A 2-kb c-mpl promoter fragment is sufficient to direct expression to the megakaryocytic lineage and sites of embryonic hematopoiesis in transgenic mice

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-mplpromoter 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.

A 2-kb HindIII –ApaI genomic fragment containing the 5′ portion of the mouse c-mpl promoter was subcloned into pBluescript KSII (Stratagene) cut withHindIII 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′ (Figure1A). 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 (Figure1B). This construct was excised as a 5-kbXhoI–NotI fragment and was used to generate transgenic mice in the B6D2F1 background by standard oocyte pronucleus injection.

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.

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 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-mplcDNA 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 

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.

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 (Figure1A). 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.

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.16Our 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.

We thank Dr Christoph Berger for help with the mouse embryo dissections and the interpretation of embryonic expression data.