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Independent origins of fetal liver haematopoietic stem and progenitor cells

Nature volume 609, pages 779–784 (2022)Cite this article

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Abstract

Self-renewal and differentiation are tightly controlled to maintain haematopoietic stem cell (HSC) homeostasis in the adult bone marrow1,2. During fetal development, expansion of HSCs (self-renewal) and production of differentiated haematopoietic cells (differentiation) are both required to sustain the haematopoietic system for body growth3,4. However, it remains unclear how these two seemingly opposing tasks are accomplished within the short embryonic period. Here we used in vivo genetic tracing in mice to analyse the formation of HSCs and progenitors from intra-arterial haematopoietic clusters, which contain HSC precursors and express the transcription factor hepatic leukaemia factor (HLF). Through kinetic study, we observed the simultaneous formation of HSCs and defined progenitors—previously regarded as descendants of HSCs5—from the HLF+ precursor population, followed by prompt formation of the hierarchical haematopoietic population structure in the fetal liver in an HSC-independent manner. The transcription factor EVI1 is heterogeneously expressed within the precursor population, with EVI1hi cells being predominantly localized to intra-embryonic arteries and preferentially giving rise to HSCs. By genetically manipulating EVI1 expression, we were able to alter HSC and progenitor output from precursors in vivo. Using fate tracking, we also demonstrated that fetal HSCs are slowly used to produce short-term HSCs at late gestation. These data suggest that fetal HSCs minimally contribute to the generation of progenitors and functional blood cells before birth. Stem cell-independent pathways during development thus offer a rational strategy for the rapid and simultaneous growth of tissues and stem cell pools.

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Fig. 1: Fate tracing of HCCs reveals HSC-independent progenitor formation from pre-HSPCs.
Fig. 2: Intra-embryonic EVI1hi cells preferentially generate HSCs.
Fig. 3: EVI1 expression level determines the fate of pre-HSPCs.
Fig. 4: Fetal HSCs minimally contribute to progenitors and mature cells.

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Data availability

All RNA-seq data were deposited in the Gene Expression Omnibus under accessions GSE167932 (E10.5 and E11.5 scRNA-seq), GSE168054 (E10.5 bulk RNA-seq) and GSE190011 (E14.5 bulk RNA-seq). Source data are provided with this paper.

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Acknowledgements

We thank E. Dzierzak for critical comments on this manuscript; and T. Umemoto, M. Kataoka and R. Koitabashi for assistance with RNA sequencing. This work was supported by JSPS Kakenhi Grant (20K08758, JP16H0627 (AdAMS) to T.Y. and 26221309 to T. Suda), SENSHIN Medical Research Foundation (to T.Y.), Takeda Science Foundation (to T.Y.), Japanese Society of Hematology (to T.Y.), the Sumitomo Foundation (to T.Y.), the National Medical Research Council grant of Singapore Translational Research Investigator Award (NMRC/STaR/0019/2014 to T. Suda) and the programme of the Inter-University Research Network for High Depth Omics, Institute of Molecular Embryology and Genetics, Kumamoto University (to S.M.-K. and M. Ogawa).

Author information

Authors and Affiliations

  1. International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan

    Tomomasa Yokomizo, Takako Ideue, Motomi Osato & Toshio Suda

  2. Microscopic and Developmental Anatomy, Tokyo Women’s Medical University, Tokyo, Japan

    Tomomasa Yokomizo

  3. Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan

    Saori Morino-Koga & Minetaro Ogawa

  4. Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore

    Cheng Yong Tham, Motomi Osato & Toshio Suda

  5. Department of Hematology and Oncology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Tomohiko Sato & Mineo Kurokawa

  6. Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan

    Naoki Takeda & Kimi Araki

  7. Department of Anatomy, Keio University School of Medicine, Tokyo, Japan

    Yoshiaki Kubota

  8. Department of Hematology, Juntendo University Graduate School of Medicine, Tokyo, Japan

    Norio Komatsu

  9. Center for Metabolic Regulation of Healthy Aging, Kumamoto University, Kumamoto, Japan

    Kimi Araki

  10. Department of Paediatrics, National University of Singapore, Singapore, Singapore

    Motomi Osato

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  1. Tomomasa Yokomizo
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Contributions

