Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Feb 1;106(2):469–478. doi: 10.1083/jcb.106.2.469

Regulation of acetylcholine receptor transcript expression during development in Xenopus laevis

PMCID: PMC2114983  PMID: 3339098

Abstract

The level of transcripts encoding the skeletal muscle acetylcholine receptor (AChR) was determined during embryonic development in Xenopus laevis. cDNAs encoding the alpha, gamma, and delta subunits of the Xenopus AChR were isolated from Xenopus embryo cDNA libraries using Torpedo AChR cDNAs as probes. The Xenopus AChR cDNAs have greater than 60% amino acid sequence homology to their Torpedo homologues and hybridize to transcripts that are restricted to the somites of developing embryos. Northern blot analysis demonstrates that a 2.3-kb transcript hybridizes to the alpha subunit cDNA, a 2.4-kb transcript hybridizes to the gamma subunit cDNA, and that two transcripts, of 1.9 and 2.5 kb, hybridize to the delta subunit cDNA. RNase protection assays demonstrate that transcripts encoding alpha, gamma, and delta subunits are coordinately expressed at late gastrula and that the amount of each transcript increases in parallel with muscle-specific actin mRNA during the ensuing 12 h. After the onset of muscle activity the level of actin mRNA per somite remains relatively constant, whereas the level of alpha subunit and delta subunit transcripts decrease fourfold per somite and the level of gamma subunit transcript decreases greater than 50-fold per somite. The decrease in amount of AChR transcripts per somite, however, occurs when embryos are paralyzed with local anaesthetic during their development. These results demonstrate that AChR transcripts in Xenopus are initially expressed coordinately, but that gamma subunit transcript levels are regulated differently than alpha and delta at later stages. Moreover, these results demonstrate that AChR transcript levels in Xenopus myotomal muscle cells are not responsive to electrical activity and suggest that AChR transcript levels are influenced by other regulatory controls.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Anderson M. J., Cohen M. W. Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):757–773. doi: 10.1113/jphysiol.1977.sp011880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barkas T., Mauron A., Roth B., Alliod C., Tzartos S. J., Ballivet M. Mapping the main immunogenic region and toxin-binding site of the nicotinic acetylcholine receptor. Science. 1987 Jan 2;235(4784):77–80. doi: 10.1126/science.2432658. [DOI] [PubMed] [Google Scholar]
  4. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  5. Blackshaw S., Warner A. Onset of acetylcholine sensitivity and endplate activity in developing myotome muscles of Xenopus. Nature. 1976 Jul 15;262(5565):217–218. doi: 10.1038/262217a0. [DOI] [PubMed] [Google Scholar]
  6. Brehm P., Steinbach J. H., Kidokoro Y. Channel open time of acetylcholine receptors on Xenopus muscle cells in dissociated cell culture. Dev Biol. 1982 May;91(1):93–102. doi: 10.1016/0012-1606(82)90012-4. [DOI] [PubMed] [Google Scholar]
  7. Brockes J. P., Hall Z. W. Synthesis of acetylcholine receptor by denervated rat diaphragm muscle. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1368–1372. doi: 10.1073/pnas.72.4.1368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  9. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen M. W., Greschner M., Tucci M. In vivo development of cholinesterase at a neuromuscular junction in the absence of motor activity in Xenopus laevis. J Physiol. 1984 Mar;348:57–66. doi: 10.1113/jphysiol.1984.sp015099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Devreotes P. N., Fambrough D. M. Acetylcholine receptor turnover in membranes of developing muscle fibers. J Cell Biol. 1975 May;65(2):335–358. doi: 10.1083/jcb.65.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Devreotes P. N., Fambrough D. M. Turnover of acetylcholine receptors in skeletal muscle. Cold Spring Harb Symp Quant Biol. 1976;40:237–251. doi: 10.1101/sqb.1976.040.01.025. [DOI] [PubMed] [Google Scholar]
