Figure 4. Proliferation and not de novo expression restore Claudin-2⁺ and Cyp2e1⁺ hepatocytes in the CCl₄-injured liver.

(A) Experimental strategy for Cldn2-EGFP mice injected with CCl₄. (B) ALT/AST serum levels demonstrate liver damage 2 days post-CCl₄ (n = 3). p values were determined by two-way ANOVA, NS, not significant (p>0.05), ***p<0.001. (C) Expression of Zone 3 (GS⁺), Zone 2/3 (Cyp2e1⁺, claudin-2/GFP⁺) and Zone 1 (Ecad⁺) markers and Tbx3 in the corn oil-injected Cldn2-EGFP adult liver. (D) Spatial distribution of the markers indicated above during the recovery phase that follows a CCl₄ bolus (see text for details). (Day 2, arrow, enucleated GS⁺ cell. Days 3–7, arrows, GFP double-positive hepatocytes; arrowheads, GFP^– hepatocytes. Asterisks indicate central vein lumens.) (E) Quantification of GS⁺, Cyp2e1⁺ and claudin-2/GFP⁺ areas, and the GFP⁺/Cyp2e1⁺ ratio, 2–7 days post-CCl₄. 3–4 representative fields from three individual livers dissected at the indicated time points were used for quantification. p values were determined by one-way ANOVA with Bonferroni’s multiple comparisons test, NS, not significant (p>0.05), *p<0.05, **p<0.01, ***p<0.001. (F) (Top) Schematic of EdU administration and tissue harvesting post-CCl₄. (Middle) Immunofluorescence results show EdU incorporation (arrows) in Cyp2e1⁺ hepatocytes located in the Zone 2 remnant and the undamaged Zone 1 (Ecad⁺) 2 days post-CCl₄. Both Cyp2e1⁺ hepatocytes (arrows, top) and Ecad⁺ hepatocytes located at the margins of restored Zone 2 (arrows, bottom) retain the EdU label after a 5 day chase. (Bottom) Quantification of EdU⁺ hepatocytes in Zone 2/3 and Zone 1. 3–4 representative fields from three individual livers dissected at the indicated time points were used for quantification. p values were determined by two-tailed unpaired Student’s t-test, ***p<0.001. (C,D,F) asterisks and dots indicate central vein lumens and images are from 3 to 4 individual livers. (B, F) n = 3. NS, not significant (p>0.05), *p<0.05, **p<0.01, ***p<0.001. P, portal veins. Scale bars: 100 μm (C, D) and 50 μm (F). Related data can be found in Figure 4—figure supplements 1 and 2.

Figure 4—figure supplement 1. Changes in liver histology and Zone 2/3 protein expression after a CCl₄ bolus.

(A) Schematic of CCl₄ administration and tissue harvesting. (B) H and E staining reveals broad areas of necrosis separating the central vein (asterisks) from the hepatocyte area at day 2, abundant perivenous immune infiltrates at day 3, reduced immune infiltrates and partially restored parenchyma at day 5, and normal liver architecture at day 7, following acute CCl₄ administration. (Arrow show hepatocytes, arrowheads indicate immune infiltrates). (C) Immunohistochemistry analysis reveals very low GS expression (arrows) remaining around the central veins at day 2, a few GS⁺ nucleated cells separated from the central vein by a cellular infiltrate (arrowheads) at day 3, increased GS+ cells in perivenous areas at day 5, and normal GS expression in Zone 3 at day seven post-CCl₄. Expression of Cyp2e1 and claudin-2 proteins is limited to few hepatocytes located at the margins of the perivenous immune infiltrate at days 2–3. The Cyp2e1⁺ and claudin-2⁺ cell population continues to expand pericentrally (day 5) and looks restored 7 days post- CCl₄ administration. Each image represents 2–3 individual livers. Scale bars: 50 μm (B, C), 100 μm (A).

Figure 4—figure supplement 2. Sox9⁺ hepatocytes do not contribute to Zone 2/3 restoration after CCl₄ induced acute liver injury.

(A) Schematic of CCl₄ administration and tissue harvesting. (B) Double-immunofluorescence showing expression of the lineage tracer GFP (arrows) in hepatocytes surrounding the portal veins (P) and located in Zone 1 (E-cadherin⁺, arrowhead, right). GFP is not expressed in the restored Zone 2/3 (Cyp2e1⁺, arrowheads, left) up to 9 days after CCl₄-induced injury. Scale bar: 100 μm.