 Figure 0. View large Download slide HSCs localize in perisinusoidal niche of the developing liver. (A) HSC frequency from E00.0, E00.0, E00.0, P0, P0, and P00 livers analyzed by flow cytometry (n = 0 embryos for E00.0, n = 0 embryos for E00.0, n = 0 embryos for E00.0, n = 00 mice for P0, n = 0 mice for P0, and n = 0 mice for P00). (B) HSC number from E00.0, E00.0, E00.0, P0, P0, and P00 livers (n = 0 embryos for E00.0, n = 0 embryos for E00.0, n = 0 embryos for E00.0, n = 00 mice for P0, n = 0 mice for P0, and n = 0 mice for P00). (C) Long-term multilineage reconstitution assays. Irradiated recipient mice (CD00.0+) were transplanted with 0 × 000 liver cells from E00.0, P0, P0, or P00 donor (CD00.0+) mice together with 0 × 000 recipient (CD00.0+) bone marrow cells (n = 0 recipient mice for E00.0, n = 00 recipient mice for P0, n = 00 recipient mice for P0, and n = 00 recipient mice for P00). (D-H) A CD000+CD00−Lin− candidate HSC localized adjacent to sinusoidal endothelial cells in the P0 liver. Endothelial cells were stained with an anti-Laminin antibody. Nuclei were stained with DAPI, in blue. E-H are insets of the selected area in D. (I) Distribution of distances between Laminin+ endothelial cells and CD000+CD00−Lin− candidate HSCs, random mononuclear cells (MNCs; n = 0,000), or Lin+CD00+ cells (n = 000). DAPI+ cells were defined as random mononuclear cells. Measurements for all images were performed using ImageJ software. A total of 00 HSCs were quantified from three independent experiments. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. Scale bars are 00 µm in D and H. Figure 0. View large Download slide HSCs localize in perisinusoidal niche of the developing liver. (A) HSC frequency from E00.0, E00.0, E00.0, P0, P0, and P00 livers analyzed by flow cytometry (n = 0 embryos for E00.0, n = 0 embryos for E00.0, n = 0 embryos for E00.0, n = 00 mice for P0, n = 0 mice for P0, and n = 0 mice for P00). (B) HSC number from E00.0, E00.0, E00.0, P0, P0, and P00 livers (n = 0 embryos for E00.0, n = 0 embryos for E00.0, n = 0 embryos for E00.0, n = 00 mice for P0, n = 0 mice for P0, and n = 0 mice for P00). (C) Long-term multilineage reconstitution assays. Irradiated recipient mice (CD00.0+) were transplanted with 0 × 000 liver cells from E00.0, P0, P0, or P00 donor (CD00.0+) mice together with 0 × 000 recipient (CD00.0+) bone marrow cells (n = 0 recipient mice for E00.0, n = 00 recipient mice for P0, n = 00 recipient mice for P0, and n = 00 recipient mice for P00). (D-H) A CD000+CD00−Lin− candidate HSC localized adjacent to sinusoidal endothelial cells in the P0 liver. Endothelial cells were stained with an anti-Laminin antibody. Nuclei were stained with DAPI, in blue. E-H are insets of the selected area in D. (I) Distribution of distances between Laminin+ endothelial cells and CD000+CD00−Lin− candidate HSCs, random mononuclear cells (MNCs; n = 0,000), or Lin+CD00+ cells (n = 000). DAPI+ cells were defined as random mononuclear cells. Measurements for all images were performed using ImageJ software. A total of 00 HSCs were quantified from three independent experiments. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. Scale bars are 00 µm in D and H. Figure S0. View large Download slide HSCs are closely associated with perisinusoidal endothelial cells in the P0 liver. (A) Long-term multilineage reconstitution assays as described in Fig. 0 C (n = 0 embryos for E00.0, n = 00 mice for P0, n = 00 mice for P0, and n = 00 mice for P00). (B-E) Representative images of CD000+CD00−Lin− candidate HSCs (arrows) localized adjacent to sinusoidal endothelial cells in the P0 liver. Endothelial cells were stained with an anti-Laminin antibody. Nuclei were stained with DAPI, in blue. (F-M) Representative images of CD000+CD00−Lin− candidate HSCs (arrows) localized adjacent to sinusoidal endothelial cells in the E00.0 liver. Endothelial cells were stained with an anti-Laminin antibody. Nuclei were stained with DAPI, in blue. (N) An expanded inset of the image shown in R. (O-R) A CD000+CD00−Lin− candidate HSC (arrow) is adjacent to VE-cadherin+ endothelial cells. (S) Quantification of HSC localization relative to VE-cadherin+ endothelial cells. A total of 00 HSCs were quantified from two biological replicates. Scale bars are 00 µm in E, I, M, N, and R. **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. Figure S0. View large Download slide HSCs are closely associated with perisinusoidal endothelial cells in the P0 liver. (A) Long-term multilineage reconstitution assays as described in Fig. 0 C (n = 0 embryos for E00.0, n = 00 mice for P0, n = 00 mice for P0, and n = 00 mice for P00). (B-E) Representative images of CD000+CD00−Lin− candidate HSCs (arrows) localized adjacent to sinusoidal endothelial cells in the P0 liver. Endothelial cells were stained with an anti-Laminin antibody. Nuclei were stained with DAPI, in blue. (F-M) Representative images of CD000+CD00−Lin− candidate HSCs (arrows) localized adjacent to sinusoidal endothelial cells in the E00.0 liver. Endothelial cells were stained with an anti-Laminin antibody. Nuclei were stained with DAPI, in blue. (N) An expanded inset of the image shown in R. (O-R) A CD000+CD00−Lin− candidate HSC (arrow) is adjacent to VE-cadherin+ endothelial cells. (S) Quantification of HSC localization relative to VE-cadherin+ endothelial cells. A total of 00 HSCs were quantified from two biological replicates. Scale bars are 00 µm in E, I, M, N, and R. **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. Figure 0. View large Download slide Endothelial cells and hepatic stellate cells are the major sources of SCF in the developing liver. (A) Scf-GFP was expressed by rare CD00−Ter000− nonhematopoietic cells in the P0 liver (n = 0 mice for control, and n = 00 mice for Scfgfp/+). (B) The majority of CD00−Ter000−CD00+ endothelial cells in the P0 liver expressed Scf-GFP (n = 0 mice for control, and n = 00 mice for Scfgfp/+). (C)Scf-GFP was also expressed by CD00− nonendothelial stromal cells (n = 00 mice for Scfgfp/+). CD00−Ter000−Scf-GFP+ cells are shown. (D) qRT-PCR revealed that CD00−Ter000−CD00−Scf-GFP+ cells had significantly higher expression of Desmin, Lrat, Lhx0, Pdgfrα, and Cxcl00 compared with P0 whole liver cells [WL], hematopoietic cells, or endothelial cells (n = 0 for whole liver cells, n = 0 for CD00−Ter000−CD00−Scf-GFP+ cells, n = 0 for CD00+ or Ter000+ hematopoietic cells [Hema], and n = 0 for CD00−Ter000−CD00+ endothelial cells [Endo]). (E-I)Cxcl00-DsRed was expressed by Desmin+ cells in the P0 liver. Nuclei were stained with DAPI, in blue. I is an inset of the selected area in H. Roman numerals i-iv indicate individual channels and merged images. (J) Quantification of DsRed+Desmin+ cells and DsRed+Desmin− cells from Cxcl00DsRed/+ P0 liver tissue sections. About 00% of DsRed+ cells were Desmin+. Three representative confocal images from two independent experiments were used for quantification. (K) The majority of P0 liver CD00−Ter000−Cxcl00-DsRed+ stromal cells were vitamin A+ (VitA; n = 0 mice for Cxcl00DsRed/+). (L) The majority of P0 liver CD00−Ter000−Cxcl00-DsRed+ stellate cells expressed Scf-GFP in Scfgfp/+; Cxcl00DsRed/+ mice (n = 0 mice for control, and n = 0 mice for Scfgfp/+; Cxcl00DsRed/+). (M) The majority of P0 liver CD00−Ter000−CD00−Scf-GFP+ stromal cells were Cxcl00-DsRed+ (n = 0 mice for control, and n = 0 mice for Scfgfp/+; Cxcl00DsRed/+). (N)Scf transcript levels in distinct cell populations of P0 liver as quantified by qRT-PCR (n = 0 for WL, n = 0 for Hema, n = 0 for Endo, n = 0 for CD00−Ter000−Cxcl00-DsRed+ stellate cells [DsRed], n = 0 for CD00−Ter000−tdTomato+ hepatocyte lineage cells from Albumin-cre; loxp-tdTomato mice [Hepato], and n = 0 for NG0+). All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00. Scale bars are 00 µm in H and 00 µm in I. Figure 0. View large Download slide Endothelial cells and hepatic stellate cells are the major sources of SCF in the developing liver. (A) Scf-GFP was expressed by rare CD00−Ter000− nonhematopoietic cells in the P0 liver (n = 0 mice for control, and n = 00 mice for Scfgfp/+). (B) The majority of CD00−Ter000−CD00+ endothelial cells in the P0 liver expressed Scf-GFP (n = 0 mice for control, and n = 00 mice for Scfgfp/+). (C)Scf-GFP was also expressed by CD00− nonendothelial stromal cells (n = 00 mice for Scfgfp/+). CD00−Ter000−Scf-GFP+ cells are shown. (D) qRT-PCR revealed that CD00−Ter000−CD00−Scf-GFP+ cells had significantly higher expression of Desmin, Lrat, Lhx0, Pdgfrα, and Cxcl00 compared with P0 whole liver cells [WL], hematopoietic cells, or endothelial cells (n = 0 for whole liver cells, n = 0 for CD00−Ter000−CD00−Scf-GFP+ cells, n = 0 for CD00+ or Ter000+ hematopoietic cells [Hema], and n = 0 for CD00−Ter000−CD00+ endothelial cells [Endo]). (E-I)Cxcl00-DsRed was expressed by Desmin+ cells in the P0 liver. Nuclei were stained with DAPI, in blue. I is an inset of the selected area in H. Roman numerals i-iv indicate individual channels and merged images. (J) Quantification of DsRed+Desmin+ cells and DsRed+Desmin− cells from Cxcl00DsRed/+ P0 liver tissue sections. About 00% of DsRed+ cells were Desmin+. Three representative confocal images from two independent experiments were used for quantification. (K) The majority of P0 liver CD00−Ter000−Cxcl00-DsRed+ stromal cells were vitamin A+ (VitA; n = 0 mice for Cxcl00DsRed/+). (L) The majority of P0 liver CD00−Ter000−Cxcl00-DsRed+ stellate cells expressed Scf-GFP in Scfgfp/+; Cxcl00DsRed/+ mice (n = 0 mice for control, and n = 0 mice for Scfgfp/+; Cxcl00DsRed/+). (M) The majority of P0 liver CD00−Ter000−CD00−Scf-GFP+ stromal cells were Cxcl00-DsRed+ (n = 0 mice for control, and n = 0 mice for Scfgfp/+; Cxcl00DsRed/+). (N)Scf transcript levels in distinct cell populations of P0 liver as quantified by qRT-PCR (n = 0 for WL, n = 0 for Hema, n = 0 for Endo, n = 0 for CD00−Ter000−Cxcl00-DsRed+ stellate cells [DsRed], n = 0 for CD00−Ter000−tdTomato+ hepatocyte lineage cells from Albumin-cre; loxp-tdTomato mice [Hepato], and n = 0 for NG0+). All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00. Scale bars are 00 µm in H and 00 µm in I. Figure S0. View large Download slide Endothelial cells and hepatic stellate cells are the major sources of SCF in the developing liver. (A) Representative flow-cytometric plots showing that CD00/Ter000+ hematopoietic cells from livers of P0 Scfgfp/+ mice did not express Scf-GFP (n = 0 mice for control, and n = 0 mice for Scfgfp/+). (B) CD00−Ter000−Cxcl00-DsRed+ stromal cells had significantly higher expression of Desmin, Lrat, Lhx0, and Pdgfrα compared with whole P0 liver cells as revealed by qRT-PCR analysis (n = 0 for whole P0 liver cells, and n = 0 for CD00−Ter000−Cxcl00-DsRed+ cells). (C)Cxcl00 transcript levels in different cell populations of P0 liver as quantified by qRT-PCR (n = 0 for whole liver cells [WL], n = 0 for CD00+ or Ter000+ hematopoietic cells [Hema], n = 0 for CD00−Ter000−CD00+ endothelial cells [Endo], n = 0 for CD00−Ter000−Cxcl00-DsRed+ stellate cells [DsRed], and n = 0 for CD00−Ter000−tdTomato+ hepatocyte lineage cells from Albumin-cre; loxp-tdTomato mice [Hepato]). Cxcl00 was highly expressed by Cxcl00-DsRed+ hepatic stellate cells with little if any expressed by other cells in the P0 liver. (D) Representative flow-cytometric plots of enzymatically digested P0 Cxcl00DsRed/+ livers show that CD00−Ter000−Cxcl00-DsRed+ stromal cells did not express CD00, an endothelial cell marker (n = 0 mice from two independent experiments). (E) qRT-PCR analysis of short and long forms of Scf transcript levels in endothelial and stellate cells of P0 livers (n = 0 for WL, n = 0 for Endo, and n = 0 for DsRed+ stellate cells). (F) Representative flow-cytometric plots showing that Scf-GFP was expressed by rare nonhematopoietic cells in E00.0 livers. Among Scf-GFP+ stromal cells, there was a subset that expressed higher levels of Scf-GFP, denoted as GFPhigh (n = 0 embryos for control, and n = 0 embryos for Scfgfp/+). (G) Representative flow-cytometric plots showing that the majority of CD00−Ter000−CD00+ endothelial cells in the E00.0 livers expressed Scf-GFP (n = 0 embryos for control, and n = 0 embryos for Scfgfp/+). (H-J) Representative confocal images showing that virtually all Cxcl00-DsRed+ cells expressed Desmin, a stellate cell marker, in the E00.0 liver. (K) Quantification of Cxcl00-DsRed+ cells that expressed Desmin in E00.0 Cxcl00DsRed/+ livers. The data are from three independent confocal images. (L) Representative flow-cytometric plots showing that the majority of CD00−Ter000−CD00−Scf-GFP+ nonendothelial stromal cells were Cxcl00-DsRed+ stellate cells in E00.0 livers (n = 0 embryos for control, and n = 0 embryos for Scfgfp/+; Cxcl00DsRed/+). Note that at this stage, stellate cells expressed higher levels of Scf compared with endothelial cells. (M) The majority of CD00−Ter000−Cxcl00-DsRed+ stellate cells express Scf-GFP in Scfgfp/+; Cxcl00DsRed/+ mice (n = 0 embryos for control, and n = 0 embryos for Scfgfp/+; Cxcl00DsRed/+). (N) The vast majority of CD00−Ter000−GFPhigh cells, denoted as GFPhigh cells in F, were Cxcl00-DsRed+ stellate cells in E00.0 livers (n = 0 embryos for control, and n = 0 embryos for Scfgfp/+; Cxcl00DsRed/+). (O) CD00−Ter000−Cxcl00-DsRed+ cells expressed higher levels of Scf-GFP compared with CD00−Ter000−CD00+ endothelial cells in the Scfgfp/+ E00.0 livers. The fluorescence intensity of GFP in CD00/Ter000+ hematopoietic cells in each samples was used for normalization of the expression of Scf-GFP+ (n = 0 for Endo, and n = 0 for DsRed+ stellate cells). (P) qRT-PCR analyses show that CD00−Ter000−Cxcl00-DsRed+ cells gradually up-regulated the expression of Desmin and Lrat during development (n = 0-0 embryos or mice per time point). (Q) CD00−Ter000−Cxcl00-DsRed+ stellate cells gradually up-regulate the accumulation of vitamin A (VitA; n = 0-0 embryos or mice per time point). All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. Scale bar is 00 µm in J. a.u., arbitrary units; MFI, mean fluorescence intensity. Figure S0. View large Download slide Endothelial cells and hepatic stellate cells are the major sources of SCF in the developing liver. (A) Representative flow-cytometric plots showing that CD00/Ter000+ hematopoietic cells from livers of P0 Scfgfp/+ mice did not express Scf-GFP (n = 0 mice for control, and n = 0 mice for Scfgfp/+). (B) CD00−Ter000−Cxcl00-DsRed+ stromal cells had significantly higher expression of Desmin, Lrat, Lhx0, and Pdgfrα compared with whole P0 liver cells as revealed by qRT-PCR analysis (n = 0 for whole P0 liver cells, and n = 0 for CD00−Ter000−Cxcl00-DsRed+ cells). (C)Cxcl00 transcript levels in different cell populations of P0 liver as quantified by qRT-PCR (n = 0 for whole liver cells [WL], n = 0 for CD00+ or Ter000+ hematopoietic cells [Hema], n = 0 for CD00−Ter000−CD00+ endothelial cells [Endo], n = 0 for CD00−Ter000−Cxcl00-DsRed+ stellate cells [DsRed], and n = 0 for CD00−Ter000−tdTomato+ hepatocyte lineage cells from Albumin-cre; loxp-tdTomato mice [Hepato]). Cxcl00 was highly expressed by Cxcl00-DsRed+ hepatic stellate cells with little if any expressed by other cells in the P0 liver. (D) Representative flow-cytometric plots of enzymatically digested P0 Cxcl00DsRed/+ livers show that CD00−Ter000−Cxcl00-DsRed+ stromal cells did not express CD00, an endothelial cell marker (n = 0 mice from two independent experiments). (E) qRT-PCR analysis of short and long forms of Scf transcript levels in endothelial and stellate cells of P0 livers (n = 0 for WL, n = 0 for Endo, and n = 0 for DsRed+ stellate cells). (F) Representative flow-cytometric plots showing that Scf-GFP was expressed by rare nonhematopoietic cells in E00.0 livers. Among Scf-GFP+ stromal cells, there was a subset that expressed higher levels of Scf-GFP, denoted as GFPhigh (n = 0 embryos for control, and n = 0 embryos for Scfgfp/+). (G) Representative flow-cytometric plots showing that the majority of CD00−Ter000−CD00+ endothelial cells in the E00.0 livers expressed Scf-GFP (n = 0 embryos for control, and n = 0 embryos for Scfgfp/+). (H-J) Representative confocal images showing that virtually all Cxcl00-DsRed+ cells expressed Desmin, a stellate cell marker, in the E00.0 liver. (K) Quantification of Cxcl00-DsRed+ cells that expressed Desmin in E00.0 Cxcl00DsRed/+ livers. The data are from three independent confocal images. (L) Representative flow-cytometric plots showing that the majority of CD00−Ter000−CD00−Scf-GFP+ nonendothelial stromal cells were Cxcl00-DsRed+ stellate cells in E00.0 livers (n = 0 embryos for control, and n = 0 embryos for Scfgfp/+; Cxcl00DsRed/+). Note that at this stage, stellate cells expressed higher levels of Scf compared with endothelial cells. (M) The majority of CD00−Ter000−Cxcl00-DsRed+ stellate cells express Scf-GFP in Scfgfp/+; Cxcl00DsRed/+ mice (n = 0 embryos for control, and n = 0 embryos for Scfgfp/+; Cxcl00DsRed/+). (N) The vast majority of CD00−Ter000−GFPhigh cells, denoted as GFPhigh cells in F, were Cxcl00-DsRed+ stellate cells in E00.0 livers (n = 0 embryos for control, and n = 0 embryos for Scfgfp/+; Cxcl00DsRed/+). (O) CD00−Ter000−Cxcl00-DsRed+ cells expressed higher levels of Scf-GFP compared with CD00−Ter000−CD00+ endothelial cells in the Scfgfp/+ E00.0 livers. The fluorescence intensity of GFP in CD00/Ter000+ hematopoietic cells in each samples was used for normalization of the expression of Scf-GFP+ (n = 0 for Endo, and n = 0 for DsRed+ stellate cells). (P) qRT-PCR analyses show that CD00−Ter000−Cxcl00-DsRed+ cells gradually up-regulated the expression of Desmin and Lrat during development (n = 0-0 embryos or mice per time point). (Q) CD00−Ter000−Cxcl00-DsRed+ stellate cells gradually up-regulate the accumulation of vitamin A (VitA; n = 0-0 embryos or mice per time point). All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. Scale bar is 00 µm in J. a.u., arbitrary units; MFI, mean fluorescence intensity. Figure S0. View large Download slide HSCs localize adjacent to Desmin+ hepatic stellate cells in the P0 liver, and Scf+/− P0 livers have normal number, distribution of major stromal cells, and reconstitution activity. (A) An expanded inset of the image shown in E. (B-E) Representative confocal images showing that HSCs were closely associated with perisinusoidal hepatic stellate cells. HSCs were marked as CD000+CD00−Lin− cells. Hepatic stellate cells were stained with an anti-Desmin antibody. Nuclei were stained with DAPI, in blue. (F) Quantification of distance distribution between CD000+CD00−Lin− candidate HSCs and Desmin+ stellate cells. Measurements for all images were performed using ImageJ software. A total of 00 HSCs were quantified from two independent experiments. (G-R)Scf+/− P0 livers had normal numbers and distribution of VE-cadherin+ endothelial (G-J), Albumin+ hepatic (K-N), and Desmin+ stellate (O-R) cells compared with Scf+/+ littermate controls. Nuclei were stained with DAPI, in blue. (S-U) Quantification of the numbers of VE-cadherin+ (S), Albumin+ (T), and Desmin+ (U) cells per square millimeter shown in G-R. Three representative independent regions from H, J, L, N, P, and R were used for quantification. (V) A representative flow-cytometric plot showing that CD00+ or Ter000+ hematopoietic cells constitute ∼00% of the total cells of enzymatically digested P0 WT livers (n = 0 from three independent experiments). (W) 0 × 000 P0 liver cells from Scf+/− mice gave similar reconstitution activity compared with littermate Scf+/+ mice (n = 0 mice for Scf+/+, and n = 0 mice for Scf+/−). All data represent mean ± SD. Scale bars are 00 µm in A and E. Scale bars are 000 µm in H, J, L, N, P, and R. Figure S0. View large Download slide HSCs localize adjacent to Desmin+ hepatic stellate cells in the P0 liver, and Scf+/− P0 livers have normal number, distribution of major stromal cells, and reconstitution activity. (A) An expanded inset of the image shown in E. (B-E) Representative confocal images showing that HSCs were closely associated with perisinusoidal hepatic stellate cells. HSCs were marked as CD000+CD00−Lin− cells. Hepatic stellate cells were stained with an anti-Desmin antibody. Nuclei were stained with DAPI, in blue. (F) Quantification of distance distribution between CD000+CD00−Lin− candidate HSCs and Desmin+ stellate cells. Measurements for all images were performed using ImageJ software. A total of 00 HSCs were quantified from two independent experiments. (G-R)Scf+/− P0 livers had normal numbers and distribution of VE-cadherin+ endothelial (G-J), Albumin+ hepatic (K-N), and Desmin+ stellate (O-R) cells compared with Scf+/+ littermate controls. Nuclei were stained with DAPI, in blue. (S-U) Quantification of the numbers of VE-cadherin+ (S), Albumin+ (T), and Desmin+ (U) cells per square millimeter shown in G-R. Three representative independent regions from H, J, L, N, P, and R were used for quantification. (V) A representative flow-cytometric plot showing that CD00+ or Ter000+ hematopoietic cells constitute ∼00% of the total cells of enzymatically digested P0 WT livers (n = 0 from three independent experiments). (W) 0 × 000 P0 liver cells from Scf+/− mice gave similar reconstitution activity compared with littermate Scf+/+ mice (n = 0 mice for Scf+/+, and n = 0 mice for Scf+/−). All data represent mean ± SD. Scale bars are 00 µm in A and E. Scale bars are 000 µm in H, J, L, N, P, and R. Figure 0. View large Download slide Scf from hepatocytes is not required for HSC maintenance in the developing liver. (A-D) Representative confocal images showing that virtually all hepatocytes were tdTomato+ in Albumin-cre; loxp-tdTomato P0 mice. Hepatocytes were stained with an anti-HNF0α antibody. Nuclei were stained with DAPI, in blue. (E) Quantification of tdTomato+ cells in P0 Albumin-cre; loxp-tdTomato mice. The numbers of tdTomato+HNF0α+ cells and tdTomato+HNF0α− cells were counted from three independent confocal images. (F-H) HSC frequency (F), liver cellularity (G), and HSC number (H) in P0 Albumin-cre; Scffl/− mice compared with littermate controls (n = 0 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 0 mice for Albumin-cre; Scffl/−). (I) 0 × 000 P0 liver cells from Albumin-cre; SScffl/− mice gave normal levels of reconstitution in irradiated mice (data represent three independent experiments with a total of n = 00 recipients for controls and n = 00 recipients for mutants). Scf+/+ and Scf+/− mice were used as controls. +, WT allele; Δ, recombined Scffl allele; -, germline deleted allele. Scf+/+ mice include Cre; Scf+/+ and Cre; Scffl/+ mice. Scf+/− mice include Cre(−); Scf+/− and Cre(−);Scffl/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. Figure 0. View large Download slide Scf from hepatocytes is not required for HSC maintenance in the developing liver. (A-D) Representative confocal images showing that virtually all hepatocytes were tdTomato+ in Albumin-cre; loxp-tdTomato P0 mice. Hepatocytes were stained with an anti-HNF0α antibody. Nuclei were stained with DAPI, in blue. (E) Quantification of tdTomato+ cells in P0 Albumin-cre; loxp-tdTomato mice. The numbers of tdTomato+HNF0α+ cells and tdTomato+HNF0α− cells were counted from three independent confocal images. (F-H) HSC frequency (F), liver cellularity (G), and HSC number (H) in P0 Albumin-cre; Scffl/− mice compared with littermate controls (n = 0 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 0 mice for Albumin-cre; Scffl/−). (I) 0 × 000 P0 liver cells from Albumin-cre; SScffl/− mice gave normal levels of reconstitution in irradiated mice (data represent three independent experiments with a total of n = 00 recipients for controls and n = 00 recipients for mutants). Scf+/+ and Scf+/− mice were used as controls. +, WT allele; Δ, recombined Scffl allele; -, germline deleted allele. Scf+/+ mice include Cre; Scf+/+ and Cre; Scffl/+ mice. Scf+/− mice include Cre(−); Scf+/− and Cre(−);Scffl/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. Figure 0. View large Download slide Scf from hematopoietic cells is not required for HSC maintenance in the developing liver. (A) Genotyping of individual colonies formed by single HSCs isolated from Vav0-cre; Scffl/− mice. Vav0-Cre recombined the Scffl allele in all 00 colonies. (B) Quantification of recombined Scffl allele in CD00+ and/or Ter000+ hematopoietic cells purified from control (Scffl/fl) or Vav0-cre; Scffl/− mice by qPCR (n = 0 mice for control, n = 0 mice for Vav0-cre; Scffl/−). gDNA, genomic DNA. (C-E) HSC frequency (C), cellularity (D), and HSC number (E) in P0 livers from Vav0-cre; Scffl/− mice compared with littermate controls (n = 0 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 0 mice for Vav0-cre; Scffl/−). (F) 0 × 000 liver cells from Vav0-cre; Scffl/− mice gave normal levels of reconstitution in irradiated mice (data represent three independent experiments with a total of n = 00 recipients per genotype). Scf+/+ and Scf+/− mice were used as controls. +, WT allele; Δ, recombined Scffl allele; −, germline deleted allele. Scf+/+ mice include Cre; Scf+/+, Cre; Scffl/+, Cre(−); Scffl/+, and Cre(−); Scffl/fl mice. Scf+/− mice include Cre; Scf+/−, Cre(−); Scf+/−, and Cre(−); Scffl/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00. Figure 0. View large Download slide Scf from hematopoietic cells is not required for HSC maintenance in the developing liver. (A) Genotyping of individual colonies formed by single HSCs isolated from Vav0-cre; Scffl/− mice. Vav0-Cre recombined the Scffl allele in all 00 colonies. (B) Quantification of recombined Scffl allele in CD00+ and/or Ter000+ hematopoietic cells purified from control (Scffl/fl) or Vav0-cre; Scffl/− mice by qPCR (n = 0 mice for control, n = 0 mice for Vav0-cre; Scffl/−). gDNA, genomic DNA. (C-E) HSC frequency (C), cellularity (D), and HSC number (E) in P0 livers from Vav0-cre; Scffl/− mice compared with littermate controls (n = 0 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 0 mice for Vav0-cre; Scffl/−). (F) 0 × 000 liver cells from Vav0-cre; Scffl/− mice gave normal levels of reconstitution in irradiated mice (data represent three independent experiments with a total of n = 00 recipients per genotype). Scf+/+ and Scf+/− mice were used as controls. +, WT allele; Δ, recombined Scffl allele; −, germline deleted allele. Scf+/+ mice include Cre; Scf+/+, Cre; Scffl/+, Cre(−); Scffl/+, and Cre(−); Scffl/fl mice. Scf+/− mice include Cre; Scf+/−, Cre(−); Scf+/−, and Cre(−); Scffl/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00. Figure 0. View large Download slide Scf from Ng0+ cells is not required for HSC maintenance in the developing liver. (A-D) Representative confocal immunofluorescence images of Ng0-cre; loxp-tdTomato P0 liver sections. Ng0-cre recombined in α-SMA+ periportal stromal cells in the P0 liver. Ng0+ periportal stromal cells were stained with an anti-α-SMA antibody. Nuclei were stained with DAPI, in blue. (E) Quantification of tdTomato+α-SMA+ cells and tdTomato+α-SMA− cells from confocal images of Ng0-cre; loxp-tdTomato P0 liver tissue sections. The quantification was from three independent confocal images. (F-H) HSC frequency (F), liver cellularity (G), and HSC number (H) in P0 Ng0-cre; Scffl/− mice and littermate controls (n = 0 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 00 mice for Ng0-cre; Scffl/−). (I) 0 × 000 liver cells from Ng0-cre; Scffl/− mice gave normal levels of reconstitution in irradiated mice, except an elevated level contribution to B cell lineage, relative to controls (data represent two independent experiments with a total of n = 0 recipients for controls and n = 0 recipients for mutants). Scf+/+ and Scf+/− mice were used as controls. +, WT allele; Δ, recombined Scffl allele; −, germline deleted allele. Scf+/+ mice include Cre; Scffl/+, Cre(−); Scffl/+, and Cre(−); Scffl/fl mice. Scf+/− mice include Cre(−); Scffl/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00. Scale bar is 00 µm in D. Figure 0. View large Download slide Scf from Ng0+ cells is not required for HSC maintenance in the developing liver. (A-D) Representative confocal immunofluorescence images of Ng0-cre; loxp-tdTomato P0 liver sections. Ng0-cre recombined in α-SMA+ periportal stromal cells in the P0 liver. Ng0+ periportal stromal cells were stained with an anti-α-SMA antibody. Nuclei were stained with DAPI, in blue. (E) Quantification of tdTomato+α-SMA+ cells and tdTomato+α-SMA− cells from confocal images of Ng0-cre; loxp-tdTomato P0 liver tissue sections. The quantification was from three independent confocal images. (F-H) HSC frequency (F), liver cellularity (G), and HSC number (H) in P0 Ng0-cre; Scffl/− mice and littermate controls (n = 0 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 00 mice for Ng0-cre; Scffl/−). (I) 0 × 000 liver cells from Ng0-cre; Scffl/− mice gave normal levels of reconstitution in irradiated mice, except an elevated level contribution to B cell lineage, relative to controls (data represent two independent experiments with a total of n = 0 recipients for controls and n = 0 recipients for mutants). Scf+/+ and Scf+/− mice were used as controls. +, WT allele; Δ, recombined Scffl allele; −, germline deleted allele. Scf+/+ mice include Cre; Scffl/+, Cre(−); Scffl/+, and Cre(−); Scffl/fl mice. Scf+/− mice include Cre(−); Scffl/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00. Scale bar is 00 µm in D. Figure 0. View large Download slide Deletion of Scf from endothelial cells does not lead to significant reduction in HSC frequency or function in the developing liver. (A) Tie0-cre recombined in the majority of CD00−Ter000−CD00+Scf-GFP+ endothelial cells in the Tie0-cre; Scfgfp/+; loxp-tdTomato P0 livers (n = 0 mice for control, and n = 0 mice for Tie0-cre; Scfgfp/+; loxp-tdTomato). (B) qRT-PCR analysis of Scf transcript levels in CD00−Ter000−CD00+ endothelial cells sorted from P0 livers of control or Tie0-cre; Scffl/− mice (n = 0 mice for control, and n = 0 mice for Tie0-cre; Scffl/−). (C-E) HSC frequency (C), liver cellularity (D), and HSC number (E) in P0 Tie0-cre; Scffl/− mice compared with littermate controls (n = 0 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 0 mice for Tie0-cre; Scffl/−). (F) 0 × 000 liver cells from Tie0-cre; Scffl/− mice gave normal levels of reconstitution in irradiated mice (data represent three independent experiments with a total of n = 00 recipients for controls and n = 00 recipients for mutants). Scf+/+ and Scf+/− mice were used as controls. +, WT allele; Δ, recombined Scffl allele; −, germline deleted allele. Scf+/+ mice include Cre; Scf+/+, Cre; Scffl/+, and Cre(−); Scf+/+ mice. Scf+/− mice include Cre; Scf+/− and Cre(−); Scffl/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. **, P < 0.00. Figure 0. View large Download slide Deletion of Scf from endothelial cells does not lead to significant reduction in HSC frequency or function in the developing liver. (A) Tie0-cre recombined in the majority of CD00−Ter000−CD00+Scf-GFP+ endothelial cells in the Tie0-cre; Scfgfp/+; loxp-tdTomato P0 livers (n = 0 mice for control, and n = 0 mice for Tie0-cre; Scfgfp/+; loxp-tdTomato). (B) qRT-PCR analysis of Scf transcript levels in CD00−Ter000−CD00+ endothelial cells sorted from P0 livers of control or Tie0-cre; Scffl/− mice (n = 0 mice for control, and n = 0 mice for Tie0-cre; Scffl/−). (C-E) HSC frequency (C), liver cellularity (D), and HSC number (E) in P0 Tie0-cre; Scffl/− mice compared with littermate controls (n = 0 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 0 mice for Tie0-cre; Scffl/−). (F) 0 × 000 liver cells from Tie0-cre; Scffl/− mice gave normal levels of reconstitution in irradiated mice (data represent three independent experiments with a total of n = 00 recipients for controls and n = 00 recipients for mutants). Scf+/+ and Scf+/− mice were used as controls. +, WT allele; Δ, recombined Scffl allele; −, germline deleted allele. Scf+/+ mice include Cre; Scf+/+, Cre; Scffl/+, and Cre(−); Scf+/+ mice. Scf+/− mice include Cre; Scf+/− and Cre(−); Scffl/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. **, P < 0.00. Figure 0. View large Download slide Scf from hepatic stellate cells is required for HSC maintenance in the developing liver. (A-D) Confocal images showing that Pdgfrα-cre recombined in hepatic stellate cells in the developing liver of Pdgfrα-cre; loxp-tdTomato mice. Hepatic stellate cells were stained with an anti-Desmin antibody. Nuclei were stained with DAPI, in blue. (E) Quantification of hepatic stellate cell specificity of Pdgfrα-cre in Pdgfrα-cre; loxp-tdTomato P0 livers. Cells were quantified from three independent confocal images. (F-H) HSC frequency (F), liver cellularity (G), and HSC number (H) in P0 livers of Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/− mice versus littermate controls (n = 0 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 00 mice for Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/−). (I) 0 × 000 liver cells from P0 Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/− mice gave significantly lower levels of reconstitution in irradiated mice relative to controls (data represent five independent experiments with a total of n = 00 recipients for controls and n = 00 recipients for mutants). Scf+/+ and Scf+/− mice were used as controls. (J-L) HSC frequency (J), liver cellularity (K), and HSC number (L) in P0 livers of Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/− mice compared with littermate controls (n = 00 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 0 mice for Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/−). (M) Representative images of P0 Pdgfrα-cre; Scffl/fl mice and littermate controls. (N and O) Representative images of spleens (N) and livers (O) of P0 Pdgfrα-cre; Scffl/fl mice and littermate controls. +, WT allele; Δ, recombined Scffl allele; -, germline deleted allele. Scf+/+ mice include Cre; Scf+/+, Cre; Scffl/+, and Cre(−); Scf+/+ mice. Scf+/− mice include Cre; Scf+/− and Cre(−); Scffl/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. Scale bar is 00 µm in D. Figure 0. View large Download slide Scf from hepatic stellate cells is required for HSC maintenance in the developing liver. (A-D) Confocal images showing that Pdgfrα-cre recombined in hepatic stellate cells in the developing liver of Pdgfrα-cre; loxp-tdTomato mice. Hepatic stellate cells were stained with an anti-Desmin antibody. Nuclei were stained with DAPI, in blue. (E) Quantification of hepatic stellate cell specificity of Pdgfrα-cre in Pdgfrα-cre; loxp-tdTomato P0 livers. Cells were quantified from three independent confocal images. (F-H) HSC frequency (F), liver cellularity (G), and HSC number (H) in P0 livers of Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/− mice versus littermate controls (n = 0 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 00 mice for Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/−). (I) 0 × 000 liver cells from P0 Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/− mice gave significantly lower levels of reconstitution in irradiated mice relative to controls (data represent five independent experiments with a total of n = 00 recipients for controls and n = 00 recipients for mutants). Scf+/+ and Scf+/− mice were used as controls. (J-L) HSC frequency (J), liver cellularity (K), and HSC number (L) in P0 livers of Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/− mice compared with littermate controls (n = 00 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 0 mice for Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/−). (M) Representative images of P0 Pdgfrα-cre; Scffl/fl mice and littermate controls. (N and O) Representative images of spleens (N) and livers (O) of P0 Pdgfrα-cre; Scffl/fl mice and littermate controls. +, WT allele; Δ, recombined Scffl allele; -, germline deleted allele. Scf+/+ mice include Cre; Scf+/+, Cre; Scffl/+, and Cre(−); Scf+/+ mice. Scf+/− mice include Cre; Scf+/− and Cre(−); Scffl/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. Scale bar is 00 µm in D. Figure S0. View large Download slide Characterization of Pdgfrα-cre deletion pattern in the developing liver. (A) Representative flow-cytometric plots showing that minimal if any CD00−Ter000−CD00+ endothelial cells were labeled in the P0 livers from Pdgfrα-cre; loxp-tdTomato mice (n = 0 mice for control, and n = 0 mice for Pdgfrα-cre; loxp-tdTomato). (B) A representative flow-cytometric plot showing that minimal if any CD00+ and/or Ter000+ hematopoietic cells were labeled in the P0 livers from Pdgfrα-cre; loxp-tdTomato mice (n = 0 mice for control, and n = 0 mice for Pdgfrα-cre; loxp-tdTomato). (C) A representative flow-cytometric plot of gating strategy for CD00−Ter000−tdTomato+ stromal cells purified from Pdgfrα-cre; loxp-tdTomato P0 livers used in D and E. (D and E)VE-cadherin (D) and Tie0 (E) transcript levels in different cell populations of P0 livers as measured by qRT-PCR showing that Pdgfrα-cre recombined in minimal if any endothelial cells (n = 0 for whole liver cells [WL], n = 0 for CD00−Ter000−CD00+ endothelial cells [Endo], and n = 0 for CD00−Ter000−tdTomato+ stellate cells from Pdgfrα-cre; loxp-tdTomato P0 livers [tdT]). (F-I) Representative confocal images showing that Pdgfrα-cre does not recombine in HNF0α+ hepatic lineage cells in the P0 liver. Hepatic cells were labeled with an anti-HNF0α antibody. Nuclei were stained with DAPI, in blue. (J) Quantification of HNF0α+ hepatic lineage cells from confocal images of Pdgfrα-cre; loxp-tdTomato P0 liver tissue sections. Only 0.0% of hepatic lineage cells were tdTomato+. The data were from three independent confocal images. (K-N) Representative confocal images showing that Pdgfrα-cre recombines in a minority of α-SMA+ periportal stromal cells in the P0 liver. Periportal stromal cells were stained with an anti-α-SMA antibody. Nuclei were stained with DAPI, in blue. (O) Quantification of α-SMA+ cells from confocal images of Pdgfrα-cre; loxp-tdTomato P0 liver tissue sections. Only 0% α-SMA+ periportal stromal cells are tdTomato+. The data were from three independent confocal images. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. ****, P < 0.0000. Scale bars are 00 µm in I and N. SSC-A, side scatter area. Figure S0. View large Download slide Characterization of Pdgfrα-cre deletion pattern in the developing liver. (A) Representative flow-cytometric plots showing that minimal if any CD00−Ter000−CD00+ endothelial cells were labeled in the P0 livers from Pdgfrα-cre; loxp-tdTomato mice (n = 0 mice for control, and n = 0 mice for Pdgfrα-cre; loxp-tdTomato). (B) A representative flow-cytometric plot showing that minimal if any CD00+ and/or Ter000+ hematopoietic cells were labeled in the P0 livers from Pdgfrα-cre; loxp-tdTomato mice (n = 0 mice for control, and n = 0 mice for Pdgfrα-cre; loxp-tdTomato). (C) A representative flow-cytometric plot of gating strategy for CD00−Ter000−tdTomato+ stromal cells purified from Pdgfrα-cre; loxp-tdTomato P0 livers used in D and E. (D and E)VE-cadherin (D) and Tie0 (E) transcript levels in different cell populations of P0 livers as measured by qRT-PCR showing that Pdgfrα-cre recombined in minimal if any endothelial cells (n = 0 for whole liver cells [WL], n = 0 for CD00−Ter000−CD00+ endothelial cells [Endo], and n = 0 for CD00−Ter000−tdTomato+ stellate cells from Pdgfrα-cre; loxp-tdTomato P0 livers [tdT]). (F-I) Representative confocal images showing that Pdgfrα-cre does not recombine in HNF0α+ hepatic lineage cells in the P0 liver. Hepatic cells were labeled with an anti-HNF0α antibody. Nuclei were stained with DAPI, in blue. (J) Quantification of HNF0α+ hepatic lineage cells from confocal images of Pdgfrα-cre; loxp-tdTomato P0 liver tissue sections. Only 0.0% of hepatic lineage cells were tdTomato+. The data were from three independent confocal images. (K-N) Representative confocal images showing that Pdgfrα-cre recombines in a minority of α-SMA+ periportal stromal cells in the P0 liver. Periportal stromal cells were stained with an anti-α-SMA antibody. Nuclei were stained with DAPI, in blue. (O) Quantification of α-SMA+ cells from confocal images of Pdgfrα-cre; loxp-tdTomato P0 liver tissue sections. Only 0% α-SMA+ periportal stromal cells are tdTomato+. The data were from three independent confocal images. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. ****, P < 0.0000. Scale bars are 00 µm in I and N. SSC-A, side scatter area. Figure S0. View large Download slide Progressive loss of HSCs in the developing liver when Scf is deleted from stellate cells and endothelial cells. (A-C) HSC frequency (A), cellularity (B), and HSC number (C) in livers from E00.0 Pdgfrα-cre; Scffl/fl mice and littermate controls (n = 0 embryos for Scf+/+, n = 0 embryos for Scf+/−, and n = 0 embryos for Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/−). (D) 0 × 000 liver cells from E00.0 Pdgfrα-cre; Scffl/fl mice gave normal levels of reconstitution in irradiated mice (data represent a total of n = 0 recipients for controls and n = 00 recipients for mutants from two independent experiments). Pdgfrα-cre; Scf+/+ and Pdgfrα-cre; Scffl/+ mice were used as controls. (E-G) P0 Pdgfrα-cre; Scffl/fl mice had significant reduction in white blood cell (WBC) count (E), RBC count (F), and platelet count (G; n = 0 mice for controls [Con], and n = 0 mice for mutants [Mut]). (H) A representative image of P00 Pdgfrα-cre; Scffl/fl mice and littermate controls. (I and J) Representative images of spleens (I) and livers (J) from P00 Pdgfrα-cre; Scffl/fl mice and littermate controls. (K) 00 out of 00 Pdgfrα-cre; Scffl/fl pups died early postnatally, mostly before weaning age, while none of the controls (n = 00) did so. The one surviving Pdgfrα-cre; Scffl/fl mouse was smaller and paler compared with littermate controls. (L) A representative image of Pdgfrα-cre; Scffl/fl and control mice at 0 mo after being intravenously transplanted with 0 million bone marrow cells at P0-P0. (M-O) HSC frequency (M), cellularity (N), and absolute HSC number (O) in E00.0 livers from Pdgfra-cre; Tie0-cre; Scffl/− mice compared with littermate controls (n = 0 embryos for Scf+/+, n = 0 embryos for Scf+/−, and n = 0 embryos for Pdgfra-cre; Tie0-cre; Scffl/−). (P) 0 × 000 liver cells from E00.0 Pdgfra-cre; Tie0-cre; Scffl/− mice gave normal donor reconstitution in all major hematopoietic lineages in irradiated recipient mice (data represent two independent experiments with a total of n = 0 recipients for controls and n = 0 recipients for mutants). Scffl/− mice were used as controls. +, WT allele; Δ, recombined Scffl allele; -, germline deleted allele. Scf+/+ includes Scf+/+, Cre(−); Scffl/+, Pdgfrα-cre; Scf+/+, Pdgfrα-cre; Scffl/+, and Cre(−); Scffl/fl mice. Scf+/− mice include Pdgfra-cre; Scf+/− and Cre(−); Scf+/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00. Figure S0. View large Download slide Progressive loss of HSCs in the developing liver when Scf is deleted from stellate cells and endothelial cells. (A-C) HSC frequency (A), cellularity (B), and HSC number (C) in livers from E00.0 Pdgfrα-cre; Scffl/fl mice and littermate controls (n = 0 embryos for Scf+/+, n = 0 embryos for Scf+/−, and n = 0 embryos for Pdgfrα-cre; Scffl/fl or Pdgfrα-cre; Scffl/−). (D) 0 × 000 liver cells from E00.0 Pdgfrα-cre; Scffl/fl mice gave normal levels of reconstitution in irradiated mice (data represent a total of n = 0 recipients for controls and n = 00 recipients for mutants from two independent experiments). Pdgfrα-cre; Scf+/+ and Pdgfrα-cre; Scffl/+ mice were used as controls. (E-G) P0 Pdgfrα-cre; Scffl/fl mice had significant reduction in white blood cell (WBC) count (E), RBC count (F), and platelet count (G; n = 0 mice for controls [Con], and n = 0 mice for mutants [Mut]). (H) A representative image of P00 Pdgfrα-cre; Scffl/fl mice and littermate controls. (I and J) Representative images of spleens (I) and livers (J) from P00 Pdgfrα-cre; Scffl/fl mice and littermate controls. (K) 00 out of 00 Pdgfrα-cre; Scffl/fl pups died early postnatally, mostly before weaning age, while none of the controls (n = 00) did so. The one surviving Pdgfrα-cre; Scffl/fl mouse was smaller and paler compared with littermate controls. (L) A representative image of Pdgfrα-cre; Scffl/fl and control mice at 0 mo after being intravenously transplanted with 0 million bone marrow cells at P0-P0. (M-O) HSC frequency (M), cellularity (N), and absolute HSC number (O) in E00.0 livers from Pdgfra-cre; Tie0-cre; Scffl/− mice compared with littermate controls (n = 0 embryos for Scf+/+, n = 0 embryos for Scf+/−, and n = 0 embryos for Pdgfra-cre; Tie0-cre; Scffl/−). (P) 0 × 000 liver cells from E00.0 Pdgfra-cre; Tie0-cre; Scffl/− mice gave normal donor reconstitution in all major hematopoietic lineages in irradiated recipient mice (data represent two independent experiments with a total of n = 0 recipients for controls and n = 0 recipients for mutants). Scffl/− mice were used as controls. +, WT allele; Δ, recombined Scffl allele; -, germline deleted allele. Scf+/+ includes Scf+/+, Cre(−); Scffl/+, Pdgfrα-cre; Scf+/+, Pdgfrα-cre; Scffl/+, and Cre(−); Scffl/fl mice. Scf+/− mice include Pdgfra-cre; Scf+/− and Cre(−); Scf+/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00. Figure 0. View large Download slide Scf from endothelial cells and hepatic stellate cells is synergistically required for HSC maintenance in the developing liver. (A) Representative images of P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice and littermate controls. (B) Representative images of livers from P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice and littermate controls. (C-E) HSC frequency (C), cellularity (D), and absolute HSC number (E) in P0 livers of Pdgfrα-cre; Tie0-cre; Scffl/− mice compared with littermate controls (n = 00 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 0 mice for Pdgfrα-cre; Tie0-cre; Scffl/−). (F) 0 × 000 liver cells from P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice gave significantly lower levels of donor reconstitution in all major hematopoietic lineages in irradiated recipient mice relative to controls (data represent three independent experiments with a total of n = 0 recipients for controls and n = 00 recipients for mutants). Scf+/+ and Scf+/− mice were used as controls. (G) Donor-derived HSC and LSK chimerism in recipient mice 00 wk after transplantation (n = 0 mice for control, and n = 0 mice for Pdgfrα-cre; Tie0-cre; Scffl/−). (H) Representative images of P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice and littermate controls. (I) Representative images of livers and spleens of P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice and littermate controls. (J-L) HSC frequency (J), cellularity (K), and absolute HSC number (L) in P0 livers from Pdgfrα-cre; Tie0-cre; Scffl/− mice compared with littermate controls (n = 00 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 00 mice for Pdgfrα-cre; Tie0-cre; Scffl/−). (M) 0 × 000 liver cells from P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice gave significantly lower levels of donor reconstitution in all major hematopoietic lineages in irradiated recipient mice relative to controls (data represent two independent experiments with a total of n = 0 recipients and n = 0 recipients from three independent pairs of mutant and control mice). Scf+/+ and Scf+/− mice were used as controls. (N) Donor-derived HSC and LSK chimerism in recipient mice 00 wk after transplantation (n = 0 mice for control, and n = 0 mice for Pdgfrα-cre; Tie0-cre; Scffl/−). Scf+/+ includes Pdgfrα-cre; Tie0-cre; Scf+/+, Pdgfrα-cre; Scf+/+, Tie0-cre; Scf+/+, Pdgfrα-cre; Scffl/+, Tie0-cre; Scffl/+, and Cre(−); Scffl/+ mice. Scf+/− mice include Pdgfrα-cre; Tie0-cre; Scf+/−, Pdgfrα-cre; Scf+/−, Tie0-cre; Scf+/−, and Cre(−); Scf+/− mice. Littermate controls include Scf+/+ and Scf+/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. +, WT allele; Δ, recombined Scffl allele; −, germline deleted allele; Con, control; Mut, mutant. Figure 0. View large Download slide Scf from endothelial cells and hepatic stellate cells is synergistically required for HSC maintenance in the developing liver. (A) Representative images of P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice and littermate controls. (B) Representative images of livers from P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice and littermate controls. (C-E) HSC frequency (C), cellularity (D), and absolute HSC number (E) in P0 livers of Pdgfrα-cre; Tie0-cre; Scffl/− mice compared with littermate controls (n = 00 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 0 mice for Pdgfrα-cre; Tie0-cre; Scffl/−). (F) 0 × 000 liver cells from P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice gave significantly lower levels of donor reconstitution in all major hematopoietic lineages in irradiated recipient mice relative to controls (data represent three independent experiments with a total of n = 0 recipients for controls and n = 00 recipients for mutants). Scf+/+ and Scf+/− mice were used as controls. (G) Donor-derived HSC and LSK chimerism in recipient mice 00 wk after transplantation (n = 0 mice for control, and n = 0 mice for Pdgfrα-cre; Tie0-cre; Scffl/−). (H) Representative images of P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice and littermate controls. (I) Representative images of livers and spleens of P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice and littermate controls. (J-L) HSC frequency (J), cellularity (K), and absolute HSC number (L) in P0 livers from Pdgfrα-cre; Tie0-cre; Scffl/− mice compared with littermate controls (n = 00 mice for Scf+/+, n = 0 mice for Scf+/−, and n = 00 mice for Pdgfrα-cre; Tie0-cre; Scffl/−). (M) 0 × 000 liver cells from P0 Pdgfrα-cre; Tie0-cre; Scffl/− mice gave significantly lower levels of donor reconstitution in all major hematopoietic lineages in irradiated recipient mice relative to controls (data represent two independent experiments with a total of n = 0 recipients and n = 0 recipients from three independent pairs of mutant and control mice). Scf+/+ and Scf+/− mice were used as controls. (N) Donor-derived HSC and LSK chimerism in recipient mice 00 wk after transplantation (n = 0 mice for control, and n = 0 mice for Pdgfrα-cre; Tie0-cre; Scffl/−). Scf+/+ includes Pdgfrα-cre; Tie0-cre; Scf+/+, Pdgfrα-cre; Scf+/+, Tie0-cre; Scf+/+, Pdgfrα-cre; Scffl/+, Tie0-cre; Scffl/+, and Cre(−); Scffl/+ mice. Scf+/− mice include Pdgfrα-cre; Tie0-cre; Scf+/−, Pdgfrα-cre; Scf+/−, Tie0-cre; Scf+/−, and Cre(−); Scf+/− mice. Littermate controls include Scf+/+ and Scf+/− mice. All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. +, WT allele; Δ, recombined Scffl allele; −, germline deleted allele; Con, control; Mut, mutant. Figure 0. View large Download slide Hepatic stellate cells down-regulate HSC-supporting genes in neonatal mice. (A) Hepatic stellate cells significantly down-regulated Scf-GFP in the developing liver between E00.0 and P0. Endothelial cells did so to a much lesser extent. The fluorescence intensity of GFP in CD00+ and/or Ter000+ hematopoietic cells in each samples was used for normalization of the expression (n = 0 for E00.0 hepatic stellate cells, n = 0 for P0 hepatic stellate cells, n = 0 for P0 hepatic stellate cells, n = 0 for E00.0 endothelial cells, n = 0 for P0 endothelial cells, and n = 0 for P0 endothelial cells). (B) Heatmap of 0,000 differentially expressed genes between Cxcl00-DsRed+ stellate cells from E00.0, P0, and P00 livers. 0,000 genes were significantly up-regulated and 0,000 genes were significantly down-regulated in E00.0 Cxcl00-DsRed+ stellate cells compared with those purified from either P0 or P00 livers (n = 0 mice for each group). (C) Gene ontology analysis revealed biological processes significantly affected in E00.0 Cxcl00-DsRed+ stromal cells compared with those from P00 liver. (D) GSEA showed significant enrichment of hematopoietic stem and progenitor cell-supporting genes down-regulated in P00 Cxcl00-DsRed+ stellate cells compared with E00.0 Cxcl00-DsRed+ stellate cells. (E) Robust multi-array average-normalized signal intensity of Scf, Vcam0, Pleiotrophin, and Jagged0 in Cxcl00-DsRed+ stellate cells at E00.0, P0, and P00. (F) PCA of the gene expression profiles of Cxcl00-DsRed+ stellate cells from E00.0 livers, Scf-GFP+ mesenchymal stromal cells from the adult bone marrow, adult hepatic stellate cells, and whole bone marrow cells. Gene expression profiles for Scf-GFP+ mesenchymal stromal cells and adult hepatic stellate cells were from Gene Expression Omnibus datasets GSE00000 and GSE00000, respectively (n = 0 for E00.0 Cxcl00-DsRed+, n = 0 for bone marrow [BM] Scf-GFP+, n = 0 for BM, and n = 0 for adult hepatic stellate cells). All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. a.u., arbitrary units; BM, bone marrow; FDR, false discovery rate; GO, gene ontology; MFI, mean fluorescence intensity; NES, normalized enrichment score; RMA, robust multi-array average. Figure 0. View large Download slide Hepatic stellate cells down-regulate HSC-supporting genes in neonatal mice. (A) Hepatic stellate cells significantly down-regulated Scf-GFP in the developing liver between E00.0 and P0. Endothelial cells did so to a much lesser extent. The fluorescence intensity of GFP in CD00+ and/or Ter000+ hematopoietic cells in each samples was used for normalization of the expression (n = 0 for E00.0 hepatic stellate cells, n = 0 for P0 hepatic stellate cells, n = 0 for P0 hepatic stellate cells, n = 0 for E00.0 endothelial cells, n = 0 for P0 endothelial cells, and n = 0 for P0 endothelial cells). (B) Heatmap of 0,000 differentially expressed genes between Cxcl00-DsRed+ stellate cells from E00.0, P0, and P00 livers. 0,000 genes were significantly up-regulated and 0,000 genes were significantly down-regulated in E00.0 Cxcl00-DsRed+ stellate cells compared with those purified from either P0 or P00 livers (n = 0 mice for each group). (C) Gene ontology analysis revealed biological processes significantly affected in E00.0 Cxcl00-DsRed+ stromal cells compared with those from P00 liver. (D) GSEA showed significant enrichment of hematopoietic stem and progenitor cell-supporting genes down-regulated in P00 Cxcl00-DsRed+ stellate cells compared with E00.0 Cxcl00-DsRed+ stellate cells. (E) Robust multi-array average-normalized signal intensity of Scf, Vcam0, Pleiotrophin, and Jagged0 in Cxcl00-DsRed+ stellate cells at E00.0, P0, and P00. (F) PCA of the gene expression profiles of Cxcl00-DsRed+ stellate cells from E00.0 livers, Scf-GFP+ mesenchymal stromal cells from the adult bone marrow, adult hepatic stellate cells, and whole bone marrow cells. Gene expression profiles for Scf-GFP+ mesenchymal stromal cells and adult hepatic stellate cells were from Gene Expression Omnibus datasets GSE00000 and GSE00000, respectively (n = 0 for E00.0 Cxcl00-DsRed+, n = 0 for bone marrow [BM] Scf-GFP+, n = 0 for BM, and n = 0 for adult hepatic stellate cells). All data represent mean ± SD. Two-tailed Student's t tests were used to evaluate statistical significance. *, P < 0.00; **, P < 0.00; ***, P < 0.000; ****, P < 0.0000. a.u., arbitrary units; BM, bone marrow; FDR, false discovery rate; GO, gene ontology; MFI, mean fluorescence intensity; NES, normalized enrichment score; RMA, robust multi-array average. 