Epcam regulates intrahepatic bile duct reconstruction in zebrafish, providing a potential model for primary cholangitis model.

Epithelial cell adhesion molecules (EpCAMs) have been identified as surface markers of proliferating ductal cells, which are referred to as liver progenitor cells (LPCs), during liver regeneration and correspond to malignancies. These cells can differentiate into hepatocytes and biliary epithelial cells (BECs) in vitro. EpCAM-positive LPCs are involved in liver regeneration following severe liver injury; however, the in vivo function of EpCAMs in the regenerating liver remains unclear. In the present study, we used a zebrafish model of LPC-driven liver regeneration to elucidate the function of EpCAMs in the regenerating liver in vivo. Proliferating ductal cells were observed after severe hepatocyte loss in the zebrafish model. Analyses of the liver size as well as hepatocyte and BEC markers revealed successful conversion of LPCs to hepatocytes and BECs in epcam mutants. Notably, epcam mutants exhibited severe defects in intrahepatic duct maturation and bile acid secretion in regenerating hepatocytes, suggesting that epcam plays a critical role in intrahepatic duct reconstruction during LPC-driven liver regeneration. Our findings provide insights into human diseases involving non-parenchymal cells, such as primary biliary cholangitis, by highlighting the regulatory effect of epcam on intrahepatic duct reconstruction.

Hepatocyte-to-cholangiocyte conversion occurs through transdifferentiation independently of proliferation in zebrafish.

BACKGROUND AND AIMS: Injury to biliary epithelial cells (BECs) lining the hepatic bile ducts leads to cholestatic liver diseases. Upon severe biliary damage, hepatocytes can convert to BECs, thereby contributing to liver recovery. Given a potential of augmenting this hepatocyte-to-BEC conversion as a therapeutic option for cholestatic liver diseases, it will be important to thoroughly understand the cellular and molecular mechanisms of the conversion process. APPROACH AND RESULTS: Towards this aim, we have established a zebrafish model for hepatocyte-to-BEC conversion by employing Tg(fabp10a:CFP-NTR) zebrafish with a temporal inhibition of Notch signaling during regeneration. Cre/loxP-mediated permanent and H2B-mCherry-mediated short-term lineage tracing revealed that in the model, all BECs originate from hepatocytes. During the conversion, BEC markers are sequentially induced in the order of Sox9b, Yap/Taz, Notch activity/ epcam , and Alcama/ krt18 ; the expression of the hepatocyte marker Bhmt disappears between the Sox9b and Yap/Taz induction. Importantly, live time-lapse imaging unambiguously revealed transdifferentiation of hepatocytes into BECs: hepatocytes convert to BECs without transitioning through a proliferative intermediate state. In addition, using compounds and transgenic and mutant lines that modulate Notch and Yap signaling, we found that both Notch and Yap signaling are required for the conversion even in Notch- and Yap-overactivating settings. CONCLUSIONS: Hepatocyte-to-BEC conversion occurs through transdifferentiation independently of proliferation, and Notch and Yap signaling control the process in parallel with a mutually positive interaction. The new zebrafish model will further contribute to a thorough understanding of the mechanisms of the conversion process.

In vivo imaging of calcium dynamics in zebrafish hepatocytes.

BACKGROUND AND AIMS: Hepatocytes were the first cell type for which oscillations of cytoplasmic calcium levels in response to hormones were described. Since then, investigation of calcium dynamics in liver explants and culture has greatly increased our understanding of calcium signaling. A bottleneck, however, exists in observing calcium dynamics in a noninvasive manner because of the optical inaccessibility of the mammalian liver. Here, we aimed to take advantage of the transparency of the zebrafish larvae to image hepatocyte calcium dynamics in vivo at cellular resolution. APPROACH AND RESULTS: We developed a transgenic model expressing a calcium sensor, GCaMP6s, specifically in zebrafish hepatocytes. Using this, we provide a quantitative assessment of intracellular calcium dynamics during multiple contexts, including growth, feeding, ethanol-induced stress, and cell ablation. Specifically, we show that synchronized calcium oscillations are present in vivo , which are lost upon starvation. Starvation induces lipid accumulation in the liver. Feeding recommences calcium waves in the liver, but in a spatially restricted manner, as well as resolves starvation-induced hepatic steatosis. By using a genetically encoded scavenger for calcium, we show that dampening of calcium signaling accelerates the accumulation of starvation-related lipid droplets in the liver. Furthermore, ethanol treatment, as well as cell ablation, induces calcium flux, but with different dynamics. The former causes asynchronous calcium oscillations, whereas the latter leads to a single calcium spike. CONCLUSIONS: We demonstrate the presence of oscillations, waves, and spikes in vivo . Calcium waves are present in response to nutrition and negatively regulate starvation-induced accumulation of lipid droplets.

Update on Hepatobiliary Plasticity.

The liver field has been debating for decades the contribution of the plasticity of the two epithelial compartments in the liver, hepatocytes and biliary epithelial cells (BECs), to derive each other as a repair mechanism. The hepatobiliary plasticity has been first observed in diseased human livers by the presence of biphenotypic cells expressing hepatocyte and BEC markers within bile ducts and regenerative nodules or budding from strings of proliferative BECs in septa. These observations are not surprising as hepatocytes and BECs derive from a common fetal progenitor, the hepatoblast, and, as such, they are expected to compensate for each other's loss in adults. To investigate the cell origin of regenerated cell compartments and associated molecular mechanisms, numerous murine and zebrafish models with ability to trace cell fates have been extensively developed. This short review summarizes the clinical and preclinical studies illustrating the hepatobiliary plasticity and its potential therapeutic application.

Biliary atresia is associated with polygenic susceptibility in ciliogenesis and planar polarity effector genes.

BACKGROUND & AIMS: Biliary atresia (BA) is poorly understood and leads to liver transplantation (LT), with the requirement for and associated risks of lifelong immunosuppression, in most children. We performed a genome-wide association study (GWAS) to determine the genetic basis of BA. METHODS: We performed a GWAS in 811 European BA cases treated with LT in US, Canadian and UK centers, and 4,654 genetically matched controls. Whole-genome sequencing of 100 cases evaluated synthetic association with rare variants. Functional studies included whole liver transcriptome analysis of 64 BA cases and perturbations in experimental models. RESULTS: A GWAS of common single nucleotide polymorphisms (SNPs), i.e. allele frequencies >1%, identified intronic SNPs rs6446628 in AFAP1 with genome-wide significance (p = 3.93E-8) and rs34599046 in TUSC3 at sub-threshold genome-wide significance (p = 1.34E-7), both supported by credible peaks of neighboring SNPs. Like other previously reported BA-associated genes, AFAP1 and TUSC3 are ciliogenesis and planar polarity effectors (CPLANE). In gene-set-based GWAS, BA was associated with 6,005 SNPs in 102 CPLANE genes (p = 5.84E-15). Compared with non-CPLANE genes, more CPLANE genes harbored rare variants (allele frequency <1%) that were assigned Human Phenotype Ontology terms related to hepatobiliary anomalies by predictive algorithms, 87% vs. 40%, p <0.0001. Rare variants were present in multiple genes distinct from those with BA-associated common variants in most BA cases. AFAP1 and TUSC3 knockdown blocked ciliogenesis in mouse tracheal cells. Inhibition of ciliogenesis caused biliary dysgenesis in zebrafish. AFAP1 and TUSC3 were expressed in fetal liver organoids, as well as fetal and BA livers, but not in normal or disease-control livers. Integrative analysis of BA-associated variants and liver transcripts revealed abnormal vasculogenesis and epithelial tube formation, explaining portal vein anomalies that co-exist with BA. CONCLUSIONS: BA is associated with polygenic susceptibility in CPLANE genes. Rare variants contribute to polygenic risk in vulnerable pathways via unique genes. IMPACT AND IMPLICATIONS: Liver transplantation is needed to cure most children born with biliary atresia, a poorly understood rare disease. Transplant immunosuppression increases the likelihood of life-threatening infections and cancers. To improve care by preventing this disease and its progression to transplantation, we examined its genetic basis. We find that this disease is associated with both common and rare mutations in highly specialized genes which maintain normal communication and movement of cells, and their organization into bile ducts and blood vessels during early development of the human embryo. Because defects in these genes also cause other birth defects, our findings could lead to preventive strategies to lower the incidence of biliary atresia and potentially other birth defects.

Follistatin-controlled activin-HNF4α-coagulation factor axis in liver progenitor cells determines outcome of acute liver failure.

BACKGROUND AND AIMS: In patients with acute liver failure (ALF) who suffer from massive hepatocyte loss, liver progenitor cells (LPCs) take over key hepatocyte functions, which ultimately determines survival. This study investigated how the expression of hepatocyte nuclear factor 4α (HNF4α), its regulators, and targets in LPCs determines clinical outcome of patients with ALF. APPROACH AND RESULTS: Clinicopathological associations were scrutinized in 19 patients with ALF (9 recovered and 10 receiving liver transplantation). Regulatory mechanisms between follistatin, activin, HNF4α, and coagulation factor expression in LPC were investigated in vitro and in metronidazole-treated zebrafish. A prospective clinical study followed up 186 patients with cirrhosis for 80 months to observe the relevance of follistatin levels in prevalence and mortality of acute-on-chronic liver failure. Recovered patients with ALF robustly express HNF4α in either LPCs or remaining hepatocytes. As in hepatocytes, HNF4α controls the expression of coagulation factors by binding to their promoters in LPC. HNF4α expression in LPCs requires the forkhead box protein H1-Sma and Mad homolog 2/3/4 transcription factor complex, which is promoted by the TGF-ß superfamily member activin. Activin signaling in LPCs is negatively regulated by follistatin, a hepatocyte-derived hormone controlled by insulin and glucagon. In contrast to patients requiring liver transplantation, recovered patients demonstrate a normal activin/follistatin ratio, robust abundance of the activin effectors phosphorylated Sma and Mad homolog 2 and HNF4α in LPCs, leading to significantly improved coagulation function. A follow-up study indicated that serum follistatin levels could predict the incidence and mortality of acute-on-chronic liver failure. CONCLUSIONS: These results highlight a crucial role of the follistatin-controlled activin-HNF4α-coagulation axis in determining the clinical outcome of massive hepatocyte loss-induced ALF. The effects of insulin and glucagon on follistatin suggest a key role of the systemic metabolic state in ALF.

Farnesoid X Receptor Activation Impairs Liver Progenitor Cell-Mediated Liver Regeneration via the PTEN-PI3K-AKT-mTOR Axis in Zebrafish.

BACKGROUND AND AIMS: Following mild liver injury, pre-existing hepatocytes replicate. However, if hepatocyte proliferation is compromised, such as in chronic liver diseases, biliary epithelial cells (BECs) contribute to hepatocytes through liver progenitor cells (LPCs), thereby restoring hepatic mass and function. Recently, augmenting innate BEC-driven liver regeneration has garnered attention as an alternative to liver transplantation, the only reliable treatment for patients with end-stage liver diseases. Despite this attention, the molecular basis of BEC-driven liver regeneration remains poorly understood. APPROACH AND RESULTS: By performing a chemical screen with the zebrafish hepatocyte ablation model, in which BECs robustly contribute to hepatocytes, we identified farnesoid X receptor (FXR) agonists as inhibitors of BEC-driven liver regeneration. Here we show that FXR activation blocks the process through the FXR-PTEN (phosphatase and tensin homolog)-PI3K (phosphoinositide 3-kinase)-AKT-mTOR (mammalian target of rapamycin) axis. We found that FXR activation blocked LPC-to-hepatocyte differentiation, but not BEC-to-LPC dedifferentiation. FXR activation also suppressed LPC proliferation and increased its death. These defects were rescued by suppressing PTEN activity with its chemical inhibitor and ptena/b mutants, indicating PTEN as a critical downstream mediator of FXR signaling in BEC-driven liver regeneration. Consistent with the role of PTEN in inhibiting the PI3K-AKT-mTOR pathway, FXR activation reduced the expression of pS6, a marker of mTORC1 activation, in LPCs of regenerating livers. Importantly, suppressing PI3K and mTORC1 activities with their chemical inhibitors blocked BEC-driven liver regeneration, as did FXR activation. CONCLUSIONS: FXR activation impairs BEC-driven liver regeneration by enhancing PTEN activity; the PI3K-AKT-mTOR pathway controls the regeneration process. Given the clinical trials and use of FXR agonists for multiple liver diseases due to their beneficial effects on steatosis and fibrosis, the detrimental effects of FXR activation on LPCs suggest a rather personalized use of the agonists in the clinic.

Attenuating the Epidermal Growth Factor Receptor-Extracellular Signal-Regulated Kinase-Sex-Determining Region Y-Box 9 Axis Promotes Liver Progenitor Cell-Mediated Liver Regeneration in Zebrafish.

BACKGROUND AND AIMS: The liver is a highly regenerative organ, but its regenerative capacity is compromised in severe liver injury settings. In chronic liver diseases, the number of liver progenitor cells (LPCs) correlates proportionally to disease severity, implying that their inefficient differentiation into hepatocytes exacerbates the disease. Moreover, LPCs secrete proinflammatory cytokines; thus, their prolonged presence worsens inflammation and induces fibrosis. Promoting LPC-to-hepatocyte differentiation in patients with advanced liver disease, for whom liver transplantation is currently the only therapeutic option, may be a feasible clinical approach because such promotion generates more functional hepatocytes and concomitantly reduces inflammation and fibrosis. APPROACH AND RESULTS: Here, using zebrafish models of LPC-mediated liver regeneration, we present a proof of principle of such therapeutics by demonstrating a role for the epidermal growth factor receptor (EGFR) signaling pathway in differentiation of LPCs into hepatocytes. We found that suppression of EGFR signaling promoted LPC-to-hepatocyte differentiation through the mitogen-activated ERK kinase (MEK)-extracellular signal-regulated kinase (ERK)-sex-determining region Y-box 9 (SOX9) cascade. Pharmacological inhibition of EGFR or MEK/ERK promoted LPC-to-hepatocyte differentiation as well as genetic suppression of the EGFR-ERK-SOX9 axis. Moreover, Sox9b overexpression in LPCs blocked their differentiation into hepatocytes. In the zebrafish liver injury model, both hepatocytes and biliary epithelial cells contributed to LPCs. EGFR inhibition promoted the differentiation of LPCs regardless of their origin. Notably, short-term treatment with EGFR inhibitors resulted in better liver recovery over the long term. CONCLUSIONS: The EGFR-ERK-SOX9 axis suppresses LPC-to-hepatocyte differentiation during LPC-mediated liver regeneration. We suggest EGFR inhibitors as a proregenerative therapeutic drug for patients with advanced liver disease.

Environment-Adaptive Object Detection Framework for Autonomous Mobile Robots.

Object detection is an essential function for mobile robots, allowing them to carry out missions efficiently. In recent years, various deep learning models based on convolutional neural networks have achieved good performance in object detection. However, in cases in which robots have to carry out missions in a particular environment, utilizing a model that has been trained without considering the environment in which robots must conduct their tasks degrades their object detection performance, leading to failed missions. This poor model accuracy occurs because of the class imbalance problem, in which the occurrence frequencies of the object classes in the training dataset are significantly different. In this study, we propose a systematic solution that can solve the class imbalance problem by training multiple object detection models and using these models effectively for robots that move through various environments to carry out missions. Moreover, we show through experiments that the proposed multi-model-based object detection framework with environment-context awareness can effectively overcome the class imbalance problem. As a result of the experiment, CPU usage decreased by 45.49% and latency decreased by more than 60%, while object detection accuracy increased by 6.6% on average.

Hdac1 Regulates Differentiation of Bipotent Liver Progenitor Cells During Regeneration via Sox9b and Cdk8.

BACKGROUND & AIMS: Upon liver injury in which hepatocyte proliferation is compromised, liver progenitor cells (LPCs), derived from biliary epithelial cells (BECs), differentiate into hepatocytes. Little is known about the mechanisms of LPC differentiation. We used zebrafish and mouse models of liver injury to study the mechanisms. METHODS: We used transgenic zebrafish, Tg(fabp10a:CFP-NTR), to study the effects of compounds that alter epigenetic factors on BEC-mediated liver regeneration. We analyzed zebrafish with disruptions of the histone deacetylase 1 gene (hdac1) or exposed to MS-275 (an inhibitor of Hdac1, Hdac2, and Hdac3). We also analyzed zebrafish with mutations in sox9b, fbxw7, kdm1a, and notch3. Zebrafish larvae were collected and analyzed by whole-mount immunostaining and in situ hybridization; their liver tissues were collected for quantitative reverse transcription polymerase chain reaction. We studied mice in which hepatocyte-specific deletion of ß-catenin (Ctnnb1flox/flox mice injected with Adeno-associated virus serotype 8 [AAV8]-TBG-Cre) induces differentiation of LPCs into hepatocytes after a choline-deficient, ethionine-supplemented (CDE) diet. Liver tissues were collected and analyzed by immunohistochemistry and immunoblots. We performed immunohistochemical analyses of liver tissues from patients with compensated or decompensated cirrhosis or acute on chronic liver failure (n = 15). RESULTS: Loss of Hdac1 activity in zebrafish blocked differentiation of LPCs into hepatocytes by increasing levels of sox9b mRNA and reduced differentiation of LPCs into BECs by increasing levels of cdk8 mRNA, which encodes a negative regulator gene of Notch signaling. We identified Notch3 as the receptor that regulates differentiation of LPCs into BECs. Loss of activity of Kdm1a, a lysine demethylase that forms repressive complexes with Hdac1, produced the same defects in differentiation of LPCs into hepatocytes and BECs as observed in zebrafish with loss of Hdac1 activity. Administration of MS-275 to mice with hepatocyte-specific loss of ß-catenin impaired differentiation of LPCs into hepatocytes after the CDE diet. HDAC1 was expressed in reactive ducts and hepatocyte buds of liver tissues from patients with cirrhosis. CONCLUSIONS: Hdac1 regulates differentiation of LPCs into hepatocytes via Sox9b and differentiation of LPCs into BECs via Cdk8, Fbxw7, and Notch3 in zebrafish with severe hepatocyte loss. HDAC1 activity was also required for differentiation of LPCs into hepatocytes in mice with liver injury after the CDE diet. These pathways might be manipulated to induce LPC differentiation for treatment of patients with advanced liver diseases.

Recovering cavity effects in corrugated organic light emitting diodes.

Cavity effects play an important role in determining the out-coupling efficiency of an OLED. By fabricating OLEDs on corrugated substrates, the waveguide and SPP modes can be extracted by diffraction. However, corrugation does not always lead to an enhancement in out-coupling efficiency due to the reduction of the electrode reflectance and hence the cavity effects. Based on the results of our rigorous couple-wave analysis (RCWA) simulation, we found that the cavity effects can be partially recovered using a low index Teflon layer inserted between the ITO anode and the substrate due to the enhancement of the reflectance of the corrugated electrodes. To verify the simulation results, we fabricated corrugated OLEDs having a low-index Teflon interlayer with an EQE of 36%, which is 29% higher than an optimized planar OLED. By experimentally measuring the OLED air mode dispersion, we confirm the cavity emission of a corrugated OLED is enhanced by the low index layer.

Bromodomain and Extraterminal (BET) Proteins Regulate Hepatocyte Proliferation in Hepatocyte-Driven Liver Regeneration.

Bromodomain and extraterminal (BET) proteins recruit key components of basic transcriptional machinery to promote gene expression. Aberrant expression and mutations in BET genes have been identified in many malignancies. Small molecule inhibitors of BET proteins such as JQ1 have shown efficacy in preclinical cancer models, including affecting growth of hepatocellular carcinoma. BET proteins also regulate cell proliferation in nontumor settings. We recently showed that BET proteins regulate cholangiocyte-driven liver regeneration. Here, we studied the role of BET proteins in hepatocyte-driven liver regeneration in partial hepatectomy (PHx) and acetaminophen-induced liver injury models in mice and zebrafish. JQ1 was injected 2 or 16 hours after PHx in mice to determine effect on hepatic injury, regeneration, and signaling. Mice treated with JQ1 after PHx displayed increased liver injury and a near-complete inhibition of hepatocyte proliferation. Levels of Ccnd1 mRNA and Cyclin D1 protein were reduced in animals injected with JQ1 16 hours after PHx and were even further reduced in animals injected with JQ1 2 hours after PHx. JQ1-treated zebrafish larvae after acetaminophen-induced injury also displayed notably impaired hepatocyte proliferation. In both models, Wnt signaling was prominently suppressed by JQ1. Our results show that BET proteins regulate hepatocyte proliferation-driven liver regeneration, and Wnt signaling is particularly sensitive to BET protein inhibition.

Wnt/ß-catenin signaling controls intrahepatic biliary network formation in zebrafish by regulating notch activity.

Malformations of the intrahepatic biliary structure cause cholestasis, a liver pathology that corresponds to poor bile flow, which leads to inflammation, fibrosis, and cirrhosis. Although the specification of biliary epithelial cells (BECs) that line the bile ducts is fairly well understood, the molecular mechanisms underlying intrahepatic biliary morphogenesis remain largely unknown. Wnt/ß-catenin signaling plays multiple roles in liver biology; however, its role in intrahepatic biliary morphogenesis remains unclear. Using pharmacological and genetic tools that allow one to manipulate Wnt/ß-catenin signaling, we show that in zebrafish both suppression and overactivation of Wnt/ß-catenin signaling impaired intrahepatic biliary morphogenesis. Hepatocytes, but not BECs, exhibited Wnt/ß-catenin activity; and the global suppression of Wnt/ß-catenin signaling reduced Notch activity in BECs. Hepatocyte-specific suppression of Wnt/ß-catenin signaling also reduced Notch activity in BECs, indicating a cell nonautonomous role for Wnt/ß-catenin signaling in regulating hepatic Notch activity. Reducing Notch activity to the same level as that observed in Wnt-suppressed livers also impaired biliary morphogenesis. Intriguingly, expression of the Notch ligand genes jag1b and jag2b in hepatocytes was reduced in Wnt-suppressed livers and enhanced in Wnt-overactivated livers, revealing their regulation by Wnt/ß-catenin signaling. Importantly, restoring Notch activity rescued the biliary defects observed in Wnt-suppressed livers. CONCLUSION: Wnt/ß-catenin signaling cell nonautonomously controls Notch activity in BECs by regulating the expression of Notch ligand genes in hepatocytes, thereby regulating biliary morphogenesis. (Hepatology 2018;67:2352-2366).

Bone morphogenetic protein signaling governs biliary-driven liver regeneration in zebrafish through tbx2b and id2a.

Upon mild liver injury, new hepatocytes originate from preexisting hepatocytes. However, if hepatocyte proliferation is impaired, a manifestation of severe liver injury, biliary epithelial cells (BECs) contribute to new hepatocytes through BEC dedifferentiation into liver progenitor cells (LPCs), also termed oval cells or hepatoblast-like cells (HB-LCs), and subsequent differentiation into hepatocytes. Despite the identification of several factors regulating BEC dedifferentiation and activation, little is known about factors involved in the regulation of LPC differentiation into hepatocytes during liver regeneration. Using a zebrafish model of near-complete hepatocyte ablation, we show that bone morphogenetic protein (Bmp) signaling is required for BEC conversion to hepatocytes, particularly for LPC differentiation into hepatocytes. We found that severe liver injury led to the up-regulation of genes involved in Bmp signaling, including smad5, tbx2b, and id2a, in the liver. Bmp suppression did not block BEC dedifferentiation into HB-LCs; however, the differentiation of HB-LCs into hepatocytes was impaired due to the maintenance of HB-LCs in an undifferentiated state. Later Bmp suppression did not affect HB-LC differentiation but increased BEC number through proliferation. Notably, smad5, tbx2b, and id2a mutants exhibited similar liver regeneration defects as those observed in Bmp-suppressed livers. Moreover, BMP2 addition promoted the differentiation of a murine LPC line into hepatocytes in vitro. CONCLUSIONS: Bmp signaling regulates BEC-driven liver regeneration through smad5, tbx2b, and id2a: it regulates HB-LC differentiation into hepatocytes through tbx2b and BEC proliferation through id2a; our findings provide insights into promoting innate liver regeneration as a novel therapy. (Hepatology 2017;66:1616-1630).

Stat3 Regulates Liver Progenitor Cell-Driven Liver Regeneration in Zebrafish.

After liver injury, regeneration manifests as either (1) hepatocytes proliferating to restore the lost hepatocyte mass or (2) if hepatocyte proliferation is compromised, biliary epithelial cells (BECs) dedifferentiating into liver progenitor cells (LPCs), which subsequently differentiate into hepatocytes. Following pharmacogenetic ablation of hepatocytes in Tg(fabp10a:CFP-NTR) zebrafish, resulting in severe liver injury, signal transducer and activator of transcription 3 (Stat3) and its target gene and negative regulator, socs3a, were upregulated in regenerating livers. Using either Stat3 inhibitors, JSI-124 and S3I-201, or stat3 zebrafish mutants, we investigated the role of Stat3 in LPC-driven liver regeneration. Although Stat3 suppression reduced the size of regenerating livers, BEC dedifferentiation into LPCs was unaffected. However, regenerating livers displayed a delay in LPC-to-hepatocyte differentiation and a significant reduction in the number of BECs. While no difference in cell death was detected, Stat3 inhibition significantly reduced LPC proliferation. Notably, stat3 mutants phenocopied the effects of Stat3 chemical inhibitors, although the mutant phenotype was incompletely penetrant. Intriguingly, a subset of socs3a mutants also displayed a lower number of BECs in regenerating livers. We conclude that the Stat3/Socs3a pathway is necessary for the proper timing of LPC-to-hepatocyte differentiation and establishing the proper number of BECs during LPC-driven liver regeneration.

Incidence, risk and prognostic role of anti-epidermal growth factor receptor-induced skin rash in biliary cancer: a meta-analysis.

BACKGROUND: Anti-epidermal growth factor receptor (EGFR)-induced skin rash is a common adverse event and is considered a prognostic factor of various cancers. However, the role of rash is rarely known in biliary cancer, possibly owing to the low incidence of this frequently fatal malignancy. We thus performed a meta-analysis to investigate the incidence, risk and prognostic significance of skin rash related to anti-EGFR treatment for biliary cancer. METHODS: Eligible studies were enrolled after a systematic search of electronic databases. A fixed-effects or random-effects model was utilized according to the heterogeneity. RESULTS: Fourteen clinical trials published between 2006 and 2017 comprising 1,106 patients with advanced biliary cancer were included. The overall incidence of all-grade and high-grade (grade ≥3) rash was 78.2% [95% confidence interval (CI) 70.4-84.3] and 11.3% (7.6-16.5), respectively. Anti-EGFR treatment correlates with a significantly increased risk of all-grade [risk ratio (RR) 7.37, 95% CI 5.11-10.64, p < 0.0001] and high-grade (RR 6.94, 95% CI 1.89-25.45, p = 0.0035) rash compared with control medication. Higher grades of skin rash correlate with a higher objective response rate (RR 3.50, 95% CI 1.47-8.33, p = 0.0048), and a longer overall [hazard ratio (HR) 0.47, 95% CI 0.31-0.71, p = 0.0003) and progression-free survival (HR 0.51, 95% CI 0.36-0.72, p = 0.0001) compared with lower grades or no rash in patients who received anti-EGFR treatment. CONCLUSIONS: Anti-EGFR treatment correlates with an increased risk of skin rash in advanced biliary cancer. Stratifying patients by the severity of rash may have major implications for survival benefit regarding anti-EGFR treatment for biliary cancer.

Glutathione antioxidant pathway activity and reserve determine toxicity and specificity of the biliary toxin biliatresone in zebrafish.

UNLABELLED: Biliatresone is an electrophilic isoflavone isolated from Dysphania species plants that has been causatively linked to naturally occurring outbreaks of a biliary atresia (BA)-like disease in livestock. Biliatresone has selective toxicity for extrahepatic cholangiocytes (EHCs) in zebrafish larvae. To better understand its mechanism of toxicity, we performed transcriptional profiling of liver cells isolated from zebrafish larvae at the earliest stage of biliatresone-mediated biliary injury, with subsequent comparison of biliary and hepatocyte gene expression profiles. Transcripts encoded by genes involved in redox stress response, particularly those involved in glutathione (GSH) metabolism, were among the most prominently up-regulated in both cholangiocytes and hepatocytes of biliatresone-treated larvae. Consistent with these findings, hepatic GSH was depleted at the onset of biliary injury, and in situ mapping of the hepatic GSH redox potential using a redox-sensitive green fluorescent protein biosensor showed that it was significantly more oxidized in EHCs both before and after treatment with biliatresone. Pharmacological and genetic manipulation of GSH redox homeostasis confirmed the importance of GSH in modulating biliatresone-induced injury given that GSH depletion sensitized both EHCs and the otherwise resistant intrahepatic cholangiocytes to the toxin, whereas replenishing GSH level by N-acetylcysteine administration or activation of nuclear factor erythroid 2-like 2 (Nrf2), a transcriptional regulator of GSH synthesis, inhibited EHC injury. CONCLUSION: These findings strongly support redox stress as a critical contributing factor in biliatresone-induced cholangiocyte injury, and suggest that variations in intrinsic stress responses underlie the susceptibility profile. Insufficient antioxidant capacity of EHCs may be critical to early pathogenesis of human BA. (Hepatology 2016;64:894-907).