T.Y. conceived the project, designed and performed the research, and wrote the manuscript. T.I. made the targeting constructs and performed whole-mount immunostaining. S.M.-K. and M. Ogawa performed the scRNA-seq analysis. C.Y.T. and M. Osato performed the bioinformatic analysis. N.T. and K.A. generated mouse lines. T. Sato, Y.K., M.K. and N.K. contributed analytical tools. T.Y., M. Osato and T. Suda participated in project planning.

Corresponding authors

Correspondence to Tomomasa Yokomizo or Toshio Suda.

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Extended data figures and tables

Extended Data Fig. 1 Hematopoietic stem/progenitor cells in the late gestational fetal liver.

a, Disappearance of EMPs at mid-late gestation. Since Hlf is not expressed in EMPs (red rectangle), EMPs and other progenitors can be distinguished by HlftdTomato reporter23. Top right, Frequency of Hlf-tdTomato+ cells in c-Kithi cells (E11.5, n = 6; E14.5, n = 6; E18.5, n = 5; adult, n = 3). b, Waves of hematopoietic progenitors in the fetal liver. Schematic is drawn based on previous reports24,60,61 and our data (a). At E14.5, most c-Kit+ progenitors in the fetal liver are classical HSC-dependent progenitors. c, Representative flow cytometry plots and hematopoietic stem/progenitor cell hierarchy in the bone marrow. Circle size represents the population size of the defined fractions. d, Representative flow cytometry plots and hematopoietic stem/progenitor cell hierarchy in the fetal liver (E14.5). Relative population size of progenitor fractions is similar to that of bone marrow progenitors. The hematopoietic stem/progenitor cell hierarchy in the late gestational fetal liver is believed to be established through the differentiation of fetal HSCs (arrows in the hierarchy) in the standard model. e, Transplantation experiments. Irradiated mice were transplanted with 100 donor cells (HSC, ST-HSC, or MPP) sorted from E14.5 fetal liver. n = 10.

Source data

Extended Data Fig. 2 Generation and characterization of HlfCreERT2 mice.

a, Experimental design for precursor labeling by CreERT2 lines. 1; Labeling of endothelial cells (e.g. Runx1-CreERT2 and VEcad-CreERT2). Labeled endothelial cells may transform into hematopoietic cells at a later time, leading to the mixture of cells at various stage of maturation. 2; Labeling of hematopoietic clusters. EHT, endothelial-to-hematopoietic transition. b, Whole-mount immunostaining analysis of E10.5 (35 sp) HlftdTomato embryos for tdTomato (red), c-Kit (green) and CD31 (white). Scale bar, 100 μm. c, Whole-mount immunostaining analysis of E10.5 (33 sp) HlftdTomato yolk sac for tdTomato (red), c-Kit (green) and CD31 (white). Scale bar, 50 μm. d, Targeting strategy of the HlfCreERT2 mouse. e, White blood cell (WBC), red blood cell (RBC), and platelet (PLT) fractions in the blood of adult Hlf+/+ and HlfCreERT2/+ mice (Hlf+/+ mice, n = 7; HlfCreERT2/+ mice, n = 9). f, Bone marrow (BM) cellularity (Hlf+/+ mice, n = 5; HlfCreERT2/+ mice, n = 6). g, HSC and progenitor fractions in HlfCreERT2/+ mice (Hlf+/+ mice, n = 4; HlfCreERT2/+ mice, n = 5). h, Whole-mount immunostaining analysis of E10.75 (tamoxifen at E10.0) HlfCreERT2ROSAtdTomato embryos. Scale bars, 50 μm. i, Frequency of tdTomato+ cells in c-Kit+ cells (Dorsal aorta, n = 6; Yolk sac, n = 3; Vitelline artery, n = 6; Umbilical artery, n = 6). j, HSC analysis. HlfCreERT2ROSAtdTomato embryos were administered with tamoxifen at E10.5 and were analyzed at E14.5. k, EMP analysis. HlfCreERT2ROSAtdTomato embryos were administered with tamoxifen at E8.0 and were analyzed at E10.5. l, Contribution of HlfCreEERT2-labeled cells to adult hematopoiesis. HlfCreERT2ROSAtdTomato embryos were administered with tamoxifen at E9.75. n = 5. m, Lympho-myeloid progenitor (LMP) analysis. HlfCreERT2ROSAtdTomato embryos were administered with tamoxifen at E9.75 and were analyzed at E11.5. n = 8. All error bars represent means ± SD. Statistical analysis was performed using two-sided unpaired Student’s t-test (e–g).

Source data

Extended Data Fig. 3 Fate tracing of Hlf+ cells.

a, Phenotypic comparison between E10.5 Hlf+c-Kit+ hematopoietic cluster cells and HlfCreERT2 -labeled cells in E11.5 fetal livers (tamoxifen at E9.75). b, Representative flow cytometry plots of HlfCreERT2-labeled cells in the AGM and yolk sac (red dots). c, Representative flow cytometry plots of tdTomato− fraction in the fetal liver and AGM of HlfCreERT2ROSAtdTomato embryos. d, Top, Schematic of tamoxifen treatment and analysis. Middle, Lineage tracing of Hlf+ cells. HlfCreERT2ROSAtdTomato embryos were administered with tamoxifen at various stages (E9.0–11.5) and were analyzed at E14.5. Each graph represents data from one litter. The highest frequency population is shaded (KL, gray; MPP, blue; HSC, orange). Bottom, Proportion of dominant-cell types.

Source data

Extended Data Fig. 4 Heterogeneity within pre-HSPCs.

a, Schematic of single-cell (sc)RNA-seq analysis. Middle segment of the dorsal aorta region and a lobe of the fetal liver were dissected to generate single cells from E10.5 and E11.5 embryos. b, Uniform manifold approximation and projection (UMAP) visualization of 13,193 cells isolated from the dorsal aorta and its surrounding tissues, including the fetal liver. Combined data from E10.5 (6,608 cells) and E11.5 (6,585 cells) are plotted. PGC, primordial germ cell; SMC, smooth muscle cell; Im, intermediate mesoderm. c, UMAP visualization of hematopoietic/endothelial clusters colored by representative lineage-specific genes (EC, endothelial cells; Ery, erythroid lineage; Meg, megakaryocytic lineage; My, myeloid lineage). d, Identification of Hlfhic-Kithi pre-HSPC population. e, UMAP visualization of stem-associated genes. f, UMAP visualization of committed hematopoietic marker genes. g, Hierarchical clustering of the Hlfhic-Kithi pre-HSPC population from E10.5 and E11.5 (marked by black circles) embryos. h, Identification of pre-HSPCs (Hlf+c-Kit+) in CD31+c-KithiGata2med AGM single cells. SPRING visualization of selected genes from the interactive website (https://gottgens-lab.stemcells.cam.ac.uk/DZIERZAK/). i, HSC score. j, SPRING visualization of stem-associated genes. k, SPRING visualization of committed hematopoietic marker genes. l, Hierarchical clustering of the pre-HSPC population. Hlf+c-Kit+ cells (n = 52) were extracted from the Vink et al. dataset (GSE143637). m, Hierarchical clustering of the pre-HSPC population. Hlf+c-Kit+ cells (n = 27) were extracted from the Fadlullah et al. dataset (GSE150412).

Source data

Extended Data Fig. 5 Evi1 expression in E10.5-11.5 embryos.

a, b, Whole-mount immunostaining analysis of E10.5 (36 sp) Evi1GFP embryos for GFP (green) and CD31 (white). a, Representative immunofluorescent images of the embryo proper and yolk sac. White arrows indicate the entrance of the yolk sac artery. GFPlo cells are detected around this region. b, Representative immunofluorescent images of the umbilical artery, vitelline artery, and umbilical vein. Orange arrows indicate hematopoietic clusters. White arrows indicate the walls of the umbilical vein. Scale bars, 50 μm. DA, dorsal aorta; VA, vitelline artery; UA, umbilical artery; UV, umbilical vein. c, Flow cytometry analysis of Evi1-GFP expression in Evi1GFPHlftdTomato embryos. Left, Representative flow cytometry plots of the E11.5 Evi1GFP/+HlftdTomato/+ AGM region. Right, Mean fluorescence intensity (MFI) of Evi1-GFP in the HCC fraction of the AGM region (E10.5, n = 3; E11.5, n = 4). HCC, hematopoietic cluster cell. All error bars represent means ± SD. Statistical analysis was performed using two-sided unpaired Student’s t-test (c).

Source data

Extended Data Fig. 6 Fate tracing of Evi1hi cells.

a, Targeting strategy of the Evi1CreERT2 mouse. b, Fetal liver (FL) cellularity (Evi1+/+ mice, n = 7; Evi1CreERT2/+ mice, n = 10). c, HSC and progenitor fractions in Evi1CreERT2/+ (Evi1+/+ mice, n = 7; Evi1CreERT2/+ mice, n = 10; Evi1GFP/+ mice, n = 5). See Supplementary Discussion for HSC reduction in Evi1CreERT2 and Evi1GFP embryos. d, Labeling of Evi1-GFPhi cells by Evi1CreERT2. Evi1CreERT2 mice were crossed with Evi1GFPROSAtdTomato mice to obtain Evi1CreERT2/GFPROSAtdTomato embryos (tamoxifen at E10.5). n = 6. e, Whole-mount immunostaining analysis of E11.25 (tamoxifen at E10.5) Evi1CreERT2ROSAtdTomato embryos. Labeled endothelial cells and hematopoietic clusters are observed in the dorsal aorta. Scale bars, 100 μm. f, Whole-mount immunostaining analysis of E11.25 (tamoxifen at E10.5) Evi1CreERT2ROSAtdTomato embryos. In contrast to the vitelline and umbilical arteries, tdTomato+c-Kit+ cells are rarely observed in the yolk sac or fetal liver (orange arrows). Scale bars, 100 μm. g, Lineage tracing of Evi1+ cells. Evi1CreERT2ROSAtdTomato embryos were administered with tamoxifen at various stages (E8.5 and E9.5) and were analyzed at E14.5 (E8.5, n = 4; E9.5, n = 4). h, Whole-mount immunostaining analysis of E9.5 (tamoxifen at E8.5) Evi1CreERT2ROSAtdTomato embryo. Flat-shaped endothelial cells are labeled. Scale bar, 200 μm. All error bars represent means ± SD. Statistical analysis was performed two-sided unpaired Student’s t-test (b and c).

Source data

Extended Data Fig. 7 Evi1extreme hi cells preferentially generate HSCs.

a, Gene set enrichment analysis (GSEA) of an Evi1+ subset (Hlf+c-Kit+CD45− cells from caudal half region) compared with an Evi1lo/− subset (Hlf+c-Kit+CD45− cells from yolk sac) for HSC, MPP, and HSPC signatures. NES, normalized enrichment score; FDR, false discovery rate. b, Experimental design to trace the fate of Evi1extreme hi cells and Evi1hi cells within the same mouse (Evi1CreERT2ROSAtdTomato/YFP dual-reporter embryo). Given that the recombination rate of the ROSA26 locus is correlated with the expression level of Evi1 (Extended Data Fig. 6d), it can be inferred that tdTomato+YFP+ cells (recombination of two ROSA26 loci) originate from extreme Evi1 expressors. c, Lineage tracing of Evi1extreme hi (tdTomato+YFP+) and Evi1hi (tdTomato+YFP− or tdTomato−YFP+) cells. Evi1CreERT2ROSAtdTomato/YFP embryos were administered with tamoxifen at E10.5 and were analyzed at E14.5. Top, Representative flow cytometry plots of E14.5 Evi1CreERT2ROSAtdTomato/YFP fetal liver. Bottom, Comparison of cell labeling between tdTomato+YFP+ cells (Evi1extreme hi-derived) and tdTomato+YFP− or tdTomato−YFP+ cells (Evi1hi-derived). n = 7. d, Models and expected outputs of tracing experiments. In the standard model, label frequencies of progenitors would be similar to that of HSCs. In the HSC-independent model, label frequencies of progenitors would be significantly lower than that of HSCs. e, Number of Hlf+c-Kit+ cells in E10.5 (32–36 sp) HlftdTomato embryos (AGM, n = 3; yolk sac, n = 4). Number was calculated from flow cytometry data. f, Number of KSL (HSCs, ST-HSCs, MPPs) cells and KL (CMPs, GMPs, MEPs) cells in E14.5 fetal liver. n = 8. Number was calculated from flow cytometry data. g, Heterogeneity of Hlf+ hematopoietic clusters in the embryo. Whole-mount immunostaining analysis of E10.5 (32 sp) Evi1GFPHlftdTomato embryo for tdTomato (red), GFP (green) and CD31 (white). Scale bar, 50 μm. All error bars represent means ± SD. Statistical analysis was performed two-sided unpaired Student’s t-test (c).

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Extended Data Fig. 8 Characterization of Evi1+/− embryos.

a, Analysis of E12.5 Evi1+/− embryos. Left, Representative images of E12.5 embryos. Scale bars, 200 μm. Right, Quantitation of c-Kit+ cells in the fetal liver (Evi1+/+ mice, n = 5 ; Evi1+/− mice, n = 7). b, Normal hematopoietic cluster formation in Evi1+/− embryos. Left, Whole-mount immunostaining of Evi1+/+ (34 sp) and Evi1+/− (33 sp) embryos for c-Kit (green) and CD31 (magenta) expression. Scale bars, 100 μm. Right, Number of c-Kit+ cells localized in the middle segment of the dorsal aorta (DA). Middle segment is 7 somite-lengths18. Evi1+/+ (n = 3, 33–35 sp). Evi1+/− (n = 3, 33 and 34 sp). c, Specific decrease in stem cell fraction in Evi1 heterozygous embryos at E14.5. Left, Representative flow cytometry plots. Right, Frequency of HSC and progenitor fractions (Evi1+/+ mice, n = 8 ; Evi1+/− mice, n = 6). Similar to the results at the E12.5 stage (Fig. 3b), severe defects were observed in the HSC fractions (15-fold decrease) in Evi1+/− embryos at this time point. d, Kinetic analysis of HSC and progenitor formation from HlfCreERT2-labeled cells in Evi1+/− embryos. Top left, Schematic of tamoxifen treatment and analysis. Bottom left, Representative flow cytometry plots of HlfCreERT2-labeled cells (red dots). Right, Quantitation of tdTomato+ cells in the fetal liver (E11.5 Evi1+/+, n = 6; E12.5, Evi1+/+ n = 5; E11.5, Evi1+/− n = 5; E12.5 Evi1+/−, n = 5). All error bars represent means ± SD. Statistical analysis was performed two-sided unpaired Student’s t-test (a–c).

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Extended Data Fig. 9 Ectopic expression of Evi1.

a, Targeting strategy of the ROSAEvi1-IRES-GFP mouse. b, Schematic of tamoxifen treatment and analysis. c, Ectopic expression of Evi1 in VE-cadherin+ cells. Left, Representative flow cytometry plots. Right, Frequency of HSC and progenitor fractions (control, VEcad-CreERT2 embryos, n = 8; VEcad-CreERT2::ROSAEvi1-IRES-GFP embryos, n = 7). d, Fate tracing of Evi1-induced VE-cadherin+ cells. Left, Representative flow cytometry plots of E14.5 fetal liver cells from a VEcad-CreERT2::ROSAtdTomato embryo (control) and a VEcad-CreERT2::ROSAEvi1-IRES-GFP embryo. Right, Frequency of HSC and KSL in E14.5 fetal livers (VEcad-CreERT2::ROSAtdTomato embryos, n = 7; VEcad-CreERT2::ROSAEvi1-IRES-GFP embryos, n = 7). e–h, Characterization of HSCs in Tie2-Cre::ROSAEvi1-IRES-GFP mice. e, Flow cytometry analysis of EPCR and CD86 expression. f, Transcriptome analysis. Left, Principal component analysis (PCA). Right, Expression of HSC-related genes, presented as transcripts per kilobase million (TPM). g, Transplantation experiments. Irradiated mice were transplanted with 100 GFP+ HSCs isolated from E14.5 Tie2-Cre::ROSAEvi1-IRES-GFP fetal liver (n = 6). Donor chimerism of myeloid, B cells, and T cells in peripheral blood was analyzed by the frequency of GFP+ cells. Chimerism of CD45+ cells is shown in Fig. 3e. h, Contribution of Evi1-induced cells (GFP+ cells) to adult hematopoiesis. Although the majority (about 70%) of Tie2-Cre::ROSAEvi1-IRES-GFP mice died soon after birth for unknown reasons, the peripheral blood from the five surviving mice was analyzed (6-month-old, n = 2; 8-month old, n = 2; 1-year-old, n = 1). i, Targeting strategy of the ROSACAG-Evi1-IRES-GFP mouse. j, Frequency of GFP+ cells in fetal liver cells from HlfCreERT2ROSAEvi1-IRES-GFP (n = 8) and HlfCreERT2ROSACAG-Evi1-IRES-GFP (n = 5) embryos. Embryos were administered with tamoxifen at E9.75 and were analyzed at E14.5. All error bars represent means ± SD. Statistical analysis was performed two-sided unpaired Student’s t-test (c,d and j) and one-way ANOVA with Tukey-Kramer test (e and f).

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Extended Data Fig. 10 Decrease in ST-HSC at late gestation.

a, Analysis of the KSL fraction. Top, Representative flow cytometry plots of the KSL fraction in E14.5 fetal livers (FL), E18.5 FL and 1-week bone marrow (BM). Middle, Comparison of HSPC fractions between E14.5 and E18.5. Number of HSCs, ST-HSCs, MPP, and c-Kit+Lin− cells. HSPC, hematopoietic stem and progenitor cells; KSL, c-Kit+Sca-1+Lineage−. Bottom, Frequency of HSC and progenitors (E14.5 embryos, n = 6; E18.5 embryos, n = 5; 1w mice, n = 6). b, Representative images of E18.5 embryos. Scale bars, 300 μm. All error bars represent means ± SD. Statistical analysis was performed two-sided unpaired Student’s t-test (a).

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Supplementary information

Supplementary Discussion

Discussion on Evi1creERT2 tracing.

Reporting Summary

Supplementary Figure 1

Gating strategies to sort nascent haematopoietic cluster cells and GFP+ HSCs. a, b, Gating strategy to sort nascent haematopoietic cluster cells (Hlf+c-Kit+CD45- cells) from E10.5 HlftdTomato embryo proper and yolk sac presented on Fig. 2a. c, Gating strategy to sort GFP+ HSCs (GFP+CD150+CD48-LSK cells) from E14.5 Tie2-Cre::ROSAEvi1-IRES-GFP embryos presented on Fig. 3e.

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Supplementary Table 1

Markers for cell type identification in scRNA-seq (Extended Data Fig. 4b). Cell type-specific marker genes (2–5 genes) were obtained from literatures.

Supplementary Table 2

Sample size information for whole-mount immunostaining. Representative micrographs from at least two biological replicates are shown in Figures and Extended Data Figures.

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Yokomizo, T., Ideue, T., Morino-Koga, S. et al. Independent origins of fetal liver haematopoietic stem and progenitor cells. Nature 609, 779–784 (2022). https://doi.org/10.1038/s41586-022-05203-0

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