Title: Antagonistic interaction between Wnt and Notch activity modulates the regenerative capacity of a zebrafish fibrotic liver model
Abstract: HepatologyVolume 60, Issue 5 p. 1753-1766 Liver Injury/RegenerationFree Access Antagonistic interaction between Wnt and Notch activity modulates the regenerative capacity of a zebrafish fibrotic liver model Mianbo Huang, Mianbo Huang School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GASearch for more papers by this authorAngela Chang, Angela Chang School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GASearch for more papers by this authorMinna Choi, Minna Choi School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GASearch for more papers by this authorDavid Zhou, David Zhou School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GASearch for more papers by this authorFrank A. Anania, Frank A. Anania Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GASearch for more papers by this authorChong Hyun Shin, Corresponding Author Chong Hyun Shin School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GAAddress reprint requests to: Chong Hyun Shin, Ph.D., School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Interdisciplinary Bioengineering Graduate Program, Georgia Institute of Technology, 315 Ferst Dr. NW, Rm. 1313, Atlanta, GA 30332. E-mail: [email protected]; fax: 404-894-0519.Search for more papers by this author Mianbo Huang, Mianbo Huang School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GASearch for more papers by this authorAngela Chang, Angela Chang School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GASearch for more papers by this authorMinna Choi, Minna Choi School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GASearch for more papers by this authorDavid Zhou, David Zhou School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GASearch for more papers by this authorFrank A. Anania, Frank A. Anania Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GASearch for more papers by this authorChong Hyun Shin, Corresponding Author Chong Hyun Shin School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GAAddress reprint requests to: Chong Hyun Shin, Ph.D., School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Interdisciplinary Bioengineering Graduate Program, Georgia Institute of Technology, 315 Ferst Dr. NW, Rm. 1313, Atlanta, GA 30332. E-mail: [email protected]; fax: 404-894-0519.Search for more papers by this author First published: 04 July 2014 https://doi.org/10.1002/hep.27285Citations: 54 Potential conflict of interest: Nothing to report. Chong Hyun Shin is supported by grant number K01DK081351 from the National Institutes of Health (NIH), the Regenerative Engineering and Medicine Research Center Pilot Award (GTEC 2731336), and a start-up package from the School of Biology, Georgia Institute of Technology. Frank A. Anania is supported by grants from the NIH (R01DK062092) and Veterans Affairs (I01BX001746). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract In chronic liver failure patients with sustained fibrosis, excessive accumulation of extracellular matrix proteins substantially dampens the regenerative capacity of the hepatocytes, resulting in poor prognosis and high mortality. Currently, the mechanisms and the strategies of inducing endogenous cellular sources such as hepatic progenitor cells (HPCs) to regenerate hepatocytes in various contexts of fibrogenic stimuli remain elusive. Here we aim to understand the molecular and cellular mechanisms that mediate the effects of sustained fibrosis on hepatocyte regeneration using the zebrafish as a model. In the ethanol-induced fibrotic zebrafish model, we identified a subset of HPCs, responsive to Notch signaling, that retains its capacity to regenerate as hepatocytes. Discrete levels of Notch signaling modulate distinct cellular outcomes of these Notch-responsive HPCs in hepatocyte regeneration. Lower levels of Notch signaling promote amplification and subsequent differentiation of these cells into hepatocytes, while high levels of Notch signaling suppress these processes. To identify small molecules facilitating hepatocyte regeneration in the fibrotic liver, we performed chemical screens and identified a number of Wnt agonists and Notch antagonists. Further analyses demonstrated that these Wnt agonists are capable of attenuating Notch signaling by inducing Numb, a membrane-associated protein that inhibits Notch signaling. This suggests that the antagonistic interplay between Wnt and Notch signaling crucially affects