Abstract
Hepatic zonation is critical for most metabolic functions in liver. Wnt signaling plays an important role in establishing and maintaining liver zonation. Yet, the anatomic expression of Wnt signaling components, especially all 10 Frizzled (Fzd) receptors, has not been characterized in adult liver. To address this, the spatial expression of Fzd receptors was quantitatively mapped in adult mouse liver via multiplex fluorescent in situ hybridization. Although all 10 Fzd receptors were expressed within a metabolic unit, Fzd receptors 1, 4, and 6 were the highest expressed. Although most Wnt signaling occurs in zone 3, expression of most Fzd receptors was not zonated. In contrast, Fzd receptor 6 was preferentially expressed in zone 1. Wnt2 and Wnt9b expression was highly zonated and primarily found in zone 3. Therefore, the current results suggest that zonated Wnt/β-catenin signaling at baseline occurs primarily due to Wnt2 and Wnt9b rather than zonation of Fzd mRNA expression. Finally, the study showed that Fzd receptors and Wnts are not uniformly expressed by all hepatic cell types. Instead, there is broad distribution among both hepatocytes and nonparenchymal cells, including endothelial cells. Overall, this establishment of a definitive mRNA expression atlas, especially of Fzd receptors, opens the door to future functional characterization in healthy and diseased liver states.
Liver performs several functions critical for metabolic health, including cholesterol metabolism, drug detoxification, and synthetic functions. The ability to compartmentalize these various metabolic functions is an essential aspect of hepatic physiology.1 Anatomically, this compartmentalization is represented by zonation of the hepatic lobule into distinct zones (zones 1 to 3) along the periportal–central vein axis. Functionally, zonation guides hepatocytes to metabolic processes specific to each zone (ie, metabolic zonation).2 These differences depend on differential expression of enzymes, proteins, transcription factors, and oxygenation, which occur along a gradient. For example, periportal hepatocytes in zone 1 predominantly catalyze the oxidative catabolism of fatty and amino acids as well as gluconeogenesis-driven glucose release and glycogen formation.1 In contrast, pericentral cells in zone 3 preferentially engage in glucose uptake for glycogen synthesis and glycolysis coupled to de novo lipogenesis.3 Significantly, evidence suggests that zone-specific differences in gene expression are essential for establishing and maintaining metabolic zonation.4,5 Such zonal division of labor makes liver an efficient metabolic, synthetic, and detoxifying organ.
Wnt signaling is a fundamental regulator of hepatic zonation by controlling zone-specific gene expression.6 Consistent with this, Wnt signaling is zone-dependent and follows the metabolic gradient, activating gene expression in pericentral hepatocytes (zone 3), while suppressing genes in its periportal counterparts (zone 1). Wnt2- and Wnt9b-dependent signaling is essential to maintain zonation.7, 8, 9 More recently, Wnt2 and Wnt9b expression was conditionally eliminated from hepatic endothelial cells, including those lining the central vein and sinusoidal endothelial cells in zones 3 and 2.7 This led to complete disruption of β-catenin activation in these zones and a notable defect in pericentral hepatocyte gene expression. Investigations of the liver's capacity for injury recovery and regeneration have yielded key insights into the roles of Wnt signaling in metabolic zonation, not only in development, but also in adult tissue homeostasis.6,10 mRNA expression of 11 Wnts and 8 Frizzleds (Fzds) has been uncovered in normal liver, suggesting a function for Wnt signaling after development, including maintenance of metabolic zonation.11 Although much is known regarding hepatic Wnt signaling, the precise anatomic expression of these signaling components in adult liver remains poorly characterized.
Defining the expression patterns of the Wnt signaling machinery is a crucial first step in better understanding the postdevelopmental roles of a pathway traditionally associated with development. Wnt/β-catenin signaling is largely quiescent in adult liver, except in the pericentral region of the hepatic lobule (zone 3). Therefore, we hypothesized that Wnt signaling components, especially the Fzd receptors, may be expressed along a gradient, with most expression in pericentral zone 3. To test this, newly developed multiplex RNAscope fluorescent in situ hybridization was utilized to spatially characterize the mRNA expression of Frizzled receptors 1 to 10 and coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (Lrp5 and Lrp6, respectively). Because Wnt2 and Wnt9b have been established to be primarily expressed in zone 3,7,9 these Wnts and some additional prototypical targets of Wnt/β-catenin signaling were used as controls. More importantly, this helped bypass past limitations in mapping some of these important Wnt signaling components at the protein level due to the lack of specific antibodies.
Materials and Methods
Animals
Animals were housed and handled in accordance with appropriate NIH guidelines12 through the University of Pittsburgh Institutional Animal Care and Use Committee (protocol number 19126451). The authors abided by all appropriate animal care guidelines for reporting animal research. Mice were housed in cages with a 12:12-hour light/dark cycle and had access to food and water ad libitum at all times.
Liver Preparation
Livers were harvested from male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) between the ages of 2.5 and 3 months. Liver tissue was washed in ice-cold phosphate-buffered saline and processed into chunks no larger than 2 mm2. Liver chunks were fixed in 4% paraformaldehyde (overnight, 4°C), incubated in 30% sucrose in phosphate-buffered saline (overnight, 4°C) until fully submerged, and were OCT embedded and frozen on dry ice. Tissue blocks were stored at −80°C until used.
Multiplex Fluorescent in Situ Hybridization
In situ detection of mRNA expression was attained via fluorescent multiplex RNAscope (Advanced Cell Diagnostics, Hayward, CA), according to manufacturer's instructions. Fixed frozen mouse liver sections (5 μm thick) were first mounted onto slides and then submerged in target retrieval solution (5 minutes, 100°C), followed by protease pretreatment (30 minutes, 40°C). Slides were then incubated with probes targeting Fzd receptors 1 to 10, Wnt2, Wnt9b, Lrp5, and Lrp6 (2 hours, 40°C). The authors defined metabolic zones using additional probes glutamine synthetase and glutaminase 2. Slides were then treated with an amplification kit (Advanced Cell Diagnostics) to hybridize each probe to its respective mRNA target, followed by fluorescent labeling (90 minutes, 40°C). Finally, slides were treated with DAPI (Advanced Cell Diagnostics) to visualize cell nuclei and mounted with Prolong Gold Antifade (Invitrogen, Waltham, MA).
Confocal Microscopy
Images were acquired with an Olympus VS120 whole slide scanner microscope (Olympus America, Center Valley, PA) equipped with an Olympus 20× objective (0.75 numerical aperture) and a Hamamatsu ORCA Flash4.0 V2 digital camera (Hamamatsu Corp., Bridgewater, NJ; 0.33 μm/pixel). Image stacks were acquired during scanning.
Image Analysis
Using Olympus VS-ASW software version 2.9.2 (Olympus America), image stacks were converted into maximum intensity projections and exported as TIFF files. Image analysis was performed using the HALO image analysis platform equipped with a fluorescent in situ hybridization plug-in version 3.0 (Indica Labs, Albuquerque, NM). Nuclei were quantified as DAPI-stained objects within 30 to 150 μm2; the minimum cytoplasmic radius was set at 5 μm. Puncta corresponding to the respective mRNA probes were quantified as any 0.01- to 0.15-μm2 object. To account for background, the number of identified puncta for each probe was divided by the total area analyzed. This number was then multiplied by the average cell area, providing an average cellular density per mRNA probe (positive cells/mm2). Thus, these thresholds represented three times the smaller observed density level and were used as a cutoff to identify positive cells.
Cell-Type Analysis
Cell-type analyses were conducted using data from a recently published data set of a single-cell RNA sequencing atlas of healthy adult mouse liver.13
Statistical Analysis
GraphPad Prism version 9.20 (GraphPad Software, San Diego, CA) was used for all statistical analyses. One-way analysis of variance, followed by Tukey multiple comparison tests, was used to analyze differences between zonal expression.
