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. 1991 Oct 2;115(2):423–434. doi: 10.1083/jcb.115.2.423

Fiber type- and position-dependent expression of a myosin light chain- CAT transgene detected with a novel histochemical stain for CAT

PMCID: PMC2289162  PMID: 1717485

Abstract

We recently generated and characterized transgenic mice in which regulatory sequences from a myosin light chain gene (MLC1f/3f) are linked to the chloramphenicol acetyltransferase (CAT) gene. Transgene expression in these mice is specific to skeletal muscle and graded along the rostrocaudal axis: adult muscles derived from successively more caudal somites express successively higher levels of CAT. To investigate the cellular basis of these patterns of expression, we developed and used a histochemical stain that allows detection of CAT in individual cells. Our main results are as follows: (a) Within muscles, CAT is detected only in muscle fibers and not in associated connective tissue, blood vessels, or nerves. Thus, the tissue specificity of transgene expression observed by biochemical assay reflects a cell-type specificity demonstrable histochemically. (b) Within individual muscles, CAT levels vary with fiber type. Like the endogenous MLC1f/3f gene, the transgene is expressed at higher levels in fast-twitch (type II) than in slow-twitch (type I) muscle fibers. In addition, CAT levels vary among type II fiber subtypes, in the order IIB greater than IIX greater than IIA. (c) Among muscles that are similar in fiber type composition, the average level of CAT per fiber varies with rostrocaudal position. This position-dependent variation in CAT level is apparent even when fibers of a single type are compared. From these results, we conclude that fiber type and position affect CAT expression independently. We therefore infer the existence of separate fiber type-specific and positionally graded transcriptional regulators that act together to determine levels of transgene expression.

