Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Jun;13(6):3311–3323. doi: 10.1128/mcb.13.6.3311

Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.

M Muthuchamy 1, L Pajak 1, P Howles 1, T Doetschman 1, D F Wieczorek 1
PMCID: PMC359786  PMID: 7684495

Abstract

Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development.

Full text

PDF
3311

Images in this article

3314Image
on p.3314
3315Image
on p.3315
3316Image
on p.3316
3317Image
on p.3317
3317Image
on p.3317
3318Image
on p.3318
3318Image
on p.3318
3319Image
on p.3319

Selected References

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

  1. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  2. Bretscher A., Lynch W. Identification and localization of immunoreactive forms of caldesmon in smooth and nonmuscle cells: a comparison with the distributions of tropomyosin and alpha-actinin. J Cell Biol. 1985 May;100(5):1656–1663. doi: 10.1083/jcb.100.5.1656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bretscher A. Thin filament regulatory proteins of smooth- and non-muscle cells. Nature. 1986 Jun 19;321(6072):726–727. doi: 10.1038/321726b0. [DOI] [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Clayton L., Reinach F. C., Chumbley G. M., MacLeod A. R. Organization of the hTMnm gene. Implications for the evolution of muscle and non-muscle tropomyosins. J Mol Biol. 1988 Jun 5;201(3):507–515. doi: 10.1016/0022-2836(88)90633-x. [DOI] [PubMed] [Google Scholar]
  6. Clouet d'Orval B., d'Aubenton Carafa Y., Sirand-Pugnet P., Gallego M., Brody E., Marie J. RNA secondary structure repression of a muscle-specific exon in HeLa cell nuclear extracts. Science. 1991 Jun 28;252(5014):1823–1828. doi: 10.1126/science.2063195. [DOI] [PubMed] [Google Scholar]
  7. Cummins P., Perry S. V. Chemical and immunochemical characteristics of tropomyosins from striated and smooth muscle. Biochem J. 1974 Jul;141(1):43–49. doi: 10.1042/bj1410043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cummins P., Perry S. V. The subunits and biological activity of polymorphic forms of tropomyosin. Biochem J. 1973 Aug;133(4):765–777. doi: 10.1042/bj1330765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Doetschman T. C., Eistetter H., Katz M., Schmidt W., Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol. 1985 Jun;87:27–45. [PubMed] [Google Scholar]
  10. Ebert J. D. An Analysis of the Synthesis and Distribution of the Contractile Protein, Myosin, in the Development of the Heart. Proc Natl Acad Sci U S A. 1953 Apr;39(4):333–344. doi: 10.1073/pnas.39.4.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. GOSS C. M. Development of the median coordinated ventricle from the lateral hearts in rat embryos with three to six somites. Anat Rec. 1952 Apr;112(4):761–796. doi: 10.1002/ar.1091120405. [DOI] [PubMed] [Google Scholar]
  12. Gallego M. E., Balvay L., Brody E. cis-acting sequences involved in exon selection in the chicken beta-tropomyosin gene. Mol Cell Biol. 1992 Dec;12(12):5415–5425. doi: 10.1128/mcb.12.12.5415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goux-Pelletan M., Libri D., d'Aubenton-Carafa Y., Fiszman M., Brody E., Marie J. In vitro splicing of mutually exclusive exons from the chicken beta-tropomyosin gene: role of the branch point location and very long pyrimidine stretch. EMBO J. 1990 Jan;9(1):241–249. doi: 10.1002/j.1460-2075.1990.tb08101.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gunning P., Gordon M., Wade R., Gahlmann R., Lin C. S., Hardeman E. Differential control of tropomyosin mRNA levels during myogenesis suggests the existence of an isoform competition-autoregulatory compensation control mechanism. Dev Biol. 1990 Apr;138(2):443–453. doi: 10.1016/0012-1606(90)90210-a. [DOI] [PubMed] [Google Scholar]
