The oxygen gradient along the porto-central axis of the hepatic lobule establishes a hypoxic microenvironment that activates hypoxia-inducible factors (HIFs) (33). Among these, HIF-1α is preferentially stabilized in pericentral hepatocytes, where it directly regulates glycolytic gene programs through binding to hypoxia-responsive elements (HREs) (33). This includes upregulation of glucose transporters GLUT1 (SLC2A1) and GLUT3 (SLC2A3), as well as key rate-limiting glycolytic enzymes such as HK2, PFK1, PKM2 and LDHA. Concurrently, HIF-1α transcriptionally activates PDK1, thereby inhibiting pyruvate entry into mitochondrial oxidative metabolism, collectively reinforcing the glycolytic phenotype characteristic of zone 3 hepatocytes (34,35). By contrast, HIF-2α primarily governs antioxidant defense and lipid metabolic regulation, directly inducing genes such as SOD2 and HMOX1 (36). However, sustained activation of HIF-2α can impair lipid homeostasis by transcriptionally suppressing PPARα-dependent fatty acid oxidation (FAO) genes, including CPT1A and ACOX1, thereby promoting steatosis (37). HIF-3α splice variants function as dominant-negative regulators by competing with HIF-1α for HRE binding or forming low-activity heterodimers, ultimately attenuating HIF-1α signaling (38,39).

Beyond oxygen gradients, the spatial distribution of hormonal signals represents another key regulatory axis shaping liver zonation. Although glucagon receptors are uniformly distributed across the hepatic lobule, glucagon itself forms a functional gradient along the porto-central axis (40). In glucagon-deficient mice, expression of the periportal marker gene Gls2 is markedly reduced, whereas the pericentral marker Glul, which is normally restricted to 1 to 3 hepatocyte layers surrounding the central vein, expands to 5 to 6 layers (41). This abnormal pattern can be reversed by exogenous glucagon supplementation. Functionally, glucagon differentially regulates periportal urea cycle enzymes and pericentral xenobiotic metabolizing enzymes, such as members of the Cyp450 family. In addition, sexual dimorphisms further increases regulatory complexity. The sex-specific patterns of growth hormone (GH) secretion directly influence liver zonation by regulating the activity of transcription factors in distinct regions. In males, pulsatile GH in zone 1 activates SREBF1, promoting gluconeogenesis-related pathways, whereas in females, continuous GH in zone 3 supports TRIM24, enhancing hepatic progenitor cell-related pathways (42). These differential transcriptional programs restrict specific hepatocyte functions to defined zones, creating a sex-biased metabolic and regenerative landscape (42). Continuous GH infusion in males can abolish this zonal dimorphism, indicating that the mode of hormone secretion determines sex-specific liver zonation (43). These findings indicate that liver zonation functions as a dynamic interface integrating metabolic, endocrine and oxygen-dependent signals.

The Wnt/β-catenin pathway is a key regulator of pericentral hepatocyte identity (44). Its activity depends on tight regulation of the β-catenin destruction complex and phosphorylation events. Under basal conditions, β-catenin is continuously degraded by a destruction complex composed of Axin, adenomatous polyposis coli (APC), CK1 and GSK3β. CK1 initiates phosphorylation at Ser45, followed by sequential phosphorylation by GSK3β at Thr41, Ser37 and Ser33, which marks β-catenin for recognition by the β-TrCP E3 ubiquitin ligase and subsequent proteasomal degradation (45). Upon Wnt stimulation, including Wnt2 and Wnt9b secreted by central vein endothelial cells, the binding of these ligands to Frizzled receptors and the co-receptors LRP5/6 recruits CK1 and GSK3β to the membrane (46,47). This leads to phosphorylation of LRP5/6, inhibition of GSK3β activity and inactivation of the destruction complex (44). Stabilized β-catenin accumulates and translocates into the nucleus, where it binds T-cell factor/lymphoid enhancer-binding factor transcription factors to activate pericentral gene programs such as glutamine synthetase and Cyp2e1.

