Loss of Hepatic Leucine-Rich Repeat-Containing G-Protein Coupled Receptors 4 and 5 Promotes Nonalcoholic Fatty Liver Disease.

The roof plate-specific spondin-leucine-rich repeat-containing G-protein coupled receptor 4/5 (LGR4/5)-zinc and ring finger 3 (ZNRF3)/ring finger protein 43 (RNF43) module is a master regulator of hepatic Wnt/ß-catenin signaling and metabolic zonation. However, its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. The current study investigated whether hepatic epithelial cell-specific loss of the Wnt/ß-catenin modulator Lgr4/5 promoted NAFLD. The 3- and 6-month-old mice with hepatic epithelial cell-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were compared with control mice fed with normal diet (ND) or high-fat diet (HFD). Six-month-old HFD-fed Lgr4/5dLKO mice developed hepatic steatosis and fibrosis but the control mice did not. Serum cholesterol-high-density lipoprotein and total cholesterol levels in 3- and 6-month-old HFD-fed Lgr4/5dLKO mice were decreased compared with those in control mice. An ex vivo primary hepatocyte culture assay and a comprehensive bile acid (BA) characterization in liver, plasma, bile, and feces demonstrated that ND-fed Lgr4/5dLKO mice had impaired BA secretion, predisposing them to develop cholestatic characteristics. Lipidome and RNA-sequencing analyses demonstrated severe alterations in several lipid species and pathways controlling lipid metabolism in the livers of Lgr4/5dLKO mice. In conclusion, loss of hepatic Wnt/ß-catenin activity by Lgr4/5 deletion led to loss of BA secretion, cholestatic features, altered lipid homeostasis, and deregulation of lipoprotein pathways. Both BA and intrinsic lipid alterations contributed to the onset of NAFLD.

The RSPO-LGR4/5-ZNRF3/RNF43 module in liver homeostasis, regeneration, and disease.

WNT/ß-catenin signaling plays pivotal roles during liver development, homeostasis, and regeneration. Likewise, its deregulation disturbs metabolic liver zonation and is responsible for the development of a large number of hepatic tumors. Liver fibrosis, which has become a major health burden for society and a hallmark of NASH, can also be promoted by WNT/ß-catenin signaling. Upstream regulatory mechanisms controlling hepatic WNT/ß-catenin activity may constitute targets for the development of novel therapies addressing these life-threatening conditions. The R-spondin (RSPO)-leucine-rich repeat-containing G protein-coupled receptor (LGR) 4/5-zinc and ring finger (ZNRF) 3/ring finger 43 (RNF43) module is fine-tuning WNT/ß-catenin signaling in several tissues and is essential for hepatic WNT/ß-catenin activity. In this review article, we recapitulate the role of the RSPO-LGR4/5-ZNRF3/RNF43 module during liver development, homeostasis, metabolic zonation, regeneration, and disease. We further discuss the controversy around LGR5 as a liver stem cell marker.

mTORC1 signaling suppresses Wnt/ß-catenin signaling through DVL-dependent regulation of Wnt receptor FZD level.

Wnt/ß-catenin signaling plays pivotal roles in cell proliferation and tissue homeostasis by maintaining somatic stem cell functions. The mammalian target of rapamycin (mTOR) signaling functions as an integrative rheostat that orchestrates various cellular and metabolic activities that shape tissue homeostasis. Whether these two fundamental signaling pathways couple to exert physiological functions still remains mysterious. Using a genome-wide CRISPR-Cas9 screening, we discover that mTOR complex 1 (mTORC1) signaling suppresses canonical Wnt/ß-catenin signaling. Deficiency in tuberous sclerosis complex 1/2 (TSC1/2), core negative regulators of mTORC1 activity, represses Wnt/ß-catenin target gene expression, which can be rescued by RAD001. Mechanistically, mTORC1 signaling regulates the cell surface level of Wnt receptor Frizzled (FZD) in a Dishevelled (DVL)-dependent manner by influencing the association of DVL and clathrin AP-2 adaptor. Sustained mTORC1 activation impairs Wnt/ß-catenin signaling and causes loss of stemness in intestinal organoids ex vivo and primitive intestinal progenitors in vivo. Wnt/ß-catenin-dependent liver metabolic zonation gene expression program is also down-regulated by mTORC1 activation. Our study provides a paradigm that mTORC1 signaling cell autonomously regulates Wnt/ß-catenin pathway to influence stem cell maintenance.

