1. The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis
by Joanna Tober, Anne Koniski, Kathleen E. McGrath, Radhika Vemishetti, Rachael Emerson, Karen K. L. de Mesy-Bentley, Richard Waugh, and James Palis
Blood
Volume 109(4):
February 15, 2007
©2007 by American Society of Hematology

2. Megakaryocyte potential is derived from hemangioblast precursors.
Megakaryocyte potential is derived from hemangioblast precursors. (A) Hemangioblast-derived colony from E7.5 mouse embryo (i). Adherent and nonadherent cells from a hemangioblast-derived colony after 3 days in liquid expansion media (ii). (B) Proplatelet formation of cells differentiated from a hemangioblast-derived colony after 3 days in liquid media (i-ii). (C) PECAM-positive cells (arrowheads) in the adherent cell population after 10 days in liquid expansion media. (D) Primitive erythroid and megakaryocyte colony formation from expanded, nonadherent cells placed into progenitor assays. An EryP-CFC–derived colony contains cells positive for βH1-globin (i) and a Meg-CFC–derived colony contains cells and proplatelet extensions (arrowheads) positive for GP1bβ (ii). (E) Hematopoietic potential of 10 individual hemangioblast colonies expanded in liquid culture. The number of EryP-CFCs, Meg-CFCs, and megakaryocyte cells (meg cells) is shown. Scale bars represent 10 μm.
Joanna Tober et al. Blood 2007;109:
©2007 by American Society of Hematology

3. Meg-CFCs and 2 novel bipotential MEPs emerge during embryonic hematopoiesis.
Meg-CFCs and 2 novel bipotential MEPs emerge during embryonic hematopoiesis. (A) Number (+SEM) of acetylcholinesterase-positive (megakaryocyte) colonies grown from individual embryonic tissues by stage. The inset (i) shows an acetylcholinesterase-positive colony. (B) Immunohistochemical staining of erythroid and megakaryocyte progenitor–derived colonies. (Top) Primitive erythroid (i), megakaryocyte (ii), and bipotential primitive MEP (iii) colonies from neural plate–stage cultures stained with primitive erythroid-specific βH1-globin (blue) and megakaryocyte-specific GP1bβ (pink) antibodies. (Bottom) Erythroid (v), megakaryocyte (vi), and bipotential definitive MEP (vii) colonies from E9.5 to E10.5 yolk sac stained with panerythroid Ter119 (blue) and with GP1bβ (pink) antibodies. Boxed areas in panels iii and vii highlight proplatelet formation at 100 × shown in panels iv and viii, respectively. Arrowheads indicate proplatelet formation in panels ii and vi. Scale bars represent 10 μm. (C) Average number (+SEM) of primitive erythroid, megakaryocyte, and bipotential primitive MEP colonies from plated whole embryos (PS-LNP) and yolk sac (HF-46 sp) as determined by immunohistochemistry. (D) Primitive MEP–derived colony derived from a neural plate–staged embryo contains cells positive for βH1-globin and CD41. (E) Number (+SEM) of βH1-globin/GP1bβ-double positive colonies from a dilution series of 4 to 5 sp yolk sacs, in triplicate. (F) Spatial and temporal distribution of Meg-CFCs as determined by GP1bβ-stained colonies per tissue. (G) Spatial and temporal distribution of definitive MEPs, as determined by Ter119/GP1bβ double-positive colonies per tissue. sp indicates somite pair; PS, preprimitive streak; MS, mid primitive streak; LS, late primitive streak; ENP, early neural plate; MNP, mid neural plate; LNP, late neural plate; HF, head fold; and LHF, late head fold. Approximate embryonic day (E) is provided below the developmental stages.
Joanna Tober et al. Blood 2007;109:
©2007 by American Society of Hematology

4. Enrichment of progenitors in extraembryonic (yolk sac) versus intraembryonic regions.
Enrichment of progenitors in extraembryonic (yolk sac) versus intraembryonic regions. Numbers of primitive MEP, definitive MEP, or unipotential Meg-CFC hematopoietic progenitors (as determined by immunohistochemistry) and erythroblasts in all intraembryonic tissues versus yolk sac. Fold enrichment was calculated as the ratio of intraembryonic progenitors to erythroblasts divided by the ratio of yolk sac progenitors to erythroblasts.35 Tissues from individual experiments were grouped and averaged by stage (± SEM).
Joanna Tober et al. Blood 2007;109:
©2007 by American Society of Hematology

5. Localization of megakaryocytes (GP1bβ-positive cells) in mouse embryos.
Localization of megakaryocytes (GP1bβ-positive cells) in mouse embryos. Individual megakaryocytes were first identified in the yolk sac of E9.5 conceptuses (arrows, top row, left panel). No GP1bβ-positive cells were detected in the E9.5 embryo proper (top row, middle panel). Maternally derived GP1bβ-positive platelets were detected in the surrounding E9.5 decidua (top, right panel). At E10.5 (middle row), clusters of GP1bβ-positive cells were evident in the yolk sac and intraembryonic megakaryocytes were seen in the circulation (arrowhead), associated with the wall of the aorta (arrows), and in the fetal liver (arrowhead). GP1bβ antibodies label both small (open arrowhead) and large cells in the E11.5 to E12.5 fetal liver, and predominantly large cells in the E13.5 to E14.5 fetal liver (lower row). Platelets are seen in E11.5 to 14.5 livers (arrows). Scale bars represent 50 μm.
Joanna Tober et al. Blood 2007;109:
©2007 by American Society of Hematology

6. Large platelets circulate in the embryonic and fetal vasculature.
Large platelets circulate in the embryonic and fetal vasculature. (A) Platelet-enriched blood was stained with anti–GPV-FITC antibodies (E11.5 shown, left panel), which specifically label platelets but not red blood cells. Shown to the right are examples of platelets from E11.5, E15.5, and adult peripheral blood, depicting the broad face (top) and narrow side (bottom). Regardless of cell diameter, all platelets were narrow and biconvex. Scale bar equals 5 μm. (B) Mean platelet diameter (± SEM) in E11.5 to E15.5 fetuses, in neonates, and in adult mice. A minimum of 100 platelets was measured per peripheral blood sample and at least 3 independent samples were studied, except for E10.5, of which 2 pooled blood samples were examined and a total of 11 platelets was measured. (C) Transmission electron microscopic images of E11.5, E15.5, and adult platelets. All images taken at × 10 000. Open canalicular system (arrows), dense granules (arrowheads), and alpha granules (α) are evident.
Joanna Tober et al. Blood 2007;109:
©2007 by American Society of Hematology

7. Fetal platelets are highly reticulated.
Fetal platelets are highly reticulated. (A) Flow cytometric analysis of adult and fetal blood. Platelet fractions were identified by forward and side scatter parameters (left panels), lack of Ter119 and Hoechst staining (middle panels), and CD41 staining (right panels). Thiazole orange–positive platelets were gated as described in “Materials and methods.” (B) The mean percentage (+SEM) of reticulated platelets in E11.5 to E15.5 fetuses (▪), in maternal blood (⊡), in neonates (), and in nonpregnant adult mice (□). Fetal blood samples were analyzed from 3 littermates (E11.5, E14.5, and neonate), 3 fetuses from 3 separate experiments (E12.5), 4 embryos from 2 separate experiments (E13.5), or taken from a single fetus (E15.5). Adult blood was analyzed from a single sample at gestational days 13.5 to 15.5, in triplicate at gestational days 11.5 to 12.5, and from 5 independent nonpregnant adult samples. (C) Mean fluorescence (± SEM) of thiazole orange in untreated platelets and platelets treated with 10 μg/mL RNase A.
Joanna Tober et al. Blood 2007;109:
©2007 by American Society of Hematology