Delineating CD150/CD55 expression allows enhanced purification of immunophenotypic MEP and CMP fractions in E14.5 fetal liver

The existence of a bipotent MEP has long been posited in mid-gestation fetal liver, in addition to adult bone marrow [3,31]. The multipotent CMP is more controversial, however, with various groups reporting that it is actually a heterogeneous population of megakaryocytic/erythroid/granulocytic/monocytic progenitors, capable of varying lineage output [5,40]. More recently, CD150 and CD55 expression have been associated with Mk/Ery potential in adult hematopoietic progenitors [41]. Therefore, we analyzed CD55/CD150 expression in the E14.5 fetal liver MEP (Lin^- SCA1^- C-KIT⁺ (LK) CD16/32^low CD34^-) and CMP (LK CD16/32^low CD34⁺) progenitor fractions (Fig 1A and 1B). Additionally, to remove the previously identified CD41⁺ MkP fraction [33,42], we analyzed solely CD41^- MEPs and CMPs.

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Fig 1. Delineating CD150/CD55 expression allows enhanced purification of immunophenotypic MEP and CMP fractions in E14.5 fetal liver.

A-B. Expression of CD150 and CD55 in wild type (WT) E14.5 fetal liver immunophenotypic MEP and CMP fractions. A. Representative FACS plots of Lin^- Sca1^- cKit⁺ (LK) hematopoietic progenitors (1^st panel); LK hematopoietic progenitors: CD16/32^low CD34^- MEPs, CD16/32^low CD34⁺ CMPs and CD16/32^high CD34⁺ GMPs (2^nd panel); CD41^- MEPs (3^rd panel); CD150/CD55 expression in MEPs (4^th panel); CD41^- CMPs (5^th panel);; and CD150/CD55 expression in CMPs (6^th panel). B. Quantitation of the proportion of CD55^- CD150^-, CD55⁺ CD150^- and CD55⁺ CD150⁺ MEPs (left) and CMPs (right) as a percentage of total live red blood cell lysed fetal liver cells. N = 3 C-E. CFU-C activity of CD55^- CD150^-, CD55⁺ CD150^- and CD55⁺ CD150⁺ MEPs and CMPs. C. Total CFU-C numbers per 100 plated cells. D. Differential (CFUe, BFUe, CFU-Mk and CFU-MkE) activity of MEPs. E. Differential (CFU-G, CFU-M, CFU-GM, BFUe, CFU-Mk, CFU-MkE and CFU-GEMM) activity of CMPs. N = 3 F-I. Lineage output of OP9 co-cultured MEPs and CMPs. F. Representative FACS plots of TER119 and CD41 expression of day 7 OP9 co-cultured MEPs. G. Proportion of TER119⁺ erythroid cells and CD41⁺ megakaryocyte cells in day 7 OP9 MEP co-cultures. H. Representative FACS plots of CD11b, GR1, TER119 and CD41 expression of day 7 OP9 co-cultured CMPs. I. Proportion of CD11b⁺ GR1⁺ granulocyte/monocyte cells, TER119⁺ erythroid cells and CD41⁺ megakaryocyte cells in day 7 OP9 CMPs co-cultures. N = 3.

https://doi.org/10.1371/journal.pgen.1007127.g001

In both the CD41^- MEP and CMP fractions, three populations were discernible: CD55^- CD150^- (4% of MEPs, 52% CMPs); CD55⁺ CD150^- (69% MEPs, 12% CMPs); and CD55⁺ CD150⁺ (26% MEPs, 30% CMPs). By contrast, we observed the GMP and MkP populations were more homogeneous with respect to both CFU-C activity (S1A Fig) and CD150/CD55 expression: 95% of MkPs were CD55⁺ CD150⁺ and 91% of GMPs were CD55^low CD150^low (S1B–S1F Fig). We also confirmed that negligible (<1%) CFU-C activity resided in the remaining cKit^- fetal liver fraction (S1A Fig). We therefore proceeded to characterize the differentiation potentials of the various CD55/CD150 MEP and CMP subfractions.

Interestingly, CD55^- CD150^- MEPs did not possess any CFU-C activity in semi-solid myeloid MethoCult medium (Fig 1C). Additionally, they did not yield cells expressing either the erythroid marker TER119, the megakaryocytic marker CD41, the granulocytic/monocytic markers CD11b/GR1 or the mast/progenitor cell-associated C-KIT following OP9 co-culture in pro-myeloid medium (Fig 1F and 1G, S1G and S1H Fig). By contrast, CD55^- CD150^- CMPs yielded CFU-Cs at 20% plating efficiency, which solely comprised granulocytic (CFU-G), monocytic/macrophage (CFU-M) and GM (CFU-GM) colonies (Fig 1C and 1E). Additionally, 98% of OP9 co-cultured CD55^- CD150^- CMP-derived cells were CD11b⁺ GR1⁺ granulocytes/monocytes (Fig 1H and 1I), with just 0.01% being TER119⁺ erythrocytes and 0.11% CD41⁺ megakaryocytes.

CD55⁺ CD150^- cells had increased CFU-C activity by comparison (25% for MEPs, 40% for CMPs, Fig 1C). CD55⁺ CD150^- MEPs solely comprised erythroid colony-forming units (CFUe, Fig 1D) and produced 92% TER119⁺ erythroid cells on OP9 (Fig 1F and 1G). CD55⁺ CD150^- CMPs chiefly yielded CFU-G, M and GM colonies, but also comprised a small number (approximately 1%) of erythroid burst-forming units (BFUe), bipotent megakaryocytic/erythroid colony-forming units (CFU-MkE) and multipotential granulocytic/erythroid/monocytic/megakaryocytic colony-forming units (CFU-GEMM, Fig 1E). Accordingly 93% CD11b⁺ GR1⁺ granulocytic/monocytic and 1.8% CD41⁺ megakaryocytic cells resulted from OP9 co-culture (Fig 1H ang 1I).

Finally, CFU-C activity was chiefly observed in the CD55⁺ CD150⁺ fractions (50% for MEPs, 70% for CMPs, Fig 1C). Crucially, the CD55⁺ CD150⁺ subpopulation was the only MEP fraction to demonstrate megakaryocytic/erythroid bipotentiality (yielding 1.2% CFU-Mks and 0.5% CFU-MkEs, Fig 1D; 79% TER119⁺ erythrocytes and 14% CD41⁺ megakaryocytes, Fig 1F and 1G). Additionally, the CD55⁺ CD150⁺ CMP fraction had the broadest range of CFU-C activity, yielding approximately equal numbers of CFU-M, GM, Mk, MkE, GEMM and BFUe colonies (Fig 1E) and all three Mk/Ery/GM cell fractions in OP9 co-culture (Fig 1H and 1I). We therefore demonstrated that inclusion of anti-CD150 and, to a lesser extent, anti-CD55 antibodies in MEP/CMP isolation protocols aids the purification of highly clonogenic multipotent myeloid progenitors.

The FL Lin^- cKit^high Sca1^high (LSK) is a highly clonogenic fraction, which can be separated into long-term repopulating HSC and short-term repopulating Multipotent Progenitor (MPP) fractions on the basis of CD48 and CD150 expression [32,43] (S1I and S1J Fig). To determine whether fetal liver LSK hematopoietic stem and progenitor cells are the likely ancestors of the fetal liver CMPs, MEPs, GMPs and MkPs, we cultured E14.5 fetal liver HSCs and LSK CD48^-/+ MPPs in pro-myeloid medium for 20 hours (S1K and S1L Fig). The majority of cultured HSCs (60%) retained an LSK immunophenotype, but had upregulated CD48. Only 6% of cultured HSCs had an LK immunophenotype, but within this fraction CD150^+/- CMP, CD150^+/- MEP, GMP and MkP fractions were discernible. More convincingly, a large fraction (27%) of cultured LSK CD48⁺ CD150⁺ MPPs had acquired an LK immunophenotype, yielding mostly CD150⁺ MEPs and MkPs. By contrast, 12% of cultured LSK CD48⁺ CD150^- MPPs had an MkP, GMP, CD150^- MEP or CD150^- CMP immunophenotype. This therefore suggests that fetal liver HSCs are capable of establishing a myeloid progenitor hierarchy, as their LSK CD48⁺ MPP progeny give rise to the Sca1^- cKit⁺ MkP, GMP, MEP and CMP progenitors.

The observation that fetal liver HSCs are capable of establishing a hematopoietic stem and progenitor cell hierarchy does not preclude the possibility that yolk sac erythro-myeloid progenitors (EMPs) could also contribute to these cell populations. To determine whether EMPs could directly give rise to a hematopoietic progenitor cell profile comparable to that observed in the fetal liver, we isolated yolk sac cells from E9.5 embryos and cultured them in pro-myeloid medium for up to 24 hours (with or without prior explant culture (S1M and S1N Fig)). We observed that a small proportion of cells (up to 15% in the explant cultures) had the LK immunophenotype. The majority of these cells were either immunophenotypic GMPs or were LK CD16/32^- CD150^- CD41⁺. No CD150⁺ LK progenitors were produced in these cultures. This therefore suggests that yolk sac EMPs are incapable of directly establishing our observed FL myeloid hematopoietic progenitor hierarchy.

Runx1-P2 reporter expression demarcates mono and bipotent subsets in the CD150⁺ MEP

We previously used Runx1-P1-GFP::P2-hCD4 dual-reporter mice to demonstrate that Runx1-P2 expression coincides with enhanced megakaryocytic specification in adult bone marrow PreMegEs [18]. Having established that CD150 expression enhances megakaryocyte/erythroid progenitor isolation in fetal liver MEPs, we tried to improve this further by characterizing Runx1-P1-GFP/P2-hCD4-expressing MEPs (Fig 2A and 2B). In E12.5, E13.5 and E14.5 fetal liver, the CD41^- CD150⁺ MEP fractions were dominated by P1-GFP⁺ P2-hCD4^- cells (66%, 72% and 80% respectively). P1-GFP^- P2-hCD4^- populations were also present (25%, 25% and 13% respectively). However, P1-GFP⁺ P2-hCD4⁺ populations were considerably smaller, at 8%, 3% and 7% of CD41^- CD150⁺ MEPs respectively. Importantly, at each timepoint this restricted P2-hCD4⁺ fraction possessed the vast majority of BFUe, CFU-Mk and CFU-MkE activity (Fig 2C, S2A and S2B Fig). This was particularly marked at E14.5, with this population yielding 20% CD41⁺ megakaryocytes following OP9 co-culture, compared to <5% in E12.5 and E13.5 cultures (Fig 2D and 2E, S2C–S2F Fig).

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Fig 2. Runx1-P2 reporter expression demarcates mono and bipotent subsets in the CD150⁺ MEP.

A-B. Expression of Runx1-P1-GFP and P2-hCD4 in P1-GFP::P2-hCD4/+ E12.5, E13.5 and E14.5 FL CD41^- CD150⁺ MEPs. A. Representative FACS plots. B. Quantitation of the proportions of P1^- P2^-, P1⁺ P2^-, P1⁺ P2⁺ and P1^- P2⁺ MEPs as a percentage of total live red blood cell lysed fetal liver cells. N = 3 C. Differential CFU-C activity of wild type, P2-hCD4^- and P2-hCD4⁺ E14.5 FL CD41^- CD150⁺ MEPs. N = 6 D-E. Lineage output of day 7 OP9 co-cultured wild type, P2-hCD4^- and P2-hCD4⁺ CD41^- CD150⁺ MEPs. D. Representative FACS plots of TER119 and CD41. E. Proportion of erythroid and megakaryocyte cells. N = 4 F-G. Lineage output of single cultured P2^- and P2⁺ MEPs in OP9 co-cultures (F) and MethoCult semi-solid myeloid culture medium (G). H-I. Short-term (14 hours) differentiation of P2^- and P2⁺ CD41^- CD150⁺ MEPs in pro-myeloid liquid culture. H. Representative FACS plots of CD16/32/CD34 (top) and CD150/CD41 (bottom) expression in the LK gate of cultured MEPs. I. Proportions of immunophenotypic MkP, CD41^- CD150^- MEPs and CD41^- CD150⁺ MEPs in short-term cultures. N = 5.

https://doi.org/10.1371/journal.pgen.1007127.g002

To achieve a more accurate estimate of the potentiality of P2-hCD4^- and P2-hCD4⁺ CD41^- CD150⁺ MEPs, we performed single cell OP9 co-culture assays (Fig 2F). Single P2-hCD4^- and P2-hCD4⁺ CD41^- CD150⁺ MEPs displayed plating efficiencies of 33.3% and 28.5% respectively in these conditions. Alternatively, replating in MethoCult yielded 60.4% and 51.0% plating efficiencies for P2-hCD4^- and P2-hCD4⁺ CD41^- CD150⁺ MEPs respectively (Fig 2G). Under both culture conditions, approximately 15% of positive wells derived from P2-hCD4⁺ CD41^- CD150⁺ MEPs yielded both megakaryocytic and erythroid cells. By contrast, no single P2-hCD4^- CD41^- CD150⁺ MEPs demonstrated dual lineage specification. We therefore established that MEP bipotentiality is restricted to the CD41^- CD150⁺ P2-hCD4⁺ MEP subfraction, comprising approximately 1% of immunophenotypic MEPs and fewer than 0.05% of total live cells in E14.5 fetal liver (Fig 2A and 2B).