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Selected References

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  1. Alam J., Cook J. L. Reporter genes: application to the study of mammalian gene transcription. Anal Biochem. 1990 Aug 1;188(2):245–254. doi: 10.1016/0003-2697(90)90601-5. [DOI] [PubMed] [Google Scholar]
  2. Ariano M. A., Armstrong R. B., Edgerton V. R. Hindlimb muscle fiber populations of five mammals. J Histochem Cytochem. 1973 Jan;21(1):51–55. doi: 10.1177/21.1.51. [DOI] [PubMed] [Google Scholar]
  3. Armstrong R. B., Phelps R. O. Muscle fiber type composition of the rat hindlimb. Am J Anat. 1984 Nov;171(3):259–272. doi: 10.1002/aja.1001710303. [DOI] [PubMed] [Google Scholar]
  4. Ausoni S., Gorza L., Schiaffino S., Gundersen K., Lømo T. Expression of myosin heavy chain isoforms in stimulated fast and slow rat muscles. J Neurosci. 1990 Jan;10(1):153–160. doi: 10.1523/JNEUROSCI.10-01-00153.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barrnett R. J., Mazurkiewicz J. E., Addis J. S. Avian salt gland: a model for the study of membrane biogenesis. Methods Enzymol. 1983;96:627–659. doi: 10.1016/s0076-6879(83)96055-x. [DOI] [PubMed] [Google Scholar]
  6. Bateson D. S., Parry D. J. Motor units in a fast-twitch muscle of normal and dystrophic mice. J Physiol. 1983 Dec;345:515–523. doi: 10.1113/jphysiol.1983.sp014993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Billeter R., Heizmann C. W., Howald H., Jenny E. Analysis of myosin light and heavy chain types in single human skeletal muscle fibers. Eur J Biochem. 1981 May 15;116(2):389–395. doi: 10.1111/j.1432-1033.1981.tb05347.x. [DOI] [PubMed] [Google Scholar]
  8. Bunge M. B., Wood P. M., Tynan L. B., Bates M. L., Sanes J. R. Perineurium originates from fibroblasts: demonstration in vitro with a retroviral marker. Science. 1989 Jan 13;243(4888):229–231. doi: 10.1126/science.2492115. [DOI] [PubMed] [Google Scholar]
  9. Burt A. M. A histochemical procedure for the localization of choline acetyltransferase activity. J Histochem Cytochem. 1970 Jun;18(6):408–415. doi: 10.1177/18.6.408. [DOI] [PubMed] [Google Scholar]
  10. Crow M. T., Olson P. S., Stockdale F. E. Myosin light-chain expression during avian muscle development. J Cell Biol. 1983 Mar;96(3):736–744. doi: 10.1083/jcb.96.3.736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Donoghue M. J., Merlie J. P., Rosenthal N., Sanes J. R. Rostrocaudal gradient of transgene expression in adult skeletal muscle. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5847–5851. doi: 10.1073/pnas.88.13.5847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Donoghue M., Ernst H., Wentworth B., Nadal-Ginard B., Rosenthal N. A muscle-specific enhancer is located at the 3' end of the myosin light-chain 1/3 gene locus. Genes Dev. 1988 Dec;2(12B):1779–1790. doi: 10.1101/gad.2.12b.1779. [DOI] [PubMed] [Google Scholar]
  13. Düsterhöft S., Yablonka-Reuveni Z., Pette D. Characterization of myosin isoforms in satellite cell cultures from adult rat diaphragm, soleus and tibialis anterior muscles. Differentiation. 1990 Dec;45(3):185–191. doi: 10.1111/j.1432-0436.1990.tb00472.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galileo D. S., Gray G. E., Owens G. C., Majors J., Sanes J. R. Neurons and glia arise from a common progenitor in chicken optic tectum: demonstration with two retroviruses and cell type-specific antibodies. Proc Natl Acad Sci U S A. 1990 Jan;87(1):458–462. doi: 10.1073/pnas.87.1.458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goring D. R., Rossant J., Clapoff S., Breitman M. L., Tsui L. C. In situ detection of beta-galactosidase in lenses of transgenic mice with a gamma-crystallin/lacZ gene. Science. 1987 Jan 23;235(4787):456–458. doi: 10.1126/science.3099390. [DOI] [PubMed] [Google Scholar]
  16. Gorza L. Identification of a novel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies. J Histochem Cytochem. 1990 Feb;38(2):257–265. doi: 10.1177/38.2.2137154. [DOI] [PubMed] [Google Scholar]
  17. Hallauer P. L., Hastings K. E., Peterson A. C. Fast skeletal muscle-specific expression of a quail troponin I gene in transgenic mice. Mol Cell Biol. 1988 Dec;8(12):5072–5079. doi: 10.1128/mcb.8.12.5072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Higgins J. A., Barrnett R. J. Cytochemical localization of transferase activities: carnitine acetyltransferase. J Cell Sci. 1970 Jan;6(1):29–51. doi: 10.1242/jcs.6.1.29. [DOI] [PubMed] [Google Scholar]
  19. Hiromi Y., Kuroiwa A., Gehring W. J. Control elements of the Drosophila segmentation gene fushi tarazu. Cell. 1985 Dec;43(3 Pt 2):603–613. doi: 10.1016/0092-8674(85)90232-6. [DOI] [PubMed] [Google Scholar]
  20. Johnson J. E., Wold B. J., Hauschka S. D. Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice. Mol Cell Biol. 1989 Aug;9(8):3393–3399. doi: 10.1128/mcb.9.8.3393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. KARNOVSKY M. J., ROOTS L. A "DIRECT-COLORING" THIOCHOLINE METHOD FOR CHOLINESTERASES. J Histochem Cytochem. 1964 Mar;12:219–221. doi: 10.1177/12.3.219. [DOI] [PubMed] [Google Scholar]
