Abstract
A polyclonal antibody against a glutathione S-transferase fusion protein containing the 76 COOH-terminal amino acids of Hex, a divergent homeobox gene, was raised in rabbits. Western blot and immunofluorescence reveal that Hex is a 35–37-kD soluble protein present both in the nucleus and cytoplasm of transfected and nontransfected cultured cells as well as in whole mouse embryo. Confocal microscopy of whole mount immunostained mouse embryos at E7.5 and E8.5 demonstrates that Hex is differentially localized in the cytoplasm and nucleus of definitive endoderm, developing blood islands, and hepatic diverticulum. In particular, in the region of the foregut that gives rise to the liver, Hex expression is nuclear in the endodermal cells of the hepatic diverticulum, whereas expression is primarily cytoplasmic in cells lateral to the liver-forming region. This suggests that nuclear localization of Hex is involved in early hepatic specification and that compartmentalization of Hex protein plays an important role in its function during mouse development.
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Abbreviations
- GST:
-
glutathione S-transferas
- IPTG:
-
isopropyl b-D-thiogalactopyranosid
- PMSF:
-
phenylmethylsulfonyl fluorid
- PBST:
-
0.1% Triton X-100 in PB
- NLS:
-
nuclear localization signa
- NES:
-
nuclear export signa
References
Gehring WJ 1987 Homeo boxes in the study of development. Science 236: 1245–1252
Duboule D 1994 Guidebook to the Homeobox Genes. Oxford University Press, New York
Bedford FK, Ashworth A, Enver T, Wiedemann LM 1993 HEX: a novel homeobox gene expressed during haematopoiesis and conserved between mouse and human. Nucleic Acids Res 21: 1245–1249
Crompton MR, Bartlett TJ, MacGregor AD, Manfioletti G, Buratti E, Giancotti V, Goodwin GH 1992 Identification of a novel vertebrate homeobox gene expressed in haematopoietic cells. Nucleic Acids Res 20: 5661–5667
Thomas PQ, Brown A, Beddington RS 1998 Hex: a homeobox gene revealing peri-implantation asymmetry in the mouse embryo and an early transient marker of endothelial cell precursors. Development 125: 85–94
Keng VW, Fujimori KE, Myint Z, Tamamaki N, Nojyo Y, Noguchi T 1998 Expression of Hex mRNA in early murine postimplantation embryo development. FEBS Lett 426: 183–186
Corsetti MT, Levi G, Lancia F, Sanseverino L, Ferrini S, Boncinelli E, Corte G 1995 Nuclear localization of three Hox homeoproteins. J Cell Sci 108: 187–193
Driever W, Nusslein-Volhard C 1988 A gradient of bicoid protein in Drosophila embryos. Cell 54: 83–93
Mlodzik M, Gehring WJ 1987 Expression of the caudal gene in the germ line of Drosophila: formation of an RNA and protein gradient during early embryogenesis. Cell 48: 465–478
Kasahara H, Bartunkova S, Schinke M, Tanaka M, Izumo S 1998 Cardiac and extracardiac expression of Csx/Nkx2.5 homeodomain protein. Circ Res 82: 936–946
Aspland SE, White RAH 1997 Nucleocytoplasmic localisation of extradenticle protein is spatially regulated throughout development in Drosophila. Development 124: 741–747
Mann RS, Abu-Shaar M 1996 Nuclear import of the homeodomain protein extradenticle in response to Wg and Dpp signalling. Nature 383: 630–633
Joliot A, Trembleau A, Raposo G, Calvet S, Volovitch M, Prochiantz A 1997 Association of engrailed homeoproteins with vesicles presenting caveolae-like properties. Development 124: 1865–1875
Newman CS, Chia F, Krieg PA 1997 The XHex homeobox gene is expressed during development of the vascular endothelium: overexpression leads to an increase in vascular endothelial cell number. Mech Dev 66: 83–93
Bogue CW, Ganea GR, Sturn E, Ianucci R, Jacobs HC 2000 Hex expression suggests a role in the development and function of organs derived from foregut endoderm. Dev Dyn 219: 84–89
Harlow E, Lane D 1988 Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Wilkinson DG 1992 Whole mount in situ hybridization of vertebrate embryos. In: Wilkinson DG (ed) In Situ Hybridization: A Practical Approach, Vol 109. IRL Press, Oxford, UK, pp 75–83
Beddington RS, Robertson EJ 1999 Axis development and early asymmetry in mammals. Cell 96: 195–209
Hromas R, Radich J, Collins S 1993 PCR cloning of an orphan homeobox gene (PRH) preferentially expressed in myeloid and liver cells. Biochem Biophys Res Commun 195: 976–983
Tanaka T, Inazu T, Yamada K, Myint Z, Keng VW, Inoue Y, Taniguchi N, Noguchi T 1999 cDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein. Biochem J 339: 111–117
Hirata J, Nakagoshi H, Nabeshima Y, Matsuzaki F 1995 Asymmetric segregation of the homeodomain protein Prospero during Drosophila development. Nature 377: 627–630
Hassan B, Li L, Bremer KA, Chang W, Pinsonneault J, Vaessin H 1997 Prospero is a panneural transcription factor that modulates homeodomain protein activity. Proc Natl Acad Sci USA 94: 10991–10996
Casares F, Mann RS 1998 Control of antennal versus leg development in Drosophila. Nature 392: 723–726
Mercader N, Leonardo E, Azpiazu N, Serrano A, Morata G, Martinez C, Torres M 1999 Conserved regulation of proximodistal limb axis development by Meis1/Hth. Nature 402: 425–429
Azpiazu N, Morata G 1998 Functional and regulatory interactions between Hox and extradenticle genes. Genes Dev 12: 261–273
Moede T, Leibiger B, Pour HG, Berggren P, Leibiger IB 1999 Identification of a nuclear localization signal, RRMKWKK, in the homeodomain transcription factor PDX-1. FEBS Lett 461: 229–234
Gorlich D, Mattaj IW 1996 Nucleocytoplasmic transport. Science 271: 1513–1518
Maizel A, Bensaude O, Prochiantz A, Joliot A 1999 A short region of its homeodomain is necessary for engrailed nuclear export and secretion. Development 126: 3183–3190
Hicks GR, Raikhel NV 1995 Protein import into the nucleus: an integrated view. Annu Rev Cell Dev Biol 11: 155–188
Matunis MJ, Coutavas E, Blobel G 1996 A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol 135: 1457–1470
Macfarlane WM, McKinnon CM, Felton-Edkins ZA, Cragg H, James RF, Docherty K 1999 Glucose stimulates translocation of the homeodomain transcription factor PDX1 from the cytoplasm to the nucleus in pancreatic beta-cells. J Biol Chem 274: 1011–1016
Duverger E, Pellerin-Mendes C, Mayer R, Roche AC, Monsigny M 1995 Nuclear import of glycoconjugates is distinct from the classical NLS pathway. J Cell Sci 108: 1325–1332
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Ghosh, B., Ganea, G., Denson, L. et al. Immunocytochemical Characterization of Murine Hex, a Homeobox-Containing Protein. Pediatr Res 48, 634–638 (2000). https://doi.org/10.1203/00006450-200011000-00014
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DOI: https://doi.org/10.1203/00006450-200011000-00014
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