Results
Establishing Metabolic Zones Using Multiplex RNAscope
mRNA expression of key components of the Wnt signaling machinery were mapped in a zone-specific manner via multiplex RNAscope. First, RNAscope's capability to measure zonated expression was confirmed by focusing on zonated markers, glutaminase 2 and glutamine synthetase, in adult mouse liver. Expression of these markers was quantified in 20-μm increments across a metabolic unit, beginning at the midline of the central vein (labeled 0 μm) and ending at the midline of the portal vein (labeled 360 μm) (Figure 1). A gradient of glutaminase 2 mRNA expression was observed to be progressively increasing toward the portal vein (Figure 1A). By contrast, glutamine synthetase mRNA expression was most enriched around the central vein (Figure 1B). Dividing metabolic units into thirds according to the 20-μm increments relative to the central vein accurately defined the zonated expression of glutaminase 2 and glutamine synthetase as zone 1 and zone 3 markers, respectively (eg, 20 to 120 μm, zone 1; 140 to 260 μm, zone 2; and 280 to 360 μm, zone 3) (Figure 1, C–E). These data are consistent with prior immunohistochemical and functional studies of these enzymes.14
Figure 1.
Establishment of metabolic zones using multiplex RNAscope. A and B: Quantification of cells positive for glutaminase 2 (Gls2) and glutamine synthetase (GS) mRNA expression within a metabolic unit is quantified in 20-μm increments, starting at 20 μm from the midline of the central vein (CV; labeled 20 μm) and terminating at the midline of the portal vein (PV; labeled 360 μm). A and B: Gls2 expression progressively increases toward the periportal region (A), whereas GS expression is restricted to the pericentral region (B). C: Representative image of a metabolic unit with GS (purple) and Gls2 (green) outlining the CV and PV, respectively. Cell nuclei are labeled with DAPI stain (blue). D: mRNA expression via multiplex RNAscope of GS reveals strongly zonated expression in zone 3. One-way analysis of variance with post hoc analyses. F (2, 79) = 70.64. E: Gls2 is expressed along a gradient, significantly decreasing from zone 1 toward zone 3. One-way analysis of variance with post hoc analyses. F (2, 77) = 25.10. F and G: mRNA expression of Wnt ligands Wnt2 (F) and Wnt9b (G) is strongly zonated, with highest expression in zone 3. One-way analysis of variance with post hoc analyses. Wnt2: F (2, 84) = 13.54; Wnt9b: F (2, 81) = 39.76. Data are given as means ± SEM (A, B, and D–G). n = 3 mice for all conditions (A–G). ∗∗P < 0.001, ∗∗∗P < 0.0001, and ∗∗∗∗P < 0.00001. Scale bar = 100 μm (C).
The study focused on Wnt2 and Wnt9b because they are critical for pericentral β-catenin activation and maintenance of zonation.7,9 Wnt2 and Wnt9b mRNA expression was significantly elevated in zone 3 relative to the other zones, consistent with zonation of both Wnts (Figure 1, F and G, and Supplemental Figure S1, A and B). Moreover, Wnt2 expression was markedly higher versus Wnt9b, including in zone 3, as also reported previously.7,9
Together, these results demonstrate that multiplex RNAscope is a valid approach to measure zonated expression in situ.
Quantification of Hepatic Frizzled Receptor and Lrp5/6 Coreceptor Expression
Although zonated Wnt signaling is established in adult liver, the precise identities of the cognate Fzd receptors responsible for this signaling have remained unclear. Therefore, RNAscope was used to determine Fzd receptor expression at the mRNA level, and zonation of these receptors. The study measured the number of cells positive for each respective Fzd receptor and found that all 10 Fzd receptors were expressed in adult mouse liver, albeit with varying levels of expression (Figure 2A). Fzd receptors 1, 4, 6, and 8 demonstrated highest levels of expression (Figure 2B and Supplemental Figure S2), whereas Fzd receptors 3, 9, and 10 were expressed at low, but detectable, levels (Figure 2A and Supplemental Figure S3). Fzd receptors 2, 5, and 7 were expressed at levels intermediate to either extreme (Figure 2A and Supplemental Figure S4). Surprisingly, all Fzd receptors, with the exception of Fzd receptor 6, were not zonated. By contrast, Fzd receptor 6 showed significantly higher expression in zone 1 compared with both zones 2 and 3 (Figure 2, A and B), whereas Fzd receptors 1, 4, and 5 demonstrated preferential expression in zone 1 over zone 2 only (Figure 2A).
Figure 2.
Hepatic expression of Wnt receptors and co-receptors in liver. A: mRNA expression of hepatic Frizzled (Fzd) receptors 1 to 10 via multiplex RNAscope. There is significantly higher zone 1 expression of the following: Fzd receptor 1 [F (2, 84) = 5.884, P = 0.004]; Fzd receptor 4 [F (2, 87) = 3.587, P = 0.031]; and Fzd receptor 5 [F (2, 84) = 4.234, P = 0.017] compared with zone 2 (one-way analysis of variance with post hoc analyses). There is no significant zonation of Fzd receptors 2, 3, 7, 8, 9, and 10 (P > 0.05). In contrast, Fzd receptor 6 displays zonation, with highest expression in zone 1 (one-way analysis of variance with post hoc analyses). F (2, 84) = 14.19. B: Representative image of Fzd receptor 6 mRNA alongside glutamine synthetase (GS) and glutaminase 2 (Gls2), marking the central vein and portal veins, respectively. Imaging reveals Fzd receptor 6 is most expressed in zone 1. Cells expressing Fzd receptor 6 are indicated by arrows. Inset: A zoomed-in Fzd6+ cell is featured. C: mRNA expression of Fzd co-receptors low-density lipoprotein receptor-related proteins 5 and 6 (Lrp5/6). Neither Lrp5 nor Lrp6 show zonated expression. Data are given as means ± SEM (A and C). n = 3 mice for all conditions (A and C). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001. Scale bar = 100 μm (B).
The study also analyzed mRNA expression of Fzd coreceptors Lrp5 and Lrp6. Although Lrp5 and Lrp6 were both expressed, neither displayed zonation. Interestingly, Lrp6 expression in all three zones was threefold higher compared with Lrp5 (Figure 2C and Supplemental Figure S1, C and D). Consistent with this, the levels of Lrp6 mRNA per cell were also approximately twofold higher versus Lrp5 (data not shown).
The average mRNA grains per cell for each respective Fzd receptor were measured (Figure 3). mRNA grain number reflected the same patterns as Fzd+ cell number (Figure 3), suggesting that levels of expression per cell are not directly influenced in a zone-specific manner.
Figure 3.
Quantification of Frizzled (Fzd) mRNA grains per cell. Quantitative analysis of mRNA grain number corresponding to Fzd receptors 1 to 10. Data are given as means ± SEM. n = 3 mice for all conditions. ∗P < 0.01, ∗∗P < 0.001, and ∗∗∗P < 0.0001.
Analysis of Wnt/Fzd Signaling Machinery by Hepatic Cell Type
To broadly determine the identities of hepatic cell types that express the machinery of Wnt-Fzd signaling in the liver, the study analyzed previously published single-cell RNA-sequencing data of adult mouse liver.13 Previously, this data set was used to ascertain the cell types responsible for the expression and secretion of Wnt2 and Wnt9b.7 Analysis of cell-type specificity for expression of Fzd receptors 1 through 10 indicated that each Fzd receptor possessed a distinct cell-type profile (Figure 4). Among the highest expressed Fzd receptors (1, 4, and 6), Fzd receptors 4 and 6 were expressed in hepatocytes, alongside nonparenchymal cells, including endothelial cells, cholangiocytes, and fibroblasts (Figure 4, D and F). In contrast, Fzd receptor 1 was not expressed in hepatocytes, but was robustly expressed in conventional type 1 dendritic cells (Figure 4A). Among the least expressed Fzd receptors (3, 9, and 10), Fzd receptors 3 and 9 were mainly expressed in cholangiocytes, whereas negligible amounts of Fzd receptor 10 were found in stromal cells and plasmacytoid dendritic cells (Figure 4, C, I, and J). Fzd receptors with intermediate expression (2, 5, 7, and 8) displayed the broadest cell-type distribution with expression in hepatocytes, as well as a variety of nonparenchymal cells (Figure 4, B, E, G, and H).
Figure 4.
Cell-specific expression of Frizzled (Fzd) receptors in various liver cells. Single-cell RNA-sequencing analysis of a previously published data set13 reveals expression of Fzd receptor throughout different liver cell types (A–H), except for Fzd receptor 9 (I), which is solely expressed in cholangiocytes, and Fzd receptor 10 (J), which is expressed in stromal cells and plasmacytoid dendritic cells (pDCs) only. cDC, conventional dendritic cell; cDC1, conventional type 1 dendritic cell; cDC2, conventional type 2 dendritic cell; ILC1, innate lymphoid cell 1; Macs, macrophages; MD, monocyte derived; Mig. cDCs, migratory conventional dendritic cells; Mono, monocytes; NK, natural killer.
Discussion
Wnt signaling is increasingly recognized as critical for regulation of hepatic zonation.15 Studies of this signaling pathway at the protein level have been restricted because of a limited repertoire of antibodies capable of recognizing endogenously expressed Wnts and Fzd receptors. The current study established multiplex RNAscope in liver to define chiefly hepatic Fzd receptor and Wnt coreceptor Lrp5 and Lrp6 expression. All 10 Fzd receptors and Lrp5/6 were seen to be expressed within a hepatic metabolic unit. However, the expression varied widely, in both the magnitude and cell-type specificity.
All Fzd receptors, except Fzd receptor 6, expressed in a nonzonated manner. Interestingly, Fzd receptor 6 was preferentially expressed in zone 1, the region exhibiting the least Wnt/β-catenin signaling activity. How does one reconcile this apparent discrepancy (ie, expressing high levels of a Fzd receptor in a region with the least Wnt signaling activity)? Prior work has shown that, unlike the other Fzd receptors, Fzd receptor 6 does not transduce canonical Wnt signaling. Rather, Fzd receptor 6 functions as a negative regulator of Wnt signaling as it lacks a C-terminal PDZ-binding domain responsible for transducing Wnt-dependent downstream signaling.16,17 We postulate that Fzd receptor 6's repressive properties enhance zonation by further restricting Wnt signaling to zone 3. Conversely, it is possible that the expression of the other Fzd receptors in adult liver becomes increasingly zonated in response to hepatic injury to facilitate cellular regeneration. Further work is required to explore such possibilities.
In contrast to the nonzonated expression patterns of most Fzd receptors, Wnt2 and Wnt9b were both highly zonated, as previously shown.7,9 This provides the mechanism by which basal activation of the Wnt/β-catenin pathway is evident in zone 3.6,15 This also suggests that maintenance of hepatic zonation is driven by ligand availability rather than the expression of the receptors themselves under physiological conditions.
The current results also show that cell-type specific mRNA expression of Fzd receptors 1 through 10 as well as Wnt2 and Wnt9b was not solely restricted to hepatocytes but was additionally found in a variety of nonparenchymal cells. Endothelial cells predominantly secrete Wnt2 and Wnt9b.7,9 Eliminating expression of Wnt2 and Wnt9b from hepatic endothelial cells mimicked liver-specific knockouts of β-catenin and Lrp5/6 (double knockout) as well as endothelial-specific Wntless knockout in terms of loss of zone 3 gene expression and delay in liver regeneration after partial hepatectomy.7,8,18, 19, 20, 21 Additional cell types in the liver expressed these Wnt genes, albeit at varying levels. Although Wnt9b expression was primarily restricted to endothelial cells, Wnt2 was also expressed in hepatocytes and monocytes. This suggests that Wnt2 and Wnt9b may play distinct roles in the maintenance of zonation based on the spatial distribution of these cell types. Cell-type–specific genetic manipulation could further elucidate the respective contributions of these Wnts. Many Fzd receptors were robustly expressed in endothelial cells, suggesting cell-cell cross talk or autocrine signaling within the hepatic vasculature. Indeed, local endothelial Wnt signaling maintains endothelial cell identity and zonation.22 Moreover, these data are consistent with earlier work in brain, demonstrating a role for Wnt/β-catenin signaling in vascular development and maintenance after development.23
The findings presented here demonstrating the broad expression of Wnt receptors throughout the liver may provide a mechanistic rationale for why the liver is capable of rapidly responding to Wnt ligands in response to injury. Increasing evidence shows that liver similarly responds quickly to R-spondin (Rspo) proteins during injury.24 Indeed, Rspo proteins amplify Wnt signaling to stimulate hepatic tissue regeneration.25 Consistent with this, periportal hepatocytes can be reprogrammed into pericentral hepatocytes by activating Wnt signaling via Rspo injections.26 We therefore posit that Wnt and Rspo ligands cooperate to induce Wnt signaling and drive zonation.6,27 However, although these data show the functional importance of Rspo proteins in liver injury response, the precise spatial distribution of Rspo ligands has yet to be comprehensively characterized in adult liver in either healthy states or response to injury; future work will explore this question.
More attention is being paid to the relevance of zonation in disease development and progression.2,15 For example, nonalcoholic steatohepatitis often starts as steatosis in zone 3 in adults28 and in zone 1 in pediatric cases.29 Autoimmune hepatitis impacts zone 1,30 whereas acetaminophen overdose causes zone 3 necrosis.31 These observations suggest that zonation is likely an important factor in compartmentalizing injury and the liver's response. Furthermore, we also posit that zonation may play key roles in the underlying mechanisms in liver disease pathogenesis. How changes in Fzd receptor expression may be a cause or an effect to such injuries needs to be directly and more specifically investigated, given the levels and location in the resting state of adult liver. Likewise, cholangiocyte-specific Fzd receptors are likely to display notable changes in cholangiopathies as well as in pathologic phenotypes, such as ductular reaction, and this needs to be directly investigated. Wnt7a, Wnt7b, and Wnt10a become uniquely up-regulated during cholestasis and have been shown to work in a paracrine manner on hepatocytes or by an autocrine mechanism on cholangiocytes.32,33 It will therefore be relevant to study whether specific Fzd receptors play a role in such cell-cell cross talk.
In summary, the current results provide a comprehensive map of Fzd receptor mRNA expression in adult liver, alongside additional components of the Wnt signaling pathway. All 10 Fzd receptors were expressed within metabolic units, alongside Lrp5 and Lrp6. However, only Fzd receptor 6 was expressed in a zone-specific manner. Furthermore, Wnt2 and Wnt9b were strongly zonated, which points to the importance of ligand availability in driving zonated expression.
Author Contributions
Z.F., S.P.M., and J.G. designed and conceptualized the study; J.G., S.H., E.D., and P.N.J. conducted experiments; J.G., S.L., and Z.F. interpreted data; and J.G., Z.F., and S.P.M. wrote the manuscript with input from the other authors.
Footnotes
Supported by NIH grants R01DK62277 and R01DK100287 and Endowed Chair for Experimental Pathology (S.P.M.), NIH grants P30DK120531 (Pittsburgh Liver Research Center, Pilot and Feasibility grant), R21DA052419, R21AA028800, and R01DK124219 (Z.F.), and US Department of Defense PR210207 (Z.F.) and T32GM133353 (J.G.). Additional funding was provided by NIH grants 1R01CA258449, 1P30DK120531 (Pittsburgh Liver Research Center), the Center for Metabolism and Mitochondrial Medicine, and The Pittsburgh FoundationMR2020 109502.
Disclosures: None declared.
Supplemental material for this article can be found at http://doi.org/10.1016/j.ajpath.2023.01.011.
Contributor Information
Satdarshan P. Monga, Email: smonga@pitt.edu.
Zachary Freyberg, Email: freyberg@pitt.edu.
Supplemental Data
Representative images of low-density lipoprotein receptor-related proteins 5 and 6 (Lrp5 and Lrp6, respectively), Wnt2, and Wnt9b. Representative images of Wnt2 (A) and Wnt9b (B) mRNA expression, as well as the mRNA expression of co-receptors Lrp5 (C) and Lrp6 (D). Glutaminase 2 (Gls2) and glutamine synthetase (GS) mRNAs mark the portal and central veins, respectively. Arrows indicate cells positive for mRNA expression. Scale bar = 100 μm (A–D).
Representative images of highly expressed Frizzled (Fzd) receptors. Representative images of Fzd receptor 1 (A), Fzd receptor 4 (B), and Fzd receptor 8 (C) Wnt receptors, which exhibit abundant mRNA expression alongside glutaminase 2 (Gls2) and glutamine synthetase (GS) mRNA expression, marking the portal and central veins, respectively. Arrows indicate Fzd+ cells, and insets highlight clusters of Fzd+ cells. Scale bar = 100 μm (A–C).
Representative images of low expressed Frizzled (Fzd) receptors. Representative images of Fzd receptor 3 (A), Fzd receptor 9 (B), and Fzd receptor 10 (C) Wnt receptors, which show a paucity of mRNA expression; glutaminase 2 (Gls2) and glutamine synthetase (GS) mRNA expression levels mark the portal and central veins, respectively. Imaging reveals that there are few positive cells for these Fzd receptors throughout the liver tissue. Arrow indicates a Fzd+ cell. Scale bar = 100 μm (A–C).
Representative images of moderately expressed Frizzled (Fzd) receptors. Representative images of Fzd receptor 2 (A), Fzd receptor 5 (B), and Fzd receptor 7 (C) mRNA expression, demonstrating moderate expression of these Wnt receptors; glutaminase 2 (Gls2) and glutamine synthetase (GS) mRNA levels mark the portal and central veins, respectively. Arrows indicate Fzd+ cells. Scale bar = 100 μm (A–C).
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Supplementary Materials
Representative images of low-density lipoprotein receptor-related proteins 5 and 6 (Lrp5 and Lrp6, respectively), Wnt2, and Wnt9b. Representative images of Wnt2 (A) and Wnt9b (B) mRNA expression, as well as the mRNA expression of co-receptors Lrp5 (C) and Lrp6 (D). Glutaminase 2 (Gls2) and glutamine synthetase (GS) mRNAs mark the portal and central veins, respectively. Arrows indicate cells positive for mRNA expression. Scale bar = 100 μm (A–D).
Representative images of highly expressed Frizzled (Fzd) receptors. Representative images of Fzd receptor 1 (A), Fzd receptor 4 (B), and Fzd receptor 8 (C) Wnt receptors, which exhibit abundant mRNA expression alongside glutaminase 2 (Gls2) and glutamine synthetase (GS) mRNA expression, marking the portal and central veins, respectively. Arrows indicate Fzd+ cells, and insets highlight clusters of Fzd+ cells. Scale bar = 100 μm (A–C).
Representative images of low expressed Frizzled (Fzd) receptors. Representative images of Fzd receptor 3 (A), Fzd receptor 9 (B), and Fzd receptor 10 (C) Wnt receptors, which show a paucity of mRNA expression; glutaminase 2 (Gls2) and glutamine synthetase (GS) mRNA expression levels mark the portal and central veins, respectively. Imaging reveals that there are few positive cells for these Fzd receptors throughout the liver tissue. Arrow indicates a Fzd+ cell. Scale bar = 100 μm (A–C).
Representative images of moderately expressed Frizzled (Fzd) receptors. Representative images of Fzd receptor 2 (A), Fzd receptor 5 (B), and Fzd receptor 7 (C) mRNA expression, demonstrating moderate expression of these Wnt receptors; glutaminase 2 (Gls2) and glutamine synthetase (GS) mRNA levels mark the portal and central veins, respectively. Arrows indicate Fzd+ cells. Scale bar = 100 μm (A–C).