  15. Guo W., Mulligan G. J., Wormsley S., Helfman D. M. Alternative splicing of beta-tropomyosin pre-mRNA: cis-acting elements and cellular factors that block the use of a skeletal muscle exon in nonmuscle cells. Genes Dev. 1991 Nov;5(11):2096–2107. doi: 10.1101/gad.5.11.2096. [DOI] [PubMed] [Google Scholar]
  16. Helfman D. M., Cheley S., Kuismanen E., Finn L. A., Yamawaki-Kataoka Y. Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation. Mol Cell Biol. 1986 Nov;6(11):3582–3595. doi: 10.1128/mcb.6.11.3582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Helfman D. M., Ricci W. M. Branch point selection in alternative splicing of tropomyosin pre-mRNAs. Nucleic Acids Res. 1989 Jul 25;17(14):5633–5650. doi: 10.1093/nar/17.14.5633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Helfman D. M., Roscigno R. F., Mulligan G. J., Finn L. A., Weber K. S. Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA. Genes Dev. 1990 Jan;4(1):98–110. doi: 10.1101/gad.4.1.98. [DOI] [PubMed] [Google Scholar]
  19. Icardo J. M. Heart anatomy and developmental biology. Experientia. 1988 Dec 1;44(11-12):910–919. doi: 10.1007/BF01939884. [DOI] [PubMed] [Google Scholar]
  20. Izumo S., Nadal-Ginard B., Mahdavi V. Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload. Proc Natl Acad Sci U S A. 1988 Jan;85(2):339–343. doi: 10.1073/pnas.85.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Karlik C. C., Fyrberg E. A. An insertion within a variably spliced Drosophila tropomyosin gene blocks accumulation of only one encoded isoform. Cell. 1985 May;41(1):57–66. doi: 10.1016/0092-8674(85)90061-3. [DOI] [PubMed] [Google Scholar]
  22. Latham K. E., Garrels J. I., Chang C., Solter D. Quantitative analysis of protein synthesis in mouse embryos. I. Extensive reprogramming at the one- and two-cell stages. Development. 1991 Aug;112(4):921–932. doi: 10.1242/dev.112.4.921. [DOI] [PubMed] [Google Scholar]
  23. Lazarides E. Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells. J Cell Biol. 1976 Feb;68(2):202–219. doi: 10.1083/jcb.68.2.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lees-Miller J. P., Goodwin L. O., Helfman D. M. Three novel brain tropomyosin isoforms are expressed from the rat alpha-tropomyosin gene through the use of alternative promoters and alternative RNA processing. Mol Cell Biol. 1990 Apr;10(4):1729–1742. doi: 10.1128/mcb.10.4.1729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lees-Miller J. P., Helfman D. M. The molecular basis for tropomyosin isoform diversity. Bioessays. 1991 Sep;13(9):429–437. doi: 10.1002/bies.950130902. [DOI] [PubMed] [Google Scholar]
  26. Lees-Miller J. P., Yan A., Helfman D. M. Structure and complete nucleotide sequence of the gene encoding rat fibroblast tropomyosin 4. J Mol Biol. 1990 Jun 5;213(3):399–405. doi: 10.1016/S0022-2836(05)80202-5. [DOI] [PubMed] [Google Scholar]
  27. Lemanski L. F. Morphology of developing heart in cardiac lethal mutant Mexican axolotls, Ambystoma mexicanum. Dev Biol. 1973 Aug;33(2):312–333. doi: 10.1016/0012-1606(73)90140-1. [DOI] [PubMed] [Google Scholar]
  28. Libri D., Balvay L., Fiszman M. Y. In vivo splicing of the beta tropomyosin pre-mRNA: a role for branch point and donor site competition. Mol Cell Biol. 1992 Jul;12(7):3204–3215. doi: 10.1128/mcb.12.7.3204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Libri D., Piseri A., Fiszman M. Y. Tissue-specific splicing in vivo of the beta-tropomyosin gene: dependence on an RNA secondary structure. Science. 1991 Jun 28;252(5014):1842–1845. doi: 10.1126/science.2063196. [DOI] [PubMed] [Google Scholar]
  30. Lindenbaum M. H., Grosveld F. An in vitro globin gene switching model based on differentiated embryonic stem cells. Genes Dev. 1990 Dec;4(12A):2075–2085. doi: 10.1101/gad.4.12a.2075. [DOI] [PubMed] [Google Scholar]
