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Prepublished online as a Blood First Edition Paper on June 28, 2002; DOI 10.1182/blood-2002-01-0281. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-01-0281v1 100/3/1072    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ziegler, S. Articles by Skoda, R. C. Search for Related Content PubMed PubMed Citation Articles by Ziegler, S. Articles by Skoda, R. C. Related Collections Hematopoiesis and Stem Cells Gene Expression Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 August 2002, Vol. 100, No. 3, pp. 1072-1074 BRIEF REPORT A 2-kb c-mpl promoter fragment is sufficient to direct expression to the megakaryocytic lineage and sites of embryonic hematopoiesis in transgenic mice Sandra Ziegler, Kurt Bürki, and Radek C. Skoda From the Biozentrum, University of Basel, DKF2, Heidelberg, Germany, and Institute for Laboratory Animal Sciences, University of Zurich, Switzerland.     Abstract Top Abstract Introduction Study design Results and discussion References Thrombopoietin receptor c-mpl is expressed on hematopoietic progenitors and cells of the megakaryocytic lineage. The c-mpl promoter may, therefore, be useful for directing the expression of transgenes. We tested whether a 2-kb genomic DNA fragment comprising the putative c-mpl regulatory elements and most of the 5'-untranslated region of mouse c-mpl is able to direct the expression of a reporter gene to hematopoietic cells in transgenic mice. As a reporter gene we used the human placental alkaline phosphatase (PLAP). In adult transgenic mice, PLAP expression was specifically detected in megakaryocytes and platelets. Embryos showed PLAP reporter gene expression already in the yolk sac at embryonic day 6.5 (E6.5) and in blood islands at E7.5. At E9.5, expression was found in blood vessels of the yolk sac and the embryo proper, followed by high levels of expression in the fetal liver at E11.5. Expression in E6.5 yolk sac is compatible with a function of c-mpl and its ligand, thrombopoietin, in the earliest stages of embryonic hematopoiesis. (Blood. 2002;100:1072-1074) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References Thrombopoietin (TPO) is the primary regulator of megakaryopoiesis and platelet production.1-4 In addition, TPO promotes the expansion of pluripotent stem cells and early hematopoietic progenitors without preference for a particular lineage.5 The effects of TPO are mediated by its cell surface receptor, c-mpl, which was initially described as the cellular homologue of the retroviral oncogene, myeloproliferative leukemia (v-mpl)6; c-mpl is a member of the cytokine receptor superfamily7,8 and its expression is restricted to cells of the megakaryocytic lineage and to CD34+ hematopoietic progenitors and stem cells.5 We tested whether a 2-kb genomic DNA fragment containing the promoter and regulatory sequences of the mouse c-mpl gene might be useful to direct expression to the megakaryocytic lineage in transgenic mice. A 2-kb genomic DNA fragment containing promoter and regulatory sequences of the mouse c-mpl gene was derived. As a reporter gene, we chose the human placental alkaline phosphatase (PLAP), a glycosylphosphatidylinositol (GPI)-linked cell surface protein. PLAP allows sensitive and specific detection of protein expression because of its heat-stable alkaline phosphatase (AP) activity.9 We found that the 2-kb c-mpl promoter fragment was sufficient to specifically direct expression of the PLAP reporter gene to the megakaryocytic lineage in the adult and to the sites of early hematopoiesis in the embryo.     Study design Top Abstract Introduction Study design Results and discussion References DNA constructs A 2-kb HindIII -ApaI genomic fragment containing the 5' portion of the mouse c-mpl promoter was subcloned into pBluescript KSII (Stratagene) cut with HindIII and ApaI. The unique ApaI site was blunted using T4 polymerase and was ligated to a 120-base pair (bp) fragment generated by polymerase chain reaction using the primers 5'-GGCCCCAGAGGGGCAGAG-3' and 5'-TGTGTGCCTGCCTTAGCC-3' (Figure 1A). This fragment corresponds to the region between positions 132 and 13 relative to the c-mpl translational start site and contains the 3' portion of the c-mpl promoter and part of the 5'-untranslated region (Figure 1A). SV40 polyadenylation signal and the PLAP cDNA were