Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Dec 2;115(6):1675–1684. doi: 10.1083/jcb.115.6.1675

Polarized and functional epithelia can form after the targeted inactivation of both mouse keratin 8 alleles

PMCID: PMC2289210  PMID: 1721911

Abstract

We have tested the requirement of keratin intermediate filaments for the formation and function of a simple epithelium. We disrupted both alleles of the mouse keratin 8 (mK8) gene in embryonic stem cells, and subsequently analyzed the phenotype in developing embryoid bodies in suspension culture. After the inactivation of the mouse keratin 8 (mK8) gene by a targeted insertion, mK8 protein synthesis was undetectable. In the absence of mK8 its complementary partners mK18 and mK19 were unable to form filaments within differentiated cells. Surprisingly, these ES cells differentiate to both simple and cystic embryoid bodies with apparently normal epithelia. Ultrastructural analysis shows an apparently normal epithelium with microvilli on the apical membrane, tight junctions and desmosomes on the lateral membrane, and an underlying basal membrane. No significant differences in the synthesis or secretion of alpha 1-fetoprotein and laminin were observed between the mK8- or wild-type embryoid bodies. Our data show that mK8 is not required for simple epithelium formation of extraembryonic endoderm.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. Baribault H., Kemler R. Embryonic stem cell culture and gene targeting in transgenic mice. Mol Biol Med. 1989 Dec;6(6):481–492. [PubMed] [Google Scholar]
  2. Boller K., Kemler R., Baribault H., Doetschman T. Differential distribution of cytokeratins after microinjection of anti-cytokeratin monoclonal antibodies. Eur J Cell Biol. 1987 Jun;43(3):459–468. [PubMed] [Google Scholar]
  3. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  4. Campos-Ortega J. A., Knust E. Genetics of early neurogenesis in Drosophila melanogaster. Annu Rev Genet. 1990;24:387–407. doi: 10.1146/annurev.ge.24.120190.002131. [DOI] [PubMed] [Google Scholar]
  5. Capecchi M. R. Altering the genome by homologous recombination. Science. 1989 Jun 16;244(4910):1288–1292. doi: 10.1126/science.2660260. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Emerson J. A. Disruption of the cytokeratin filament network in the preimplantation mouse embryo. Development. 1988 Oct;104(2):219–234. doi: 10.1242/dev.104.2.219. [DOI] [PubMed] [Google Scholar]
  8. Evans M. J., Kaufman M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981 Jul 9;292(5819):154–156. doi: 10.1038/292154a0. [DOI] [PubMed] [Google Scholar]
  9. Grover A., Oshima R. G., Adamson E. D. Epithelial layer formation in differentiating aggregates of F9 embryonal carcinoma cells. J Cell Biol. 1983 Jun;96(6):1690–1696. doi: 10.1083/jcb.96.6.1690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hatzfeld M., Franke W. W. Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides. J Cell Biol. 1985 Nov;101(5 Pt 1):1826–1841. doi: 10.1083/jcb.101.5.1826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hatzfeld M., Weber K. The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression. J Cell Biol. 1990 Apr;110(4):1199–1210. doi: 10.1083/jcb.110.4.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hooper M., Hardy K., Handyside A., Hunter S., Monk M. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature. 1987 Mar 19;326(6110):292–295. doi: 10.1038/326292a0. [DOI] [PubMed] [Google Scholar]
  13. Jackson B. W., Grund C., Schmid E., Bürki K., Franke W. W., Illmensee K. Formation of cytoskeletal elements during mouse embryogenesis. Intermediate filaments of the cytokeratin type and desmosomes in preimplantation embryos. Differentiation. 1980;17(3):161–179. doi: 10.1111/j.1432-0436.1980.tb01093.x. [DOI] [PubMed] [Google Scholar]
