Compensatory hepatic adaptation accompanies permanent absence of intrahepatic biliary network due to YAP1 loss in liver progenitors

Abstract

Yes-associated protein 1 (YAP1) regulates cell plasticity during liver injury, regeneration, and cancer, but its role in liver development is unknown. We detect YAP1 activity in biliary cells and in cells at the hepatobiliary bifurcation in single-cell RNA sequencing analysis of developing livers. Deletion of Yap1 in hepatoblasts does not impair Notch-driven SOX9+ ductal plate formation but does prevent the formation of the abutting second layer of SOX9+ ductal cells, blocking the formation of a patent intrahepatic biliary tree. Intriguingly, these mice survive for 8 months with severe cholestatic injury and without hepatocyte-to-biliary transdifferentiation. Ductular reaction in the perihilar region suggests extrahepatic biliary proliferation, likely seeking the missing intrahepatic biliary network. Long-term survival of these mice occurs through hepatocyte adaptation via reduced metabolic and synthetic function, including altered bile acid metabolism and transport. Overall, we show YAP1 as a key regulator of bile duct development while highlighting a profound adaptive capability of hepatocytes.

Keywords: 3D-tissue clearing and imaging; Hippo signaling; bile acid metabolism; bile duct development; intrahepatic bile duct; intravital microscopy; liver adaptation; liver development; liver regeneration.

Figure 1.. Loss of YAP1 in HBs leads to failure of intrahepatic bile duct formation

(A) Gross image of WT and YAP1 KO mouse. The arrow shows jaundiced ears. (B and C) Body weight (B) and liver weight-to-body weight (LW/BW) ratio (C) of WT and KO mice over time. (D–H) Serum levels of (D) alanine aminotransferase (ALT), (E) aspartate aminotransferase (AST), (F) total bilirubin, (G) direct bilirubin, and (H) alkaline phosphatase in WT and KO mice over time. Graphs show mean ± SD. Data were analyzed by 2-way ANOVA with Sidak multiple comparison test, n = 2–5 mice per group (*p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (I) IHC for YAP1 in WT mice. Arrows show nuclear YAP1 in bile ducts. (J and K) CK19 marks bile ducts in the liver (J) spanning the three main regions described in (K). (L) IHC for YAP1 in KO mice. (M) CK19 staining in various lobes of YAP1 KO mice. Asterisks mark the gallbladders. (I)–(M) represent adult mice age 3–4 months old. Scale bars are 500 μm for whole lobes, 100 μm for insets.

Figure 2.. YAP1 KO mice show no long-term regeneration of bile ducts and no transdifferentiation of hepatocytes into cholangiocytes

(A–D) IF for CK19 followed by ribbon-confocal scanning microscopy illustrates in 3D the mature biliary tree of WT mice at (A) P21 and (C) 8 months of age and shows the absence of bile ducts in YAP1 KO mice at (B) P21 and (D) 8 months of age. Scale bars are 2 mm. Regions 1, 2, and 3 refer approximately to the expected positions of perihilar ducts, intrahepatic large ducts, and intrahepatic small ducts, respectively. (E) IHC for YAP1 in adult WT mice. Arrows highlight bile ducts. (F) IHC for YAP1 in adult KO mice. Arrows highlight ductular reaction. Scale bars are 100 μm. (G) IF co-staining for YAP1, CK19, and HNF4α in WT and YAP1 KO mice (203 magnification). (H) IF co-staining for HNF4α and EYFP in YAP1 KO mice (20× magnification). (I) Quantification of EYFP-positive, HNF4α-positive cells in adult YAP1 KO mice (mean ± SD, n = 5 mice, representing the average of 3–5 20× fields per mouse).

Figure 3.. YAP1 KO mice adapt to chronic cholestasis by reducing bile acid toxicity and secreting them into the bloodstream

(A) Cannulation of the common bile duct was used to measure baseline bile flow in adult WT and YAP1 KO mice (3–5 mice per group, two-tailed Mann-Whitney test, *p < 0.05). (B) GSEA revealed several metabolic pathways negatively enriched in YAP1 KO mice versus WT. (C–F) RNA-seq analysis shows altered gene expression of genes related to bile acid synthesis and excretion (*q < 0.05, **q < 0.01, ***q < 0.001, ****q < 0.0001). (G) Mass spectrometry was used to measure the abundance of bile acid species in liver tissue and serum from WT and YAP1 KO mice (n of 8 WT with 4 males and 4 females and 7 KO with 4 males and 3 females; data show mean ± SD; 2-way ANOVA with Sidak multiple comparison test, *p < 0.05, ****p < 0.0001). (H) Hydrophobicity index of the bile acid pool in liver and serum was calculated based on the Heuman index values for each bile acid species (Heuman, 1989) (mean ± SD, t test, ****p < 0.0001). Underneath, we include the percentages of total bile acids used in each calculation, because the index values for certain species are unavailable. (I) Average distribution and abundance of murine bile acid species are shown for WT and YAP1 mice. Asterisks refer to conjugated bile acids. (J) Still shots taken from live movies (Video S2) from intravital microscopy, showing the circulation of blood and bile in both WT and YAP1 KO mice.

Figure 4.. YAP1 KO leads to defective bile duct morphogenesis in late liver development

(A–D) IHC for SOX9 in WT and YAP1 KO livers at (A) E14.5, (B) E16.5, (C) E17.5, and (D) P9. Arrows and insets point to various stages of bile duct development as described in the text. (E) IF co-staining for CK8 and acetylated tubulin at E17.5 in WT and YAP1 KO livers, showing bile ducts at intermediate stages of maturation. Yellow arrows highlight punctate tubulin staining marking primary cilia. (F) IF co-staining for CK8, pan-laminin, and HNF4α at E17.5 in WT and YAP1 KO livers, showing bile ducts at intermediate stages of maturation. Yellow arrows mark laminin deposition in the basement membrane of maturing WT ducts, whereas white boxes show an immature duct in KO mice with no laminin deposition. Scale bars are (A, E, and F) 50 μm and (B–D) 100 μm. Representative images chosen after analyzing n = 3–4 mice in each group.