hepatocyte regeneration in the fibrotic liver. Conclusion: Our findings not only elucidate how signaling pathways and cell-cell communications direct the cellular response of HPCs to fibrogenic stimuli, but also identify novel potential therapeutic strategies for chronic liver disease. (Hepatology 2014;60:1753–1766) Abbreviations Alcam activated leukocyte cell adhesion molecule BPDEC bile preductular epithelial cell CCl4 carbon tetrachloride CFP cyan fluorescent protein DAPT N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester DMSO dimethyl sulfoxide dpa days-post-ablation dpf days-post-fertilization ECM extracellular matrix EdU 5-ethynyl-2 ′-deoxyuridine EGFP enhanced green fluorescent protein EtOH ethanol fabp10 fatty acid binding protein 10 hpa hours-post-ablation HPC hepatic progenitor cell hpf hours-post-fertilization HSC hepatic stellate cell IHBP intrahepatic biliary passageway mpa month-post-ablation MTZ metronidazole NRC Notch-responsive cell 4-OHT 4-hydroxytamoxifen NTR nitroreductase qPCR quantitative polymerase chain reaction Sustained liver fibrosis associated with the accumulation of extracellular matrix (ECM) proteins leads to cirrhosis with high morbidity and mortality.1 Currently, antifibrotic therapies mainly focus on targeting profibrogenic cytokines and activated myofibroblasts,2 which are primarily derived from hepatic stellate cells (HSCs), the liver-specific mesenchymal cells essential for liver physiology and fibrosis.1 However, mechanisms and strategies on how to activate hepatic resident cellular sources such as hepatic progenitor cells (HPCs) to regenerate hepatocytes in the presence of fibrogenic insults in vivo have not been adequately investigated. The liver has a remarkable capacity for regeneration upon injury, a process that is driven primarily by the proliferation of mature hepatocytes.3 Hepatocytes are metabolically active cells in the liver that make up 70-80% of the liver mass. The other differentiated epithelial cell type in the liver, cholangiocytes, form the biliary network. When the ability of hepatocyte proliferation is compromised, liver repopulation occurs through the activation of “oval cells,” the quiescent HPCs residing within the canals of Hering, which are extensions of the portal bile ductules.4 Genetic lineage tracing suggested that Sox9, Foxl1, and Lgr5 expression marks these progenitor cells that give rise to both hepatocytes and cholangiocytes in vivo.5-7 Furthermore, by using ductal cell surface-marking antibodies, subpopulations of liver cells from normal adult mice or those undergoing an oval cell response were isolated and their capacity to form bilineage colonies in vitro was confirmed.8 Nonetheless, whether oval cells are species-specific and/or hepatic insults-specific, or conserved across these variations remains unresolved. Challenges in studying these cells in vivo deter full comprehension of their cellular behavior. It has been shown that interactions between signaling pathways are critical for the fate commitment of HPCs during liver regeneration.9 In the case of biliary damage, a cell-cell interaction between Notch-expressing HPCs and Jagged1-expressing myofibroblasts acts as the default pathway to specify biliary cell fate in HPCs.9 In parallel, Wnt3a, secreted by macrophages in reaction to phagocytosis of apoptotic hepatocytes, suppresses a default Notch signaling in HPCs through an induction of Notch antagonist NUMB,10 a direct target of the canonical-Wnt signaling,11 leading HPCs to attain the hepatocyte lineage.9 Furthermore, activity of Wnt reporter Axin-lacZ was up-regulated upon liver injury by carbon tetrachloride (CCl4) injection. Subsequently, many Wnt target genes, including Lgr5, were induced in the groups of small cells near the biliary ducts, which contribute to hepatocytes and cholangiocytes during the repair phase.7 Nevertheless, the pivotal question of how the underlying signaling pathways and cell-cell communications orchestrate the distinct cellular responses of the HPCs in different contexts of hepatic insults remain elusive. In particular, the link between the signal(s) responsible for specification of HPCs towards the biliary or hepatocyte lineage and the one(s) leading to the expansion of the HPCs awaits further investigation. The zebrafish (Danio rerio) has emerged as an invaluable vertebrate model system for studying regeneration, possessing a range of molecular, cellular, and genetic tools needed to thoroughly investigate underlying processes and/or mechanisms of regeneration.12 The