Cell Biology

BMP9 and BMP10 coordinate liver cellular crosstalk to maintain liver health

Dianyuan Zhao author has email address
Ziwei Huang
Xiaoyu Li
Huan Wang
Qingwei Hou
Yuyao Wang
Fang Yan
Wenting Yang
Di Liu
Shaoqiong Yi
Chunguang Han
Yanan Hao
Li Tang author has email address

State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China
Department of Immunology, School of Basic Medical Sciences, Anhui Medical University, Hefei, China
School of Basic Medicine, Qingdao University, Qingdao, China

https://doi.org/10.7554/eLife.95811.2

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Figures and data

HSCs are the major source of BMP9 and BMP10 in the liver.
(a) Quantitative PCR analysis of Bmp9 and Bmp10 expression in the liver from the indicated mice at the age of 8-14 weeks (n=4-6/group).
(b) Representative flow cytometric expression of tdTomato in HSCs, HCs, ECs, and KCs from Lrat^CreH11^tdTomato mice at the age of 8-9 weeks (n=3/group). Gating strategies were shown in Supplementary figure 1.
(c) Quantitative PCR analysis of Bmp10 expression in the right atrium from Bmp9^fl/flBmp10^fl/flLrat^Cre and their littermate controls at the age of 8-12 weeks (n=4/group).
(d) Representative flow cytometric expression of tdTomato in HSCs, HCs, ECs, and KCs from Alk1^tdTomato mice at the age of 8-12 weeks (n=3/group).
(e) Immunofluorescence images of liver sections from Bmp9^fl/flBmp10^fl/flLrat^Cre and their littermate controls at the age of 3-12 weeks (n=6/group). Scale bars: 100μm.
(f) Flow cytometric expression of Tim4 and VSIG4 in KCs (pregated on CD45⁺Ly6C^-F4/80⁺CD64⁺) and percentage of Tim4⁺VSIG4⁺ KCs from Bmp9^fl/flBmp10^fl/flLrat^Cre (n=5) and their littermate controls (n=3) at the age of 8-12 weeks.
(g) Flow cytometric expression of LYVE1 and CLEC2 in ECs (pregated on CD45^-CD31⁺) and percentage of LYVE1 ⁺CLEC2⁺ ECs from Bmp9^fl/flBmp10^fl/flLrat^Cre (n=3) and their littermate controls (n=3) at the age of 8-12 weeks. Results represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P <0.0001, by Mann-Whitney test (a,c,e-g).

The differentiation of liver macrophages was inhibited in Bmp9^fl/flBmp10^fl/flLrat^Cre mice.
(a,b) PCA and volcano plot of the RNA-seq data of sorted liver macrophages from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice at the age of 8-10 weeks (n=3/group). Genes upregulated and downregulated are shown in red and blue, respectively (fold change [FC] > 2, adjusted p[p-adj] < 0.05).
(c) Heatmap showing signature genes expressed differentially in liver macrophages from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice.
(d) Expression counts of indicated genes in liver macrophages from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice.***p-adj < 0.001.
(e) Immunofluorescence images of F4/80⁺ and CD64⁺ liver macrophages in sections from Bmp9^fl/flBmp10^fl/flLrat^Cre mice and their controls at the age of 12 weeks (n=5/group). Liver macrophages number was measured (right).
(f) Representative flow cytometric expression of CLEC2 and TREML4 in liver macrophages from the indicated mice at the age of 8-12 weeks (n=3-4/group).
(g,h) PCA and volcano plot of the RNA-seq data of sorted liver macrophages from Smad4^fl/flVav1^Cre and control mice at the age of 8-10 weeks (n=3/group). Genes upregulated and downregulated are shown in red and blue, respectively (fold change [FC] > 2, adjusted p[p-adj] < 0.05).
(i) Heatmap showing signature genes expressed differentially in liver macrophages from Smad4^fl/flVav1^Cre and control mice.
(j, k) Venn diagram showing DE genes (j) and transcription factors (k) specific to liver macrophages of Bmp9^fl/flBmp10^fl/flLrat^Cre, Smad4-deficient liver macrophages or shared between both mac populations. Results represent the mean ± SEM. *P < 0.05, by Mann-Whitney test (e).

Endothelial cells from Bmp9^fl/flBmp10^fl/flLrat^Cre mice are transdifferentiated to continuous endothelial cells.
(a,b) PCA and Volcano plot of the RNA-seq data of sorted hepatic ECs from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice at the age of 8-10 weeks (n=3/group).
(c) Expression counts of Gata4 and Maf genes in ECs from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice. ***p-adj < 0.001
(d, e) Heatmap showing sinusoidal EC-associated genes (d) and continuous EC-associated genes (e) expressed differentially in hepatic ECs from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice.
(f) Immunofluorescence images in liver sections from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice at the age of 12 weeks (n=4/group). CD34⁺ area was measured (right). Scale bars: 100μm.
(g) Immunofluorescence images in liver sections from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice at the age of 8 weeks (n=4-7/group). LYVE1⁺ and EMCN⁺ cells were measured (right). Scale bars: 100μm.
(h,i) Flow cytometric expression of CD32b, C-Maf, CD206 and CD117 in hepatic ECs from Bmp9^fl/flBmp10^fl/flLrat^Cre and littermate controls at the age of 8-10 weeks (n=4-6/group).
(j) Heatmap showing the indicated genes expressed differentially in hepatic ECs from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice. Results represent the mean± SEM. *P < 0.05 and **P <0.01, by Mann-Whitney test (f-i).

Liver metabolic zonation and iron metabolism are disrupted in Bmp9^fl/flBmp10^fl/flLrat^Cre mice.
(a) Heatmap showing the indicated genes expressed differentially in hepatic ECs from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice.
(b) Prussian blue in livers from Bmp9^fl/flBmp10^fl/flLrat^Cre mice and their controls at the age of 12 weeks (n=7/group).
(c) Immunofluorescence images in liver sections from Bmp9^fl/flBmp10^fl/flLrat^Cre mice and their controls at the age of 12 weeks (n=5/ group). Scale bars: 200μm.
(d) Heatmap showing the indicated genes expressed differentially in liver tissues from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice at the age of 12 weeks.
(e, f) Immunofluorescence images in liver sections from Bmp9^fl/flLrat^Cre (e) and Bmp10^fl/flLrat^Cre (f) mice and their controls at the age of 13-21 weeks (n=2/group). Scale bars: 200μm. Results represent the mean± SEM. **P < 0.01, by Mann-Whitney test (c, e, f).

Liver fibrosis occurs in Bmp9^fl/flBmp10^fl/flLrat^Cre mice.
(a) Liver weight and liver to body weight ratio of Bmp9^fl/flBmp10^fl/flLrat^Cre mice and their littermate controls at the age of 12 weeks.
(b) PSR staining of liver sections from Bmp9^fl/flBmp10^fl/flLrat^Cre mice and their controls at the age of 12 weeks (n=9-10/group). Scale bars: 100μm.
(c) Expression counts of Pdgfb gene in liver tissues (left), endothelial cells (middle) and liver macrophages (right) from Bmp9^fl/flBmp10f^{l/ fl}Lrat^Cre mice and their control mice. ***p-adj < 0.001.
(d) Immunofluorescence images of Desmin and collagen I in liver sections in liver tissues from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice at the age of 12-28 weeks (n=4-6/group).
(e) Collagen IV immunohistochemistry staining of liver sections from Bmp9^fl/flBmp10^fl/flLrat^Cre mice and their controls at the age of 12 weeks (n=4-5/group). Scale bars: 100μm.
(f) Expression counts of indicated genes in endothelial cells from Bmp9^fl/flBmp10^fl/flLrat^Cre and control mice. ***p-adj < 0.001. Results represent the mean± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P <0.0001, by Mann-Whitney test (a, b, d, e).

Gating strategies.
(a-d) Flow cytometry plots show representative pre-gating strategies for live CD45⁺Ly6C^-CD64⁺F4/80⁺ KCs from WT and Bmp9/10^HSC-KO mice (a), live CD45^-CD31⁺ ECs (b), live CD45^-CD31^-CD61⁺ HSCs (c), and live SSC^hiFSC^hi HCs (d) in the liver.

The phenotypes of KCs were not affected in the liver from Bmp9^fl/flBmp10^fl/flAlb^Cre, Bmp9^fl/flBmp10^fl/flTek^Cre and Bmp9^fl/flBmp10^fl/flClec4f^Cre mice.
(a) Representative immunofluorescence images of liver sections from the indicated mice at the age of 8-14 weeks (n=3-5/group). Scale bars: 100μm.
(b) Representative flow cytometric expression of Tim4 and VSIG4 in KCs from the indicated mice at the age of 8-12 weeks (n=3-5/group).

BMP10 can compensate the role of BMP9 in the liver.
(a) Quantitative PCR analysis of Bmp9 and Bmp10 expression in the liver from Bmp10^fl/flLrat^Cre and their littermate controls at the age of 12 weeks (n=4-6/group).
(b) Representative immunofluorescence images of liver sections from the indicated mice at the age of 14 weeks (n=2/group). Scale bars: 100μm.
(c, d) Representative flow cytometric expression of the indicated markers in KCs (c) and ECs (d) from Bmp10^fl/flLrat^Cre and their littermate controls at the age of 6-13 weeks (n=2-4/group). Results represent the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001, by Mann-Whitney test (a).

RXRα is required for the differentiation from blood monocytes to MoKCs.
(a) Representative flow cytometric expression of YFP in blood monocytes from Csf1r^CreR26^YFP and control mice at the age of 8-12 weeks (n=2/group).
(b, c) Representative flow cytometric expression (b) of CLEC2 and VSIG4 in liver macrophages, and percentage (c) of F4/80⁺, VSIG4⁺ and CLEC2⁺ liver macrophages from Rxra^fl/flCsf1r^CreClec4f^DTR mice and controls at the age of 11-12 weeks. The indicated mice were injected with 200ng/mice diphtheria toxin via IP. After 7 days, VSIG4 and CLEC2 expression in CD45⁺Ly6C^-CD64⁺F4/80⁺ liver macrophages were assessed. Results represent the mean ± SEM. *P<0.05, by Mann-Whitney test (c).

Transcription factors (TFs) downregulated in liver macrophages from BMP9/10-HSC KO mice and Smad4fl/fl Vav1-Cre mice.

Primers used for real-time PCR.

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