To fully demonstrate the increased megakaryocytic lineage commitment in E14.5 fetal liver P2-hCD4⁺ CD41^- CD150⁺ MEPs compared to their P2-hCD4^- counterparts, we cultured both cell types in pro-myeloid medium for 14 hours (Fig 2H and 2I). P2-hCD4⁺ CD41^- CD150⁺ MEPs produced more MkPs than the P2-hCD4^- population, which yielded greater proportions of erythroid-dominated CD150^- MEPs. We therefore demonstrated that the P2-hCD4⁺ fraction represents a pro-megakaryocyte subpopulation of immunophenotypic MEPs in E14.5 FL.

Runx1-P2-hCD4⁺ CMPs have enriched multilineage output

Having established that Runx1-P2-hCD4 expression heterogeneity in CD41^- CD150⁺ MEPs is associated with different lineages, we turned to CD41^- CD150⁺ CMPs. E12.5, E13.5 and E14.5 fetal liver CD41^- CD150⁺ CMPs mostly express Runx1-P1-GFP (81%, 86% and 94% respectively, Fig 3A and 3B). Additionally, most CD150⁺ CMPs co-express P2-hCD4 (74% in E12.5, 81% in E13.5 and 73% in E14.5 fetal liver). At all three stages, colony-forming output was skewed in favor of CFU-Ms and away from BFUes in P2-hCD4⁺ CMPs compared to P2-hCD4^- CMPs (Fig 3C, S3A and S3B Fig). Moreover, CFU-GM output was enriched at E13.5 and E14.5, and multipotent CFU-GEMM activity was enriched in P2-hCD4⁺ CMPs at E13.5 compared to the P2-hCD4^- populations (Fig 3C, S3B Fig).

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Fig 3. Runx1-P2-hCD4⁺ CMPs have enriched multilineage output.

A-B. Expression of Runx1-P1-GFP and P2-hCD4 in P1-GFP::P2-hCD4/+ E12.5, E13.5 and E14.5 FL CD41^- CD150⁺ CMPs. A. Representative FACS plots. B. Quantitation of the proportions of P1^- P2^-, P1⁺ P2^-, P1⁺ P2⁺ and P1^- P2⁺ CMPs as a percentage of total live red blood cell lysed fetal liver cells. N = 3 C. Differential CFU-C activity of wild type, P2-hCD4^- and P2-hCD4⁺ E14.5 FL CD41^- CD150⁺ CMPs. N = 7 D-E. Lineage output of day 7 OP9 co-cultured wild type, P2-hCD4^- and P2-hCD4⁺ CD41^- CD150⁺ CMPs. D. Representative FACS plots of CD11b, GR1, TER119 and CD41. E. Proportion of granulocyte/monocyte, erythroid and megakaryocyte cells. N = 7 F-G. Lineage output of single cultured P2^- and P2⁺ CMPs in OP9 co-cultures. F. Table of plating efficiency. G. Ternary plots displaying proportions of granulocyte/monocyte, erythroid and megakaryocyte cells in each positive well for wild type, P2-hCD4^- and P2-hCD4⁺ CD41^- CD150⁺ CMPs. H-I. Short-term (14 hours) differentiation of P2^- and P2⁺ CD41^- CD150⁺ CMPs in pro-myeloid liquid culture. H. Representative FACS plots of CD16/32/CD34 (top) and CD150/CD41 (middle and bottom) expression in the LK gate of cultured CMPs. I. Proportions of immunophenotypic MkPs, CD41^- CD150^- MEPs, CD41^- CD150⁺ MEPs, GMPs, CD41^- CD150^- CMPs and CD41^- CD150⁺ CMPs in short-term cultures. N = 3.

https://doi.org/10.1371/journal.pgen.1007127.g003

OP9 co-culture of the bulk P2-hCD4^- and P2-hCD4⁺ CD41^- CD150⁺ CMP populations revealed changing lineage output in E12.5, E13.5 and E14.5 fetal liver (Fig 3D and 3E, S3C–S3F Fig). Notably, pro-GM/anti-Erythroid bias was evident in E14.5 P2-hCD4⁺ CMPs. Analysis of single co-cultured CMPs (Fig 3F and 3G) confirmed this, as almost 60% of E14.5 P2-hCD4^- CMPs produced solely erythroid cells, compared to 12.5% of P2-hCD4⁺ CMPs. The granulocyte/monocyte-restricted fraction (“GM only”) represented 3% and 16% of the P2-hCD4^- and P2-hCD4⁺ CMPs respectively. Megakaryocytic output was similarly enhanced in P2-hCD4⁺ CMPs, with 38% possessing Mk-restricted and 9% demonstrating bipotential megakaryocytic/erythroid (Mk+Ery) activity, (compared to 28% and 6% of P2-hCD4^- CMPs). Importantly, we observed multipotent (balanced Mk+Ery+GM) lineage output in P2-hCD4⁺ CMPs only, albeit in <10% of positive wells. This suggests multilineage activity is highly restricted to Runx1-P2-hCD4⁺ CMPs in E14.5 fetal liver.

Following culture in pro-myeloid medium for 14 hours, the majority of P2-hCD4^- CMPs had acquired a CD34^- MEP phenotype (90% of total cells, Fig 3H and 3I). Of these, 71% had downregulated CD150 expression, suggesting erythroid commitment. By contrast, >20% of cultured P2-hCD4⁺ CMPs had acquired an MkP immunophenotype (upregulating CD41), 10% had a CD41^- CD150^- MEP immunophenotype, and 10% expressed CD16/32, thus acquiring a GMP immunophenotype. Runx1-P2-hCD4⁺ CMPs can therefore efficiently produce immunophenotypic MkPs, MEPs and GMPs, whereas P2-hCD4^- CMPs appear committed to the generation of immunophenotypic MEPs.

CD31 expression distinguishes multilineage HPCs from more restricted progenitors

Having established that Runx1-P2-hCD4⁺ MEPs and CMPs are enriched in multilineage progenitors, we endeavored to identify cell surface markers which correlated with P2-hCD4 expression in E14.5 fetal liver. The aim was to achieve the prospective isolation of these populations from wild type (WT) or other transgenic mouse lines in the absence of the Runx1-P2-hCD4 reporter. Firstly, we performed Single Cell RNA Sequencing on CD41^- CD150⁺ P2-hCD4^- and P2-hCD4⁺ MEPs and CMPs. By performing a principal component analysis, we observed a clear transition in transcriptomic activity from the P2- MEP to the P2+ CMP (S4A Fig). In particular, erythroid gene expression (for example, Klf1) was highly upregulated in the P2- MEPs being downregulated with the upregulation of P2-hCD4 and also sharing an inverse relationship with the megakaryocytic transcription factor Fli1 and the granulocyte/monocyte transcription factor Spi1 (Pu.1). (Full lists of differentially expressed genes between P2-/+ MEPs and P2-/+ CMPs can be found in S5 Table and S6 Table respectively.)

In order to identify markers which may aid the isolation of P2-hCD4^- and P2-hCD4⁺ MEPs, we analyzed the expression of various cell surface markers, the aim being to identify genes which are upregulated or downregulated in some or all P2-hCD4⁺ MEPs compared to P2-hCD4^- MEPs. Promising candidates included Cd48, Pecam1 (Cd31), Ptprc (Cd45), Eng (Endoglin), Itgb1 (Cd29) and Tek (Tie2) (S4B Fig). We therefore screened antibodies which had been raised against these markers and other heterogeneously-expressed markers in fetal liver (S4C Fig).

Promising candidates included the leukocyte common antigen, protein tyrosine phosphatase receptor type C (PTPRC) or CD45; CD48 antigen; and platelet/endothelial cell adhesion molecule 1 (PECAM1) or CD31. All three markers correlated positively with Runx1-P2-hCD4 in LK hematopoietic progenitors (Fig 4A). Immunofluorescence staining, performed on total fetal liver sections from P1-GFP::P2-RFP E14.5 embryos, demonstrated that although P2-RFP cells are comparatively rare (2.89% of total DAPI⁺ cells, Fig 4B, S4D Fig) in E14.5 FL, a large proportion (64.4%) have cell surface CD31 staining. We therefore proceeded to characterize CD31 expression in CD41^- CD150⁺ MEPs and CMPs.

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Fig 4. CD31 expression distinguishes multilineage HPCs from more restricted progenitors.

A. Representative FACS plots of CD45, CD31 and CD48 expression in P1-GFP::P2-hCD4/+ E14.5 FL P2-hCD4^- and P2-hCD4⁺ LK cells. B. Representative micrographs of DAPI, P2-RFP, CD31 and P1-GFP Immunofluorescence staining in Runx1-P1-GFP::P2-RFP E14.5 fetal liver. Examples of P1-GFP^- P2-RFP⁺ (white arrows), P1-GFP⁺ P2-RFP⁺ (white arrowheads) and P1-GFP⁺ P2-RFP^- (white asterisks) cells are indicated. C. Representative FACS plots of CD31/CD45 expression in wild type E14.5 fetal liver CD41^- CD150⁺ MEPs and CMPs. D-E. Differential CFU-C activity in wild type E14.5 fetal liver CD31^-/+ MEPs (D, N = 7) and CD31^low/high CMPs (E, N = 6). F-I. Lineage output of OP9 co-cultured MEPs and CMPs. F. Representative FACS plots of TER119 and CD41 expression of day 7 OP9 co-cultured MEPs. G. Representative FACS plots of CD11b, GR1, TER119 and CD41 expression of day 7 OP9 co-cultured CMPs. H. Proportion of TER119⁺ erythroid cells and CD41⁺ megakaryocyte cells in day 7 OP9 MEP co-cultures. I. Proportion of granulocytes/monocytes, erythroid cells and megakaryocytes in day 7 OP9 CMPs co-cultures. N = 8.

https://doi.org/10.1371/journal.pgen.1007127.g004

In wild type fetal liver, 20% of CD41^- CD150⁺ MEPs expressed CD31 (Fig 4C). By comparison, >90% of CD41^- CD150⁺ CMPs were CD31⁺, although CD31^low and CD31^high populations were discernible. We therefore separated CD41^- CD150⁺ CMPs into the 50% lowest and 50% highest CD31-expressing subfractions (CD31^low and CD31^high CMPs respectively) for further characterization.

All MEPs and CMPs expressed CD45 and CD48 (Fig 4C, S4E Fig). Expression of the RUNX1 target genes Gfi1b and Gfi1 in MEPs and CMPs was also assessed, utilizing the Gfi1b-GFP and Gfi1-GFP reporter mouse lines (S4F and S4G Fig). The megakaryocytic/erythroid transcription factor Gfi1b was expressed in all CD41^- CD150⁺ MEPs and CMPs, although the Gfi1b-GFP median fluorescence intensity (MFI) was decreased in some CD31^high CMPs (S4F Fig). In contrast, granulocyte/monocyte-associated Gfi1-GFP was expressed in only 8% of CD41^- CD150⁺ MEPs and 39% of CD41^- CD150⁺ CMPs, the highest Gfi1-GFP MFI being associated with high CD31 expression (S4G Fig).

Assessing the CFU-C activity of bulk and single MEP cells in MethoCult culture (Fig 4D, S4H Fig) revealed that all CFU-Mk and CFU-MkE activity resided with CD31⁺ MEPs; CD31^- MEPs possessed CFUe and BFUe activity only. OP9 co-culture confirmed this lineage output (Fig 4F and 4H; S4I Fig), as CD41⁺ megakaryocyte cells were absent in CD31^- MEP co-cultures but present in 7% of positive wells derived from CD31⁺ MEPs (S4I Fig). This suggested that CD31⁺ MEPs possess some Mk lineage specificity (albeit in only 7–20% of cells), whereas CD31^- MEPs solely constitute erythroid progenitors.

Erythroid lineage output was enriched in CD31^low CMPs compared to CD31^high CMPs (Fig 4E, 4G and 4I; S4J Fig); 20% of CD31^low CMPs produced BFUes and 54% of single co-cultured cells yielded solely TER119⁺ erythroid cells. CD11b⁺ Gr1⁺ granulocyte/monocyte and CD41⁺ megakaryocyte cell output was enhanced considerably in CD31^high CMPs. Interestingly, although CFU-GEMM activity was enriched in CD31^high CMPs, the proportion of positive co-cultured wells yielding megakaryocyte/erythroid/granulocyte/monocyte cells was higher for CD31^low CMPs. This was probably due to the overall plating efficiencies being higher for CD31^high CMPs. Therefore, both CD31^low CMPs and CD31^high CMP populations are heterogeneous, albeit skewed towards erythroid and GM/megakaryocytic specification respectively.

The short-term culture of these populations revealed hierarchical relationships; CD31⁺ MEPs gave rise to CD31^- MEPs and MkPs, whereas CD31^- MEPs rapidly downregulated CD150 (S4K and S4M Fig). The CD31^low CMPs are skewed to a pro-MEP fate, whereas CD31^high CMPs efficiently produce GMPs and CD31⁺ MEPs (S4L and S4N Fig). CD31^low CMPs therefore comprise a more advanced pro-erythroid fraction, but can also produce CD31^high CMPs, suggesting some granulocyte/monocyte commitment.

As previously indicated, the provenance of the fetal liver myeloid progenitors is not entirely clear. Following initial fetal liver colonization at E11.5, the HSCs expand exponentially and the numbers of repopulating units (per embryo equivalent) peak by E16, as fetal bone marrow colonization is underway (having begun from E15) [29,30,44]. To add weight to the hypothesis that the CD150⁺ MEP and CMP populations are fetal liver HSC-derived, we analyzed these fractions in E16.5 fetal liver. Firstly, upon analyzing the LK hematopoietic progenitor compartment of the Runx1 P1-GFP::P2-hCD4 E16.5 fetal liver, we observed that the frequencies of P2^+/- CD41^- CD150⁺ MEPs and CMPs closely resembled that observed in E14.5 fetal liver (S5A and S5B Fig). Upon isolating these populations, we observed that the P2⁺ CD41^- CD150⁺ MEPs and CMPs displayed decreased erythroid and increased megakaryocytic/GM output compared to the P2^- fractions (S5C–S5H Fig). We also confirmed that Runx1 P2-hCD4 expression correlated well with CD31 expression in the E16.5 fetal liver LK fractions (S5I-J) and that wild type E16.5 CD31^+/- MEPs and CD31^low/high CMPs displayed similar lineage specificities, particular concerning erythroid output, compared to their P2^+/- equivalents (S5K-P). This therefore suggests that similar MEP and CMP populations can be discerned in E16.5 fetal liver as in E14.5 fetal liver and therefore that the hematopoietic hierarchy is maintained 5 days after fetal liver colonization, even after the shift to bone marrow colonization has begun.

Runx1-null CD31^low CMPs have impaired erythroid cell production

Defining restricted fetal liver hematopoietic progenitor cell compartments should provide the ability to identify the impact of genetic alterations with greater precision. For example, Runx1-flox::Vav1-Cre conditional knockout mice display impaired fetal liver erythroid and megakaryocytic maturation (S6A–S6H Fig).

The absence of RUNX1 protein in Runx1-flox/flox::Vav1-Cre (Runx1-del/del, S6A Fig) apparently caused a block in the upregulation of TER119 expression. Consequently, there was an accumulation of the immature S0-S2 erythroid lineage subsets, but a decrease in the CD71^high TER119^high S3 population (S6B-E) [45]. Following megakaryocytic culture, Runx1-del/del fetal liver samples produced more CD41^high megakaryocytes than their WT and Runx1-del/+ littermates, but the majority of these did not upregulate CD42d, reflecting the previously described megakaryocytic maturation block (S6F and S6G Fig). This was confirmed by the complete absence of morphologically mature megakaryocytes, following staining with May-Grünwald Giemsa reagent (S6H Fig). This megakaryocyte maturation block is reminiscent of Familial Platelet Disorder with Predisposition to Acute Myeloid Leukemia (FPD/AML), almost exclusively the result of germline heterozygous RUNX1 gene mutations/deletions [46,47].

To further investigate the impact of the absence of RUNX1, we performed a detailed characterization of the hematopoietic progenitor composition of Runx1-del/del E14.5 fetal liver using our CD31 staining protocol and compared it to wild type and Runx1-del/+ heterozygous littermates (Fig 5A–5C, S7A and S7B Fig). CD41^- CD150⁺ CD31^+/- MEP and CD31^low/high CMP populations were all expanded in Runx1-del/del fetal liver, as was the pro-GM CD41^- CD150^- CMP fraction (Fig 5B–5C, S7B Fig). By contrast, pro-erythroid CD41^- CD150^- MEPs were depleted (Fig 5A, S7B Fig), suggesting the absence of RUNX1 may block erythroid differentiation during fetal liver hematopoiesis.

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Fig 5. Runx1-null CD31^low CMPs have impaired erythroid cell production.

A-C. Identifying CD31-expressing CD41^- CD150⁺ MEPs and CMPs in Runx1 wild type, heterozygous (Del/+) and null (Del/Del) E14.5 fetal liver. Representative FACS plots of CD150/CD41 and CD31/CD45 expression in MEPs (A) and CMPs (B). C. Quantitation of CD31^-/+ MEPs and CD31^low/high CMPs. N = 6 D-E. Lineage output of OP9 co-cultured Runx1 wild type, heterozygous and null CD31^low CMPs. D. Representative FACS plots of CD11b, GR1, TER119 and CD41 expression of day 7 OP9 co-cultured CD31^low CMPs. E. Proportion of granulocyte/monocyte, erythroid and megakaryocyte cells in day 7 OP9/CD31^low CMPs co-cultures. N = 4 F-H. Lineage output of single OP9 co-cultured Runx1 wild type, heterozygous and null CD31^low and CD31^high CMPs. F. Plating efficiency of CD31^low and CD31^high CMPs. G. Ternary plots displaying proportions of granulocyte/monocyte, erythroid and megakaryocyte cells in each positive well for CD31^low CMPs. H. Proportions of unilineage (Ery, Mk and GM only) and multilineage (Mk+Ery, Mk+GM, Ery+GM and GEMM) wells derived from CD31^low CMPs. N = 4.

https://doi.org/10.1371/journal.pgen.1007127.g005

Assessment of CFU-C activities (S7C–S7F Fig) uncovered a modest increase in CFUe production by Runx1-del/del CD41^- CD150⁺ CD31⁺ MEPs but decreased CFU-MkE and CFU-GEMM activity in Runx1-del/del CD41^- CD150⁺ CD31^high CMPs. Total CFU-Mk output did not appear to be decreased, but defective megakaryocytic maturation was evident as morphologically mature CFU-Mks were absent from Runx1-del/del hematopoietic progenitor cultures (S7E and S7F Fig).

Bulk OP9 co-culture revealed decreased mature TER119⁺ erythroid cell production by Runx1-del/del CD31^low CMPs and CD31^-/+ MEPs, but not CD31^high CMPs (Fig 5D and 5E, S7G and S7K Fig), confirming an erythroid maturation block. Runx1-del/del CD31^low CMPs had increased CD41⁺ megakaryocytic output, in line with the previously reported increased proliferation/reduced maturation in this lineage [48]. However, this did not answer the question of whether lineage specification was altered by the absence of RUNX1 causing a perturbation of the numbers of pro-erythroid, pro-megakaryocyte and/or pro-granulocyte/monocyte progenitors in hematopoietic progenitor pools.

To address this question, we performed single cell OP9 co-cultures and observed similar total plating efficiencies for wild type, Runx1-del/+ and Runx1-del/del populations (Fig 5F). Whereas >50% of wild type and Runx1-del/+ CD31^low CMPs solely produced TER119⁺ Erythroid cells, this was reduced 4-fold in Runx1-del/del CD31^low CMPs (Fig 5G and 5H, S7L Fig), with a concurrent increase in pro-GM progenitors. Erythroid cells were more modestly reduced in CD31^high CMP cultures (S7M–S7O Fig), but this was not very impactful as the population has a lower pro-erythroid pool in wild type fetal liver compared to its CD31^low counterpart. Moreover, the Median Fluorescent Intensity of the TER119⁺ erythroid cells was decreased in Runx1-del/del CD31^low CMP cultures compared to that of wild type littermates (S7P Fig). This confirmed that the proportion of pro-erythroid progenitors residing in the fetal liver CD31^low CMP fraction is diminished in Runx1-null E14.5 embryos, and that differentiation of these progenitors is impaired.

A key advantage of identifying these highly purified CD31^low and CD31^high CMP fractions in Runx1 null fetal liver is the ability to analyze underlying cell-intrinsic changes driving the lineage specification and maturation defects with greater precision, particularly at a transcriptome level. We therefore analyzed the expression of key HSC and lineage-associated transcriptional regulators in wild type, Runx1-del/+ and Runx1-del/del CD31^low and CD31^high CMPs (S7Q Fig). One key observation was that several key HSC and megakaryocyte/erythrocyte-associated transcription factors (Tal1, Gfi1b and Klf1) were downregulated in wild type CD31^high CMPs compared to wild type CD31^low CMPs, reflecting the megakaryocytic/erythroid lineage commitment which accompanies CD31 downregulation in the CMP compartment and demonstrating that CD31^low and CD31^high CMPs represent transcriptionally distinct progenitor subsets.

Notably, expression of Tal1, Gfi1b and Klf1 did not differ between wild type, Runx1-del/+ and Runx1-del/del CD31^high CMPs. However, they were substantially downregulated in Runx1-del/del CD31^low CMPs compared to their wild type and Runx1-del/+ equivalents. In fact, the transcription factors Tal1, Gfi1b, Klf1 and Gata2, plus the megakaryocytic/erythroid lineage markers Itga2b, Pf4 and Epor (S7R Fig), were all expressed at comparable levels in Runx1-del/del CD31^low CMPs to CD31^high CMPs (of all genotypes). This supports the hypothesis that the absence of RUNX1 results in a differentiation block between the CD31^high and CD31^low CMPs. Additionally, it indicates that the megakaryocytic/erythroid maturation defects are established even at this early stage, with a failure to upregulate key maturation-associated transcripts.

Interestingly, unlike the pro-erythroid transcription factor Klf1, the pro-megakaryocytic Fli1 and the pro-GM Spi1 and Gfi1 were not significantly different in CD31^low and CD31^high CMPs; nor were they impacted by the absence of RUNX1. This may indicate that commitment to the erythroid lineage is the default position in the FL CD31^high-to-CD31^low CMP transition, which was hindered in the absence of RUNX1. We therefore clearly demonstrate the benefits of isolating highly purified myeloid progenitors to aid in the understanding of the mechanistic basis of congenital hematopoietic defects in the fetal liver.

Runx1-P1-MRIPV CD31^high CMPs have impaired Mk specification

We recently reported that deleting the RUNX1C isoform in adult mice, whilst maintaining total RUNX1 expression, resulted in mild thrombocytopenia, due to impaired megakaryocytic specification but with normal megakaryocyte maturation. To achieve this we utilized the Runx1-P1-MRIPV line, in which the P1-encoded RUNX1C isoform is replaced by the P2-encoded RUNX1B isoform [39]. Given the contrast between this phenotype and that of the Runx1-del/del adult model, we decided to interrogate P1-MRIPV fetal liver hematopoiesis.

Having confirmed P1-MRIPV/MRIPV and P1-MRIPV/+ fetal liver cells maintain RUNX1 protein expression at wild type levels (S8A Fig), we examined erythroid and megakaryocytic maturation in these samples (S8B–S8G Fig). Unlike in Runx1-null fetal liver, P1-MRIPV/MRIPV fetal liver displayed only a modest increase in the CD71^-/low TER119^- S0 fraction and corresponding decrease in the CD71^high TER119^high S3 fraction. This suggested that the absence of RUNX1C/overexpression of RUNX1B does not significantly impact erythroid maturation (S8B–S8E Fig). Following megakaryocyte culture, we observed that megakaryocytic maturation was also unimpaired, as the proportions of mature CD41^high CD42d⁺ megakaryocytes were similar in wild type, P1-MRIPV/+ and P1-MRIPV/MRIPV cultures (S8F and S8G Fig).

We next proceeded to characterize fetal liver myeloid hematopoietic progenitor compartments using our CD31 staining protocol (Fig 6A–6C, S9A and S9B Fig). The impact of deleting RUNX1C alone was far more restricted, the CD41^- CD150⁺ CD31^- MEP being the only expanded population identified in P1-MRIPV/MRIPV fetal liver (Fig 6A–6C). As the CD31^- MEP is highly erythroid-biased, we investigated whether P1-MRIPV/MRIPV fetal liver progenitors displayed pro-erythroid/anti-megakaryocytic bias. CFU-C assays revealed increased CFUe activity in the P1-MRIPV/MRIPV CD31⁺ MEP, but decreased CFU-Mk and increased CFU-M activity in the CD31^high CMP (S9C–S9F Fig).

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Fig 6. Runx1-P1-MRIPV CD31^high CMPs have impaired Mk specification.

A-C. CD31-expressing CD41^- CD150⁺ MEPs and CMPs in Runx1 wild type, P1-MRIPV/+ and P1-MRIPV/MRIPV E14.5 fetal liver. Representative FACS plots of CD150/CD41 and CD31/CD45 expression in MEPs (A) and CMPs (B). C. Quantitation of CD31^-/+ MEPs and CD31^low/high CMPs. N = 7 D-G. Lineage output of OP9 co-cultured Runx1 wild type, P1-MRIPV/+ and P1-MRIPV/MRIPV CD31^low and CD31^high CMPs. D-E. Representative FACS plots of CD11b/GR1 and TER119/CD41 expression of day 7 OP9 co-cultured CD31^low (D) and CD31^high CMPs (E). F-G. Proportions of granulocyte/monocyte, erythroid and megakaryocytic cells in day 7 OP9 co-cultured CD31^low (F) and CD31^high (G) CMPs. N = 5.

https://doi.org/10.1371/journal.pgen.1007127.g006

OP9 co-culture suggested the absence of RUNX1C did not impair the lineage output of CD31^- or CD31⁺ MEPs (S9G–S9I Fig). Whilst the P1-MRIPV/MRIPV CD31^low CMP was modestly affected, substantially decreased CD41⁺ megakaryocyte output and increased CD11b⁺ GR1⁺ granulocyte/monocyte output were observed for P1-MRIPV/MRIPV CD31^high CMPs (Fig 6D–6G). We therefore hypothesized that the dominant impact of deleting RUNX1C (or overexpressing RUNX1B) on megakaryocytic versus granulocyte/monocyte specification occurs in the CD31^high CMP fraction.

This hypothesis was confirmed through short-term culture of the CMP populations (S9J–S9M Fig), as P1-MRIPV/MRIPV CD31^high CMPs produced fewer MkPs and more GMPs than their wild type counterparts, but the lineage specification of CD31^low CMPs was largely unaffected. Therefore, in contrast to the impaired erythroid specification of Runx1-del/del CD31^low CMPs, P1-MRIPV/MRIPV CD31^high CMPs display impaired megakaryocytic specification. This demonstrates that the RUNX1B and RUNX1C isoforms have distinct roles during fetal liver hematopoiesis, akin to adult bone marrow hematopoiesis, which can be uncovered using our enhanced fetal liver HPC purification strategies.

Animal husbandry, timed matings and tissue collection

Runx1-P1-GFP::P2-hCD4, Runx1-P1-MRIPV, Runx1-flox::Vav1-Cre, Gf1i-GFP and Gfi1b-GFP mice have been described [17,38,39,84,85]. Dual-reporter Runx1-P1-GFP::P2-RFP chimeric mouse lines were generated by transfecting P1-GFP embryonic stem cells (ESCs) [17] with a P2-RFP targeting construct [86,87], screening for heterozygote knock-in lines targeting the same allele, and injecting correctly targeted ESCs into C57BL6J blastocysts.

Runx1 P1-GFP::P2-hCD4, P1-GFP::P2-RFP, P1-MRIPV, Runx1 flox::Vav1-Cre, Gfi1-GFP and Gfi1b-GFP mice were backcrossed with C57BL/6 mice for at least 10 generations and were housed in specific pathogen free cages with environmental enrichment. To trace Runx1 promoter activity, timed matings were set up between Runx1 P1-GFP::P2-hCD4 or P1-GFP::P2-RFP male mice and wild type (WT) ICR female mice. To produce Runx1 P1-MRIPV/MRIPV or Runx1 flox/flox::Vav1-Cre embryos (plus wild type and heterozygous control littermates), timed matings were set up between Runx1 P1-MRIPV/+ mice or between Runx1 flox/+::Vav1-Cre males and Runx1 flox/+ females. To trace Gfi1 and Gfi1b expression, timed matings were set up between Gfi1-GFP or Gfi1b-GFP male mice and WT ICR female mice. Embryos were harvested on embryonic day (E) 12.5, 13.5, 14.5 or 16.5 and the fetal livers dissected for flow cytometric or histological analysis. For yolk sac experiments, embryos were harvested on E9.5 and the yolk sacs dissected for flow cytometric analysis, or for explant or pro-myeloid culture (as described below). Approximately 10⁴ cells were kept for genotyping by PCR (oligonucleotides listed in S1 Table).

All animal work was performed under regulations governed by UK Home Office Legislation under the Animals (Scientific Procedures) Act 1986 and was approved by the Animal Welfare and Ethics Review Body of the Cancer Research UK Manchester Institute.

Fluorescence Activated Cell Sorting (FACS)

Details of FACS reagents and combinations used for each analysis are listed in S2 and S3 Tables. Prior to flow sorting or analysis of HPCs in E12.5, E13.5, E14.5 or E16.5 fetal liver, red blood cell lysis was performed as described [18]. Dead cells were excluded using 1μg/ml Hoechst 33258 (ThermoFisher Scientific); gates were positioned based on Full Minus One controls. Cells were analyzed using a LSR-II or LSR-II Fortessa analyzer (BD Biosciences) or a NovoCyte (ACEA Biosciences Inc.). Cells were sorted using a FACSAria-II, FACSAria-III or Influx cell sorter (BD Biosciences).

Cell culture

Cytospin analysis

Up to 50,000 cultured fetal liver cells were suspended in 150μl PBS and immobilized on twin frosted glass microscope slides (Fisher Scientific) by cytospin at 200rpm, low acceleration for 5 minutes in a Shandon Cytospin3 (Thermo Scientific). Air-dried slides were submerged in May-Grünwald Eosin methylene blue Q Path stain (VWR) for 3 minutes, rinsed in tap water and submerged in 5% Giemsa’s stain (VWR) for 20 minutes. Slides were subsequently air dried, mounted and scanned using the Pannoramic 250 Flash III (3DHISTECH). Images were acquired with the Pannoramic 250 software and analyzed with the Pannoramic Viewer software (3DHISTECH).

Immunofluorescent staining and imaging of fetal liver sections

Dissected fetal livers were fixed in 4% Paraformaldehyde (PFA) overnight, before they were soaked in 30% sucrose and mounted in OCT compound. 10μm sections were prepared using a cryostat. The sections were incubated in blocking buffer (PBS with 10% FBS, 0.05% Tween20 and 10% goat serum (DAKO)) for 1 hour before the sections were incubated with primary antibodies at 4°C overnight in blocking buffer.

Primary antibodies used in this study were rabbit anti-GFP (598, polyclonal, MBL) (1/200); and purified rat anti-mouse CD31 (553370, MEC13.3, BD Biosciences) (1/100).

Sections were washed three times in PBST (PBS with 0.05% Tween20) for 15 minutes each and then incubated with fluorochrome-conjugated secondary antibody at room temperature for 1 hour.

Secondary antibodies used in this study include Alexa Fluor 488 Goat Anti-Rat IgG (A11006, Life Technologies); and Alexa Fluor 647 F(ab')₂ Fragment of Goat Anti-Rabbit IgG (H+L) (A21246, Life Technologies). All secondary antibodies were used at 1/400 dilution.

Sections were further washed three times in PBS and mounted using Prolong Gold anti-fade medium with DAPI (Life Technologies). Images (of Alexa Fluor 488, Alexa Fluor 647, DAPI and endogenous RFP) were taken using a low-light time lapse microscope (Leica) using the Metamorph imaging software and processed using ImageJ.

Western blot analysis

Whole cell protein extracts, purified using RIPA lysis buffer, were quantitated by the Bradford assay (Protein Assay Dye Reagent, Bio-Rad) on the Glomax Multi Detection System (Promega) and loaded alongside the SeeBlue Plus2 Prestained Standard (ThermoFisher Scientific) for electrophoretic separation on NuPAGE 4–12% Bis-Tris gels using the Novex Mini Cell system (ThermoFisher Scientific). Protein transfer was performed to nitrocellulose membranes using the iBlot system (ThermoFisher Scientific) and membranes were probed with anti-RUNX (EPR3099, Abcam) and anti-beta-actin (AC-15, Sigma-Aldrich) primary antibodies, followed by Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse (ThermoFisher Scientific) secondary antibodies respectively, in the iBind Western System (ThermoFisher Scientific). HRP activity was detected using Amersham ECL Prime Western Blotting Detection Reagent, imaged using the BioRad ChemiDoc Touch Imaging System and analyzed with BioRad Image Lab Software Version 6.

Gene expression by quantitative PCR

RNA was extracted using the RNeasy Plus Micro Kit (QIAGEN) and complementary DNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Quantitative PCR (qPCR) was performed using Universal ProbeLibrary assays (Roche); primers and probes are listed in S4 Table. Expression values were normalized to beta-actin (Actb).

Single cell RNA sequencing

E14.5 P1-GFP::P2-hCD4/+ fetal livers were prepared for flow sorting as described above. Single P2-hCD4^-/+ MEPs and CMPs were sorted into 384-well plates containing lysis buffer and snap frozen. Libraries were prepared using a modified version of the Smart-Seq2 protocol [89]. Briefly, cDNA was prepared using a Mantis platform (Formulatrix) and quantified with quantIT picogreen reagent (Thermo Fisher). Dual indexed sequencing libraries were prepared from 0.1ng cDNA using an Echo525 automation system (Labcyte) in miniaturized reaction volumes. The library pool was quantified by qPCR using a Library Quantification Kit for Illumina sequencing platforms (Kapa Biosystems). Paired-end 75bp sequencing was carried out by clustering 1.5pM of the library pool on a NextSeq 500 sequencer (Illumina).

Base call files generated from the NextSeq 500 sequencing run were converted to the FASTQ format with the bcl2fast converter (Illumina). All FASTQ files corresponding to the same sample (derived from separately sequenced lanes) were then merged into a single FASTQ file (one per sample). Read trimming was performed with trimmomatic (v0.36) with the following settings “CROP:75 HEADCROP:5 SLIDINGWINDOW:20:20 MINLEN:36”. The mouse reference genome GRCm38 (version M12, Ensembl release 87) and the ERCC reference sequence (Thermo Fisher) were combined and used as the reference genome for sequencing alignment, performed using STAR (version 2.4.2a) [90]. The expression levels of 49,585 features annotated in the GENCODE mouse genome and 92 ERCC features were determined using HTSeq (version 0.6.1p1) [91]. The following parameters were specified for the HTSeq quantification: ‘—format = bam–stranded = no–type = exon’.

Single cell count data were loaded into the R environment (R version 3.4.0) as a SCESet object using the Scater package (version 1.4.0) [92]. Normalized gene expression values were taken from the default normalization performed by scater. Cells with fewer than 250,000 sequencing reads, more than 25% unmapped reads, and more than 15% ERCC content were removed. Low abundance genes (mean count <1) were excluded, as were overrepresented genes (>20% of total sequencing reads). Differentially expressed genes were identified using DESeq2 (version 1.16.1, Bioconductor). Prior to differential expression analysis, the data were filtered to remove genes with a dropout rate of higher than 75%; differential expression analysis was then performed using the function “DESeqDataSetFromMatrix” and by specifying the contrast of interest. The full scripts for the analysis of these data are available at https://github.com/m-zaki/CRUKMI_github/tree/master/JuliaDraper_PLOSgenetics.

The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus [93,94] and are accessible through GEO series accession number GSE107653.

Statistical analysis

For FACS purified populations (MEPs, CMPs) a sample size of n = 1 refers to tissues pooled from embryos from one litter. For total fetal liver analyses (Western blot, total FL culture), a sample size of n = 1 refers to one embryo.

Data were evaluated using an Ordinary 2-way ANOVA and expressed as mean ± standard error of the mean (SEM). P<0.05 was considered statistically significant.

*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001