Kolm PJ and Sive HL (1995), Efficient hormone-inducible protein function in...

XB-ART-19318

Dev Biol 1995 Sep 01;1711:267-72. doi: 10.1006/dbio.1995.1279.

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Efficient hormone-inducible protein function in Xenopus laevis.

Kolm PJ , Sive HL .

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A major problem in analyzing gene function during Xenopus development has been the inability to induce gene expression in a temporally controlled manner. We have attempted to solve this problem with a system of hormone-activated protein function, using the myogenic gene MyoD as a paradigm. We show that microinjection of RNA for MyoD fused to the ligand-binding domain of either the estrogen or glucocorticoid receptor results in hormone-dependent activation of MyoD function, as assayed by ectopic induction of muscle-specific actin mRNA. Induction is tightly regulated in both isolated animal caps and intact embryos, with ectopic muscle-specific actin expression inducible after 2 hr of hormone treatment. Higher levels of MyoD-receptor fusion proteins that native MyoD protein are present in embryos, apparently a result of increased fusion protein stability. This is the first demonstration that hormone-inducible fusion proteins can work effectively in a complex embryo.

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Species referenced: Xenopus laevis
Genes referenced: acta4 actl6a myod1 nr3c1

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FIG. 1. (A) Muscle-specific actin induction in animal caps. Mouse MyoD, MyoD-GR, or MyoDÂ·ER mRNA was microinjected into each cell of 2-cell embryos and animal caps were isolated at mid-blastula [stage 8.5) and allowed to develop to the equivalent of early neurula !stage 18) in the presence or absence of 10- 6 M ~-estradiol (E) or w-; M dexamethasone (D). Muscle-spec ific actin RNA was visualized by Northern blot analysis. Lanes 1-3, uninjected; lanes 4-6, unmodified MyoD; lanes 7- 9, MyoD-GR; lanes 10- 12., MyoD-ER. Muscle specific actin is indicated by an asterisk. The blot was repro bed f01 EF la as a loading control. (B) Muscle-specific actin induction in whole embryos. Mouse MyoD-ER or MyoD-GR mRNA was microinjected into one cell of 2-cell albino embryos together with mRNA for nuclearly localized ,13-galactosidase (light blue). Embryos developed without hormone or with hormone treatment from mid-blastula !stage 8.5). At late neurula (stage 18), muscle-specific actin (MSA) was visualized by whole-mount in situ hybridization (purple). ,6-Galactosidase staining was obscured by strong ectopic MSA expression. Arrows indicate endogenous MSA expression. (I) MyoD-E.RÂ·injected, no hormone treatment, lateral view, anterior to right; (2) MyoD-ER-injccted, )3-estradiol treatment, lateral view, anterior to left; (3) MyoD-GR-injected, no treatment, ventrolateral view, ante.rior to right; 141 MyoD-CR-injected, dexamethasone (DEX) treatment, ventral view, anterior to right. (C) Western blot analysis of MyoD protein levels in microinjected embryos. Mouse MyoD or MyoD-GR mRNA was microinjected into each cell of 2-cell embryos. Embryos were treated with dexamethasone {DEX) from mid-blastula (stage 81 onward and lysed at the stage indicated. One embryo equivalent oÂ£ protein was analyzed using the 5.2F anti mouse-MyoD monoclonal antibody. Lanes 1-4, harvest at early gastrula I stage 10.5): lane 1, MyoD-injected, untreated; lane 2, MyoD-injected, DEX treatment; lane 3, MyoD-GR-injected, untreated; lane 4, MyoD-GR-injecrcd, DEX treatment; lanes 5- 9, lu!rvest at late gastrula (stage 12.5]; lane 5, MyoD-injected, untreated; lane 6, MyoD injected, DEX treatment; lane 7, MyoD-GR-injectcd, untreated; lane 8, MyoD-GR-injected, DEX treatment; lane 9, uninjected embryo. The Ponceau S-stained gel indicated equivalent loading in all lanes (not shown). I D) Northern blot analysis of mMyoD RNA levels in microinjected embryos. Mouse MyoD or MyoD-GR mRNA was microinjccted into each cell of 2-cell embryos. Embryos were treated with DEX from mid-blastula I stage 8) onward and lysed at the stage indicated. One embryo equivalent of RNA was analyzed by Northern blotti ng, using a mouse MyoD p robe. Under the hybridization conditions used, Xenopus MyoD transcripts were not detected. Ethidium bromide-stained 28S ribosomal RNA is shown as a loading control. Lanes 1 and 2, embryos harvested 90 min after injection (stage 5); lanes 3-6, harvest at early gastrula (stage 10.5); lanes 7 and 8, harvest at late gastrula (stage 12.5). Lane 1, MyoDÂ·injected, untreated; lane 2, MyoDÂ·GR-injected, untreated; lane 3, MyoD-injected, untreated; lane 4, MyoD-injected, DEX treatment; lane 5, MyoD-GR-injectcd, untreated; lane 6, MyoD-GR-injected, DEX treatment; lane 7, MyoD-injected, untreated; lane 8, MyoD-GR-injected, untreated.