added to yield the c-mpl-PLAP-SV40 transgene construct (Figure 1B). This construct was excised as a 5-kb XhoI-NotI fragment and was used to generate transgenic mice in the B6D2F1 background by standard oocyte pronucleus injection. View larger version (46K): [in this window] [in a new window]   Figure 1. Transgene construct and expression of PLAP mRNA and protein in adult mice. (A) Mouse c-mpl gene (top) with sequences of the 5'-upstream region (enlarged) aligned with the c-mpl/PLAP transgene. Black box represents c-mpl exon 1. Start codons for c-mpl and PLAP are highlighted. The transgene retains most of the c-mpl 5'-untranslated region. (B) Transgenic construct and position of the riboprobe used for the detection of PLAP mRNA expression. Tissues of mice heterozygous for the PLAP transgene were analyzed: bm, bone marrow; ln, mesenteric lymph node; pa, pancreas; br, brain; lu, lung; li, liver; sp, spleen; ki, kidney; ht, heart; th, thymus. Riboprobes for PLAP, c-mpl, and HPRT were mixed to allow the simultaneous detection of PLAP, endogenous c-mpl, and HPRT mRNAs. (C) PLAP protein expression in platelets and megakaryocytes of wild-type (wt) and transgenic (TG) mice. Dark staining indicates PLAP activity. Note that platelets have aggregated in the blood smear from TG. Magnification × 500. Blood and bone marrow Blood was obtained by cardiac puncture, and blood counts were taken with an automated blood counter (Technicon H-1; Miles, Tarrytown, NY). Femur bones were flushed with Iscoves modified Dulbecco medium supplemented with 10% fetal bovine serum (Gibco), and bone marrow cells were concentrated on a glass slide by cytospin centrifugation. Staining for alkaline phosphatase Blood or bone marrow cells were heated to 65°C for 5 minutes, fixed in 4% paraformaldehyde, washed in HEPES buffered saline (HBS) (150 mM NaCl, 20 mM HEPES), and incubated for 5 to 10 minutes at room temperature in AP staining solution containing 0.17 mg/mL BCIP and 0.33 mg/mL NBT, 100 mM Tris, pH 9.5, 100 mM NaCl, and 5 mM MgCl2. Alkaline phosphatase activity results in blue staining.10 RNA isolation and ribonuclease protection assay RNA from mouse tissues was isolated by the acid phenol method.11 To assess the expression of PLAP mRNA, a ribonuclease protection assay was performed.12 Total RNA (30 µg) was simultaneously hybridized with the following 3 riboprobes: (1) a riboprobe specific for PLAP (generated with the polymerase chain reaction primers 5'-TGTTCGACGACGCCATTGAG-3' (sense) and 5'-CTTCGTCCAGGGGCACTGCT-3' (antisense) that resulted in the protection of a 290-nucleotide (nt) fragment; (2) a riboprobe for mouse c-mpl (nucleotides 6 to +233 of the mouse c-mpl cDNA sequence7) that protects a 239-nt fragment was added; and (3) a riboprobe for mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT) to normalize for RNA loading that protected a 162-nt fragment.13 Analysis of transgene expression during embryogenesis Timed pregnancies were obtained by natural mating in a 6 am to 7 pm light cycle. Plugs were checked in the morning, and 12 am was taken as the time of fertilization. Pregnant females were killed at embryonic days 6.5, 7.5, 8.5, 9.5, 10.5, and 11.5. Embryos with their yolk sacs were isolated in phosphate-buffered saline and immediately fixed for 4 hours in 4% paraformaldehyde. Endogenous AP activity was inactivated by heating the embryos to 65°C for 1 hour. PLAP activity in whole mounts was detected by AP staining for 4 hours.     Results and discussion Top Abstract Introduction Study design Results and discussion References PLAP cDNA, as a reporter gene, was placed under the control of a 2-kb fragment containing the promoter and the regulatory sequences of the mouse c-mpl gene (Figure 1). Because the transcriptional start site of mouse c-mpl has not been precisely mapped, we included most of the c-mpl 5'-untranslated region (Figure 1A). Transgenic mice were generated and analyzed for expression of the PLAP reporter gene. Of 2 transgenic lines obtained, one showed expression and was chosen for further analysis. Using an RNase protection assay, we detected the expression of PLAP mRNA in bone marrow and spleen at levels comparable with endogenous c-mpl (Figure 1B). Furthermore, PLAP protein was detectable in megakaryocytes and platelets from transgenic mice but not in wild-type controls (Figure 1C). Mice expressing