  14. Jahn L., Fouquet B., Rohe K., Franke W. W. Cytokeratins in certain endothelial and smooth muscle cells of two taxonomically distant vertebrate species, Xenopus laevis and man. Differentiation. 1987;36(3):234–254. doi: 10.1111/j.1432-0436.1987.tb00198.x. [DOI] [PubMed] [Google Scholar]
  15. Jones J. C., Goldman R. D. Intermediate filaments and the initiation of desmosome assembly. J Cell Biol. 1985 Aug;101(2):506–517. doi: 10.1083/jcb.101.2.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Joyner A. L., Herrup K., Auerbach B. A., Davis C. A., Rossant J. Subtle cerebellar phenotype in mice homozygous for a targeted deletion of the En-2 homeobox. Science. 1991 Mar 8;251(4998):1239–1243. doi: 10.1126/science.1672471. [DOI] [PubMed] [Google Scholar]
  17. Kemler R., Brûlet P., Schnebelen M. T., Gaillard J., Jacob F. Reactivity of monoclonal antibodies against intermediate filament proteins during embryonic development. J Embryol Exp Morphol. 1981 Aug;64:45–60. [PubMed] [Google Scholar]
  18. Kim H. S., Smithies O. Recombinant fragment assay for gene targetting based on the polymerase chain reaction. Nucleic Acids Res. 1988 Sep 26;16(18):8887–8903. doi: 10.1093/nar/16.18.8887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Klatte D. H., Kurpakus M. A., Grelling K. A., Jones J. C. Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells. J Cell Biol. 1989 Dec;109(6 Pt 2):3377–3390. doi: 10.1083/jcb.109.6.3377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klymkowsky M. W., Miller R. H., Lane E. B. Morphology, behavior, and interaction of cultured epithelial cells after the antibody-induced disruption of keratin filament organization. J Cell Biol. 1983 Feb;96(2):494–509. doi: 10.1083/jcb.96.2.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Koller B. H., Hagemann L. J., Doetschman T., Hagaman J. R., Huang S., Williams P. J., First N. L., Maeda N., Smithies O. Germ-line transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8927–8931. doi: 10.1073/pnas.86.22.8927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kulesh D. A., Ceceña G., Darmon Y. M., Vasseur M., Oshima R. G. Posttranslational regulation of keratins: degradation of mouse and human keratins 18 and 8. Mol Cell Biol. 1989 Apr;9(4):1553–1565. doi: 10.1128/mcb.9.4.1553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kuruc N., Franke W. W. Transient coexpression of desmin and cytokeratins 8 and 18 in developing myocardial cells of some vertebrate species. Differentiation. 1988 Sep;38(3):177–193. doi: 10.1111/j.1432-0436.1988.tb00212.x. [DOI] [PubMed] [Google Scholar]
  24. Lu X., Lane E. B. Retrovirus-mediated transgenic keratin expression in cultured fibroblasts: specific domain functions in keratin stabilization and filament formation. Cell. 1990 Aug 24;62(4):681–696. doi: 10.1016/0092-8674(90)90114-t. [DOI] [PubMed] [Google Scholar]
  25. Mansour S. L., Thomas K. R., Capecchi M. R. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988 Nov 24;336(6197):348–352. doi: 10.1038/336348a0. [DOI] [PubMed] [Google Scholar]
  26. Mansour S. L., Thomas K. R., Deng C. X., Capecchi M. R. Introduction of a lacZ reporter gene into the mouse int-2 locus by homologous recombination. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7688–7692. doi: 10.1073/pnas.87.19.7688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Markl J., Franke W. W. Localization of cytokeratins in tissues of the rainbow trout: fundamental differences in expression pattern between fish and higher vertebrates. Differentiation. 1988 Dec;39(2):97–122. doi: 10.1111/j.1432-0436.1988.tb00086.x. [DOI] [PubMed] [Google Scholar]
