Mixed signals at tumor margins: The Hippo signaling pathway has been implicated in tumor growth, sparking interest in the pathway as a potential therapeutic target. In a study of liver cancer in genetically manipulated mice, Moya et al. discovered that the role of this pathway in tumorigenesis is more complex than previously appreciated. They confirmed that activation of the Hippo pathway within tumor cells drives tumor growth; however, they also found that activation of the pathway in adjacent healthy cells has the opposite effect, suppressing tumor growth. Whether tumor cells survive or are eliminated thus appears to depend on competing signals produced by the tumor and surrounding tissue. Science , this issue p. 1029

In the mammalian liver, hepatocytes exhibit diverse metabolic and functional profiles based on their location within the liver lobule. However, it is unclear whether this spatial variation, called zonation, is governed by a well-defined gene regulatory code. Here, using a combination of single-cell multiomics, spatial omics, massively parallel reporter assays and deep learning, we mapped enhancer-gene regulatory networks across mouse liver cell types. We found that zonation affects gene expression and chromatin accessibility in hepatocytes, among other cell types. These states are driven by the repressors TCF7L1 and TBX3, alongside other core hepatocyte transcription factors, such as HNF4A, CEBPA, FOXA1 and ONECUT1. To examine the architecture of the enhancers driving these cell states, we trained a hierarchical deep learning model called DeepLiver. Our study provides a multimodal understanding of the regulatory code underlying hepatocyte identity and their zonation state that can be used to engineer enhancers with specific activity levels and zonation patterns.

The Hippo signaling pathway is widely considered a master regulator of organ growth because of the prominent overgrowth phenotypes caused by experimental manipulation of its activity. Contrary to this model, we show here that removing Hippo transcriptional output did not impair the ability of the mouse liver and Drosophila eyes to grow to their normal size. Moreover, the transcriptional activity of the Hippo pathway effectors Yap/Taz/Yki did not correlate with cell proliferation, and hyperactivation of these effectors induced gene expression programs that did not recapitulate normal development. Concordantly, a functional screen in Drosophila identified several Hippo pathway target genes that were required for ectopic overgrowth but not normal growth. Thus, Hippo signaling does not instruct normal growth, and the Hippo-induced overgrowth phenotypes are caused by the activation of abnormal genetic programs.

The Hippo pathway and its downstream effectors YAP and TAZ (YAP/TAZ) are heralded as important regulators of organ growth and regeneration. However, different studies provided contradictory conclusions about their role during regeneration of different organs ranging from promoting proliferation to inhibiting it. Here, we resolve the function of YAP/TAZ during regeneration of the liver, where Hippo's role in growth control has been studied most intensely. We evaluated liver regeneration after CCl4 toxic liver injury in mice with conditional deletion of Yap/Taz in hepatocytes and/or biliary epithelial cells and measured the behavior of different cell types during regeneration by histology, RNA-sequencing and flow cytometry. We found that YAP/TAZ were activated in hepatocytes in response to CCl4 toxic injury. However, their targeted deletion in adult hepatocytes did not noticeably impair liver regeneration. In contrast, Yap/Taz deletion in adult bile ducts caused severe defects and delay in liver regeneration. Mechanistically, we show that Yap/Taz mutant bile ducts degenerated, causing cholestasis which stalled the recruitment of phagocytic macrophages and the removal of cellular corpses from injury sites. Elevated bile acids activated PXR, which was sufficient to recapitulate the phenotype observed in mutant mice. Our data show that YAP/TAZ are practically dispensable in hepatocytes for liver development and regeneration. Rather, YAP/TAZ play an indirect role in liver regeneration by preserving bile duct integrity and securing immune cell recruitment and function.

Inducible cyclization recombinase (Cre) transgenic mouse strains are powerful tools for cell lineage tracing and tissue-specific knockout experiments. However, low efficiency or leaky expression can be important pitfalls. Here, we compared the efficiency and specificity of two commonly used cholangiocyte-specific Cre drivers, the Opn-iCreER^T2 and Ck19-CreER^T drivers, using a tdTomato reporter strain. We found that Opn-iCreER^T2 triggered recombination of the tdTomato reporter in 99.9% of all cholangiocytes while Ck19-CreER^T only had 32% recombination efficiency after tamoxifen injection. In the absence of tamoxifen, recombination was also induced in 2% of cholangiocytes for the Opn-iCreER^T2 driver and in 13% for the Ck19-CreER^T driver. For both drivers, Cre recombination was highly specific for cholangiocytes since recombination was rare in other liver cell types. Toxic liver injury ectopically activated Opn-iCreER^T2 but not Ck19-CreER^T expression in hepatocytes. However, ectopic recombination in hepatocytes could be avoided by applying a three-day long wash-out period between tamoxifen treatment and toxin injection. Therefore, the Opn-iCreER^T2 driver is best suited for the generation of mutant bile ducts, while the Ck19-CreER^T driver has near absolute specificity for bile duct cells and is therefore favorable for lineage tracing experiments.

Fibrosis contributes to tissue repair, but excessive fibrosis disrupts organ function. Alagille syndrome (ALGS, caused by mutations in JAGGED1 ) results in liver disease and characteristic fibrosis. Here, we show that Jag1 ^Ndr/Ndr mice, a model for ALGS, recapitulate ALGS-like fibrosis. Single-cell RNA-seq and multi-color flow cytometry of the liver revealed immature hepatocytes and paradoxically low intrahepatic T cell infiltration despite cholestasis in Jag1 ^Ndr/Ndr mice. Thymic and splenic regulatory T cells (Tregs) were enriched and Jag1 ^Ndr/Ndr lymphocyte immune and fibrotic capacity was tested with adoptive transfer into Rag1 ^−/− mice, challenged with dextran sulfate sodium (DSS) or bile duct ligation (BDL). Transplanted Jag1 ^Ndr/Ndr lymphocytes were less inflammatory with fewer activated T cells than Jag1 ^+/+ lymphocytes in response to DSS. Cholestasis induced by BDL in Rag1 ^−/− mice with Jag1 ^Ndr/Ndr lymphocytes resulted in periportal Treg accumulation and three-fold less periportal fibrosis than in Rag1 ^−/− mice with Jag1 ^+/+ lymphocytes. Finally, the Jag1 ^Ndr/Ndr hepatocyte expression profile and Treg overrepresentation were corroborated in patients’ liver samples. Jag1-dependent hepatic and immune defects thus interact to determine the fibrotic process in ALGS.