the reporter gene had normal peripheral blood counts (not shown); thus, expression of the PLAP protein had no unexpected adverse effects. The alkaline phosphatase activity of PLAP allowed us to follow expression of the PLAP reporter gene during embryonic development. Already at 6.5 days after coitus (E6.5), PLAP was expressed as a ring in the yolk sac (Figure 2A). At E7.5 this staining was even more pronounced, and sections through the yolk sac revealed PLAP-positive cells within blood islands (Figure 2B). From E8.5 to E9.5, a major expansion of PLAP-positive cells was detectable in the extra-embryonic blood vessels, along with the appearance of staining within the embryo proper. Cross-sections through blood vessels of day E9.5 yolk sac showed staining of cells associated with the endothelium and with blood cells in the lumen (Figure 2B). Furthermore, PLAP-positive cells were also detectable in blood vessels of the embryo, including the aorta. At E10.5, staining of the extra-embryonic blood vessels markedly decreased, PLAP activity was found in a punctate pattern throughout the embryo, and strong expression of PLAP was detected in the fetal liver of E11.5 embryos. View larger version (58K): [in this window] [in a new window]   Figure 2. Transgene expression in mouse embryos. (A) Wild-type (wt) and transgenic (TG) embryos from E6.5 through E11.5 were collected and assayed in situ for PLAP enzymatic activity (blue staining). Embryos from E9.5 to E11.5 are shown with yolk sac (left column) and without yolk sac (right column). a indicates allantois; br, brain; e, embryo proper; ey, eye; fl, fetal liver; h, heart; lb, limb bud; n, notochord; p, placenta; t, tail; ys, yolk sac. Filled arrowheads point to blood islands or blood vessels in the yolk sac. Magnification × 10-20. (B) Paraffin sections at E7.5 and E9.5 show transgene expression in blood islands or blood vessels of the yolk sac. Annotations are as in panel A. Magnification × 200. In summary, embryonic expression of the PLAP reporter driven by the 2-kb c-mpl promoter followed the known sites of embryonic hematopoiesis, starting in blood islands and later appearing in the fetal liver. TPO/c-mpl signaling was implicated as playing a role in early yolk sac hematopoiesis,14,15 and a recent abstract also suggests a function in the hemangioblast.16 Our finding of PLAP expression as early as day 6.5 further strengthens this possibility. At later stages, expression was found in blood cells and in endothelial cells. In accordance, the expression of c-mpl was previously reported in a mouse liver-derived endothelial cell line, LEC-1,17 and in human umbilical cord vein-derived endothelial cells.18 Additional transgenic lines will be necessary to confirm these results and to rule out the effects of integration site on the pattern and the levels of expression. We expect that this c-mpl promoter will be useful for directing the expression of other transgenes to sites of hematopoiesis in embryos and to the megakaryocytic lineage in adult mice.     Acknowledgments We thank Dr Christoph Berger for help with the mouse embryo dissections and the interpretation of embryonic expression data.     Footnotes Submitted January 30, 2002; accepted March 21, 2002. Prepublished online as Blood First Edition Paper, June 28, 2002; DOI 10.1182/blood-2002-01-0281. Supported by grants from the Swiss National Science Foundation to R.C.S. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Radek C. Skoda, Department of Research, Experimental Hematology, Basel University Hospitals, Hebelstrasse 20, 4031 Basel, Switzerland; e-mail: radek.skoda{at}unibas.ch.     References Top Abstract Introduction Study design Results and discussion References 1. de Sauvage FJ, Hass PE, Spencer SD, et al. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-mpl ligand. Nature. 1994;369:533-538[CrossRef][Medline] [Order article via Infotrieve]. 2. Lok S, Kaushansky K, Holly RD, et al. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature. 1994;369:565-568[CrossRef][Medline] [Order article via Infotrieve]. 3. Kaushansky K, Lok S, Holly RD, et al. Promotion of megakaryocyte progenitor expansion and differentiation by the c-mpl ligand thrombopoietin. Nature. 1994;369:568-571[CrossRef][Medline] [Order article via Infotrieve]. 4. Bartley TD, Bogenberger J, Hunt P, et al. Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor mpl. Cell. 1994;77:1117-1124[CrossRef][Medline] [Order article via Infotrieve]. 5. Kaushansky K. Thrombopoietin and hematopoietic stem cell development. Ann N Y Acad Sci. 1999;872:314-319[CrossRef][Medline] [Order article via Infotrieve]. 6. Souyri M, Vigon I, Penciolelli JF, Heard JM, Tambourin P, Wendling F. A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors. Cell. 1990;63:1137-1147[CrossRef][Medline] [Order article via Infotrieve]. 7. Skoda RC, Seldin DC, Chiang MK, Peichel CL, Vogt TF, Leder P. Murine c-mpl: a member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal. EMBO J. 1993;12:2645-2653[Medline] [Order article via Infotrieve]. 8. Vigon I, Florindo C, Fichelson S, et al. Characterization of the murine Mpl proto-oncogene, a member of the hematopoietic cytokine receptor family: molecular cloning, chromosomal location and evidence for a function in cell growth. Oncogene. 1993;8:2607-2615[Medline] [Order article via Infotrieve]. 9. DePrimo SE, Stambrook PJ, Stringer JR. Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice. Transgenic Res. 1996;5:459-466[CrossRef][Medline] [Order article via Infotrieve]. 10. Harlow E, Lane D. Antibodies: A Laboratory Manual. 1st ed. Cold Spring Harbor, NY: Cold Spring Harbor Press; 1988. 11. Chomczynski P, Sachhi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159[Medline] [Order article via Infotrieve]. 12. Krieg PA, Melton DA. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 1987;155:397-415[Medline] [Order article via Infotrieve]. 13. Stoffel R, Wiestner A, Skoda RC. Thrombopoietin in thrombocytopenic mice: evidence against regulation at the mRNA level and for a direct regulatory role of platelets. Blood. 1996;87:567-573[Abstract/Free Full Text]. 14. Kieran MW, Perkins AC, Orkin SH, Zon LI. Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor. Proc Natl Acad Sci U S A. 1996;93:9126-9131[Abstract/Free Full Text]. 15. Xie X, Yoder MC. The role of thrombopoietin in promoting yolk sac hematopoietic progenitor cell growth and differentiation [abstract]. Blood. 2001;98:72. 16. Perlingeiro RCR, Kyba M, Daley GQ. Thrombopoietin is a growth factor for the hemangioblast [abstract]. Blood. 2001;98:71. 17. Cardier JE, Dempsey J. Thrombopoietin and its receptor, c-mpl, are constitutively expressed by mouse liver endothelial cells: evidence of thrombopoietin as a growth factor for liver endothelial cells. Blood. 1998;91:923-929[Abstract/Free Full Text]. 18. Brizzi MF, Battaglia E, Montrucchio G, et al. Thrombopoietin stimulates endothelial cell motility and neoangiogenesis by a platelet-activating factor-dependent mechanism. Circ Res. 1999;84:785-796[Abstract/Free Full Text]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: S. N. Constantinescu Mpl and thrombocytosis: levels matter Blood, February 19, 2009; 113(8): 1617 - 1618. [Full Text] [PDF] B. J. Lannutti, A. Epp, J. Roy, J. Chen, and N. C. Josephson Incomplete restoration of Mpl expression in the mpl-/- mouse produces partial correction of the stem cell-repopulating defect and paradoxical thrombocytosis Blood, February 19, 2009; 113(8): 1778 - 1785. [Abstract] [Full Text] [PDF] R. Tiedt, J. Coers, S. Ziegler, A. Wiestner, H. Hao-Shen, C. Bornmann, J. Schenkel, S. Karakhanova, F. J. de Sauvage, C. W. Jackson, et al. Pronounced thrombocytosis in transgenic mice expressing reduced levels of Mpl in platelets and terminally differentiated megakaryocytes Blood, February 19, 2009; 113(8): 1768 - 1777. [Abstract] [Full Text] [PDF] L. Petit-Cocault, C. Volle-Challier, M. Fleury, B. Peault, and M. Souyri Dual role of Mpl receptor during the establishment of definitive hematopoiesis Development, August 15, 2007; 134(16): 3031 - 3040. [Abstract] [Full Text] [PDF] L. Silberstein, M.-J. Sanchez, M. Socolovsky, Y. Liu, G. Hoffman, S. Kinston, S. Piltz, M. Bowen, L. Gambardella, A. R. Green, et al. 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2002-01-0281v1 100/3/1072    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ziegler, S. Articles by Skoda, R. C. Search for Related Content PubMed PubMed Citation Articles by Ziegler, S. Articles by Skoda, R. C. Related Collections Hematopoiesis and Stem Cells Gene Expression Brief Reports Social Bookmarking                   What's this?

	Copyright © 2002 by American Society of Hematology         Online ISSN: 1528-0020