  28. Martin G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634–7638. doi: 10.1073/pnas.78.12.7634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moll R., Franke W. W., Schiller D. L., Geiger B., Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982 Nov;31(1):11–24. doi: 10.1016/0092-8674(82)90400-7. [DOI] [PubMed] [Google Scholar]
  30. Moreau J. F., Donaldson D. D., Bennett F., Witek-Giannotti J., Clark S. C., Wong G. G. Leukaemia inhibitory factor is identical to the myeloid growth factor human interleukin for DA cells. Nature. 1988 Dec 15;336(6200):690–692. doi: 10.1038/336690a0. [DOI] [PubMed] [Google Scholar]
  31. Mortensen R. M., Zubiaur M., Neer E. J., Seidman J. G. Embryonic stem cells lacking a functional inhibitory G-protein subunit (alpha i2) produced by gene targeting of both alleles. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7036–7040. doi: 10.1073/pnas.88.16.7036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nagy A., Gócza E., Diaz E. M., Prideaux V. R., Iványi E., Markkula M., Rossant J. Embryonic stem cells alone are able to support fetal development in the mouse. Development. 1990 Nov;110(3):815–821. doi: 10.1242/dev.110.3.815. [DOI] [PubMed] [Google Scholar]
  33. Oshima R. G. Developmental expression of murine extra-embryonic endodermal cytoskeletal proteins. J Biol Chem. 1982 Apr 10;257(7):3414–3421. [PubMed] [Google Scholar]
  34. Oshima R. G., Howe W. E., Klier F. G., Adamson E. D., Shevinsky L. H. Intermediate filament protein synthesis in preimplantation murine embryos. Dev Biol. 1983 Oct;99(2):447–455. doi: 10.1016/0012-1606(83)90294-4. [DOI] [PubMed] [Google Scholar]
  35. Oshima R. G. Identification and immunoprecipitation of cytoskeletal proteins from murine extra-embryonic endodermal cells. J Biol Chem. 1981 Aug 10;256(15):8124–8133. [PubMed] [Google Scholar]
  36. Oshima R., Linney E. Identification of murine extra-embryonic endodermal cells by reaction with teratocarcinoma basement membrane antiserum. Exp Cell Res. 1980 Apr;126(2):485–490. doi: 10.1016/0014-4827(80)90294-3. [DOI] [PubMed] [Google Scholar]
  37. Owaribe K., Kartenbeck J., Rungger-Brändle E., Franke W. W. Cytoskeletons of retinal pigment epithelial cells: interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins. Cell Tissue Res. 1988 Nov;254(2):301–315. doi: 10.1007/BF00225803. [DOI] [PubMed] [Google Scholar]
  38. Pasdar M., Krzeminski K. A., Nelson W. J. Regulation of desmosome assembly in MDCK epithelial cells: coordination of membrane core and cytoplasmic plaque domain assembly at the plasma membrane. J Cell Biol. 1991 May;113(3):645–655. doi: 10.1083/jcb.113.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Paulin D., Jakob H., Jacob F., Weber K., Osborn M. In vitro differentiation of mouse teratocarcinoma cells monitored by intermediate filament expression. Differentiation. 1982;22(2):90–99. doi: 10.1111/j.1432-0436.1982.tb01231.x. [DOI] [PubMed] [Google Scholar]
  40. Pevny L., Simon M. C., Robertson E., Klein W. H., Tsai S. F., D'Agati V., Orkin S. H., Costantini F. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature. 1991 Jan 17;349(6306):257–260. doi: 10.1038/349257a0. [DOI] [PubMed] [Google Scholar]
  41. Plancha C. E., Carmo-Fonseca M., David-Ferreira J. F. Cytokeratin filaments are present in golden hamster oocytes and early embryos. Differentiation. 1989 Oct;42(1):1–9. doi: 10.1111/j.1432-0436.1989.tb00601.x. [DOI] [PubMed] [Google Scholar]
  42. Reid L. H., Gregg R. G., Smithies O., Koller B. H. Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4299–4303. doi: 10.1073/pnas.87.11.4299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Rossant J., Joyner A. L. Towards a molecular-genetic analysis of mammalian development. Trends Genet. 1989 Aug;5(8):277–283. doi: 10.1016/0168-9525(89)90102-9. [DOI] [PubMed] [Google Scholar]
