new-born rats and its elimination during maturation. J Physiol. 1976 Oct;261(2):387–422. doi: 10.1113/jphysiol.1976.sp011565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burden S. J., Sargent P. B., McMahan U. J. Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve. J Cell Biol. 1979 Aug;82(2):412–425. doi: 10.1083/jcb.82.2.412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burden S. Acetylcholine receptors at the neuromuscular junction: developmental change in receptor turnover. Dev Biol. 1977 Nov;61(1):79–85. doi: 10.1016/0012-1606(77)90343-8. [DOI] [PubMed] [Google Scholar]
  6. Burden S. Development of the neuromuscular junction in the chick embryo: the number, distribution, and stability of acetylcholine receptors. Dev Biol. 1977 Jun;57(2):317–329. doi: 10.1016/0012-1606(77)90218-4. [DOI] [PubMed] [Google Scholar]
  7. Crow M. T., Stockdale F. E. Myosin expression and specialization among the earliest muscle fibers of the developing avian limb. Dev Biol. 1986 Jan;113(1):238–254. doi: 10.1016/0012-1606(86)90126-0. [DOI] [PubMed] [Google Scholar]
  8. Dahm L. M., Landmesser L. T. The regulation of intramuscular nerve branching during normal development and following activity blockade. Dev Biol. 1988 Dec;130(2):621–644. doi: 10.1016/0012-1606(88)90357-0. [DOI] [PubMed] [Google Scholar]
  9. Dennis M. J. Development of the neuromuscular junction: inductive interactions between cells. Annu Rev Neurosci. 1981;4:43–68. doi: 10.1146/annurev.ne.04.030181.000355. [DOI] [PubMed] [Google Scholar]
  10. Fallon J. R., Nitkin R. M., Reist N. E., Wallace B. G., McMahan U. J. Acetylcholine receptor-aggregating factor is similar to molecules concentrated at neuromuscular junctions. Nature. 1985 Jun 13;315(6020):571–574. doi: 10.1038/315571a0. [DOI] [PubMed] [Google Scholar]
  11. Godfrey E. W., Dietz M. E., Morstad A. L., Wallskog P. A., Yorde D. E. Acetylcholine receptor-aggregating proteins are associated with the extracellular matrix of many tissues in Torpedo. J Cell Biol. 1988 Apr;106(4):1263–1272. doi: 10.1083/jcb.106.4.1263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Godfrey E. W., Siebenlist R. E., Wallskog P. A., Walters L. M., Bolender D. L., Yorde D. E. Basal lamina components are concentrated in premuscle masses and at early acetylcholine receptor clusters in chick embryo hindlimb muscles. Dev Biol. 1988 Dec;130(2):471–486. doi: 10.1016/0012-1606(88)90343-0. [DOI] [PubMed] [Google Scholar]
  13. Jacob M., Lentz T. L. Localization of acetylcholine receptors by means of horseradish peroxidase-alpha-bungarotoxin during formation and development of the neuromuscular junction in the chick embryo. J Cell Biol. 1979 Jul;82(1):195–211. doi: 10.1083/jcb.82.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnson G. D., Davidson R. S., McNamee K. C., Russell G., Goodwin D., Holborow E. J. Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy. J Immunol Methods. 1982 Dec 17;55(2):231–242. doi: 10.1016/0022-1759(82)90035-7. [DOI] [PubMed] [Google Scholar]
  15. Lance-Jones C., Landmesser L. Motoneurone projection patterns in embryonic chick limbs following partial deletions of the spinal cord. J Physiol. 1980 May;302:559–580. doi: 10.1113/jphysiol.1980.sp013261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Landmesser L., Morris D. G. The development of functional innervation in the hind limb of the chick embryo. J Physiol. 1975 Jul;249(2):301–326. doi: 10.1113/jphysiol.1975.sp011017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Magill-Solc C., McMahan U. J. Motor neurons contain agrin-like molecules. J Cell Biol. 1988 Nov;107(5):1825–1833. doi: 10.1083/jcb.107.5.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McLennan I. S. Differentiation of muscle fiber types in the chicken hindlimb. Dev Biol. 1983 May;97(1):222–228. doi: 10.1016/0012-1606(83)90079-9. [DOI] [PubMed] [Google Scholar]
  19. McMahan U. J., Slater C. R. The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle. J Cell Biol. 1984 Apr;98(4):1453–1473. doi: 10.1083/jcb.98.4.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nitkin R. M., Smith M. A., Magill C., Fallon J. R., Yao Y. M., Wallace B. G., McMahan U. J. Identification of agrin, a synaptic organizing protein from Torpedo electric organ. J Cell Biol. 1987 Dec;105(6 Pt 1):2471–2478. doi: 10.1083/jcb.105.6.2471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pittman R., Oppenheim R. W. Cell death of motoneurons in the chick embryo spinal cord. IV. Evidence that a functional neuromuscular interaction is involved in the regulation of naturally occurring cell death and the stabilization of synapses. J Comp Neurol. 1979 Sep 15;187(2):425–446. doi: 10.1002/cne.901870210. [DOI] [PubMed] [Google Scholar]
  22. Pockett S. Elimination of polyneuronal innervation in proximal and distal leg muscles of chick embryos. Brain Res. 1981 Apr;227(2):299–302. doi: 10.1016/0165-3806(81)90117-6. [DOI] [PubMed] [Google Scholar]
  23. Ravdin P., Axelrod D. Fluorescent tetramethyl rhodamine derivatives of alpha-bungarotoxin: preparation, separation, and characterization. Anal Biochem. 1977 Jun;80(2):585–592. doi: 10.1016/0003-2697(77)90682-0. [DOI] [PubMed] [Google Scholar]
  24. Reist N. E., Magill C., McMahan U. J. Agrin-like molecules at synaptic sites in normal, denervated, and damaged skeletal muscles. J Cell Biol. 1987 Dec;105(6 Pt 1):2457–2469. doi: 10.1083/jcb.105.6.2457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanes J. R., Marshall L. M., McMahan U. J. Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites. J Cell Biol. 1978 Jul;78(1):176–198. doi: 10.1083/jcb.78.1.176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smith M. A., Slater C. R. Spatial distribution of acetylcholine receptors at developing chick neuromuscular junctions. J Neurocytol. 1983 Dec;12(6):993–1005. doi: 10.1007/BF01153346. [DOI] [PubMed] [Google Scholar]
  27. Tosney K. W., Landmesser L. T. Development of the major pathways for neurite outgrowth in the chick hindlimb. Dev Biol. 1985 May;109(1):193–214. doi: 10.1016/0012-1606(85)90360-4. [DOI] [PubMed] [Google Scholar]
  28. Tosney K. W., Watanabe M., Landmesser L., Rutishauser U. The distribution of NCAM in the chick hindlimb during axon outgrowth and synaptogenesis. Dev Biol. 1986 Apr;114(2):437–452. doi: 10.1016/0012-1606(86)90208-3. [DOI] [PubMed] [Google Scholar]
  29. Wallace B. G. Aggregating factor from Torpedo electric organ induces patches containing acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase on cultured myotubes. J Cell Biol. 1986 Mar;102(3):783–794. doi: 10.1083/jcb.102.3.783. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES