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Foxc1 is a critical regulator of haematopoietic stem/progenitor cell niche formation

Abstract

Haematopoietic stem and progenitor cells are maintained by special microenvironments known as niches in bone marrow1,2,3,4,5,6. Many studies have identified diverse candidate cells that constitute niches for haematopoietic stem cells in the marrow, including osteoblasts7,8,9,10, endothelial cells11,12,13,14, Schwann cells15, α-smooth muscle actin-expressing macrophages16 and mesenchymal progenitors such as CXC chemokine ligand (CXCL)12-abundant reticular (CAR) cells17,18, stem cell factor-expressing cells13, nestin-expressing cells19 and platelet-derived growth factor receptor-α (PDGFR-α)+Sca-1+CD45Ter119 (PαS) cells20. However, the molecular basis of the formation of the niches remains unclear. Here we find that the transcription factor Foxc1 is preferentially expressed in the adipo-osteogenic progenitor CAR cells essential for haematopoietic stem and progenitor cell maintenance in vivo5,13,18 in the developing and adult bone marrow. When Foxc1 was deleted in all marrow mesenchymal cells or CAR cells, from embryogenesis onwards, osteoblasts appeared normal, but haematopoietic stem and progenitor cells were markedly reduced and marrow cavities were occupied by adipocytes (yellow adipose marrow) with reduced CAR cells. Inducible deletion of Foxc1 in adult mice depleted haematopoietic stem and progenitor cells and reduced CXCL12 and stem cell factor expression in CAR cells but did not induce a change to yellow marrow. These data suggest a role for Foxc1 in inhibiting adipogenic processes in CAR progenitors. Foxc1 might also promote CAR cell development, upregulating CXCL12 and stem cell factor expression. This study identifies Foxc1 as a specific transcriptional regulator essential for development and maintenance of the mesenchymal niches for haematopoietic stem and progenitor cells.

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Figure 1: Foxc1 is preferentially expressed in CAR cells.
Figure 2: Foxc1 deletion results in yellow marrow with reduced HSPCs and CAR cells.
Figure 3: Essential roles of Foxc1 in CAR cells.
Figure 4: Foxc1 is essential for maintenance of HSPC niche functions in adult bone marrow.

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Acknowledgements

We appreciate the technical assistance provided by K. Kawaguchi and G. Kondoh, and thank I. Sasagawa for secretarial assistance. This research was supported by JST, CREST and the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI.

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Authors

Contributions

Y.O. and T.N. designed and performed the experiments, analysed the data and prepared the paper. T.N. supervised the study. M.S. and T.S. performed the experiments. T.K. contributed materials and tools. All authors discussed results and edited the manuscript.

Corresponding author

Correspondence to Takashi Nagasawa.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Progenitors of CAR cells and their development in fetal and postnatal bone marrow.

a, b, Immunohistochemical analysis of E14.5 (a) and E16.5 (b) femurs of Osx–GFP mice with antibodies against PDGFR-β (red) and CD31 (blue). Osx–GFP+ cells (green) expressing PDGFR-β (arrowheads) were observed inside the marrow cavity at E16.5 (b with magnified views of the boxed area on the right) but not at E14.5 (a). B, cortical bone (b, right). c, Immunohistochemical analysis of the newborn femur of CXCL12–GFP mice with antibodies against PDGFR-β (red). CXCL12–GFP+ cells (green) express PDGFR-β. d, Immunofluorescent profiles of Osx–GFP+PDGFR-βhi cells (boxed area) in the limbs of E14.5 and E16.5 Osx–GFP mice. eg Relative mRNA expression levels of PDGFR-β, Lepr, CXCL12, SCF, PPARγ, C/EBPα and Osx in Osx–GFP+PDGFR-βhi, Osx–GFP+PDGFR-βlo, CXCL12–GFP+ cells, osteoblasts (Ob), PαS, Osx–GFPPDGFR-β+Sca-1CD31CD45Ter119 (Osx+) cells and endothelial cells (ECs) from femurs of E16.5 Osx–GFP mice and newborn CXCL12–GFP mice (e) as well as 14- to 20-week-old CXCL12–GFP mice (g), or in CXCL12–GFP+ cells from newborn, 1-week-old, 3-week-old and 14- to 20-week-old CXCL12–GFP mice (f) (n = 3). *P < 0.05.

Extended Data Figure 2 Expression of PDGFR-α in Sca-1+CD31 cells.

Immunohistochemical analysis of the bone marrow cavity of wild-type mice with antibodies against Sca-1 (red), CD31 (blue) and PDGFR-α (green). All Sca-1+CD31 mesenchymal cells surrounding arteries expressed PDGFR-α.

Extended Data Figure 3 Expression levels of Foxc2 were similar in CAR cells and osteoblasts.

ac, Relative mRNA expression levels of Foxc2 in CAR cells, osteoblasts (Ob), PαS cells, bone marrow endothelial cells (ECs), c-kit+Sca-1+Lin (KSL) cells, macrophages and muscle PαS cells from adult mice (a), in Osx–GFP+PDGFR-βhi, Osx–GFP+PDGFR-βlo, CXCL12–GFP+ CAR cells, osteoblasts, PαS, Osx–GFPPDGFR-β+Sca-1CD31CD45Ter119 (Osx+) cells and endothelial cells from femurs of E16.5 Osx–GFP mice (b) and newborn mice (b, c), and in CAR cells and osteoblasts from newborn, 1-week-old, 3-week-old and 14- to 20-week-old CXCL12–GFP mice (c) (n = 3). Error bars, s.d. *P < 0.05.

Extended Data Figure 4 The numbers of functional multilineage reconstituting HSCs were markedly reduced in the bone marrow of Prx1-Cre;Foxc1f/f mice.

a, C-kit+Sca-1+Lin (KSL) cells sorted from 3-week-old control or Prx1-Cre;Foxc1f/f mice as tester progenitors and those sorted from wild-type mice as competitor progenitors were mixed at a ratio of 1:1 and injected intravenously into recipient mice. The percentages of donor-derived Gr-1+ myeloid, B220+ B and CD3+ T cells in peripheral blood were analysed for 14 weeks after transplantation (n = 3). b, The numbers of KSL cells in the bone marrow of 3-week-old control or Prx1-Cre;Foxc1f/f mice (n = 3). Because KSL cell numbers were reduced, a marked decrease in the numbers of functional multilineage reconstituting HSCs in the mutants was observed. c, The numbers of LTC-ICs in the bone marrow of 3-week-old control and Prx1-Cre;Foxc1f/f mice (n = 3). LTC-IC numbers per femurs and tibiae were assayed by limiting dilution analysis. Error bars, s.d. *P < 0.05.

Extended Data Figure 5 The numbers of HSPCs were reduced in newborn Prx1-Cre;Foxc1f/f mice although to a lesser extent than juvenile mutants.

Total haematopoietic cell counts and the numbers of cells in the CD150+CD48 KSL population (HSCs), pro-B cells, pre-B cells and proerythroblasts (pro-E) in the bone marrow of newborn control and Prx1-Cre;Foxc1f/f mice (n = 5). *P < 0.05.

Extended Data Figure 6 CAR cells can be identified as S100+ cells in the marrow cavity.

Immunohistochemical analysis of bone marrow from CXCL12–GFP mice with antibodies against S100 (red). CAR cells (green) were identified as S100+ cells in the marrow cavity.

Extended Data Figure 7 Expression of PDGFR-α in Sca-1+CD31 PαS cells in the bone marrow from Prx1-Cre;Foxc1f/f or Lepr-Cre;Foxc1f/f mice.

a, b, Immunohistochemical analysis of the bone marrow cavity of control, Prx1-Cre;Foxc1f/f (a) and Lepr-Cre;Foxc1f/f mice (b) with antibodies against Sca-1 (red), CD31 (blue) and PDGFR-α (green). Sca-1+CD31 mesenchymal cells surrounding arteries expressed PDGFR-α (arrowheads).

Extended Data Figure 8 The numbers of functional multilineage reconstituting HSCs were markedly reduced in the bone marrow of Lepr-Cre;Foxc1f/f mice.

a, KSL cells sorted from 14- to 18-week-old control or Lepr-Cre;Foxc1f/f mice as tester progenitors and those sorted from wild-type mice as competitor progenitors were mixed at a ratio of 1:1 and injected intravenously into recipient mice. The percentages of donor-derived Gr-1+ myeloid, B220+ B and CD3+ T cells in peripheral blood were analysed for 14 weeks after transplantation (n = 3). b, The numbers of KSL cells in the bone marrow of 14- to 18-week-old control or Lepr-Cre;Foxc1f/f mice (n = 3). Because KSL cell numbers were reduced, a marked decrease in the numbers of functional multilineage reconstituting HSCs in the mutants was observed. c, The numbers of LTC-ICs in the bone marrow of 14- to 18-week-old control or Lepr-Cre;Foxc1f/f mice (n = 3). LTC-IC numbers per femur and tibia were assayed by limiting dilution analysis. Error bars, s.d. *P < 0.05.

Extended Data Figure 9 HSPC maintenance and haematopoiesis were not affected when Foxc1 was deleted from endothelial cells and haematopoietic cells.

Total haematopoietic cell counts and the numbers of LT-HSCs, ST-HSCs, MPPs, CLPs, pro-B cells, pre-B cells, proerythroblasts (pro-E) and granulocyte/macrophage progenitors (GMPs) in the bone marrow of 14- to 18-week-old control and Tie2-Cre;Foxc1f/f mice, in which the Foxc1 gene was deleted in endothelial cells and haematopoietic cells (n = 3).

Extended Data Table 1 Primers for RT–PCR

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Omatsu, Y., Seike, M., Sugiyama, T. et al. Foxc1 is a critical regulator of haematopoietic stem/progenitor cell niche formation. Nature 508, 536–540 (2014). https://doi.org/10.1038/nature13071

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