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I. Cortegano et al.
ture, platelet factor 4 (PF4), CD9, von Willebrand factor (VWF), are separated from erythroid progenitors, and closer to other myeloid progenitors expressing Flt3 and the macrophage colony-stimulating factor-1 receptor (Csf1r/CD115).7 Clonal unilineage megakaryocyte progeni- tors (MKP) were defined as burst-forming unit megakary- ocytes (BFU-MK) and as colony-forming unit megakary- ocytes (MK-CFU), and were Lin-c-Kit+Sca1- FcγRII/IIIloCD127-Thy1.1-CD9++CD41+ cells expressing the thrombopoietin receptor (myeloproliferative leukemia virus, MPL).1,9 Other studies revealed distinct lineage poten- tials among erythromyeloid progenitors,10 defining megakaryocyte/erythroid-committed progenitors (PreMegE) as Lin-Sca1-c-Kit+FcγR-CD105-CD150+CD41- and MKP, exclusively associated with megakaryocyte genera- tion, as Lin-Sca1-c-Kit++CD150+CD41+.
Embryo hematopoiesis proceeds in two phases, primitive and definitive, which are conserved among different species, including mice and humans.11,12 In the mouse, a primitive wave of erythromyeloid cells forms in the yolk sac (YS) at E7.5.13,14 At E8.5 erythromyeloid progenitors are generated in the YS and the intraembryonic paraaortic splanchnopleura/aorta-gonads-mesonephros region (P- Sp/AGM), the latter also containing progenitors with lym- phoid activity.15-17 Definitive HSC that are the source of all adult hematopoietic cell lineages are present in the P- Sp/AGM at E10.5.18 The emergence of these definitive HSC in the embryo is dependent on the expression of the tran- scription factor RUNX1,19 which is required for progression of CD41+ embryonic precursors into HSC.20 The fetal liver (FL) represents the major hematopoietic organ during gesta- tion, receiving extrinsic HSC and MPP from the YS, P- Sp/AGM and the placenta at E10.5. MEP involved in prim- itive and definitive megakaryopoiesis appear in the YS at E7.25 and at E9.5, respectively.21,22 RUNX1-independent diploid platelet-forming cells have been identified in the YS at E8.5/10.5.23 Moreover, CD42c+ megakaryocytes can be identified in the YS, in circulation and in the FL from E9.5 onwards, and large reticulated immature platelets circulate at E10.5.21,24
Embryo-derived megakaryocytes differ from those from the adult BM, as illustrated by the in vitro effects of throm- bopoietin,25 cell-intrinsic differences in vivo after transplanta- tion26 and the smaller size of those from YS.22 In the FL from E10.5-E11.5 mice, megakaryocytes progressively increase in size and ploidy.27 However, despite several reports on BM- derived megakaryopoiesis published recently, the interme- diate cells that appear during this process early in life, and the changes in surface phenotype, have yet to be fully defined.
We found previously that at E10.5/E11.5, FL megakaryo- cytes are c-KitDCD49f++CD41++CD9++CD42c+VWF+ and they rapidly produce, independently of thrombopoietin stimulation, proplatelet-bearing megakaryocytes (P-MK) in vitro.28 Strikingly, these FL megakaryocytes were CD41++CD45-, as were the diploid platelet-forming cells found in the YS.23 Here we show that, unlike those from BM, FL megakaryocytes remain CD45- until E13.5, as do the PreMegE and MKP. However, both CD41+CD45+ and CD41+CD45- cells are present in the FL, these populations bearing MK-CFU, megakaryocyte gene expression, and containing Lin-Sca1-c-Kit++CD150+CD41+ MKP. These cells develop into CD41++CD45-CD42c++ P-MK in vitro. The E11.5 FL also contains CD41-CD45++CD11b+ cells that produce CD41+CD45+ cells in vitro, although they do not develop
into P-MK. Accordingly, CD45++EGFP+ cells from E11.5 FL ex vivo preparations from MaFIA transgenic mice, which trace cells expressing Csf1r/CD115,29 give origin poorly to CD41++ cells both in vivo and in vitro. Interestingly, a high proportion of adult BM CD41++CD45++CD9++CD42c++ megakaryocytes from C57BL/6 mice express CD115 and are EGFP+ in MaFIA mice. Our results identify different pathways of megakaryopoiesis in the mouse embryo FL and in adult BM, driven by distinct MKP expressing or not CD45.
Methods
Mice and embryo microsurgery and cell suspensions
BALB/c, C57BL/6 and C57BL/6-Tg(Csf1r-EGFP- NGFR/FKBP1A/TNFRSF6) 2Bck/J MaFIA29 mice were maintained at the animal facilities of the Instituto de Salud Carlos III. All animal studies were approved by the Animal Health Ethics Authority from the Autonomous Government of Madrid (PROEX 080/15). Embryo microsurgery and cell suspensions were obtained as described previously28 and in the Online Supplementary Methods.
Flow cytometry and cell purification
Cells were stained as reported elsewhere,28 with the fluo- rochrome-labeled antibodies described in the Online Supplementary Methods and Online Supplementary Tables S1 and S2.
Quantitative real-time polymerase chain reaction analysis
RNA was extracted, oligo(dT)-primed cDNA samples were pre- pared and quantitative real-time polymerase chain reaction (RT- qPCR) amplifications were performed with the primers and pro- tocols described,30,31 as indicated in the Online Supplementary Methods and Online Supplementary Table S3.
Colony-forming cell assays and cell cultures
Clonal semisolid cultures and cultures of purified cell popula- tions were performed as indicated in the Online Supplementary Methods.
Immunofluorescence
Immunostaining was performed on cryosections from YS and embryos as indicated in the Online Supplementary Methods. The preparations were analyzed by confocal microscopy (Leica DMRD) and the images were processed with ImageJ software.
Statistical analysis
GraphPad Prism 4.0 software was used to calculate the means and standard error of the mean (SEM). Comparisons were per- formed with unpaired and paired Student t tests, with the χ2 test or the Kruskal-Wallis test, to obtain the P values. Data are expressed as mean ± SEM. A P-value less than 0.05 was defined as statistically significant; statistical significance is shown as *P<0.05, **P<0.01 and ***P<0.001.
Results
Megakaryocyte lineage cells are present in hematopoi- etic organs and blood vessels during post-gastrulation embryo development
Co-expression of the CD41/aIIa integrin (GPIIb) and CD42c/GPIb-β chains was used to trace megakaryocytes and platelets by flow cytometry. CD41++CD42c+
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haematologica | 2019; 104(9)