  22. KARNOVSKY M. J. THE LOCALIZATION OF CHOLINESTERASE ACTIVITY IN RAT CARDIAC MUSCLE BY ELECTRON MICROSCOPY. J Cell Biol. 1964 Nov;23:217–232. doi: 10.1083/jcb.23.2.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kása P., Mann S. P., Hebb C. Localization of choline acetyltransferase. Nature. 1970 May 30;226(5248):812–814. doi: 10.1038/226812a0. [DOI] [PubMed] [Google Scholar]
  24. Kása P. The cholinergic systems in brain and spinal cord. Prog Neurobiol. 1986;26(3):211–272. doi: 10.1016/0301-0082(86)90016-x. [DOI] [PubMed] [Google Scholar]
  25. Laskowski M. B., High J. A. Expression of nerve-muscle topography during development. J Neurosci. 1989 Jan;9(1):175–182. doi: 10.1523/JNEUROSCI.09-01-00175.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Laskowski M. B., Sanes J. R. Topographic mapping of motor pools onto skeletal muscles. J Neurosci. 1987 Jan;7(1):252–260. doi: 10.1523/JNEUROSCI.07-01-00252.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Laskowski M. B., Sanes J. R. Topographically selective reinnervation of adult mammalian skeletal muscles. J Neurosci. 1988 Aug;8(8):3094–3099. doi: 10.1523/JNEUROSCI.08-08-03094.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lewis D. M., Parry D. J., Rowlerson A. Isometric contractions of motor units and immunohistochemistry of mouse soleus muscle. J Physiol. 1982 Apr;325:393–401. doi: 10.1113/jphysiol.1982.sp014157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mabuchi K., Szvetko D., Pintér K., Sréter F. A. Type IIB to IIA fiber transformation in intermittently stimulated rabbit muscles. Am J Physiol. 1982 May;242(5):C373–C381. doi: 10.1152/ajpcell.1982.242.5.C373. [DOI] [PubMed] [Google Scholar]
  30. Merlie J. P., Kornhauser J. M. Neural regulation of gene expression by an acetylcholine receptor promoter in muscle of transgenic mice. Neuron. 1989 Apr;2(4):1295–1300. doi: 10.1016/0896-6273(89)90067-6. [DOI] [PubMed] [Google Scholar]
  31. Miller J. B., Stockdale F. E. Developmental origins of skeletal muscle fibers: clonal analysis of myogenic cell lineages based on expression of fast and slow myosin heavy chains. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3860–3864. doi: 10.1073/pnas.83.11.3860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mizusawa H., Takagi A., Sugita H., Toyokura Y. Coexistence of fast and slow types of myosin light chains in a single fiber of rat soleus muscle. J Biochem. 1982 Jan;91(1):423–425. doi: 10.1093/oxfordjournals.jbchem.a133706. [DOI] [PubMed] [Google Scholar]
  33. Moscovici C., Moscovici M. G., Jimenez H., Lai M. M., Hayman M. J., Vogt P. K. Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail. Cell. 1977 May;11(1):95–103. doi: 10.1016/0092-8674(77)90320-8. [DOI] [PubMed] [Google Scholar]
  34. Nabeshima Y., Fujii-Kuriyama Y., Muramatsu M., Ogata K. Alternative transcription and two modes of splicing results in two myosin light chains from one gene. Nature. 1984 Mar 22;308(5957):333–338. doi: 10.1038/308333a0. [DOI] [PubMed] [Google Scholar]
  35. Parry D. J., Wilkinson R. S. The effect of reinnervation on the distribution of muscle fibre types in the tibialis anterior muscle of the mouse. Can J Physiol Pharmacol. 1990 May;68(5):596–602. doi: 10.1139/y90-086. [DOI] [PubMed] [Google Scholar]
  36. Periasamy M., Strehler E. E., Garfinkel L. I., Gubits R. M., Ruiz-Opazo N., Nadal-Ginard B. Fast skeletal muscle myosin light chains 1 and 3 are produced from a single gene by a combined process of differential RNA transcription and splicing. J Biol Chem. 1984 Nov 10;259(21):13595–13604. [PubMed] [Google Scholar]
  37. Petropoulos C. J., Rosenberg M. P., Jenkins N. A., Copeland N. G., Hughes S. H. The chicken skeletal muscle alpha-actin promoter is tissue specific in transgenic mice. Mol Cell Biol. 1989 Sep;9(9):3785–3792. doi: 10.1128/mcb.9.9.3785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pette D., Schnez U. Myosin light chain patterns of individual fast and slow-twitch fibres of rabbit muscles. Histochemistry. 1977 Oct 22;54(2):97–107. doi: 10.1007/BF00489668. [DOI] [PubMed] [Google Scholar]
  39. Pette D., Staron R. S. Cellular and molecular diversities of mammalian skeletal muscle fibers. Rev Physiol Biochem Pharmacol. 1990;116:1–76. doi: 10.1007/3540528806_3. [DOI] [PubMed] [Google Scholar]
  40. Pette D., Vrbová G. Neural control of phenotypic expression in mammalian muscle fibers. Muscle Nerve. 1985 Oct;8(8):676–689. doi: 10.1002/mus.880080810. [DOI] [PubMed] [Google Scholar]
  41. Robert B., Daubas P., Akimenko M. A., Cohen A., Garner I., Guenet J. L., Buckingham M. A single locus in the mouse encodes both myosin light chains 1 and 3, a second locus corresponds to a related pseudogene. Cell. 1984 Nov;39(1):129–140. doi: 10.1016/0092-8674(84)90198-3. [DOI] [PubMed] [Google Scholar]