  13. Dworkin-Rastl E., Kelley D. B., Dworkin M. B. Localization of specific mRNA sequences in Xenopus laevis embryos by in situ hybridization. J Embryol Exp Morphol. 1986 Feb;91:153–168. [PubMed] [Google Scholar]
  14. Fambrough D. M. Control of acetylcholine receptors in skeletal muscle. Physiol Rev. 1979 Jan;59(1):165–227. doi: 10.1152/physrev.1979.59.1.165. [DOI] [PubMed] [Google Scholar]
  15. Finer-Moore J., Stroud R. M. Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor. Proc Natl Acad Sci U S A. 1984 Jan;81(1):155–159. doi: 10.1073/pnas.81.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goldman D., Boulter J., Heinemann S., Patrick J. Muscle denervation increases the levels of two mRNAs coding for the acetylcholine receptor alpha-subunit. J Neurosci. 1985 Sep;5(9):2553–2558. doi: 10.1523/JNEUROSCI.05-09-02553.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  18. Hall Z. W., Reiness C. G. Electrical stimulation of denervated muscles reduces incorporation of methionine into the ACh receptor. Nature. 1977 Aug 18;268(5621):655–657. doi: 10.1038/268655a0. [DOI] [PubMed] [Google Scholar]
  19. Hamilton L. The formation of somites in Xenopus. J Embryol Exp Morphol. 1969 Sep;22(2):253–264. [PubMed] [Google Scholar]
  20. Kidokoro Y., Gruener R. Distribution and density of alpha-bungarotoxin binding sites on innervated and noninnervated Xenopus muscle cells in culture. Dev Biol. 1982 May;91(1):78–85. doi: 10.1016/0012-1606(82)90010-0. [DOI] [PubMed] [Google Scholar]
  21. Kintner C. R., Melton D. A. Expression of Xenopus N-CAM RNA in ectoderm is an early response to neural induction. Development. 1987 Mar;99(3):311–325. doi: 10.1242/dev.99.3.311. [DOI] [PubMed] [Google Scholar]
  22. Klarsfeld A., Changeux J. P. Activity regulates the levels of acetylcholine receptor alpha-subunit mRNA in cultured chicken myotubes. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4558–4562. doi: 10.1073/pnas.82.13.4558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Krieg P. A., Melton D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kullberg R. W., Brehm P., Steinbach J. H. Nonjunctional acetylcholine receptor channel open time decreases during development of Xenopus muscle. Nature. 1981 Jan 29;289(5796):411–413. doi: 10.1038/289411a0. [DOI] [PubMed] [Google Scholar]
  25. Kullberg R. W., Lentz T. L., Cohen M. W. Development of the myotomal neuromuscular junction in Xenopus laevis: an electrophysiological and fine-structural study. Dev Biol. 1977 Oct 1;60(1):101–129. doi: 10.1016/0012-1606(77)90113-0. [DOI] [PubMed] [Google Scholar]
  26. Kullberg R., Kasprzak H. Gating kinetics of nonjunctional acetylcholine receptor channels in developing Xenopus muscle. J Neurosci. 1985 Apr;5(4):970–976. doi: 10.1523/JNEUROSCI.05-04-00970.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Levitt D., Cooper M. D. Mouse pre-B cells synthesize and secrete mu heavy chains but not light chains. Cell. 1980 Mar;19(3):617–625. doi: 10.1016/s0092-8674(80)80038-9. [DOI] [PubMed] [Google Scholar]
  28. Lomo T., Westgaard R. H. Control of ACh sensitivity in rat muscle fibers. Cold Spring Harb Symp Quant Biol. 1976;40:263–274. doi: 10.1101/sqb.1976.040.01.027. [DOI] [PubMed] [Google Scholar]
  29. MACKAY B., MUIR A. R., PETERS A. Observations on the terminal innervation of segmental muscle fibres in amphibia. Acta Anat (Basel) 1960;40:1–12. doi: 10.1159/000141568. [DOI] [PubMed] [Google Scholar]
