The first embryonic platelets are produced by a unique lineage of diploid cells not polyploid MKs.
Diploid platelet-forming cells are produced in the early mouse embryo via a progenitor cell–independent pathway.
In this study, we test the assumption that the hematopoietic progenitor/colony-forming cells of the embryonic yolk sac (YS), which are endowed with megakaryocytic potential, differentiate into the first platelet-forming cells in vivo. We demonstrate that from embryonic day (E) 8.5 all megakaryocyte (MK) colony-forming cells belong to the conventional hematopoietic progenitor cell (HPC) compartment. Although these cells are indeed capable of generating polyploid MKs, they are not the source of the first platelet-forming cells. We show that proplatelet formation first occurs in a unique and previously unrecognized lineage of diploid platelet-forming cells, which develop within the YS in parallel to HPCs but can be specified in the E8.5 Runx1-null embryo despite the absence of the progenitor cell lineage.
Blood cell formation first occurs within the embryonic yolk sac (YS), yielding primitive red cells and multiple classes of colony-forming units (CFUs)/hematopoietic progenitor cells (HPCs).1 From embryonic day (E) 7.5, the YS contains megakaryocyte (MK) CFUs2-4 and, by E8.5, is capable of generating polyploid MKs in vitro.2 Detection of circulating platelets soon follows the appearance of HPCs.4 The intuitive extrapolation is that MK-CFUs differentiate within the YS to initiate platelet formation, but this model remains untested.
To better understand how platelet production proceeds in the YS, we have employed a combination of functional assays, transcriptomics, imaging, and gene disruption to investigate the relationship between HPCs and generation of the first platelet-forming cells.
UBI-gfp,5 Runx1LacZ/LacZ,6 and Runx1Δ/Δ7 mouse lines were maintained as C57BL/6. Walter and Eliza Hall Institute Animal Ethics Committee approved the experiments. Developmental stages were determined morphologically or by counting somite pairs.
For CFU-MK culture, MegaCult-C (StemCell Technologies) was used as previously described8 ; colonies were defined as clusters of ≥10 CD41+ cells after 5 days. M3434 (StemCell Technologies) was used for myelo-erythroid assays.
An LSM 780 microscope was used for confocal imaging. Analysis was performed using Imaris (Bitplane).
For transcriptome analyses, samples were hybridized to Illumina Expression BeadChips (ArrayExpress accession: E-MTAB-2625) and analyzed using limma.
Results and discussion
Although MK-CFUs have been functionally identified in the YS,2,4 the immunophenotype of these progenitors is undescribed, making it unclear how they relate to the conventional CD45+CD41low HPC population.11,12 To address this, we isolated cells expressing CD45 and/or CD41 from E9.5 and E10.5 YS (Figure 1A) and performed in vitro colony-forming assays. At both stages, all MK-CFUs present in whole YS were accounted for by the conventional HPC population (Figure 1B; supplemental Figure 1, see the Blood Web site).
We found that platelets first entered the peripheral blood (PB) from E9.5, increasing rapidly in number by E10.5 (Figure 1C). It would therefore be expected that the cells responsible for platelet production are terminally differentiated by E10.5. Hallmarks of mature MKs are their highly polyploid nuclei and the ability to form proplatelets.13 Proplatelets can be acutely induced ex vivo from MKs within hours,14 whereas production of proplatelet-forming MKs from fetal precursors require at least 4 days.15 We found that after 72 hours in vitro HPCs generated polyploid MKs, but proplatelets were rarely formed (Figure 1D), indicating that HPCs had not differentiated into acute proplatelet-forming cells in vivo and were therefore not the source of the first platelets.
We next queried if an alternative population was responsible for platelet formation. By comparing the transcriptional profiles of E10.5 YS cells with E13.5 liver reference lineages, including MKs, we investigated if elements of an MK signature were present at E10.5. We found that a previously uncharacterized population of CD45−CD41high cells was strikingly similar to E13.5 MKs (Figure 1E, supplemental Figure 2A); this population also coexpressed MK-associated proteins including MPL (Myeloproliferative leukemia virus oncogene), CD42D, and acetylcholinesterase (supplemental Figure 2B-C). Curiously, CD45−CD41high cells did not exhibit the high ploidy range associated with conventional MKs or E10.5 YS HPC-derived MKs; rather, the majority were diploid (Figure 1F).
Approximately half of the low-ploidy CD45−CD41high cells acutely produced proplatelets in vitro (Figure 1G). Similarly to FL MKs,16 in vitro thrombopoiesis was not THPO dependent (supplemental Figure 2D), but surprisingly, proplatelets were formed while in a diploid state (Figure 1H).
Using a refined MK immunophenotype (CD41highCD42D+), we confirmed that proplatelet formation also occurred in a diploid state in vivo (Figure 1I-J). We therefore defined this lineage as DPFCs.
That HPC-derived MKs were highly polyploid yet in vivo platelet-forming cells were diploid prompted us to ask whether HPCs are the source of YS DPFCs in vivo. To address this, we investigated when DPFC commitment first occurred.
CD41highCD42D+ cells were generated in vitro from E7.5 and E7.75 YSs, but acute proplatelet formation was rarely observed, and only from E7.75 (Figure 2A). By E8.5 CD41low/−TER119+ primitive erythroid (EryP)11 and VECAD+CD41high HPC lineages can be prospectively isolated.11,17 Accompanying these, we identified a population of VECAD−CD41high cells that encompassed all CD42D+ cells (Figure 2B), indicating that they might include acute proplatelet-forming cells.
CFU and proplatelet assays revealed that VECAD+CD41high cells contained all myeloid/erythroid and MK CFUs, but only VECAD−CD41high cells were capable of acute proplatelet formation (Figure 2C-D, supplemental Figure 3). That E8.5 VECAD−CD41high cells displayed little acetylcholinesterase activity (supplemental Figure 4) suggested that they are an immature DPFC precursor. Thus, as early as E8.5, acute proplatelet-forming cells exist in the YS and are immunophenotypically distinct from the HPC lineage.
Hematopoietic commitment in the YS occurs via VECAD-expressing precursors.18,19 Consistent with this, all CD41-expressing cells in the E7.75–8.25 blood band coexpressed VECAD, but by E8.5, they had diverged into VECAD+ (HPC) and VECAD− (pre-DPFC) counterparts (supplemental Figure 5). This suggested that HPC and DPFC lineages both progressed via VECAD-expressing precursors. We questioned whether YS DPFCs arise in parallel to, but independently of, HPCs.
Determining whether a lineage is HPC derived in the YS has been problematic. The best attempts have used c-Myb−/− mice.3,20 However, multiple classes of HPCs are produced in c-Myb−/− embryos; among these are erythroid, macrophage, and MK CFUs.3,21 We reasoned that a more stringent approach would be the use of Runx1-null embryos. Without RUNX1, HPC formation is completely blocked22,23 ; yet hematopoietic specification from the mesoderm is permitted, as evidenced by EryP formation.24 Thus, studying the Runx1-null YS should allow us to test if HPCs give rise to DPFCs.
Using 2 independent Runx1-null lines6,7 (both carrying the null allele through the germ line), we found that the E8.5 Runx1-null YSs contained VECAD−CD41high pre-DPFCs and were capable of generating CD41+CD42D+ cells in vitro (Figure 2E-F). Analysis of E10.5 Runx1-null YS confirmed that despite the absence of HPCs, platelet-producing DPFCs were formed in vivo (Figure 2G-H). Although Runx1-null DPFC numbers were lower both in vitro and in vivo, which is likely a result of RUNX1 being an essential factor for megakaryopoiesis,25 these proof-of-concept experiments demonstrated that developmental specification of the DPFC lineage was not HPC dependent.
In summary, we have shown that polyploid MKs generated from YS cultures,2 which resemble MKs of fetal life, are the product of HPC differentiation. These do not represent the first in vivo platelet-forming cells of the embryo. Platelet formation in the YS is initiated by a previously unrecognized cell that we have termed DPFC, which likely develops via the primitive hematopoietic pathway.2,4 A key challenge will now be to experimentally define when HPC-derived conventional MKs supersede DPFCs to become the source of platelets during fetal life.
The online version of this article contains a data supplement.
There is an Inside Blood Commentary on this article in this issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
The authors thank Dr Julie Sheridan for critical discussions.
This work was supported by the Australian Research Council (Special Research Initiative in Stem Cell Sciences, Discovery Early Career Researcher Award Fellowship [S.T.], Strategic Australian Postgraduate Award Studentship [K.S.P.]), Program Grant (1016647), Fellowships (W.S.A., G.K.S., and D.J.H.), and Independent Research Institutes Infrastructure Support Scheme Grant (361646) from the National Health and Medical Research Council, and Victorian State Government Operational Infrastructure Support.
Contribution: K.S.P., S.T., W.S.A., and D.J.H. designed the research, analyzed data, and wrote the manuscript; J.F.M., C.B., E.C.J., L.W.W., K.L.R., A.L., B.T.K., and A.M. designed the research; and T.J.S., W.S., and G.K.S. analyzed data.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Samir Taoudi, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne, VIC 3052, Australia; e-mail: firstname.lastname@example.org.
D.J.H. and S.T. contributed equally to this study.