Livers from Fgf10−/− and Fgfr2b−/− embryos were smaller than wild-type littermate livers and were misshapen. When normalized to embryo weight, both mutant embryos exhibited statistically significant reductions in liver/embryo weight ratios than wild-type littermate livers (). Phospho-Histone H3 staining demonstrated a statistically significant reduction in overall proliferation in Fgf10−/− livers compared with Fgf10+/+ livers at E12.5 (). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling demonstrated a 4-fold increase in apoptosis in Fgf10−/− livers compared with Fgf10+/+ livers at E12.5 (). Using triple label immunofluorescence, we showed a significant reduction in the quantity of histone H3+ albumin+ pancytokeratin+ cells in E12.5 Fgf10−/− livers compared with wild-type littermates indicating a reduction in proliferating hepatoblasts ().
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 (more ...)
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 (more ...)
Kelly et al. initially characterized a transgenic mouse line, Fgf10+/LacZ
, with the β-galactosidase gene, LacZ
, under the control of endogenous Fgf10
regulatory sequences such that expression of LacZ
accurately reports Fgf10
Analysis of serial sections of X-gal stained Fgf10+/LacZ
embryo livers demonstrated Fgf10
expression in the mesenchyme of the septum transversum surrounding the hepatic diverticulum at E9 (). As the liver grows within the septum transversum, Fgf10
expression persists along the surface of the liver near the bile duct (). By E12.5, Fgf10
expression is scattered within the liver itself but is also concentrated along the mesenchyme of the extrahepatic portal structures (). By E14.5, parenchymal Fgf10
expression diminishes, persisting only along the extrahepatic portal vein and bile duct (not shown). This early peak in Fgf10
expression coincides temporally with maximal β-catenin activation as observed by β-galactosidase activity in the livers of TOPGAL
embryos (). Analyses of the temporal expression patterns of Fgf10
and genes encoding other FGFR2B ligands was performed via real-time RT-PCR on total RNA from pooled livers or individual wild-type liver embryos from E12.5 to E17.5 (); the patterns of expression were similar in either case. Fgf10
is maximally expressed at E12.5 followed by a greater than 5-fold reduction by E14.5. In comparison, Fgf1
message levels peak later at E17.5 and E15.5, respectively. Fgf3
expression was undetectable in the liver at all studied stages (data not shown.) Fgfr2b
expression increased gradually from E12.5 to E17.5.
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 (more ...)
Comparison of β-galactosidase activity in Fgf10+/LacZ and TOPGAL livers at E11.5, E12.5, and E18.5. Scale bar = 0.2 mm.
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.
To characterize the phenotypes of β-galactosidase expressing cells from Fgf10+/LacZ and TOPGAL livers, the livers were dissected at the time point when maximal β-galactosidase activity was present, respectively E12.5 in Fgf10+/LacZ and E13.5 in TOPGAL livers. Livers were digested to a single cell suspension. Cells were then stained for the hematopoietic cell marker CD45 and subjected to cell-sorting flow cytometry based on fluorescein content generated by β-galactosidase–driven conversion of FDG. Rosa 26 liver cells loaded with FDG demonstrated bright fluorescein staining (positive control), whereas FDG-loaded C57Bl/6 controls did not (negative control) (). The approximately 20% PI+ dead cells were excluded from analysis. Fifteen percent of the total cells from E12.5–13.5 livers demonstrated surface expression of CD45; these cells were removed from FACS isolation by gating techniques. 3.4 ± 2.0% of the total cells from E12.5 Fgf10+/LacZ livers were Fluorescein+ CD45− PI− (). 6.0 ± 2.5% of the total cells from E13.5 TOPGAL livers were Fluorescein+ CD45− PI− ().
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 (more ...)
To analyze these sorted cells for gene expression, total RNA was isolated for RT-PCR (). Fluorescein+
cells from E12.5 Fgf10+/LacZ
livers demonstrated expression of Fgf10
along with myofibroblastic markers, α-Sma
, and stellate cell markers, Desmin
data not shown), but display no detectable expression of the hepatoblast markers Albumin
, and Hnf4
α, nor of the hematopoietic marker CD45
, nor Fgfr2b
This expression profile indicated that we had isolated either 2 distinct cell populations (embryonic myofibroblasts and stellate cells) or a single population of cells with dual embryonic stellate/myofibroblastic phenotype, all of which express Fgf10
cells from E13.5 TOPGAL
livers expressed Albumin
α, and Fgfr2b
genes, but had no detectable expression of Fgf10
, or CD45
, indicating absence of mesenchymal cells. This expression profile is consistent with that of a bipotent hepatoblast. In addition, Fgfr2b
expression suggests that these cells may respond to FGF10.
To determine whether β-galactosidase expressing cells from TOPGAL livers truly represented hepatoblasts or a mixed population of hepatocytes and cholangiocytes, total RNA was immediately isolated from single individual DDAO+ CD45− cells isolated by FACS from E13.5 TOPGAL livers. RT-PCR was then performed for each single cell. Sixteen of 26 (61.5%) single cells demonstrated bipotent gene expression with Albumin and CK19 messenger RNA. In contrast, only 6.2% of adult hepatocytes had the same expression profile (P < 0.05). Thus, single-cell analysis confirms that at E13.5, the majority of the liver cells with active β-catenin signaling are hepatoblasts with bipotent gene expression (). However, approximately 30% of the cells yielded an amplicon for only 1 marker, suggesting that these cells had already committed to the hepatocyte or cholangiocyte lineage, respectively, unless an amplicon of the complementary marker was not generated due to sensitivity limitations of single-cell RT-PCR. Epifluorescence microscopy of sections of E12.5 livers from Fgf10+/LacZ embryos confirmed that β-galactosidase activity is present in distinct cells in close proximity to hepatoblasts coexpressing albumin and cytokeratin ().
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, (more ...)
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 (more ...)
To determine if FGF10 and FGFR2B act upstream of β-catenin signaling in the developing liver, both loss of function and gain of function experiments were performed. For loss of function, β-galactosidase activity was assessed via X-gal staining of TOPGAL
livers lacking FGFR2b signaling. Fgfr2b−/−
livers were smaller and displayed less β-galactosidase activity than livers of Fgfr2b+/+
littermates at both E11.5 () and E12.5 (not shown). Moreover, as a model of gain of function, E12.5 TOPGAL+/+
liver explants in Matrigel were treated with rFGF10 for 24 hours. They showed marked expansion of X-gal staining with more pronounced bridging of LacZ
-positive regions compared with nontreated controls (). Furthermore, treatment of E12.5 liver cells in culture with rFGF10 resulted in 2.9-fold increases in expression of CyclinD1
, a downstream target of β-catenin.19
Importantly, there was some β-galactosidase activity—albeit less in the Fgfr2b−/−
embryo livers—suggesting that other pathways for β-catenin activation are present.
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 (more ...)
To better quantify the effect of FGF10/FGFR2B signaling on the β-catenin pathway, we again used FACS analysis on liver cells from TOPGAL+/− embryos. E12.5 TOPGAL+/− liver cells in suspension were cultured overnight in DMEM, with or without rFGF10 or rWNT3A. Cells cultured with either rFGF10 or rWNT3A demonstrated a significant survival advantage compared with control cells after 24 hours as assessed via PI staining (). Additionally, the mean fluorescence index, which is a measure of the relative fluorescence of individual cells, was significantly higher in both rFGF10- and rWNT3A-treated cells compared with controls (control, 0.7 ± 0.3; rFGF10, 3.3 ± 1.0; rWNT3A, 3.7 ± 0.8 [P < 0.05]) indicating greater β-catenin activation per cell as seen by a shift of the scatter plots to the right (). Furthermore, the number of fluorescein+ cells increased significantly with treatment with rFGF10 (4.6-fold increase) or rWNT3A (5.1-fold increase) compared with controls (P < 0.05), indicating an increase in the number of cells manifesting β-catenin activation (). Using LacZ expression measured via real-time RT-PCR as a readout of β-catenin activation, treatment of TOPGAL+/− liver cells with rFGF10 for 12 hours resulted in a 1.7-fold increase in β-catenin activation, whereas treatment with rFGF9, which binds specifically to FGFR3b, had no effect. From these experiments, we infer that FGF10 can act on TOPGAL-positive hepatoblasts to promote their survival.
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 (more ...)