  30. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Merlie J. P., Isenberg K. E., Russell S. D., Sanes J. R. Denervation supersensitivity in skeletal muscle: analysis with a cloned cDNA probe. J Cell Biol. 1984 Jul;99(1 Pt 1):332–335. doi: 10.1083/jcb.99.1.332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Merlie J. P., Sanes J. R. Concentration of acetylcholine receptor mRNA in synaptic regions of adult muscle fibres. Nature. 1985 Sep 5;317(6032):66–68. doi: 10.1038/317066a0. [DOI] [PubMed] [Google Scholar]
  33. Merlie J. P., Sebbane R., Gardner S., Olson E., Lindstrom J. The regulation of acetylcholine receptor expression in mammalian muscle. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 1):135–146. doi: 10.1101/sqb.1983.048.01.016. [DOI] [PubMed] [Google Scholar]
  34. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  35. Mishina M., Takai T., Imoto K., Noda M., Takahashi T., Numa S., Methfessel C., Sakmann B. Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature. 1986 May 22;321(6068):406–411. doi: 10.1038/321406a0. [DOI] [PubMed] [Google Scholar]
  36. Mohun T. J., Brennan S., Dathan N., Fairman S., Gurdon J. B. Cell type-specific activation of actin genes in the early amphibian embryo. Nature. 1984 Oct 25;311(5988):716–721. doi: 10.1038/311716a0. [DOI] [PubMed] [Google Scholar]
  37. Newport J., Kirschner M. A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. Cell. 1982 Oct;30(3):675–686. doi: 10.1016/0092-8674(82)90272-0. [DOI] [PubMed] [Google Scholar]
  38. Noda M., Takahashi H., Tanabe T., Toyosato M., Kikyotani S., Furutani Y., Hirose T., Takashima H., Inayama S., Miyata T. Structural homology of Torpedo californica acetylcholine receptor subunits. Nature. 1983 Apr 7;302(5908):528–532. doi: 10.1038/302528a0. [DOI] [PubMed] [Google Scholar]
  39. Nudel U., Salmon J., Fibach E., Terada M., Rifkind R., Marks P. A., Bank A. Accumulation of alpha- and beta-globin messenger RNAs in mouse erythroleukemia cells. Cell. 1977 Oct;12(2):463–469. doi: 10.1016/0092-8674(77)90122-2. [DOI] [PubMed] [Google Scholar]
  40. Perry R. P., Kelley D. E., Coleclough C., Kearney J. F. Organization and expression of immunoglobulin genes in fetal liver hybridomas. Proc Natl Acad Sci U S A. 1981 Jan;78(1):247–251. doi: 10.1073/pnas.78.1.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rebagliati M. R., Weeks D. L., Harvey R. P., Melton D. A. Identification and cloning of localized maternal RNAs from Xenopus eggs. Cell. 1985 Oct;42(3):769–777. doi: 10.1016/0092-8674(85)90273-9. [DOI] [PubMed] [Google Scholar]
  42. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  43. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sargent P. B., Hedges B. E., Tsavaler L., Clemmons L., Tzartos S., Lindstrom J. M. Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies. J Cell Biol. 1984 Feb;98(2):609–618. doi: 10.1083/jcb.98.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Siden E., Alt F. W., Shinefeld L., Sato V., Baltimore D. Synthesis of immunoglobulin mu chain gene products precedes synthesis of light chains during B-lymphocyte development. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1823–1827. doi: 10.1073/pnas.78.3.1823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Takai T., Noda M., Mishina M., Shimizu S., Furutani Y., Kayano T., Ikeda T., Kubo T., Takahashi H., Takahashi T. Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle. 1985 Jun 27-Jul 3Nature. 315(6022):761–764. doi: 10.1038/315761a0. [DOI] [PubMed] [Google Scholar]
  47. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yu L., LaPolla R. J., Davidson N. Mouse muscle nicotinic acetylcholine receptor gamma subunit: cDNA sequence and gene expression. Nucleic Acids Res. 1986 Apr 25;14(8):3539–3555. doi: 10.1093/nar/14.8.3539. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES