Fibroblast growth factor 10 is critical for liver growth during embryogenesis and controls hepatoblast survival via beta-catenin activation

Abstract

Fibroblast growth factor (FGF) signaling and beta-catenin activation have been shown to be crucial for early embryonic liver development. This study determined the significance of FGF10-mediated signaling in a murine embryonic liver progenitor cell population as well as its relation to beta-catenin activation. We observed that Fgf10(-/-) and Fgfr2b(-/-) mouse embryonic livers are smaller than wild-type livers; Fgf10(-/-) livers exhibit diminished proliferation of hepatoblasts. A comparison of beta-galactosidase activity as a readout of Fgf10 expression in Fgf10(+/LacZ) mice and of beta-catenin activation in TOPGAL mice, demonstrated peak Fgf10 expression from E9 to E13.5 coinciding with peak beta-catenin activation. Flow cytometric isolation and marker gene expression analysis of LacZ(+) cells from E13.5 Fgf10(+/LacZ) and TOPGAL livers, respectively, revealed that Fgf10 expression and beta-catenin signaling occur distinctly in stellate/myofibroblastic cells and hepatoblasts, respectively. Moreover, hepatoblasts express Fgfr2b, which strongly suggests they can respond to recombinant FGF10 produced by stellate cells. Fgfr2b(-/-)/TOPGAL(+/+) embryonic livers displayed less beta-galactosidase activity than livers of Fgfr2b(+/+)/TOPGAL(+/+) littermates. In addition, cultures of whole liver explants in Matrigel or cell in suspension from E12.5 TOPGAL(+/+)mice displayed a marked increase in beta-galactosidase activity and cell survival upon treatment with recombinant FGF10, indicating that FGFR (most likely FGFR2B) activation is upstream of beta-catenin signaling and promote hepatoblast survival.

Conclusion: Embryonic stellate/myofibroblastic cells promote beta-catenin activation in and survival of hepatoblasts via FGF10-mediated signaling. We suggest a role for stellate/myofibroblastic FGF10 within the liver stem cell niche in supporting the proliferating hepatoblast.

Fig. 1

Comparison of liver mass of wild-type and Fgf10^−/− or Fgfr2b^−/− embryos. (A, B) Whole mount P0 Fgf10^+/+ and Fgf10^−/− livers. (C, D) Whole mount E18.5 Fgfr2b^+/+ and Fgfr2b^−/− livers. (E) Graphic representation of respective liver/embryo weight ratios of both null mutants and their wild-type littermates. *P < 0.05; n = 10. Scale bar = 1 mm.

Fig. 2

Comparison of cell proliferation and apoptosis in E12.5 Fgf10^−/− livers. (A–C) Immunofluorescence imaging of phosphorylated histone H3 in Fgf10^−/− and wild-type livers (n = 16). (D–F) Immunofluorescence imaging of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling in Fgf10^−/− and wild-type livers (n = 8). (G–I) Immunofluorescence imaging of phosphorylated histone H3, albumin, and pancytokeratin in Fgf10^−/− and wild-type livers (n = 8). *P < 0.05.

Fig. 3

Spatial and temporal expression pattern of β-galactosidase activity in Fgf10^+/LacZ embryos early during hepatogenesis. (A) E9 (original magnification ×20). (B) E10.5 (original magnification ×20). (C, D) BABB cleared whole mount E12.5 liver. Arrow indicates staining along extrahepatic portal structures (original magnification ×2.5, ×10). (E, F) E12.5 (original magnification ×20, ×40). Abbreviations: b, bile duct; hd, hepatic diverticulum; p, portal vein; st, septum transversum.

Fig. 4

Comparison of β-galactosidase activity in Fgf10^+/LacZ and TOPGAL livers at E11.5, E12.5, and E18.5. Scale bar = 0.2 mm.

Fig. 5

Analysis of relative expression levels of messenger RNAs encoding (A) Fgf10, (B) Fgf1, (C) Fgf7, and (D) Fgfr2b from E12.5 to E17.5 via real-time RT-PCR.

Fig. 6

Flow cytometric and marker gene analyses of fluorescein⁺ cells from Fgf10^+/LacZ and TOPGAL embryo livers. (A–C) Validation of flow cytometric cell sorting based on β-galactosidase–generated fluorescein in livers from C57Bl/6 and Rosa 26 mice. *P < 0.05. (D, E) Flow cytometic sorting of cells from Fgf10^+/LacZ and TOPGAL embryo livers. (F) Gene expression analysis of fluorescein⁺ CD45⁻ cells from either transgenic strain via RT-PCR.

Fig. 7

Gene expression analysis of single isolated TOPGAL-expressing cells. (A) A representative experiment of single-cell RT-PCR on individual fluorescein⁺ CD45⁻ cells from TOPGAL livers. Bone marrow cells and adult liver are negative and positive control, respectively. (B) Comparison of percentage of Albumin and Ck19 coexpressed by fluorescein⁺ CD45⁻ cells E13.5 TOPGAL liver and by mature murine hepatocytes.

Fig. 8

Immunofluorescence colocalization of pancytokeratin and albumin in proximity to Fgf10-expressing cells in E12.5 liver. (A) X-gal staining for β-galactosidase activity. (B, C) Immunodetection of albumin and pancytokeratin. (D) Merged image shows distinct Fgf10 expression (blue) in close proximity to putative hepatoblasts coexpressing cytokeratin and albumin (yellow).

Fig. 9

Comparison of β-galactosidase activity in E11.5 TOPGAL^+/− livers. (A) Fgfr2b^+/+. (B) Fgfr2b^−/−. (C) DMEM alone (original magnification ×2.5). (D) DMEM + rFGF10 (original magnification ×2.5). (E) Inset of panel C (original magnification ×10). (F) Inset of panel D (original magnification ×10). (F’) Additional view of liver in panel D (original magnification ×10).

Fig. 10

Treatment of E12.5 TOPGAL liver cells with rFGF10 or rWNT3A. (A) FACS scatter plots of TOPGAL E12.5 liver cells cultured overnight in DMEM (left), with presence of rFGF10 (middle) or rWNT3A (right). (B) Graphic representation showing relative number of LacZ-expressing (fluorescein⁺) cells upon rFGF10 treatment of TOPGAL liver cells. (C) Graphic representation of percentage of PI⁺ cells with either rFGF10 or rWNT3A treatment. *P < 0.05 compared with control; n = 6.