Abstract
The administration of the teratogen retinoic acid (and other retinoids) to vertebrate embryos causes a range of developmental abnormalities. It remains to be shown how these teratogenic effects are mediated, and whether or not they reflect any morphogenetic roles retinoids may have in normal development. The most intensively studied cellular action of retinoids has been the activation of retinoid receptors (RARs and RXRs), which are members of the steroid/thyroid family of ligand-modulated transcription factors. Here we report experiments designed to investigate whether the teratogenic effects of retinoic acid on early Xenopus embryos are mediated transcriptionally by receptors, and if these receptors are necessary for normal early Xenopus development. We have demonstrated transcriptional activation of injected reporter genes by exogeneously supplied retinoic acid in Xenopus embryos, presumably as a consequence of the activation of endogenous retinoid receptors. This assay system has been used to demonstrate functional expression, from injected mRNA of (1) a wild-type RARγ (2) a domain-swapped receptor in which the retinoic acid binding domain has been replaced by a thyroid hormone domain to create a thyroid hormone responsive receptor, and (3) a dominant negative from of the RARγ. The wild-type RARγ increases the severity of retinoic acid-mediated defects. In the presence of thyroid hormone the domain-swapped receptor causes abnormalities of gastrulation. The dominant negative decreases the severity of retinoic acid-mediated defects. We conclude that the teratogenic effects of exogenous retinoic acid on Xenopus embryos are mediated, at least in part, transcriptionally via retinoid receptors. It is notable that the dominant negative has no effect on normal development in the absence of exogenous retinoic acid. This is despite observations that this receptor completely blocks transcriptional activation of reporter genes by exogenous retinoic acid up to the beginning of gastrulation, and substantially relieves the teratogenic effects of retinoic acid.
Similar content being viewed by others
References
Amaya E, Musci TJ, Kirschner MW (1991) Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 66:257–270
Balling R (1991) CRABP and the teratogenic effects of retinoids. Trends Genet 7:35–36
Bernard BA, Luca LM De, Hassell JR, Yamada KM, Olden K (1984) Retinoic acid alters the proportion of high mannose to complex type oligosaccharides on fibronectin secreted by cultured chondrocytes. J Biol Chem 259:5310–5315
Blumberg B, Mangelsdorf DJ, Dyck JA, Bitner DA, Evans RM, Robertis de EM (1992) Multiple retinoid-responsive receptors in a single cell: Families of retinoid “X” receptors and retinoic acid receptors in the Xenopus egg. Proc Natl Acad Sci USA 89:2321–2325
Bolmer SD, Wolf G (1982) Retinoids and phorbol esters alter release of fibronectin from enucleated cells. Proc Natl Acad Sci USA 79:6541–6545
Brent GA, Harney JW, Chen Y, Warne RL, Moore DD, Larsen PR (1989) Mutations of the rat GH promoter which increase and decrease response to thyroid hormone define a consensus thyroid hormone response element. Mol Endocrinol 3:1996–2004
Brooks AR, Sweeney G, Old RW (1989) Structure and functional expression of a cloned Xenopus thyroid hormone receptor. Nucleic Acids Res 22:9395–9405
Cho KWY, Robertis EM de (1990) Differential activation of Xenopus imhomeo box genes by mesoderm-inducing growth factors and retinoic acid. Genes Dev 4:1910–1916
Cho KWY, Morita EA, Wright CVE, Robertis EM de (1991) Overexpression of a homeodomain protein confers axis-forming activity to uncommitted Xenopus embryonic cells. Cell 65:55–64
Dingle JT, Lucy JA (1965) Vitamin A, carotenoids and cell function. Biol Rev Cambridge Philos Soc 40:422–461
Dolle P, Ruberte E, Leroy P, Morriss-Kay G, Chambon P (1990) Retinoic acid receptors and cellular retinoid binding proteins. I. A systematic study of their differential patterns of transcription during mouse organogenesis. Development 110:1133–1151
Durston AJ, Timmermans JPM, Hage WJ, Hendriks HFJ, Vries NJ de, Heideveld M, Nieuwkoop PD (1989) Retinoic acid causes an anteroposterior transformation in the developing central nervous system. Nature 340:140–144
Ellinger-Ziegelbauer H, Dreyer C (1991) A retinoic acid receptor expressed in the early development of Xenopus laevis. Gene Dev 5:94–104
Giguere V, Ong ES, Segui P, Evans RM (1987) Identification of a receptor for the morphogen retinoic acid. Nature 330:624–629
Giguere V, Shago M, Zirngibl R, Tate P, Rossant J, Varmuza S (1990) Identification of a novel isoform of the retinoic acid receptor γ expressed in the mouse embryo. Mol Cell Biol 10:2335–2340
Glass CK, Lipkin SM, Devary OV, Rosenfeld MG (1989) Positive and negative regulation of gene transcription by a retinoic acid-thyroid hormone heterodimer. Cell 59:697–708
Glass CK, Devary OV, Rosenfeld MG (1990) Multiple cell type-specific proteins differentially regulate target gene sequence recognition by the α retinoic acid receptor. Cell 63:729–738
Gorman CM, Moffat LF, Howard BH (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2:1044–1051
Green S (1993) Promiscuous liasons, Nature 361:590–591
Heyman RA, Mangelsdorf DJ, Dyck JA, Stein RB, Eichele G, Evans RM, Thaller C (1992) 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell 68:397–406
Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68
Jones EA, Woodland HR (1986) Development of the ectoderm in Xenopus: tissue specification and the role of cell association and division. Cell 44:345–355
Kastner P, Krust A, Mendelsohn C, Garnier JM, Zelent A, Leroy P, Staub A, Chambon P (1990) Murine isoforms of retinoic acid receptor γ with specific patterns of expression. Proc Natl Acad Sci USA 87:2700–2704
Kliewer SA, Umesono K, Mangelsdorf DJ, Evans RM (1992) Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature 355:446–449
Krieg PA, Melton DA (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 12:7057–7070
Krust A, Kastner P, Petkovich M, Zelent A, Chambon P (1989) A third human retinoic acid receptor, hRAR-γ. Proc Natl Acad Sci USA 86:5310–5314
Leid M, Kastner P, Lyons R, Nakshatri H, Saunders M, Zacharewski T, Cheng J-Y, Staub A, Garnier M-M, Mader S, Chamnbon P (1992a) Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell 68:377–395
Leid M, Kaster P, Chambon P (1992b) Multiplicity generates diversity in the retinoic acid signalling pathways. Trends Biochem Sci 17:427–433
Lohnes D, Kastner P, Dierich A, Mark M, LeMeur M, Chambon P (1993) Function of retinoic acid receptor γ in the mouse. Cell 73:643–658
Luca L de (1977) The direct involvement of vitamin A in glycosyl transfer reactions of mammlian membranes. In: Munson PL, Diczfalusy E, Olson RE (eds) Vitamins and Hormones, Academic Press, New York, pp 1–57
Mangelsdorf DJ, Borgmeyer U, Heyman RA, Zhou JY, Ong ES, Oro AE, Kakizuka A, Evans RM (1992) Characterisation of three RXR genes that mediate the action of 9-cis retinoic acid. Genes Dev 6:329–344
Maniatis T, Fritsch ET, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, New York
Mavillo F (1993) Regulation of vertebrate homeobox-containing genes by morphogenes. Eur J Biochem 212:273–288
Old RW, Jones EA, Sweeney G, Smith DP (1992) Precocious synthesis of a thyroid hormone receptor in Xenopus embryos causes hormone-dependent developmental abnormalities. Roux's Arch Dev Biol 201:312–321
Pektovich M, Brand NJ, Krust A, Chambon P (1987) A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 330:444–450
Ragsdale CW, Petkovich M, Gates PB, Chambon P, Brockes JP (1989) Identification of a novel retinoic acid receptor in regenerative tissues of the newt. Nature 341:654–657
Richter K, Grunz H, Dawid IB (1988) Gene expression in the embryonic nervous system of Xenopus laevis. Proc Natl Acad Sci USA 85:8086–8090
Ruberte E, Dolle P, Chambon P, Morriss-Kay G (1991) Retinoic acid receptors and cellular retinoid binding proteins. II. Their differential pattern of transcription during early morphogenesis in mouse embryos. Development 111:45–60
Ruiz i Altaba A, Jessel TM (1991a) Retinoic acid modifies mesodermal patterning in early Xenopus embryos. Genes Dev 5:175–187
Ruiz i Altaba A, Jessell TM (1991b) Retinoic acid modifies the pattern of cell differentiation in the central nervous system of neurula stage Xenopus embryos. Development 112:945–958
Sharpe CR (1991) Retinoic acid can mimic endogenous signals involved in transformation of the Xenopus nervous system. Neuron 7:239–247
Shen S, Saag van der PT, Kruijer W (1993) Dominant negative retinoic acid receptor β. Mech Dev 40:177–189
Sive HL, Cheng PF (1991) Retinoic acid perturbs the expression of Xhox. lab genes and alters mesodermal determination in Xenopus laevis. Gene Dev 5:1321–1322
Sive HL, Draper BW, Harland RM, Weintraub H (1990) Identification of a retinoic acid sensitive period during primary axis formation in Xenopus laevis. Genes Dev 4:932–942
Sweeney GE, Old RW (1988) Trans-activation of transcription, from promoters containing immunoglobulin gene octamer sequences, by myeloma cell mRNA in Xenopus oocytes. Nucleic Acids Res 16:4903–4913
Thé H de, Vivanco-Ruiz MM, Tiollais P, Stunnenberg H, Dejean A (1990) Identification of a retinoic acid responsive element in the retinoic acid receptor β gene. Nature 343:177–180
Thomas HE, Stunnenberg HG, Stewart AF (1993) Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and Ultraspiracle. Nature 362:471–475
Umesono K, Giguere V, Glass CK, Rosenfeld MG, Evans RM (1988) Retinoic acid and thyroid hormone induce gene expression through a common responsive element. Nature 336:262–264
Yu VC, Delsert C, Andersen B, Holloway JM, Devary OV, Naar AM, Kim SY, Boutin J-M, Glass CK, Evans RM (1991) RXRβ: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell 67:1251–1266
Zelent A, Krust A, Petkovich M, Kastner P, Chambon P (1989) Cloning of murine α and β retinoic acid receptors and a novel receptor predominantly expressed in the skin. Nature 339:714–717
Zerlauth G, Wolf G (1984) Kinetics of fibronectin release from fibroblasts in response to 12-O-tetradecanoylphorbol-13-acetate and retinoic acid. Carcinogenesis 5:863–868
Zhang X-k, Hoffmann B, Tran PB-V, Gaupner G, Pfahl M (1992) Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature 355:441–146
Author information
Authors and Affiliations
Additional information
Correspondence to: R. Old
Rights and permissions
About this article
Cite this article
Smith, D.P., Mason, C.S., Jones, E. et al. Expression of a dominant negative retinoic acid receptor γ in Xenopus embryos leads to partial resistance to retinoic acid. Roux's Arch Dev Biol 203, 254–265 (1994). https://doi.org/10.1007/BF00360521
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00360521