Fig 1: Lack of Wnt ligand secretion from LSECs impairs adult zonation maintenance.(A) Double in situ hybridization for Rspo3 (green), Wnt9b (green) and Wnt2 (green) showing a few LSCEs (Lyve1+, red) expressing those transcripts (blue arrows and inset) in P2 livers. Only Wnt2 transcripts are detected in some LSECs (inset) in P60 livers. Arrows indicate central vein endothelial cells and arrowheads indicate LSECs. Scale bars: 25 µm. Each image is representative of 3 individual mice (n = 3). (B) Double-immunofluorescence results show that hepatic Zones 3 (GS+) and 2/3 (Cyp2e1+) are densely irrigated by the hepatic sinusoids (Lyve1+, arrows) in P2, P15 and P30 wildtype livers. The sinusoidal vasculature (arrows) is also in direct contact with claudin-2/GFP+ hepatocytes in P2, P15 and P30 Cldn2-EGFP livers. Scale bars: 50 µm. Each image is representative of 2–4 individual mice (n = 2–4). (C) P2, Quantitative double immunofluorescence results show that Zone 2 (Cyp2e1+, claudin-2/GFP+) is relatively unchanged and Zone 3 (GS+) is significantly reduced in Lyve1-Cre;Wlsf/f;Cldn2-GFP livers at P2. E-cadherin expression (arrows) is indistinguishable in P2 livers with or without endothelial Wls-deletion. P30, Similar quantitative results demonstrate that GS+ hepatocytes are nearly absent, Zone 2 (Cyp2e1+/GFP+) is significantly reduced and restricted to pericentral areas, and E-cadherin expression (arrows, arrowheads are GFP+ hepatocytes) is expanded towards the central veins in P30 Lyve1-Cre;Wlsf/f;Cldn2-GFP livers. 3–4 representative fields from three individual livers of each genotype were used for quantification. p values were determined by two-tailed unpaired Student’s t-test, NS, not significant (p>0.05), *p<0.05, ***p<0.001. Arrows indicate GFP-double positive hepatocytes, white arrowheads are GFP+ hepatocytes and yellow arrowheads are GFP– hepatocytes. Scale bars: 100 µm (D) Q-PCR results demonstrate reduced expression of Zone 2/3 transcripts, increased expression of Zone 1 transcripts, and normal levels of the hepatocyte transcript Prox1, in adult Lyve1-Cre;Wlsf/f livers (n = 3). p values were determined by two-way ANOVA, NS, not significant (p>0.05), *p<0.05, ***p<0.001. (E) Q-PCR results showing the effects of culturing AML-12 mouse hepatic cells with CHIR99021, Wnt2, Wntb9, or Wnt2/Wnt9b plus Rspo3 on Axin2, Cyp2e1, Glul and Cldn2 expression. p values from two-tailed unpaired Student’s t-test, *p<0.05, ***p<0.01; n = 6. (A–C) Asterisks indicate central vein lumens. Related data can be found in Figure 3—figure supplements 1–3.Figure 3—source data 1.Quantification of GS+, Cyp2e1+ and claudin-2/GFP+ areas and the relative abundance of claudin-2/GFP+ hepatocytes in P2 and P30 Lyve1-Cre;Wlsf/f;Cldn2-GFP livers, and Quantification of Wnt/ß-catenin target genes expression of P30 Lyve1-Cre;Wlsf/f;Cldn2-GFP livers.
Fig 2: Endothelial Wnt ligand secretion reestablishes metabolic zonation in the CCl4-acutely injured liver.(A) (Left) Schematic of CCl4 administration and tissue harvesting using Cldn2-EGFP mice. (Right) Double-immunofluorescence results show physical association of claudin-2/GFP+ hepatocytes (arrows) with hepatic endothelial cells (PECAM+) throughout the recovery period that follows CCl4-acute injury. (B) (Top, left) Schematic of the experimental strategy and tissue harvesting. ALT/AST serum levels demonstrate that CCl4 promotes liver damage in Cdh5-CreERT2 and Cdh5-CreERT2;Wlsf/f mice injected with tamoxifen. (n = 3). p values were determined by one-way ANOVA with Bonferroni’s multiple comparisons test, NS, not significant (p>0.05), ***p<0.001. (Right and bottom) Diagrams indicate the experimental strategy and tissue harvesting. Double-immunofluorescence and quantitative results show progressive expansion and full restoration of Zone 3 (GS+, arrows) and Zone 2 (Cyp2e1+, arrows) and transient macrophage infiltrates (F4/80+, arrows; arrowheads are Kupffer cells) in the liver of Cdh5-CreERT2 mice after acute CCl4 administration. In contrast, Zone 3 is nearly absent, Zone 2 is significantly smaller, and Zone 1 is expanded, in Cdh5-CreERT2;Wlsf/f livers 5-, 7- and 15 days post-CCl4. Perivenous macrophage infiltrates are observed in both Cdh5-CreERT2 and Cdh5-CreERT2;Wlsf/f livers 5–7 days post-CCl4 but not 15 days after CCl4 administration. p values were determined by two-tailed unpaired Student’s t-test, ***p<0.001, (n = 3). (C) (Left) Schematic of CCl4 administration and tissue harvesting. (Right) Triple-immunofluorescence results show that CD117 proteins are restricted to the Lyve-1+/Lyve-1LOW sinusoidal endothelium traversing Zones 2/3 in the normal and CCl4-injected (day 3) adult liver. (D) Schematic of CD117+ and Lyve1+ HECs isolation. Nonparenchymal liver cells (NPCs) were isolated using a two-step collagenase perfusion method and incubated with CD31-coated Dynabeads. The eluted CD31+ (HEC) fraction was incubated with CD117-coated Dynabeads to isolate CD117+CD31+ (‘pericentral/perivenous’) HECs. The unbound fraction from this step was incubated with Lyve-1-coated Dynabeads to separate Lyve+CD31+ hepatic sinusoidal cells from other HECs. (E) QRT-PCR results show comparable Lyve1 transcript expression in CD117+/CD31+ and Lyve1+/CD31+ isolates from saline-injected (‘control’) or CCl4-injected (day 3) livers, lower Kit expression in LSECs from injured livers compared to control livers, and higher Wnt2, Wnt9b and Rspo3 expression in LSECs from injured livers compared to control livers. (Three individual livers per condition were used to isolate LSECs.) (A–C) Each image represents 2–4 individual livers. Asterisks: central veins. (B) NS, not significant (p>0.05), ***p<0.001, (n = 3). Scale bars: 50 µm (C), 100 µm (A, B).Figure 6—source data 1.Quantification of ALT/AST serum levels, GS, Cyp2e1 immunofluorescence in Cdh5-CreERT2;Wlsf/f livers post-CCl4 injection.
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