Natural variation of TBR confers plant zinc toxicity tolerance through root cell wall pectin methylesterification.

Zinc (Zn) is an essential micronutrient but can be cytotoxic when present in excess. Plants have evolved mechanisms to tolerate Zn toxicity. To identify genetic loci responsible for natural variation of plant tolerance to Zn toxicity, we conduct genome-wide association studies for root growth responses to high Zn and identify 21 significant associated loci. Among these loci, we identify Trichome Birefringence (TBR) allelic variation determining root growth variation in high Zn conditions. Natural alleles of TBR determine TBR transcript and protein levels which affect pectin methylesterification in root cell walls. Together with previously published data showing that pectin methylesterification increase goes along with decreased Zn binding to cell walls in TBR mutants, our findings lead to a model in which TBR allelic variation enables Zn tolerance through modulating root cell wall pectin methylesterification. The role of TBR in Zn tolerance is conserved across dicot and monocot plant species.

Therapeutic potential of SOX9 dysruption in Combined Hepatocellular Carcinoma-Cholangiocarcinoma.

Combined hepatocellular carcinoma-cholangiocarcinoma (cHCC-CCA) represents a challenging subtype of primary liver cancer with limited treatment options and a poor prognosis. Recently, we and others have highlighted the context-dependent roles of the biliary-specific transcription factor SOX9 in the pathogenesis of liver cancers using various Cre applications in Sox9 (flox/flox) strains, to achieve elimination for exon 2 and 3 of the Sox9 gene locus as a preventive manner. Here, we reveal the contrasting responses of developmental Sox9 elimination using Alb-Cre;Sox9 (flox/flox) ( Sox9 LKO) versus CRISPR/Cas9 -based tumor specific acute Sox9 CKO in SB-HDTVI-based Akt-YAP1 and Akt-NRAS cHCC-CCA formation. Sox9 LKO specifically abrogates the Akt-YAP1 CCA region while robustly stimulating the proliferation of remaining poorly differentiated HCC pertaining liver progenitor cell characteristics, whereas Sox9 CKO potently prevents Akt-YAP1 and Akt-NRAS cHCC-CCA development irrespective of fate of tumor cells compared to respective controls. Additionally, we find that Akt-NRAS , but not Akt-YAP1 , tumor formation is partially dependent on the Sox9-Dnmt1 cascade. Pathologically, SOX9 is indispensable for Akt-YAP1 -mediated HC-to-BEC/CCA reprogramming but required for the maintenance of CCA nodules. Lastly, therapeutic elimination of Sox9 using the OPN-CreERT2 strain combined with an inducible CRISPR/Cas9 -based Sox9 iKO significantly reduces Akt-YAP1 cHCC-CCA tumor burden, similar to Sox9 CKO. Thus, we contrast the outcomes of acute Sox9 deletion with developmental Sox9 knockout models, emphasizing the importance of considering adaptation mechanisms in therapeutic strategies. This necessitates the careful consideration of genetic liver cancer studies using developmental Cre and somatic mutant lines, particularly for genes involved in hepatic commitment during development. Our findings suggest that SOX9 elimination may hold promise as a therapeutic approach for cHCC-CCA and underscore the need for further investigation to translate these preclinical insights into clinical applications.

Production of grains with ultra-low heavy metal accumulation by pyramiding novel Alleles of OsNramp5 and OsLsi2 in two-line hybrid rice.

Ensuring rice yield and grain safety quality are vital for human health. In this study, we developed two-line hybrid rice (TLHR) with ultra-low grain cadmium (Cd) and arsenic (As) accumulation by pyramiding novel alleles of OsNramp5 and OsLsi2. We first generated low Cd accumulation restorer (R) lines by editing OsNramp5, OsLCD, and OsLCT in japonica and indica. After confirming that OsNramp5 was most efficient in reducing Cd, we edited this gene in C815S, a genic male sterile line (GMSL), and screened it for alleles with low Cd accumulation. Next, we generated R and GMSL lines with low As accumulation by editing OsLsi2 in a series of YK17 and C815S lines. When cultivated in soils that were heavily polluted with Cd and As, the edited R, GMSL, and TLHR plants showed significantly reduced heavy metal accumulation, while maintaining a relatively stable yield potential. This study provides an effective scheme for the safe production of grains in As- and/or Cd-polluted paddy fields.

Loss-of-function of PGL10 impairs photosynthesis and tolerance to high-temperature stress in rice.

High temperature (HT) affects the production of chlorophyll (Chl) pigment and inhibits cellular processes that impair photosynthesis, and growth and development in plants. However, the molecular mechanisms underlying heat stress in rice are not fully understood yet. In this study, we identified two mutants varying in leaf color from the ethylmethanesulfonate mutant library of indica rice cv. Zhongjiazao-17, which showed pale-green leaf color and variegated leaf phenotype under HT conditions. Mut-map revealed that both mutants were allelic, and their phenotype was controlled by a single recessive gene PALE GREEN LEAF 10 (PGL10) that encodes NADPH:protochlorophyllide oxidoreductase B, which is required for the reduction of protochlorophyllide into chlorophyllide in light-dependent tetrapyrrole biosynthetic pathway-based Chl synthesis. Overexpression-based complementation and CRISPR/Cas9-based knockout analyses confirmed the results of Mut-map. Moreover, qRT-PCR-based expression analysis of PGL10 showed that it expresses in almost all plant parts with the lowest expression in root, followed by seed, third leaf, and then other green tissues in both mutants, pgl10a and pgl10b. Its protein localizes in chloroplasts, and the first 17 amino acids from N-terminus are responsible for signals in chloroplasts. Moreover, transcriptome analysis performed under HT conditions revealed that the genes involved in the Chl biosynthesis and degradation, photosynthesis, and reactive oxygen species detoxification were differentially expressed in mutants compared to WT. Thus, these results indicate that PGL10 is required for maintaining chloroplast function and plays an important role in rice adaptation to HT stress conditions by controlling photosynthetic activity.

Photosynthesis regulates tillering bud elongation and nitrogen-use efficiency via sugar-induced NGR5 in rice.

Rice tillering is one of the most important agronomical traits largely determining grain yield. Photosynthesis and nitrogen availability are two important factors affecting rice tiller bud elongation; however, underlying mechanism and their cross-talk is poorly understood. Here, we used map-based cloning, transcriptome profiling, phenotypic analysis, and molecular genetics to understand the roles of the Decreased Tiller Number 1 (DTN1) gene that encodes the fructose-1,6-bisphosphate aldolase and involves in photosynthesis required for light-induced axillary bud elongation in rice. Deficiency of DTN1 results in the reduced photosynthetic rate and decreased contents of sucrose and other sugars in both leaves and axillary buds, and the reduced tiller number in dtn1 mutant could be partially rescued by exogenous sucrose treatment. Furthermore, we found that the expression of nitrogen-mediated tiller growth response 5 (NGR5) was remarkably decreased in shoot base of dtn1-2, which can be activated by sucrose treatment. Overexpression of NGR5 in the dtn1-2 could partially rescue the reduced tiller number, and the tiller number of dtn1-2 was insensitive to nitrogen supply. This work demonstrated that the sugar level regulated by photosynthesis and DTN1 could positively regulate NGR5 expression, which coordinates the cross-talk between carbon and nitrate to control tiller bud outgrowth in rice.

An orchestrated ethylene-gibberellin signaling cascade contributes to mesocotyl elongation and emergence of rice direct seeding.

A mechanized direct seeding of rice with less labor and water usage, has been widely adopted. However, this approach requires varieties that exhibit uniform seedling emergence. Mesocotyl elongation (ME) offers the main drive of fast emergence of rice seedlings from soils; nevertheless, its genetic basis remains unknown. Here, we identify a major rice quantitative trait locus Mesocotyl Elongation1 (qME1), an allele of the Green Revolution gene Semi-Dwarf1 (SD1), encoding GA20-oxidase for gibberellin (GA) biosynthesis. ME1 expression is strongly induced by soil depth and ethylene. When rice grains are direct-seeded in soils, the ethylene core signaling factor OsEIL1 directly promotes ME1 transcription, accelerating bioactive GA biosynthesis. The GAs further degrade the DELLA protein SLENDER RICE 1 (SLR1), alleviating its inhibition of rice PHYTOCHROME-INTERACTING FACTOR-LIKE13 (OsPIL13) to activate the downstream expansion gene OsEXPA4 and ultimately promote rice seedling ME and emergence. The ancient traits of long mesocotyl and strong emergence ability in wild rice and landrace were gradually lost in company with the Green Revolution dwarf breeding process, and an elite ME1-R allele (D349H) is found in some modern Geng varieties (long mesocotyl lengths) in northern China, which can be used in the direct seeding and dwarf breeding of Geng varieties. Furthermore, the ectopic and high expression of ME1 driven by mesocotyl-specific promoters resulted in rice plants that could be direct-seeded without obvious plant architecture or yield penalties. Collectively, we reveal the molecular mechanism of rice ME, and provide useful information for breeding new Green Revolution varieties with long mesocotyl suitable for direct-seeding practice.

OsLESV and OsESV1 promote transitory and storage starch biosynthesis to determine rice grain quality and yield.

Transitory starch is an important carbon source in leaves, and its biosynthesis and metabolism are closely related to grain quality and yield. The molecular mechanisms controlling leaf transitory starch biosynthesis and degradation and their effects on rice (Oryza sativa) quality and yield remain unclear. Here, we show that OsLESV and OsESV1, the rice orthologs of AtLESV and AtESV1, are associated with transitory starch biosynthesis in rice. The total starch and amylose contents in leaves and endosperms are significantly reduced, and the final grain quality and yield are compromised in oslesv and osesv1 single and oslesv esv1 double mutants. Furthermore, we found that OsLESV and OsESV1 bind to starch, and this binding depends on a highly conserved C-terminal tryptophan-rich region that acts as a starch-binding domain. Importantly, OsLESV and OsESV1 also interact with the key enzymes of starch biosynthesis, granule-bound starch synthase I (GBSSI), GBSSII, and pyruvate orthophosphote dikiase (PPDKB), to maintain their protein stability and activity. OsLESV and OsESV1 also facilitate the targeting of GBSSI and GBSSII from plastid stroma to starch granules. Overexpression of GBSSI, GBSSII, and PPDKB can partly rescue the phenotypic defects of the oslesv and osesv1 mutants. Thus, we demonstrate that OsLESV and OsESV1 play a key role in regulating the biosynthesis of both leaf transitory starch and endosperm storage starch in rice. These findings deepen our understanding of the molecular mechanisms underlying transitory starch biosynthesis in rice leaves and reveal how the transitory starch metabolism affects rice grain quality and yield, providing useful information for the genetic improvement of rice grain quality and yield.

A mediator of OsbZIP46 deactivation and degradation negatively regulates seed dormancy in rice.

Preharvest sprouting (PHS) is a deleterious phenotype that occurs frequently in rice-growing regions where the temperature and precipitation are high. It negatively affects yield, quality, and downstream grain processing. Seed dormancy is a trait related to PHS. Longer seed dormancy is preferred for rice production as it can prevent PHS. Here, we map QTLs associated with rice seed dormancy and clone Seed Dormancy 3.1 (SDR3.1) underlying one major QTL. SDR3.1 encodes a mediator of OsbZIP46 deactivation and degradation (MODD). We show that SDR3.1 negatively regulates seed dormancy by inhibiting the transcriptional activity of ABIs. In addition, we reveal two critical amino acids of SDR3.1 that are critical for the differences in seed dormancy between the Xian/indica and Geng/japonica cultivars. Further, SDR3.1 has been artificially selected during rice domestication. We propose a two-line model for the process of rice seed dormancy domestication from wild rice to modern cultivars. We believe the candidate gene and germplasm studied in this study would be beneficial for the genetic improvement of rice seed dormancy.

Unveiling Innovative Approaches to Mitigate Metals/Metalloids Toxicity for Sustainable Agriculture.

Due to anthropogenic activities, environmental pollution of heavy metals/metalloids (HMs) has increased and received growing attention in recent decades. Plants growing in HM-contaminated soils have slower growth and development, resulting in lower agricultural yield. Exposure to HMs leads to the generation of free radicals (oxidative stress), which alters plant morpho-physiological and biochemical pathways at the cellular and tissue levels. Plants have evolved complex defense mechanisms to avoid or tolerate the toxic effects of HMs, including HMs absorption and accumulation in cell organelles, immobilization by forming complexes with organic chelates, extraction via numerous transporters, ion channels, signaling cascades, and transcription elements, among others. Nonetheless, these internal defensive mechanisms are insufficient to overcome HMs toxicity. Therefore, unveiling HMs adaptation and tolerance mechanisms is necessary for sustainable agriculture. Recent breakthroughs in cutting-edge approaches such as phytohormone and gasotransmitters application, nanotechnology, omics, and genetic engineering tools have identified molecular regulators linked to HMs tolerance, which may be applied to generate HMs-tolerant future plants. This review summarizes numerous systems that plants have adapted to resist HMs toxicity, such as physiological, biochemical, and molecular responses. Diverse adaptation strategies have also been comprehensively presented to advance plant resilience to HMs toxicity that could enable sustainable agricultural production.

The elite eating quality alleles Wxb and ALKb are regulated by OsDOF18 and coordinately improve head rice yield.

Head rice yield (HRY) measures rice milling quality and determines final grain yield and commercial value. Here, we report that two major quantitative trait loci for milling quality in rice, qMq-1 and qMq-2, represent allelic variants of Waxylv/Waxyb (hereafter Wx) encoding Granule-Bound Starch Synthase I (GBSSI) and Alkali Spreading Value ALKc/ALKb encoding Soluble Starch Synthase IIa (SSIIa), respectively. Complementation and overexpression transgenic lines in indica and japonica backgrounds confirmed that Wx and ALK coordinately regulate HRY by affecting amylose content, the number of amylopectin branches, amyloplast size, and thus grain filling and hardness. The transcription factor OsDOF18 acts upstream of Wx and ALK by activating their transcription. Furthermore, rice accessions with Wxb and ALKb alleles showed improved HRY over those with Wxlv and ALKc. Our study not only reveals the novel molecular mechanism underlying the formation of HRY but also provides a strategy for breeding rice cultivars with improved HRY.

In-situ self-crosslinking strategy for super-tough polylactic acid/ bio-based polyurethane blends.

As a bio-based degradable plastic, polylactic acid (PLA) is highly commercialized, but its inherent brittleness limits its widespread use. In-situ polymerization techniques are effective in improving the toughness of PLA. However, the enhancement of the toughening effect in polyurethanes (PUs) through in-situ self-crosslinking still requires improvement and heavily relies on petroleum-derived feedstocks in certain approaches. In this paper, 1,3-polypropanediol (PO3G) of bio-based origin rather than conventional polyols like polyethylene glycol (PEG) and poly propylene glycol (PPG) was used. PLA/PO3G-PU blends were prepared via an in-situ self-crosslinking strategy. With a notch impact and tensile strength of 55.95 kJ/m2 and 47.77 MPa (a retention rate of 68.9 % compared with pure PLA), respectively, PLA/PO3G-PU blends achieved a better balance between stiffness and toughness. This work provides a new option for PLA to achieve a stiffness-toughness balance and get rid of dependence on petrochemical resources.

YGL3 Encoding an IPP and DMAPP Synthase Interacts with OsPIL11 to Regulate Chloroplast Development in Rice.

As the source of isoprenoid precursors, the plastidial methylerythritol phosphate (MEP) pathway plays an essential role in plant development. Here, we report a novel rice (Oryza sativa L.) mutant ygl3 (yellow-green leaf3) that exhibits yellow-green leaves and lower photosynthetic efficiency compared to the wild type due to abnormal chloroplast ultrastructure and reduced chlorophyll content. Map-based cloning showed that YGL3, one of the major genes involved in the MEP pathway, encodes 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, which is localized in the thylakoid membrane. A single base substitution in ygl3 plants resulted in lower 4-hydroxy-3-methylbut-2-enyl diphosphate reductase activity and lower contents of isopentenyl diphosphate (IPP) compared to the wild type. The transcript levels of genes involved in the syntheses of chlorophyll and thylakoid membrane proteins were significantly reduced in the ygl3 mutant compared to the wild type. The phytochrome interacting factor-like gene OsPIL11 regulated chlorophyll synthesis during the de-etiolation process by directly binding to the promoter of YGL3 to activate its expression. The findings provides a theoretical basis for understanding the molecular mechanisms by which the MEP pathway regulate chloroplast development in rice.

OsLEA1b Modulates Starch Biosynthesis at High Temperatures in Rice.

High temperatures accelerate the accumulation of storage material in seeds, often leading to defects in grain filling. However, the mechanisms regulating grain filling at high temperatures remain unknown. Here, we want to explore the quality factors influenced by the environment and have identified a LATE EMBROYGENESIS ABUNDANT gene, OsLEA1b, a heat-stress-responsive gene in rice grain filling. OsLEA1b is highly expressed in the endosperm, and its coding protein localizes to the nucleus and cytoplasm. Knock-out mutants of OsLEA1b had abnormal compound starch granules in endosperm cells and chalky endosperm with significantly decreased grain weight and grain number per panicle. The oslea1b mutants exhibited a lower proportion of short starch chains with degrees of polymerization values from 6 to 13 and a higher proportion of chains with degrees from 14 to 48, as well as significantly lower contents of starch, protein, and lipid compared to the wild type. The difference was exacerbated under high temperature conditions. Moreover, OsLEA1b was induced by drought stress. The survival rate of oslea1b mutants decreased significantly under drought stress treatment, with significant increase in ROS levels. These results indicate that OsLEA1b regulates starch biosynthesis and influences rice grain quality, especially under high temperatures. This provides a valuable resource for genetic improvement in rice grain quality.

Huntingtin loss in hepatocytes is associated with altered metabolism, adhesion, and liver zonation.

Huntington's disease arises from a toxic gain of function in the huntingtin (HTT) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological, and plasma metabolite levels. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic ß-catenin function.

Genome-wide identification and expression analysis of 3-ketoacyl-CoA synthase gene family in rice (Oryza sativa L.) under cadmium stress.

3-Ketoacyl-CoA synthase (KCS) is the key rate-limiting enzyme for the synthesis of very long-chain fatty acids (VLCFAs) in plants, which determines the carbon chain length of VLCFAs. However, a comprehensive study of KCSs in Oryza sativa has not been reported yet. In this study, we identified 22 OsKCS genes in rice, which are unevenly distributed on nine chromosomes. The OsKCS gene family is divided into six subclasses. Many cis-acting elements related to plant growth, light, hormone, and stress response were enriched in the promoters of OsKCS genes. Gene duplication played a crucial role in the expansion of the OsKCS gene family and underwent a strong purifying selection. Quantitative Real-time polymerase chain reaction (qRT-PCR) results revealed that most KCS genes are constitutively expressed. We also revealed that KCS genes responded differently to exogenous cadmium stress in japonica and indica background, and the KCS genes with higher expression in leaves and seeds may have functions under cadmium stress. This study provides a basis for further understanding the functions of KCS genes and the biosynthesis of VLCFA in rice.

Huntingtin loss in hepatocytes is associated with altered metabolism, adhesion, and liver zonation.

Huntington's disease arises from a toxic gain of function in the huntingtin ( HTT ) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological and plasma metabolite level. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic ß-catenin function.

Transcriptome and Proteome Analysis Revealed That Hormone and Reactive Oxygen Species Synergetically Regulate Dormancy of Introgression Line in Rice (Oryza sativa L.).

Dormancy is a complex agronomy phenotype controlled by multiple signaling and a key trait repressing pre-harvest sprouting (PHS). However, the signaling network of dormancy remains unclear. In this study, we used Zhonghua11 (ZH11) with a weak dormancy, and Introgression line (IL) with a strong dormancy to study the mechanism of hormones and reactive oxygen species (ROS) crosstalk regulating rice dormancy. The germination experiment showed that the germination rate of ZH11 was 76.86%, while that of IL was only 1.25%. Transcriptome analysis showed that there were 1658 differentially expressed genes (DEGs) between IL and ZH11, of which 577 were up-regulated and 1081 were down-regulated. Additionally, DEGs were mainly enriched in oxidoreductase activity, cell periphery, and plant hormone signal transduction pathways. Tandem mass tags (TMT) quantitative proteomics analysis showed 275 differentially expressed proteins (DEPs) between IL and ZH11, of which 176 proteins were up-regulated, 99 were down-regulated, and the DEPs were mainly enriched in the metabolic process and oxidation-reduction process. The comprehensive transcriptome and proteome analysis showed that their correlation was very low, and only 56 genes were co-expressed. Hormone content detection showed that IL had significantly lower abscisic acid (ABA) contents than the ZH11 while having significantly higher jasmonic acid (JA) contents than the ZH11. ROS content measurement showed that the hydrogen peroxide (H2O2) content of IL was significantly lower than the ZH11, while the production rate of superoxide anion (O2.-) was significantly higher than the ZH11. These results indicate that hormones and ROS crosstalk to regulate rice dormancy. In particular, this study has deepened our mechanism of ROS and JA crosstalk regulating rice dormancy and is conducive to our precise inhibition of PHS.

Fabrication of Polyurethane Elastomer/Hindered Phenol Composites with Tunable Damping Property.

Vibration and noise-reduction materials are indispensable in various fields. Polyurethane (PU)-based damping materials can dissipate the external mechanical and acoustic energy through molecular chain movements to mitigate the adverse effects of vibrations and noise. In this study, PU-based damping composites were obtained by compositing PU rubber prepared using 3-methyltetrahydrofuran/tetrahydrofuran copolyether glycol, 4,4'-diphenylmethane diisocyanate, and trimethylolpropane monoallyl ether as raw materials with hindered phenol, viz., and 3,9-bis{2-[3-(3-tert-butyl-4-hydroxy-5-methylphenyl)proponyloxy]-1,1-dimethylethyl}-2,4,8,10-tetraoxaspiro[5.5]undecane (AO-80). Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, dynamic mechanical analysis, and tensile tests were conducted to evaluate the properties of the resulting composites. The glass transition temperature of the composite increased from -40 to -23 °C, and the tan δMax of the PU rubber increased by 81%, from 0.86 to 1.56 when 30 phr of AO-80 was added. This study provides a new platform for the design and preparation of damping materials for industrial applications and daily life.

A Spatial Atlas of Wnt Receptors in Adult Mouse Liver.

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/ß-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.