A critical determinant of zonal regulation is the spatially restricted expression of APC (48). APC is largely absent in the pericentral zone, permitting sustained nuclear accumulation of β-catenin, whereas its high expression in periportal hepatocytes constrains Wnt signaling (48). Genetic studies demonstrate that loss of APC leads to panlobular activation of β-catenin, accompanied by ectopic expression of pericentral markers such as Cyp2e1 in periportal hepatocytes (49). In addition, oxygen gradients modulate this axis: HIF-1α suppresses APC transcription in the pericentral zone, thereby enhancing β-catenin signaling, while reactive oxygen species in the periportal region stabilize APC, establishing a redox-dependent zonal boundary (50,51). Furthermore, Wnt5a in the periportal region antagonizes β-catenin activity through calcium-dependent signaling, which reinforces zone 1 identity (52). The Ras/MAPK/ERK pathway contributes additional modulation by promoting β-catenin phosphorylation and proteasomal degradation in periportal hepatocytes, thereby balancing regional signaling intensity (53).

Non-canonical Wnt pathways further refine zonal patterning. Hypoxia-induced Wnt11 in zone 3 activates planar cell polarity signaling via the ROR2 receptor, regulating pericentral gene expression independently of β-catenin (54-56). Wnt signaling defines hepatic zonation through coordinated regulation of the β-catenin destruction complex, spatial control of APC expression and integration of canonical and non-canonical pathways. It also interacts with multiple signaling networks to precisely specify hepatocyte identity along the porto-central axis.

The Hh signaling pathway is also a key regulator of hepatic metabolic zonation. It coordinates functional specialization along the porto-central axis through spatially restricted ligand-receptor interactions (57). Spatial transcriptomics analyses have shown that Indian Hh is enriched in the pericentral region, forming a morphogen gradient that is inversely correlated with Wnt/β-catenin signaling activity in the periportal zone (31). This spatial relationship is maintained through GLI-mediated inhibition of β-catenin transcriptional activity, establishing a dynamic balance between the two pathways (58). The Hh gradient can also regulate components of the insulin-like growth factor axis (IGF-I/IGFBP-1) to coordinate zonal metabolic programming (59). Pericentral hepatocytes exhibit GLI-dependent upregulation of gluconeogenic enzymes, whereas periportal regions show enhanced lipid oxidation (32). Furthermore, hepatic stellate cells (HSCs) in the space of Disse act as spatial signal integrators; they convert Hh/Wnt crosstalk into extracellular matrix (ECM) remodeling, thereby physically maintaining the structure of metabolic zonation (60).

Emerging evidence indicates that the spatially restricted cellular niches of non-parenchymal cells, particularly the periportal vs. pericentral zonation patterns, establish microenvironments that directly modulate hepatocyte functional specialization via ECM regulation, metabolic exchange and immune surveillance, thereby driving the establishment of hepatic metabolic zonation (61). Among these, liver sinusoidal endothelial cells (LSECs) act as central regulators of zonation. Central vein associated LSECs selectively secrete Wnt2, Wnt9b and R-spondin 3, thereby activating β-catenin signaling in adjacent hepatocytes and maintaining pericentral cell identity (47,62). Consistently, combined deletion of Wnt2 and Wnt9b abolishes zone 3 specific gene expression. In addition, LSECs modulate sinusoidal blood flow and oxygen tension through the secretion of vascular endothelial growth factors, contributing to the establishment of the porto-central metabolic axis (63). Kupffer cells exhibit a preferential periportal distribution, forming an immune zonation pattern (29). By restricting infiltrating neutrophils to the periportal region, they spatially confine pro-inflammatory responses, thereby protecting the metabolically vulnerable pericentral hepatocytes. Disruption of Kupffer cell localization leads to bacterial dissemination and panlobular inflammatory injury (64-65). HSCs also display zonal heterogeneity, comprising functionally distinct subpopulations (66). Periportal-associated stellate cells express markers such as Ngfr and Tagln and are involved in rapid immune interactions, whereas pericentral-associated stellate cells specifically express Rspo3, which enhances Wnt signaling to sustain pericentral hepatocyte homeostasis (67,68).

Beyond extrinsic cues, hepatic zonation is also governed by intrinsic epigenetic programs (69). DNA methylation has been shown to form a gradient along the porto-central axis, progressively decreasing from the periportal to the pericentral zone. Key transcription factors exhibit spatially restricted expression patterns; USF1 is enriched in periportal hepatocytes, where it exerts negative feedback on periportal-specific genes while modulating lipid and glucose metabolism (70). By contrast, Nr2f2 is progressively enriched in the pericentral zone and activates pericentral identity genes during hepatocyte maturation (69,71). Although HNF4α is uniformly expressed, its DNA-binding activity is gated by methylation status, and its deletion leads to ectopic expression of pericentral genes in periportal hepatocytes (71,72).

Histone modifications further reinforce zonal specialization. H3K27ac, a histone acetylation marker of active enhancers, is enriched at regulatory elements of Wnt/β-catenin-associated pericentral metabolic genes, whereas H3K4me3, a histone trimethylation marker of active promoters, predominantly labels genes involved in periportal metabolic pathways such as gluconeogenesis and the urea cycle (73). The SWI/SNF chromatin remodeling component ARID1A maintains hepatocyte metabolic gene programs by regulating nucleosome positioning and chromatin accessibility (74); hepatocyte-specific loss of ARID1A reduces accessibility at metabolic loci, impairs binding of transcription factors such as PPARα and suppresses the expression of genes involved in fatty acid β-oxidation (75).

MicroRNAs (miRs) also provide an additional layer of fine-tuning. For example, liver-enriched miR-122 is required for the maintenance of zonation, and its deletion results in expansion of zone 3 characteristics (76,77). Collectively, DNA methylation, histone modifications, non-coding RNAs and transcription factor dynamics converge to form an integrated epigenetic-metabolic axis that spatially encodes hepatocyte identity.

Based on the principles of zonated hepatocyte renewal, pregnancy induces a spatiotemporally coordinated remodeling of the liver. Hepatocyte proliferation progresses in a zonal sequence, initiating in the periportal zone 1 during early pregnancy, shifting to zone 2 in mid-gestation and culminating in pericentral zone 3 expansion in late pregnancy. This dynamic pattern parallels the progressive increase in fetal metabolic demands (20). Lineage tracing studies indicate that estrogen coordinates this hierarchical zonal program by selectively promoting the proliferation of Ccnd1 positive hepatocytes in zone 2, whereas prolactin fine-tunes liver mass through bile acid mediated hypertrophic regulation (90,91). This spatial proliferation program is closely aligned with metabolic compartmentalization. Expansion in zones 1 and 2 supports glucose and cofactor synthesis required for fetal growth, while proliferation in zone 3 enhances detoxification capacity to counteract pregnancy associated oxidative stress (90,91). Despite these advances, critical gaps remain in understanding how maternal liver zonation adapts and synchronizes with key milestones of fetal development. This highlights the need to elucidate how the placenta liver signaling axis preserves zonation fidelity during pregnancy.

Metabolic dysfunction associated steatotic liver disease (MASLD) is a liver disorder linked to metabolic dysfunction and is characterized by a systemic collapse of lobular metabolic zonation architecture (94). This pathological cascade is driven by coordinated dysregulation of the three core metabolic processes, uptake, synthesis and export, ultimately disrupting the signaling gradients required to maintain functional compartmentalization (95). Hepatic steatosis, a hallmark feature of MASLD, shows a developmentally and spatially patterned distribution. In adult patients, lipid accumulation typically initiates in zone 3, the primary site of fatty acid synthesis (96). Lipotoxicity in this region triggers zone 3-predominant hepatocyte apoptosis and pericentral fibrosis (97). As the disease progresses to metabolic dysfunction-associated steatohepatitis (MASH), the spatial pattern of fibrosis is remodeled, with collagen deposition shifting toward zone 1 and forming porto-central bridging fibrosis, a key precursor of cirrhosis (98). But prepubertal MASLD exhibits an opposite spatial pattern, characterized by zone 1-dominant steatosis and periportal fibrosis without pericentral involvement, which is associated with developmental metabolic programming and microenvironment-specific immune responses (99).

Dysregulated zonal signaling is a key determinant of disease severity. Elevated Hh ligand levels and yes-associated protein (YAP) activation are associated with steatohepatitis and fibrosis progression, while Wnt/β-catenin-mediated pericentral homeostasis is disrupted (100,101). Kupffer cells, traditionally localized to the periportal region, amplify zonation-restricted inflammation in early MASLD by recruiting neutrophils and releasing pro-fibrotic cytokines, thereby exacerbating zone 1 injury (102). Meanwhile, periportal ductular reactions characterized by hepatic progenitor cell expansion may contribute to porto-portal fibrosis, although their direct pro-fibrotic role remains to be fully validated (103,104). Mechanistically, early MASLD retains lobular zonation, with preserved regional gene expression and DNA methylation patterns, suggesting a therapeutic window for zonation restoration (71). This reversibility highlights the potential of targeting zone-specific drivers, including Kupffer cell-mediated spatial inflammation, HSC-derived ECM imbalance and liver sinusoidal endothelial cell-driven Wnt signaling dysregulation, to block the steatosis-fibrosis axis (105-107).

HCC arises from zonation-collapsed microenvironments in cirrhotic livers, with chronic hepatitis and metabolic dysfunction driving spatially biased oncogenesis (108). It is reported that >50% of HCCs exhibit constitutive Wnt/β-catenin activation, a pathway central to pericentral zonation, alongside Hh signaling hyperactivation, constitutive YAP/TAZ activation and glycolytic reprogramming, collectively hijacking zonation-maintaining circuits to fuel tumor initiation (109,110). While HCC cellular origins remain debated, clonal tracing reveals that regeneration-competent hepatocytes within injury niches serve as dominant precursors, acquiring malignant traits through niche-derived pressures (83,111).

Hypoxia-mediated metabolic zonation collapse further shapes HCC evolution. Pericentral hypoxia stabilizes HIF-1α, synergizing with β-catenin to select for venous zone-adapted clones with glutamine addiction and invasive potential (112). Notably, HCCs retain zonation-like phenotypic diversity, as portal-like HCC with low β-catenin expression mirrors periportal metabolism, exhibiting indolent behavior and favorable post-resection outcomes due to preserved immune surveillance. However, central-like HCC recapitulates pericentral features with high β-catenin, marked by immune-cold microenvironments and resistance to checkpoint blockade, yet maintains vulnerability to Wnt/Hh-targeted therapies (108). This zonation-based classification system overcomes the limitations of conventional histopathological subtyping and enables prognostic stratification as well as compartment-specific therapeutic strategies (113). From a therapeutic perspective, restoring Wnt signaling gradients or targeting the hypoxia-HIF-β-catenin axis may reverse treatment resistance driven by spatial dedifferentiation. In addition, portal vein-like HCC may benefit from immunomodulatory approaches that exploit residual zonal differentiation integrity (114,115).

DILI, exemplified by acetaminophen (APAP) and carbon tetrachloride models, manifests zonation-biased damage, as demonstrated by the studies that pericentral hepatocytes (zone 3) with high cytochrome P450 activity (including Cyp2e1) metabolize toxins into reactive intermediates such as NAPQI, triggering oxidative stress and mitochondrial dysfunction that culminate in zone 3-predominant necrosis (116-118). Spatial reprogramming at the injury border initiates repair where damage-adjacent hepatocytes transiently reactivate fetal genes and upregulate ribosomes and proteasomes, enabling proteome remodeling to adopt pericentral identities (82). Concurrently, zonation-restricted regeneration unfolds where surviving pericentral hepatocytes initiate proliferation via mTOR signaling, followed by zone 2 hepatocyte expansion (3). A recent study demonstrated zone 2-derived hepatocytes as a main contributor to compartmentalized replenishment, where Igfbp2⁺ hepatocytes migrate toward necrotic zone 3 and restore metabolic zonation (89).

In addition, annexin A2-positive (ANXA2⁺) hepatocyte subpopulations exhibit a migration-dominant wound closure mechanism during APAP-induced injury, sealing necrotic areas prior to mitotic expansion (119). This ANXA2-mediated migratory plasticity, previously implicated in cancer cell invasion, suggests a form of region-specific reparative plasticity in which spatial remodeling, rather than proliferation alone, drives functional recovery (119,120). The interplay between migration and mitosis may help maintain a dynamic balance between lobular structural integrity and metabolic demands. However, excessive migratory pressure may contribute to ductular reactions and fibrotic scar formation (121,122).

Experimental cholestasis models, including DDC diet and bile duct ligation, precisely recapitulate human cholangiopathies while unveiling zonation-defined injury patterns (123). Cholestatic injury predominantly targets zone 1 hepatocytes, as bile acids first accumulate in the periportal region following impaired biliary excretion. Hydrophobic bile acids induce membrane damage through detergent-like effects, disrupt mitochondrial function, and trigger oxidative stress and endoplasmic reticulum stress. In parallel, bile acid-activated death receptor signaling, including Fas and TRAIL pathways, further amplifies hepatocyte injury. Zone 1 hepatocytes are particularly susceptible due to their high expression of bile acid uptake transporters such as NTCP and OATPs, together with limited adaptive capacity for detoxification compared with downstream zones (124). In addition, cholestasis induces inflammatory signaling from portal stromal and immune cells, further reinforcing periportal injury and ECM remodeling.

Mechanistically, periportal hepatocytes exposed to cholestatic stress can establish a self-limiting reparative microenvironment. Within this setting, a distinct subpopulation of hepatocytes with a unique phenotype has been identified in the periportal niche. These cells express multiple biliary-enriched genes, including low levels of Sox9, while retaining the canonical hepatocyte marker HNF4α. This population, termed hybrid hepatocytes (HybHPs), is capable of contributing to parenchymal regeneration through proliferative expansion (125). However, the origin of HybHPs remains controversial. In chronic injury models, HybHPs have been proposed to arise from embryonic progenitors located in the ductal plate, as these cells can upregulate biliary markers and differentiate into cholangiocytes. However, studies using partial hepatectomy models suggest that HybHPs originate from mature hepatocytes, as they appear as early as 3 h after resection without evidence of early activation of hepatic progenitor cells (126). Furthermore, in MASLD models, analysis of fetal liver markers indicates that these biphenotypic cells do not undergo dedifferentiation or redifferentiation (127). Taken together, these findings suggest that HybHPs most likely arise from mature hepatocytes undergoing microenvironment-driven transdifferentiation in response to alterations in the periportal niche during injury, rather than from stem cell populations or developmental progenitors. In addition, chronic cholestasis activates cholangiocyte progenitor cells through the FXR/TGR5 signaling pathway, which in turn induces collagen deposition from HSCs, leading to periportal fibrosis and consequently disrupting the architecture of zone 1 (128).

Liver regeneration necessitates dynamic metabolic adaptation, where zonation-defined specialization is transiently repurposed to resolve the energy-biosynthesis paradox (135). In the early phase of regeneration, HIF-1α-driven glycolytic flux predominates, while AKT/mTOR-mediated suppression of gluconeogenesis and FAO redirects substrates toward lipid droplet biogenesis, providing a strategic energy reserve to support proliferative expansion (137). This metabolic triage creates therapeutic opportunities through phased interventions. First, HIF stabilizers boost zone 1-2 glycolytic priming. Concurrently, PPARγ agonists sustain lipogenic competence, while mTOR modulators delay catabolic restoration to preserve energy reservoirs (138). Post-proliferative metabolic recovery, driven by PPARα-dependent FAO reactivation and mitochondrial rejuvenation, is spatially coordinated to reinstate functional zonation (139,140). This phase ensures pericentral detoxification via CYP450 restoration and zonal energy flux balancing, completing the metabolic reprogramming cycle essential for functional restoration.

Epigenetic modifiers serve as spatial regulators of metabolic reprogramming, offering druggable nodes to enhance regenerative precision. DNA methyltransferases (DNMTs) enforce FAO gene silencing during regeneration initiation, with pharmacological DNMT inhibition extending the glycolytic proliferative window (141). Conversely, histone acetylase activators accelerate oxidative phosphorylation recovery by decompacting chromatin at mitochondrial genes, enabling timely metabolic restoration (142,143). Spatial multiomics have decoded zone-specific metabolite-epigenome crosstalk, identifying serine and ketone bodies as zone 2 resolved cofactors that prime chromatin accessibility for plasticity transcription factors, a mechanism that may be exploitable through dietary or metabolite supplementation strategies (144). For instance, zone 1 targeted HIF stabilizers amplify glycolytic flux, while zone 2 directed serine supplementation reinforces epigenetic licensing of plasticity genes. By mirroring the liver's innate spatiotemporal hierarchy where metabolic priorities shift from proliferation fuel to functional specialization, these strategies transform regenerative bottlenecks into therapeutic windows.

Non-parenchymal cells have been recognized for their roles in maintaining liver zonation, and also make critical contributions to the regenerative microenvironment. Spatial transcriptomics and single-cell analyses have shown that pericentral HSC subsets enhance Wnt/β-catenin signaling through secretion of Rspo3, thereby supporting hepatocyte proliferation during regeneration (141). The loss of HSCs or Rspo3 impairs regenerative capacity, while clinical data indicate that higher Rspo3 expression is associated with improved survival and a reduced risk of hepatocellular carcinoma, supporting its potential as a therapeutic target (145).

LSECs are also key regulators of regeneration. Previous studies demonstrate that LSECs promote hepatocyte proliferation through secretion of Wnt2 and Wnt9b (47,146,147). Further work has identified c-Kit signaling in LSECs as essential for this pro-regenerative function. c-Kit⁺ LSECs upregulate Wnt2 expression following injury, thereby stimulating proliferation of adjacent hepatocytes, whereas disruption of c-Kit signaling reduces Wnt production and significantly impairs liver regeneration (147,148). Recent advances have extended these findings into therapeutic strategies. Engineered exosome-based systems have been developed to target LSECs, combining CRLTRKRGLK peptide-mediated targeting with CD47 mediated evasion of mononuclear phagocyte clearance, enabling efficient delivery of Wnt2 mRNA (149). In APAP and dimethylnitrosamine-induced liver injury models, this approach enhances Wnt signaling, promotes hepatocyte proliferation, and improves tissue repair, demonstrating the feasibility of modulating non-parenchymal cell-derived signals to reconstruct the regenerative microenvironment (149).

In addition, a recent study has shown that injury-induced downregulation of URI1 leads to glutamate accumulation, which acts as a paracrine signal to recruit bone marrow-derived monocytes. These immune cells subsequently secrete Wnt3, activating YAP1 signaling in zone 2 hepatocytes and driving their proliferation (150). This metabolic immune regenerative axis follows a well-defined spatiotemporal sequence and has been validated across multiple models of acute and chronic liver injury. Clinical analyses further suggest that this pathway is dysregulated in chronic liver disease, characterized by reduced Wnt3 expression and disrupted metabolic signaling (150). Non-parenchymal cells thus establish a spatially organized signaling network that governs liver regeneration within the framework of liver zonation. This coordinated multicellular system indicates that liver regeneration is not driven by a single cell type but instead depends on the dynamic remodeling of zonation-defined regenerative niches. Targeting non-parenchymal cells and their spatial signaling networks therefore represents a promising strategy for enhancing liver regeneration.

Hepatocyte dedifferentiation, a spatially restricted process predominantly localized to zone 2, functions as an evolutionary safeguard mechanism for hepatic repair. This regenerative phenomenon involves surviving hepatocytes reactivating fetal transcriptional programs through conserved developmental pathways, particularly Wnt/β-catenin (151), YAP (152) and TGF-β signaling cascades (153). Beyond the classical Sox9⁺/HNF4α⁺ progenitor-like transition, studies have identified multiple regenerative hepatocyte subpopulations arising from dedifferentiation or enhanced cellular plasticity of mature hepatocytes (154). For instance, AFP-positive reprogrammed hepatocytes represent a state of partial dedifferentiation, retaining lineage restriction while acquiring proliferative capacity. Hepatocytes driven by YAP or Wnt/β-catenin signaling exhibit a reversible progenitor-like phenotype (152). In addition, transitional hepatocyte populations with high transcriptional plasticity, as well as hepatocytes with migratory capacity involved in wound repair, have been identified at injury borders (119). Collectively, these distinct cellular states constitute a heterogeneous pool of regenerative sources, indicating that liver regeneration relies on multilayered cell fate reprogramming rather than a single progenitor reservoir.

Critically, liver regeneration extends beyond cell-autonomous reprogramming through spatially coordinated niche interactions. For example, portal vein injury models reveal that regionally activated Kupffer cells secrete IL-6, which induces STAT3 activation in adjacent hepatocytes to directly drive reprogramming-related gene re-expression (154). This spatial regulation is further governed by metabolic zonation dynamics that compartmentalize regenerative responses across hepatic lobules.