GABA suppresses neurogenesis in the adult hippocampus through GABAB receptors.

Adult neurogenesis is tightly regulated through the interaction of neural stem/progenitor cells (NSCs) with their niche. Neurotransmitters, including GABA activation of GABAA receptor ion channels, are important niche signals. We show that adult mouse hippocampal NSCs and their progeny express metabotropic GABAB receptors. Pharmacological inhibition of GABAB receptors stimulated NSC proliferation and genetic deletion of GABAB1 receptor subunits increased NSC proliferation and differentiation of neuroblasts in vivo. Cell-specific conditional deletion of GABAB receptors supports a cell-autonomous role in newly generated cells. Our data indicate that signaling through GABAB receptors is an inhibitor of adult neurogenesis.

YAP promotes proliferation, chemoresistance, and angiogenesis in human cholangiocarcinoma through TEAD transcription factors.

UNLABELLED: The Yes-associated protein (YAP)/Hippo pathway has been implicated in tissue development, regeneration, and tumorigenesis. However, its role in cholangiocarcinoma (CC) is not established. We show that YAP activation is a common feature in CC patient biopsies and human CC cell lines. Using microarray expression profiling of CC cells with overexpressed or down-regulated YAP, we show that YAP regulates genes involved in proliferation, apoptosis, and angiogenesis. YAP activity promotes CC growth in vitro and in vivo by functionally interacting with TEAD transcription factors (TEADs). YAP activity together with TEADs prevents apoptosis induced by cytotoxic drugs, whereas YAP knockdown sensitizes CC cells to drug-induced apoptosis. We further show that the proangiogenic microfibrillar-associated protein 5 (MFAP5) is a direct transcriptional target of YAP/TEAD in CC cells and that secreted MFAP5 promotes tube formation of human microvascular endothelial cells. High YAP activity in human CC xenografts and clinical samples correlates with increased MFAP5 expression and CD31(+) vasculature. CONCLUSIONS: These findings establish YAP as a key regulator of proliferation and antiapoptotic mechanisms in CC and provide first evidence that YAP promotes angiogenesis by regulating the expression of secreted proangiogenic proteins.

Functional roles of Lgr4 and Lgr5 in embryonic gut, kidney and skin development in mice.

Lgr4 and Lgr5 are known markers of adult and embryonic tissue stem cells in various organs. However, whether Lgr4 and Lgr5 are important for embryonic development remains unclear. To study their functions during intestinal crypt, skin and kidney development we now generated mice lacking either Lgr4 (Lgr4KO), Lgr5 (Lgr5KO) or both receptors (Lgr4/5dKO). E16.5 Lgr4KO mice displayed complete loss of Lgr5+/Olfm4+intestinal stem cells, compromised Wnt signaling and impaired proliferation and differentiation of gut epithelium. Similarly, E16.5 Lgr4KO mice showed reduced basal cell proliferation and hair follicle numbers in the developing skin, as well as dilated kidney tubules and ectatic Bowman׳s spaces. Although Lgr4KO and Lgr5KO mice both died perinatally, Lgr5 deletion did not compromise embryonic development of gut, kidney or skin. Concomitant deletion of Lgr4 and Lgr5 did not prevent perinatal lethality, in contrast to a previous report that suggested rescue of Lgr5 KO perinatal lethality by a hypomorphic Lgr4 mutant. While the double deletion did not further promote the phenotypes observed in Lgr4KO intestines, impaired kidney cell proliferation, reduced epidermal thickness, loss of Lgr5+follicular epithelium and impaired hair follicle development were only observed in Lgr4/5dKO mice. This supports complementary functions of both receptors. Our findings clearly establish the importance of Lgr4 and Lgr5 during embryonic gut, skin and kidney development, with a dominant role of Lgr4.

Constitutive Notch2 signaling induces hepatic tumors in mice.

UNLABELLED: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCC) are the most common liver tumors and a leading cause for cancer-related death in men. Notch2 regulates cellular differentiation in the developing and adult liver. Although aberrant Notch signaling is implicated in various cancers, it is still unclear whether Notch2 regulates proliferation and differentiation in liver carcinogenesis and thereby contributes to HCC and CCC formation. Here, we investigated the oncogenic potential of constitutive Notch2 signaling in the liver. We show that liver-specific expression of the intracellular domain of Notch2 (N2ICD) in mice is sufficient to induce HCC formation and biliary hyperplasia. Specifically, constitutive N2ICD signaling in the liver leads to up-regulation of pro-proliferative genes and proliferation of hepatocytes and biliary epithelial cells (BECs). Using the diethylnitrosamine (DEN) HCC carcinogenesis model, we further show that constitutive Notch2 signaling accelerates DEN-induced HCC formation. DEN-induced HCCs with constitutive Notch2 signaling (DEN(N2ICD) HCCs) exhibit a marked increase in size, proliferation, and expression of pro-proliferative genes when compared with HCCs from DEN-induced control mice (DEN(ctrl) HCCs). Moreover, DEN(N2ICD) HCCs exhibit increased Sox9 messenger RNA (mRNA) levels and reduced Albumin and Alpha-fetoprotein mRNA levels, indicating that they are less differentiated than DEN(ctrl) HCCs. Additionally, DEN(N2ICD) mice develop large hepatic cysts, dysplasia of the biliary epithelium, and eventually CCC. CCC formation in patients and DEN(N2ICD) mice is accompanied by re-expression of hepatocyte nuclear factor 4α(HNF4α), possibly indicating dedifferentiation of BECs. CONCLUSION: Our data establish an oncogenic role for constitutive Notch2 signaling in liver cancer development.

Hepatic stellate cells: From bad reputation to mediators of liver homeostasis.

Organ size and function critically depend on the tight regulation of cellular turnover. In this issue of Science Signaling, Trinh et al. reveal that hepatic stellate cells play an important role in maintaining liver homeostasis by stimulating midzonal hepatocyte proliferation through the secretion of neurotrophin-3.

Distinct hepatocyte identities in liver homeostasis and regeneration.

The process of metabolic liver zonation is spontaneously established by assigning distributed tasks to hepatocytes along the porto-central blood flow. Hepatocytes fulfil critical metabolic functions, while also maintaining hepatocyte mass by replication when needed. Recent technological advances have enabled us to fine-tune our understanding of hepatocyte identity during homeostasis and regeneration. Subsets of hepatocytes have been identified to be more regenerative and some have even been proposed to function like stem cells, challenging the long-standing view that all hepatocytes are similarly capable of regeneration. The latest data show that hepatocyte renewal during homeostasis and regeneration after liver injury is not limited to rare hepatocytes; however, hepatocytes are not exactly the same. Herein, we review the known differences that give individual hepatocytes distinct identities, recent findings demonstrating how these distinct identities correspond to differences in hepatocyte regenerative capacity, and how the plasticity of hepatocyte identity allows for division of labour among hepatocytes. We further discuss how these distinct hepatocyte identities may play a role during liver disease.

Spatiotemporal Metabolic Liver Zonation and Consequences on Pathophysiology.

Hepatocytes are the main workers in the hepatic factory, managing metabolism of nutrients and xenobiotics, production and recycling of proteins, and glucose and lipid homeostasis. Division of labor between hepatocytes is critical to coordinate complex complementary or opposing multistep processes, similar to distributed tasks at an assembly line. This so-called metabolic zonation has both spatial and temporal components. Spatial distribution of metabolic function in hepatocytes of different lobular zones is necessary to perform complex sequential multistep metabolic processes and to assign metabolic tasks to the right environment. Moreover, temporal control of metabolic processes is critical to align required metabolic processes to the feeding and fasting cycles. Disruption of this complex spatiotemporal hepatic organization impairs key metabolic processes with both local and systemic consequences. Many metabolic diseases, such as nonalcoholic steatohepatitis and diabetes, are associated with impaired metabolic liver zonation. Recent technological advances shed new light on the spatiotemporal gene expression networks controlling liver function and how their deregulation may be involved in a large variety of diseases. We summarize the current knowledge about spatiotemporal metabolic liver zonation and consequences on liver pathobiology.

Functional ProTracer identifies patterns of cell proliferation in tissues and underlying regulatory mechanisms.

A genetic system, ProTracer, has been recently developed to record cell proliferation in vivo. However, the ProTracer is initiated by an infrequently used recombinase Dre, which limits its broad application for functional studies employing floxed gene alleles. Here we generated Cre-activated functional ProTracer (fProTracer) mice, which enable simultaneous recording of cell proliferation and tissue-specific gene deletion, facilitating broad functional analysis of cell proliferation by any Cre driver.

Bipotent transitional liver progenitor cells contribute to liver regeneration.

Following severe liver injury, when hepatocyte-mediated regeneration is impaired, biliary epithelial cells (BECs) can transdifferentiate into functional hepatocytes. However, the subset of BECs with such facultative tissue stem cell potential, as well as the mechanisms enabling transdifferentiation, remains elusive. Here we identify a transitional liver progenitor cell (TLPC), which originates from BECs and differentiates into hepatocytes during regeneration from severe liver injury. By applying a dual genetic lineage tracing approach, we specifically labeled TLPCs and found that they are bipotent, as they either differentiate into hepatocytes or re-adopt BEC fate. Mechanistically, Notch and Wnt/ß-catenin signaling orchestrate BEC-to-TLPC and TLPC-to-hepatocyte conversions, respectively. Together, our study provides functional and mechanistic insights into transdifferentiation-assisted liver regeneration.

Multicellular dynamics of zonal liver regeneration mapped in space and time.

In this issue, Ben-Moshe et al. (2022) use spatiotemporally resolved single-cell and spatial transcriptomic profiling to dissect the multicellular dynamics enabling zonal liver regeneration. They highlight how pan-zonal compensatory hepatocyte proliferation, transient reprogramming of peri-injury hepatocytes, and concerted zonated action of different liver cell types orchestrate the healing process.

Genome-wide screening in human kidney organoids identifies developmental and disease-related aspects of nephrogenesis.

Human organoids allow the study of proliferation, lineage specification, and 3D tissue development. Here we present a genome-wide CRISPR screen in induced pluripotent stem cell (iPSC)-derived kidney organoids. The combination of inducible genome editing, longitudinal sampling, and endpoint sorting of tubular and stromal cells generated a complex, high-quality dataset uncovering a broad spectrum of insightful biology from early development to "adult" epithelial morphogenesis. Our functional dataset allows improving mesoderm induction by ROCK inhibition, contains monogenetic and complex trait kidney disease genes, confirms two additional congenital anomalies of the kidney and urinary tract (CAKUT) genes (CCDC170 and MYH7B), and provides a large candidate list of ciliopathy-related genes. Finally, identification of a cis-inhibitory effect of Jagged1 controlling epithelial proliferation shows how mosaic knockouts in pooled CRISPR screening can reveal ways of communication between heterogeneous cell populations in complex tissues. These data serve as a rich resource for the kidney research community and as a benchmark for future iPSC-derived organoid CRISPR screens.

Cell adhesion molecule KIRREL1 is a feedback regulator of Hippo signaling recruiting SAV1 to cell-cell contact sites.

The Hippo/YAP pathway controls cell proliferation through sensing physical and spatial organization of cells. How cell-cell contact is sensed by Hippo signaling is poorly understood. Here, we identified the cell adhesion molecule KIRREL1 as an upstream positive regulator of the mammalian Hippo pathway. KIRREL1 physically interacts with SAV1 and recruits SAV1 to cell-cell contact sites. Consistent with the hypothesis that KIRREL1-mediated cell adhesion suppresses YAP activity, knockout of KIRREL1 increases YAP activity in neighboring cells. Analyzing pan-cancer CRISPR proliferation screen data reveals KIRREL1 as the top plasma membrane protein showing strong correlation with known Hippo regulators, highlighting a critical role of KIRREL1 in regulating Hippo signaling and cell proliferation. During liver regeneration in mice, KIRREL1 is upregulated, and its genetic ablation enhances hepatic YAP activity, hepatocyte reprogramming and biliary epithelial cell proliferation. Our data suggest that KIRREL1 functions as a feedback regulator of the mammalian Hippo pathway through sensing cell-cell interaction and recruiting SAV1 to cell-cell contact sites.

Proliferation tracing reveals regional hepatocyte generation in liver homeostasis and repair.

Organ homeostasis is orchestrated by time- and spatially restricted cell proliferation. Studies identifying cells with superior proliferative capacities often rely on the lineage tracing of a subset of cell populations, which introduces a potential selective bias. In this work, we developed a genetic system [proliferation tracer (ProTracer)] by incorporating dual recombinases to seamlessly record the proliferation events of entire cell populations over time in multiple organs. In the mouse liver, ProTracer revealed more hepatocyte proliferation in distinct zones during liver homeostasis, injury repair, and regrowth. Clonal analysis showed that most of the hepatocytes labeled by ProTracer had undergone cell division. By genetically recording proliferation events of entire cell populations, ProTracer enables the unbiased detection of specific cellular compartments with enhanced regenerative capacities.

ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation.

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/ß-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/ß-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of ß-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/ß-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/ß-Catenin activity, balancing metabolic function and hepatocyte proliferation.

Notch2 signaling promotes biliary epithelial cell fate specification and tubulogenesis during bile duct development in mice.

UNLABELLED: Intrahepatic bile duct (IHBD) development begins with the differentiation of hepatoblasts into a single continuous biliary epithelial cell (BEC) layer, called the ductal plate. During ductal plate remodeling, tubular structures arise at distinct sites of the ductal plate, forming bile ducts that dilate into the biliary tree. Alagille syndrome patients, who suffer from bile duct paucity, carry Jagged1 and Notch2 mutations, indicating that Notch2 signaling is important for IHBD development. To clarify the role of Notch2 in BEC differentiation, tubulogenesis, and BEC survival, we developed a mouse model for conditional expression of activated Notch2 in the liver. We show that expression of the intracellular domain of Notch2 (Notch2ICD) differentiates hepatoblasts into BECs, which form additional bile ducts in periportal regions and ectopic ducts in lobular regions. Additional ducts in periportal regions are maintained into adulthood and connect to the biliary tight junction network, resulting in an increased number of bile ducts per portal tract. Remarkably, Notch2ICD-expressing ductal plate remnants were not eliminated during postnatal development, implicating Notch2 signaling in BEC survival. Ectopic ducts in lobular regions did not persist into adulthood, indicating that local signals in the portal environment are important for maintaining bile ducts. CONCLUSION: Notch2 signaling regulates BEC differentiation, the induction of tubulogenesis during IHBD development, and BEC survival.

The Conundrum of the Pericentral Hepatic Niche: WNT/-Catenin Signaling, Metabolic Zonation, and Many Open Questions.

WNT/-catenin signaling promotes stemness, proliferation, and cell fate decisions in various tissue stem cell compartments, which maintain organs with a high turnover of cells (e.g., skin, stomach, and gut). Thus, the -catenin target genes AXIN2 and LGR5 are widely considered as tissue stem cell markers. In contrast, AXIN2 and LGR5 are expressed in pericentral hepatocytes, which do not show overt proliferation during liver homeostasis. Given the low hepatocyte turnover, the liver does not require constant high rates of proliferation, whereas WNT/-catenin signaling is critical for metabolic zonation. Yet, WNT/-catenin pathway upregulation, including AXIN2 and LGR5 induction in hepatocytes throughout the liver, enables hepatocyte regeneration in response to various injuries. In this brief review, I discuss the role of WNT/-catenin signaling in controlling metabolic zonation and the conundrum around pericentral hepatocytes that have been proposed as liver stem cells.