  31. Liu H. P., Bretscher A. Disruption of the single tropomyosin gene in yeast results in the disappearance of actin cables from the cytoskeleton. Cell. 1989 Apr 21;57(2):233–242. doi: 10.1016/0092-8674(89)90961-6. [DOI] [PubMed] [Google Scholar]
  32. Marston S. B., Smith C. W. The thin filaments of smooth muscles. J Muscle Res Cell Motil. 1985 Dec;6(6):669–708. doi: 10.1007/BF00712237. [DOI] [PubMed] [Google Scholar]
  33. McInnes C., Leader D. P. The tropomyosin mRNAs of mouse striated muscles: molecular cloning of beta-tropomyosin. Biochim Biophys Acta. 1988 Nov 10;951(1):117–122. doi: 10.1016/0167-4781(88)90031-0. [DOI] [PubMed] [Google Scholar]
  34. Muthuchamy M., Pajak L., Wieczorek D. F. Induction of endogenous myosin light chain 1 and cardiac alpha-actin expression in L6E9 cells by MyoD1. J Biol Chem. 1992 Sep 15;267(26):18728–18734. [PubMed] [Google Scholar]
  35. Robbins J., Gulick J., Sanchez A., Howles P., Doetschman T. Mouse embryonic stem cells express the cardiac myosin heavy chain genes during development in vitro. J Biol Chem. 1990 Jul 15;265(20):11905–11909. [PubMed] [Google Scholar]
  36. Ruiz-Opazo N., Nadal-Ginard B. Alpha-tropomyosin gene organization. Alternative splicing of duplicated isotype-specific exons accounts for the production of smooth and striated muscle isoforms. J Biol Chem. 1987 Apr 5;262(10):4755–4765. [PubMed] [Google Scholar]
  37. Schultheiss T., Lin Z. X., Lu M. H., Murray J., Fischman D. A., Weber K., Masaki T., Imamura M., Holtzer H. Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils. J Cell Biol. 1990 Apr;110(4):1159–1172. doi: 10.1083/jcb.110.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sánchez A., Jones W. K., Gulick J., Doetschman T., Robbins J. Myosin heavy chain gene expression in mouse embryoid bodies. An in vitro developmental study. J Biol Chem. 1991 Nov 25;266(33):22419–22426. [PubMed] [Google Scholar]
  39. Takenaga K., Nakamura Y., Kageyama H., Sakiyama S. Nucleotide sequence of cDNA for nonmuscle tropomyosin 5 of mouse fibroblast. Biochim Biophys Acta. 1990 Sep 10;1087(1):101–103. doi: 10.1016/0167-4781(90)90129-p. [DOI] [PubMed] [Google Scholar]
  40. Tansey T., Mikus M. D., Dumoulin M., Storti R. V. Transformation and rescue of a flightless Drosophila tropomyosin mutant. EMBO J. 1987 May;6(5):1375–1385. doi: 10.1002/j.1460-2075.1987.tb02378.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wang S. M., Greaser M. L., Schultz E., Bulinski J. C., Lin J. J., Lessard J. L. Studies on cardiac myofibrillogenesis with antibodies to titin, actin, tropomyosin, and myosin. J Cell Biol. 1988 Sep;107(3):1075–1083. doi: 10.1083/jcb.107.3.1075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wang Y. C., Rubenstein P. A. Choice of 3' cleavage/polyadenylation site in beta-tropomyosin RNA processing is differentiation-dependent in mouse BC3H1 muscle cells. J Biol Chem. 1992 Feb 5;267(4):2728–2736. [PubMed] [Google Scholar]
  43. Wang Y. C., Rubenstein P. A. Splicing of two alternative exon pairs in beta-tropomyosin pre-mRNA is independently controlled during myogenesis. J Biol Chem. 1992 Jun 15;267(17):12004–12010. [PubMed] [Google Scholar]
  44. Wieczorek D. F., Periasamy M., Butler-Browne G. S., Whalen R. G., Nadal-Ginard B. Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature. J Cell Biol. 1985 Aug;101(2):618–629. doi: 10.1083/jcb.101.2.618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wieczorek D. F., Smith C. W., Nadal-Ginard B. The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing. Mol Cell Biol. 1988 Feb;8(2):679–694. doi: 10.1128/mcb.8.2.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yamawaki-Kataoka Y., Helfman D. M. Rat embryonic fibroblast tropomyosin 1. cDNA and complete primary amino acid sequence. J Biol Chem. 1985 Nov 25;260(27):14440–14445. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES