Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling

Abstract

Genetic screens in lower organisms, particularly those that identify modifiers of preexisting genetic defects, have been used successfully to order components of complex signaling pathways. To date, similar suppressor screens have not been used in vertebrates. To define the molecular pathways regulating platelet production, we have executed a large-scale modifier screen with genetically thrombocytopenic Mpl(-/-) mice by using N-ethyl-N-nitrosourea mutagenesis. Here we show that mutations in the c-Myb gene cause a myeloproliferative syndrome and supraphysiological expansion of megakaryocyte and platelet production in the absence of thrombopoietin signaling. This screen demonstrates the utility of large-scale N-ethyl-N-nitrosourea mutagenesis suppressor screens in mice for the simultaneous discovery and in vivo validation of targets for therapeutic discovery in diseases for which mouse models are available.

Fig. 1.

Identification of ENU-mutant mice with ameliorated thrombocytopenia. Peripheral blood platelet counts at 7 weeks of age for 100 Mpl^+/+mice (a), 783 Mpl^-/- mice (b), 1,575 G₁ Mpl^-/- mice harboring random ENU-induced mutations (c), the Plt3 G₁ mouse (d), the G₂ progeny derived from mating the G₁ Plt3 mouse to Mpl^-/- mice (e), the progeny derived from crossing heterozygous Plt3/+ Mpl^-/- mice (f), the G₁ Plt4 mouse (g), the G₂ progeny derived from mating the G₁ Plt4 mouse to Mpl^-/- mice (h), the progeny derived from intercrossing heterozygous Plt4/+ Mpl^-/- mice (i), the progeny derived from mating homozygous Plt4/Plt4 Mpl^-/- mice to Mpl^-/- mice (j), and the progeny derived from intercrossing homozygous Plt4/Plt4 Mpl^-/- mice (k).

Fig. 2.

Plt3 and Plt4 are tightly linked on chromosome 10 and are alleles of c-Myb. (a) To map Plt4, a (C57BL/6 × 129Sv)F₂ generation of 65 mice was produced, bled at 7 weeks of age, and categorized as having low platelets (<150 × 10⁶/ml) characteristic of +/+ Mpl^-/- mice, moderate numbers of platelets (150 × 10⁶ to 2,000 × 10⁶/ml) characteristic of Plt4/+ Mpl^-/- mice, or extremely high platelets (>2,000 × 10⁶/ml) characteristic of Plt4/Plt4 Mpl^-/- mice. Animals were then genotyped, and markers found to be homozygous 129/Sv are shown in white, markers that were heterozygous are shown in gray, and markers homozygous C57BL/6 are shown in black. The number of animals with each haplotype is shown below. The Plt4 mutation was localized to between D10Mit213 and D10Mit 214. To confirm Plt3 was located close to Plt4, we produced 68 (C57BL/6 × 129Sv)N₂ generation mice, measured platelet numbers in these animals, and genotyped them by using the markers most closely linked to Plt4. (Right) Correlation between genotype and phenotype for markers at the centromeric region of chromosome 10. (b) Sequence of PCR-amplified exons and intron boundaries of the c-Myb gene showing a single A to T transversion in Plt3 and Plt4, resulting in an Asp to Val substitution at positions 152 and 382, respectively. Representative traces are shown; a total of three Plt3/Pl3 Mpl^-/-, three Plt3/+ Mpl^-/- mice, three Plt4/Plt4 Mpl^-/- mice, three Plt4/+ Mpl^-/- mice, three +/+ Mpl^-/- mice, and three +/+ Mpl^+/+ mice were analyzed. (c) The transactivation activity of wild-type c-Myb, Plt3 and Plt4 mutant c-Myb, and a constitutively activated truncation of c-Myb (CT3; ref. 26) were compared by measuring production of chloramphenicol acetyltransferase from a c-Myb responsive promoter (28). The activity of both Plt3 and Plt4 c-Myb was significantly lower than wild type; (P = 0.017 and P = 0.0003, respectively; n = 9).

Fig. 3.

Mutation of c-Myb results in an elevation in progenitor cells, megakaryocytes, and platelets independent of Mpl. (a) The numbers of colony-forming units (spleen), a measure of multipotential progenitor cells, in the bone marrow (Far Left), clonogenic megakaryocyte progenitor cells (Left), megakaryocytes (Right) and platelets (Far Right)in c-Myb^+/+ (+/+), c-Myb ^Plt4/+ (4/+), c-Myb ^Plt4/Plt4 (4/4), c-Myb ^Plt3/+ (3/+), and c-Myb ^Plt3/Plt3 (3/3) mutants on a Mpl^-/- or Mpl^+/+ background are shown. Assays were performed as described in Materials and Methods with the error bars representing the SD from the mean of data from 3–7 [colony-forming units (spleen)], 2–6 (progenitor cell data), 2–7 (megakaryocyte data), and 4–50 (platelet data) mice. (b) Flow cytometric analysis of B lymphoid cells in the spleen and erythroid cells in the bone marrow of Plt4 mutant mice showing marked reductions in preB (B220⁺IgM^-) and B (B220⁺IgM⁺) lymphocytes and accumulation of more immature erythroid cells (CD71^hiTer119^med and CD71^hiTer119^hi; ref. 31) in Mpl^-/- c-Myb^Plt4/Plt4 mice. (c) Histological sections of spleens from Mpl^+/+ c-Myb^+/+, Mpl ^-/- c-Myb^Plt3/Plt3, Mpl^-/- c-Myb^Plt4/Plt4, and Mpl^+/+ c-Myb^Plt4/Plt4 mice. Note the poor development of lymphoid follicles and expanded red pulp displaying reduced cellularity and disrupted architecture.