  45. Schmitt R. M., Bruyns E., Snodgrass H. R. Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression. Genes Dev. 1991 May;5(5):728–740. doi: 10.1101/gad.5.5.728. [DOI] [PubMed] [Google Scholar]
  46. Schwartzberg P. L., Goff S. P., Robertson E. J. Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells. Science. 1989 Nov 10;246(4931):799–803. doi: 10.1126/science.2554496. [DOI] [PubMed] [Google Scholar]
  47. Singer P. A., Trevor K., Oshima R. G. Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos. J Biol Chem. 1986 Jan 15;261(2):538–547. [PubMed] [Google Scholar]
  48. Smith A. G., Heath J. K., Donaldson D. D., Wong G. G., Moreau J., Stahl M., Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 1988 Dec 15;336(6200):688–690. doi: 10.1038/336688a0. [DOI] [PubMed] [Google Scholar]
  49. Soriano P., Montgomery C., Geske R., Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991 Feb 22;64(4):693–702. doi: 10.1016/0092-8674(91)90499-o. [DOI] [PubMed] [Google Scholar]
  50. Steinert P. M., Roop D. R. Molecular and cellular biology of intermediate filaments. Annu Rev Biochem. 1988;57:593–625. doi: 10.1146/annurev.bi.57.070188.003113. [DOI] [PubMed] [Google Scholar]
  51. Sémat A., Vasseur M., Maillet L., Brûlet P., Darmon Y. M. Sequence analysis of murine cytokeratin endo A (no. 8) cDNA. Evidence for mRNA species initiated upstream of the normal 5' end in PCC4 cells. Differentiation. 1988;37(1):40–46. doi: 10.1111/j.1432-0436.1988.tb00794.x. [DOI] [PubMed] [Google Scholar]
  52. Thompson S., Clarke A. R., Pow A. M., Hooper M. L., Melton D. W. Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells. Cell. 1989 Jan 27;56(2):313–321. doi: 10.1016/0092-8674(89)90905-7. [DOI] [PubMed] [Google Scholar]
  53. Trevor K. T. Disruption of keratin filaments in embryonic epithelial cell types. New Biol. 1990 Nov;2(11):1004–1014. [PubMed] [Google Scholar]
  54. Valancius V., Smithies O. Testing an "in-out" targeting procedure for making subtle genomic modifications in mouse embryonic stem cells. Mol Cell Biol. 1991 Mar;11(3):1402–1408. doi: 10.1128/mcb.11.3.1402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Vassar R., Coulombe P. A., Degenstein L., Albers K., Fuchs E. Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease. Cell. 1991 Jan 25;64(2):365–380. doi: 10.1016/0092-8674(91)90645-f. [DOI] [PubMed] [Google Scholar]
  56. Vasseur M., Duprey P., Brûlet P., Jacob F. One gene and one pseudogene for the cytokeratin endo A. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1155–1159. doi: 10.1073/pnas.82.4.1155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wiles M. V., Keller G. Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development. 1991 Feb;111(2):259–267. doi: 10.1242/dev.111.2.259. [DOI] [PubMed] [Google Scholar]
  58. Williams R. L., Hilton D. J., Pease S., Willson T. A., Stewart C. L., Gearing D. P., Wagner E. F., Metcalf D., Nicola N. A., Gough N. M. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature. 1988 Dec 15;336(6200):684–687. doi: 10.1038/336684a0. [DOI] [PubMed] [Google Scholar]
  59. Zijlstra M., Li E., Sajjadi F., Subramani S., Jaenisch R. Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells. Nature. 1989 Nov 23;342(6248):435–438. doi: 10.1038/342435a0. [DOI] [PubMed] [Google Scholar]
  60. te Riele H., Maandag E. R., Clarke A., Hooper M., Berns A. Consecutive inactivation of both alleles of the pim-1 proto-oncogene by homologous recombination in embryonic stem cells. Nature. 1990 Dec 13;348(6302):649–651. doi: 10.1038/348649a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES