Page 185 - 2019_09-HaematologicaMondo-web

CD45-negative megakaryopoiesis in the mouse embryo
Discussion
The morphological, functional and molecular changes that take place in the differentiation of megakaryocytes have been assessed here using bulk in vitro cultures of megakaryocyte-committed progenitors from the E11.5 FL. One striking finding was that embryonic CD41++CD42c+CD61++CD9++ megakaryocytes are nega- tive, until E13.5, for the leukocyte common CD45 antigen, a large transmembrane glycoprotein expressed on the sur- face of all hematopoietic cells and their precursors, except mature erythrocytes and platelets.33,35,36 CD45 accounts for up to 10% of lymphocyte cell surface proteins and is involved in the dephosphorylation of the regulatory tyro- sine of Src family kinases, negatively modulating cell sig- naling.33,35,36 The CD45 protein sets the threshold for signal transduction, and CD45 deficiency produces developmen- tal defects and extended phosphorylation of the JAK/STAT cascade.37 The absence of CD45 or diminished levels of this protein have been associated with a hyper- adhesive phenotype and impairment of progenitor mobi- lization from the BM.38,39 It could be that the low expres- sion of CD45 may favor the observed accumulation of megakaryocytes in FL at E11.5, together with interactions through integrin receptors that are expressed highly by megakaryocytes.
We used CD41 expression to trace megakaryocytes, since CD41 is expressed strongly by cells of the megakaryocyte lineage, including platelets, in the adult mouse.40 CD41 was defined as a marker for the early stages of primitive and definitive hematopoiesis in the mouse embryo,13 and as a marker of HSC in mice and zebrafish,41,42 tracing the divergence of definitive hematopoiesis from endothelial cells in mouse c-Kit+ pro- genitors.40,43 CD41++CD45- megakaryocytes are found in the YS and embryo (P-Sp/AGM, FL) from E9.5 and in the circulating blood, as also reported by others.23 Interestingly, E11.5, PreMegE and MKP also display less CD45 than those from newborn and adult BM, whereas CD45 levels appear to be similar in other lineage progeni- tors, revealing a linkage of the CD45-/dim trait to embryo erythroid/megakaryocyte-lineage cells. Since CD41++ megakaryocytes remain CD45- until E13.5 in the FL, it is tempting to speculate that CD45- EMK may correspond to the primitive wave of megakaryopoiesis generating CD41+CD42c+ Runx1- diploid platelet-forming cells described in the YS at E10.5.23 The progression of primi- tive HSC to definitive HSC is dependent on RUNX1.20 At E11.5 RUNX1-deficient mice have primitive erythrocytes but lack hematopoietic cells in FL and identifiable platelets in blood.44 They also lack definitive HSC and CD45+ cells, and have very few CD41++CD45- cells.20 It would thus be conceivable that CD45- Runx1+ megakaryocytes present in the FL at E11.5 belong to the definitive wave of megakaryopoiesis. However, RUNX1 is essential for megakaryocyte maturation in the adult BM.45 Therefore, the fact that EMK in the E11.5 FL are Runx1+, and that many of them are tetraploid cells with larger size than those in the contemporaneous YS, may indicate that the local environment in the FL provides conditions allowing maturation of primitive wave CD45- megakaryocytes. It has been described that megakaryocytes require MPL in order to reach >8N maturation stages after E14.5.24 At E11.5, after 2 days in culture, the cell subpopulations iso- lated from FL produced mostly megakaryocytes with 8N
ploidy, which may represent the in vitro differentiation of MPL-independent megakaryocytes. Also, at E11.5 FL R4/CD41++CD45- megakaryocytes express the transcrip- tion factors NF-E2 and Fli1, in agreement with the findings on a megakaryocyte transcription factor core for YS diploid platelet-forming cells at E10.5 and for FL megakaryocytes at E13.5.24
In the FL, CD41 and CD45 expression define several cell subsets at E11.5. CD41++CD45- cells are already megakary- ocyte-committed CD42c+MPL+CD9+CD61++AChE+ cells that develop rapidly in culture to P-MK, whereas CD41- CD45++ cells are mostly CD11b+ myelo/monocyte-com- mitted cells. On the other hand, CD41+CD45+ and CD41+CD45- cells have a more immature phenotype than the aforementioned populations. The phenotypic data and the gene expression profile ex vivo, as well as in vitro studies of these purified populations, prompt us to propose two major pathways of megakaryocyte differentiation operat- ing in the E11.5 FL (Figure 7): (i) from CD41+CD45- iEMK, CD41 is upregulated and CD42c is expressed, producing EMK (CD41++CD45-CD42c+) that develop proplatelets with no evidence of CD45 expression (P-MK); (ii) from CD41+CD45+ iAMK (that are CD115-) (Figure 6C), cells enter a CD41+CD45+CD42c+ stage from which CD41++CD45-CD42c+ EMK arise. Therefore, CD45 dimin- ishes when the levels of CD41 of these increase and they acquire CD42c to become EMK. The first pathway is common before E13.5 but becomes rare after E15.5, and it is currently unknown whether it is even retained at low levels in the BM, while the reverse applies to the CD45- derived pathway, although in this case CD45 is retained in BM CD41++CD42c+ megakaryocytes. Moreover, our data reveal the involvement of Csf1r-expressing cells in adult BM CD45+ megakaryopoiesis, which may represent a third pathway of megakaryopoiesis (Figure 7C), minor or absent in the embryo, and opens the issue of the genera- tion of CD41++ megakaryocytes from CD45++CD115- expressing cells in the adult. Csf1r/CD115 is considered a mature monocytic differentiation receptor,46 but besides the high expression of Csf1r in monocytes, macrophages, osteoclasts and myeloid dendritic cells, it is also expressed at low levels on HSC, CMP and CLP, as well as among several non-hematopoietic embryonic cells.47 More work is needed to clarify the differential contribution of these CD45++CD11b+CD115+ cells to adult and embryo megakaryopoiesis and its relevance.
Embryo-fetal-derived megakaryocytes engraft poorly into adult mice and produce low number of platelets.48,49 It is presently unknown whether the different subsets of megakaryocyte progenitors identified in the embryo may give rise to functional or immature platelets in vivo, but they may represent new tools to uncover the mechanisms underlying the maturation of the membrane demarcation system assembly machinery that yields platelets, similarly as the recently described mechanisms by which BM megakaryocytes sense extracellular matrix rigidity to release platelets.50 In summary, we present a number of findings proving that embryo megakaryocytes are hematopoietic CD45- nucleated cells that are produced from CD45- and CD45+ progenitor cells, findings that may be extended to human cord blood samples in order to probe the existence of a human CD45- megakaryocyte counterpart. These issues have relevant implications for understanding aberrant megakaryopoiesis processes and megakaryocyte-derived tumors, and also represent a tool
haematologica | 2019; 104(9)
1863