  42. Rosenthal N., Kornhauser J. M., Donoghue M., Rosen K. M., Merlie J. P. Myosin light chain enhancer activates muscle-specific, developmentally regulated gene expression in transgenic mice. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7780–7784. doi: 10.1073/pnas.86.20.7780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rubinstein N. A., Holtzer H. Fast and slow muscles in tissue culture synthesise only fast myosin. Nature. 1979 Jul 26;280(5720):323–325. doi: 10.1038/280323a0. [DOI] [PubMed] [Google Scholar]
  44. Russo A. F., Crenshaw E. B., 3rd, Lira S. A., Simmons D. M., Swanson L. W., Rosenfeld M. G. Neuronal expression of chimeric genes in transgenic mice. Neuron. 1988 Jun;1(4):311–320. doi: 10.1016/0896-6273(88)90079-7. [DOI] [PubMed] [Google Scholar]
  45. Sanes J. R., Rubenstein J. L., Nicolas J. F. Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 1986 Dec 1;5(12):3133–3142. doi: 10.1002/j.1460-2075.1986.tb04620.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schiaffino S., Gorza L., Sartore S., Saggin L., Ausoni S., Vianello M., Gundersen K., Lømo T. Three myosin heavy chain isoforms in type 2 skeletal muscle fibres. J Muscle Res Cell Motil. 1989 Jun;10(3):197–205. doi: 10.1007/BF01739810. [DOI] [PubMed] [Google Scholar]
  47. Shani M. Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice. Mol Cell Biol. 1986 Jul;6(7):2624–2631. doi: 10.1128/mcb.6.7.2624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shani M. Tissue-specific expression of rat myosin light-chain 2 gene in transgenic mice. Nature. 1985 Mar 21;314(6008):283–286. doi: 10.1038/314283a0. [DOI] [PubMed] [Google Scholar]
  49. Staron R. S., Pette D. The multiplicity of combinations of myosin light chains and heavy chains in histochemically typed single fibres. Rabbit soleus muscle. Biochem J. 1987 May 1;243(3):687–693. doi: 10.1042/bj2430687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Staron R. S., Pette D. The multiplicity of combinations of myosin light chains and heavy chains in histochemically typed single fibres. Rabbit tibialis anterior muscle. Biochem J. 1987 May 1;243(3):695–699. doi: 10.1042/bj2430695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Stockdale F. E., Miller J. B. The cellular basis of myosin heavy chain isoform expression during development of avian skeletal muscles. Dev Biol. 1987 Sep;123(1):1–9. doi: 10.1016/0012-1606(87)90420-9. [DOI] [PubMed] [Google Scholar]
  52. Strehler E. E., Periasamy M., Strehler-Page M. A., Nadal-Ginard B. Myosin light-chain 1 and 3 gene has two structurally distinct and differentially regulated promoters evolving at different rates. Mol Cell Biol. 1985 Nov;5(11):3168–3182. doi: 10.1128/mcb.5.11.3168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Suzue T., Kaprielian Z., Patterson P. H. A monoclonal antibody that defines rostrocaudal gradients in the mammalian nervous system. Neuron. 1990 Oct;5(4):421–431. doi: 10.1016/0896-6273(90)90081-p. [DOI] [PubMed] [Google Scholar]
  54. Termin A., Staron R. S., Pette D. Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle. Histochemistry. 1989;92(6):453–457. doi: 10.1007/BF00524756. [DOI] [PubMed] [Google Scholar]
  55. Thompson W. J., Condon K., Astrow S. H. The origin and selective innervation of early muscle fiber types in the rat. J Neurobiol. 1990 Jan;21(1):212–222. doi: 10.1002/neu.480210114. [DOI] [PubMed] [Google Scholar]
  56. Tsuji S., Larabi Y. A modification of thiocholine-ferricyanide method of Karnovsky and Roots for localization of acetylcholinesterase activity without interference by Koelle's copper thiocholine iodide precipitate. Histochemistry. 1983;78(3):317–323. doi: 10.1007/BF00496619. [DOI] [PubMed] [Google Scholar]
  57. Weintraub H., Davis R., Tapscott S., Thayer M., Krause M., Benezra R., Blackwell T. K., Turner D., Rupp R., Hollenberg S. The myoD gene family: nodal point during specification of the muscle cell lineage. Science. 1991 Feb 15;251(4995):761–766. doi: 10.1126/science.1846704. [DOI] [PubMed] [Google Scholar]
  58. Wigston D. J., Sanes J. R. Selective reinnervation of adult mammalian muscle by axons from different segmental levels. Nature. 1982 Sep 30;299(5882):464–467. doi: 10.1038/299464a0. [DOI] [PubMed] [Google Scholar]
  59. Wigston D. J., Sanes J. R. Selective reinnervation of intercostal muscles transplanted from different segmental levels to a common site. J Neurosci. 1985 May;5(5):1208–1221. doi: 10.1523/JNEUROSCI.05-05-01208.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Young O. A., Davey C. L. Electrophoretic analysis of proteins from single bovine muscle fibres. Biochem J. 1981 Apr 1;195(1):317–327. doi: 10.1042/bj1950317. [DOI] [PMC free article] [PubMed] [Google Scholar]

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