by Joanna Tober, Kathleen E. McGrath, and James Palis Primitive erythropoiesis and megakaryopoiesis in the yolk sac are independent of c-myb by Joanna Tober, Kathleen E. McGrath, and James Palis Blood Volume 111(5):2636-2639 March 1, 2008 ©2008 by American Society of Hematology

Emergence of the erythroid and megakaryocyte lineages in wildtype and c-myb–null mouse fetuses. Emergence of the erythroid and megakaryocyte lineages in wildtype and c-myb–null mouse fetuses. (A) Expression of c-myb in E12.5 (i) and E8.5 (ii) mouse embryos analyzed by in situ hybridization. c-myb transcripts accumulated in E12.5 fetal liver but were not detected above background in E8.5 yolk sac blood islands. Size bars indicate 50 μm. (B) Quantitation by qPCR of c-myb transcripts in sites of hematopoiesis and in purified erythroid cell populations: (1) E8.5 yolk sac (YS), (2) E9.5 yolk sac, (3) Ter119-positive cells from E9.0 yolk sac (16-20 sp), (4) Ter119− cells from E9.0 yolk sac (16-20 sp), (5) Ter119+ cells from E15.5 fetal liver (FL), and (6) Ter119− cells from E15.5 fetal liver. (C-E) Early ontogeny of primitive erythroid progenitors (EryP-CFC), bipotential megakaryocyte/erythroid progenitors (MEP), and megakaryocyte progenitors (Meg-CFC) between E6.5 and E11.5 of gestation in c-myb–null (−/−) and combined wildtype (+/+) and c-myb-heterozygous (+/−) mouse embryos. Mean (± SEM) of each progenitor type is graphed. Wildtype and c-myb-heterozygous embryos revealed similar progenitor numbers and temporal kinetics. EryP-CFC-derived colonies contain βH1-globin+ primitive erythroid cells, MEP-derived colonies contain GP1Bβ+megakaryocytes and either primitive erythroid (βH1-globin+ cells through 19 sp) or definitive erythroid (Ter119+ cells > 19 sp), and Meg-CFC-derived colonies contain GP1Bβ+ megakaryocytes. Embryonic stages and (number of embryos examined at each stage): PS, primitive-streak (2); ES-LS, early primitive streak to late primitive streak (9); NP, neural plate (21); head fold, (16) and number of somite pairs; 0 to 3 sp (16); 4 to 7 sp (21); 8 to 10 sp (5); 15 to 19 sp (12); 20 to 24 sp (18); 28 to 32 sp, (15) 45 to 59 sp. (9). (F) Morphology of Meg-CFC-derived colonies (Meg) and primitive MEP-derived colonies (pE/Meg) from wildtype (+/+), c-myb-heterozygous (+/−), and c-myb–null (−/−) embryos. Megakaryocytes (red/pink stain) are labeled with rat anti–mouse GP1bβ (Emfret Analytics, Würzburg, Germany), and primitive erythroid cells (blue/purple stain) are labeled with rabbit anti–mouse βH1-globin16 as described in Tober et al.4 All cultures were grown for 10 days. Arrows and higher magnification insets highlight proplatelet formation. Size bars indicate 50 μm. (G) Mean percentage (± SEM) of Meg-CFC-derived colonies (composed of GP1bβ-positive cells) consisting of fewer than 10 megakaryocytes in combined wild-type (+/+) and c-myb–heterozygous (+/−; ) and c-myb–null (−/−, ▭) mouse embryos. Representative small colonies from wild-type (i) and c-myb–null (ii) E10.5 cultures are shown. Proplatelet formation and platelet-sized fragments are evident. Size bars indicate 50 μm. Images were processed with lens defect correction and brightness/contrast optimization in Photoshop (Adobe, San Jose, CA) with Fovea Pro plug-in (Reindeer Graphics, Ashville, NC). Joanna Tober et al. Blood 2008;111:2636-2639 ©2008 by American Society of Hematology

Comparison of megakaryocyte colony numbers and thrombopoiesis in c-myb–null and combined wildtype and c-myb-heterozygous embryos. Comparison of megakaryocyte colony numbers and thrombopoiesis in c-myb–null and combined wildtype and c-myb-heterozygous embryos. (A) The number of MEP (± SEM) in staged wildtype and c-myb-heterozygous mouse embryos () compared with the number of Meg-CFC (± SEM) in similarly staged c-myb–null embryos (▭). Data derived from Figures 1C and D. YS = yolk sac, FL = fetal liver. (B) Total platelet numbers (± SEM) in c-myb–null (−/−, dashed line) and combined wild-type (+/+) and c-myb-heterozygous (+/−) (solid line) mouse embryos at E12.5 and E15.5 of mouse gestation. Platelets and blood cells, obtained from 3 to 5 μL of whole blood diluted in PBS/0.1M EDTA/0.1 mg/mL heparin (Sigma-Aldrich, St Louis, MO), were counted on a hemacytometer at 40× magnification using phase optics. Total platelet counts were based on platelet concentration × total blood cell measurements.16 Joanna Tober et al. Blood 2008;111:2636-2639 ©2008 by American Society of Hematology