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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Clinical translation of liver regeneration therapies: A conceptual road map.

The increasing incidence of severe liver diseases worldwide has resulted in a high demand for curative liver transplantation. Unfortunately, the need for transplants by far eclipses the availability of suitable grafts leaving many waitlisted patients to face liver failure and often death. Routine use of smaller grafts (for example left lobes, split livers) from living or deceased donors could increase the number of life-saving transplants but is often limited by the graft versus recipient weight ratio defining the safety margins that minimize the risk of small for size syndrome (SFSS). SFSS is a severe complication characterized by failure of a small liver graft to regenerate and occurs when a donor graft is insufficient to meet the metabolic demand of the recipient, leading to liver failure as a result of insufficient liver mass. SFSS is not limited to transplantation but can also occur in the setting of hepatic surgical resections, where life-saving large resections of tumors may be limited by concerns of post-surgical liver failure. There are, as yet no available pro-regenerative therapies to enable liver regrowth and thus prevent SFSS. However, there is optimism around targeting factors and pathways that have been identified as regulators of liver regeneration to induce regrowth in vivo and ex vivo for clinical use. In this commentary, we propose a roadmap for developing such pro-regenerative therapy and for bringing it into the clinic. We summarize the clinical indications, preclinical models, pro-regenerative pathways and safety considerations necessary for developing such a drug.

AXIN2+ Pericentral Hepatocytes Have Limited Contributions to Liver Homeostasis and Regeneration.

The existence of specialized liver stem cell populations, including AXIN2+ pericentral hepatocytes, that safeguard homeostasis and repair has been controversial. Here, using AXIN2 lineage tracing in BAC-transgenic mice, we confirm the regenerative potential of intestinal stem cells (ISCs) but find limited roles for pericentral hepatocytes in liver parenchyma homeostasis. Liver regrowth following partial hepatectomy is enabled by proliferation of hepatocytes throughout the liver, rather than by a pericentral population. Periportal hepatocyte injury triggers local repair as well as auxiliary proliferation in all liver zones. DTA-mediated ablation of AXIN2+ pericentral hepatocytes transiently disrupts this zone, which is reestablished by conversion of pericentral vein-juxtaposed glutamine synthetase (GS)- hepatocytes into GS+ hepatocytes and by compensatory proliferation of hepatocytes across liver zones. These findings show hepatocytes throughout the liver can upregulate AXIN2 and LGR5 after injury and contribute to liver regeneration on demand, without zonal dominance by a putative pericentral stem cell population.