tomm22 Knockdown-Mediated Hepatocyte Damages Elicit Both the Formation of Hybrid Hepatocytes and Biliary Conversion to Hepatocytes in Zebrafish Larvae.

The liver has a highly regenerative capacity. In the normal liver, hepatocytes proliferate to restore lost liver mass. However, when hepatocyte proliferation is impaired, biliary epithelial cells (BECs) activate and contribute to hepatocytes. We previously reported in zebrafish that upon severe hepatocyte ablation, BECs extensively contribute to regenerated hepatocytes. It was also speculated that BEC-driven liver regeneration might occur in another zebrafish liver injury model in which temporary knockdown of the mitochondrial import gene tomm22 by morpholino antisense oligonucleotides (MO) induces hepatocyte death. Given the importance of multiple BEC-driven liver regeneration models for better elucidating the mechanisms underlying innate liver regeneration in the diseased liver, we hypothesized that BECs would contribute to hepatocytes in tomm22 MO-injected larvae. In this MO-based liver injury model, by tracing the lineage of BECs, we found that BECs significantly contributed to hepatocytes. Moreover, we found that surviving, preexisting hepatocytes become BEC-hepatocyte hybrid cells in tomm22 MO-injected larvae. Intriguingly, both the inhibition of Wnt/ß-catenin signaling and macrophage ablation suppressed the formation of the hybrid hepatocytes. This new liver injury model in which both hepatocytes and BECs contribute to regenerated hepatocytes will aid in better understanding the mechanisms of innate liver regeneration in the diseased liver.

Bromodomain and extraterminal (BET) proteins regulate biliary-driven liver regeneration.

BACKGROUND & AIMS: During liver regeneration, hepatocytes are derived from pre-existing hepatocytes. However, if hepatocyte proliferation is compromised, biliary epithelial cells (BECs) become the source of new hepatocytes. We recently reported on a zebrafish liver regeneration model in which BECs extensively contribute to hepatocytes. Using this model, we performed a targeted chemical screen to identify important factors that regulate BEC-driven liver regeneration, the mechanisms of which remain largely unknown. METHODS: Using Tg(fabp10a:CFP-NTR) zebrafish, we examined the effects of 44 selected compounds on BEC-driven liver regeneration. Liver size was assessed by fabp10a:DsRed expression; liver marker expression was analyzed by immunostaining, in situ hybridization and quantitative PCR. Proliferation and apoptosis were also examined. Moreover, we used a mouse liver injury model, choline-deficient, ethionine-supplemented (CDE) diet. RESULTS: We identified 10 compounds that affected regenerating liver size. Among them, only bromodomain and extraterminal domain (BET) inhibitors, JQ1 and iBET151, blocked both Prox1 and Hnf4a induction in BECs. BET inhibition during hepatocyte ablation blocked BEC dedifferentiation into hepatoblast-like cells (HB-LCs). Intriguingly, after JQ1 washout, liver regeneration resumed, indicating temporal, but not permanent, perturbation of liver regeneration by BET inhibition. BET inhibition after hepatocyte ablation suppressed the proliferation of newly generated hepatocytes and delayed hepatocyte maturation. Importantly, Myca overexpression, in part, rescued the proliferation defect. Furthermore, oval cell numbers in mice fed CDE diet were greatly reduced upon JQ1 administration, supporting the zebrafish findings. CONCLUSIONS: BET proteins regulate BEC-driven liver regeneration at multiple steps: BEC dedifferentiation, HB-LC proliferation, the proliferation of newly generated hepatocytes, and hepatocyte maturation.

Autophagy induction is a Tor- and Tp53-independent cell survival response in a zebrafish model of disrupted ribosome biogenesis.

Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.