composition, structure, function, and genetic control of the liver are relatively conserved between zebrafish and mammals.13 With transgenic strategies for expressing fluorescent proteins in different hepatic cell types, we could visualize hepatic cell behaviors at the single-cell level along with real-time in vivo imaging. Furthermore, we could perform genetic and chemical screens to discover regulators of liver development, disease, and regeneration in a cost- and time-effective way.13 Recent comprehensive studies have discovered conserved and/or novel genes and pathways that regulate liver development and pathogenesis in zebrafish.14, 15 However, few studies have effectively modeled liver regeneration in response to sustained fibrogenic stimuli of chronic liver disease with zebrafish. In this study, we established a zebrafish model to delineate the molecular and cellular mechanisms that mediate the effects of sustained fibrogenic insult on hepatocyte regeneration. We identified a subset of HPCs that experience different levels of Notch signaling, which in turn is essential for hepatocyte regeneration. Lower levels of Notch signaling promote proliferation and subsequent differentiation of these cells into hepatocytes, while high levels of Notch signaling suppress hepatocyte regeneration. Through chemical screens, we pinpointed a number of Wnt agonists and Notch antagonists that facilitate hepatocyte regeneration. We further determined that these Wnt agonists suppress Notch signaling by inducing a Notch antagonist, Numb. These data suggest an essential interplay between Wnt and Notch signaling during hepatocyte regeneration in the fibrotic liver, providing legitimate therapeutic strategies for chronic liver failure in vivo. Materials and Methods Zebrafish Lines Animals received humane care according to the criteria of the National Institutes of Health and the Georgia Institute of Technology Institutional Animal Care and Use Committee. The generation of nitroreductase constructs to establish a hepatocyte-specific genetic ablation system and zebrafish transgenic lines are described in the Supporting Materials and Methods. Ethanol and Metronidazole Treatment To ablate hepatocytes, the larvae (15 mM, 24-36 hours) or adult fish (10 mM, 8 hours) were treated with metronidazole (MTZ, Sigma-Aldrich, St. Louis, MO). The MTZ was then washed out, marking the 0 hours-post-ablation (hpa) point. For ethanol (EtOH) treatment, the larvae were pretreated with 1.5% EtOH (Sigma-Aldrich) for 24 hours and then concurrently treated with MTZ for 24 hours. For the adult fish, we pretreated the fish with 1% EtOH for 72 hours and followed with MTZ treatment. Immunohistochemistry, 5-Ethynyl-2′-Deoxyuridine (EdU) Cell-Cycle Analysis, and Chemical Treatment Immunohistochemistry, EdU cell-cycle analysis, and chemical treatment were performed as previously described.16 Further details are described in the Supporting Materials and Methods. Quantitative Real-Time Polymerase Chain Reaction, Reporter Assay, and Chemical Screens Materials and methods for these experiments are described in the Supporting Materials and Methods. Results Establishing a Model of Sustained Fibrotic Liver in Zebrafish To establish a clinically relevant fibrotic liver model in zebrafish, we induced near-complete eradication of hepatocytes in the presence of fibrogenic insult. First, we generated two transgenic lines, Tg(fabp10a:CFP-NTR)gt1 and Tg(fabp10a:mCherry-NTR)gt2, to specifically ablate hepatocytes in the liver. Nitroreductase (NTR) converts the nontoxic prodrug MTZ into a DNA interstrand cross-linking agent, which induces cell death.17-19 Treatment of MTZ from 84-90 hours-post-fertilization (hpf), when the hepatocytes and biliary epithelial cells have differentiated,20 effectively ablated the hepatocytes (Supporting Fig. 1) in the presence of hepatic endothelial cells (Supporting Fig. 2E) and HSCs (Supporting Fig. 2F). We additionally used two transgenic lines, Tg(Tp1:EGFP)um14 and Tg(Tp1:mCherry)jh11,21 which mark Notch signaling experiencing cells (Notch-responsive cells, NRCs) with EGFP and nuclear mCherry, respectively, under the control of the TP1 module containing multiple RBP-Jk-binding sites (Supporting Fig. 1B,F). Confocal analyses showed that the pattern of hepatic EGFP or mCherry expression significantly overlapped with the distribution of the 2F11 epitope, which marks intrahepatic biliary epithelia22 (data not shown). At 0 hours-post-ablation (hpa), MTZ-mediated damaged livers showed substantial reduction or complete absence of hepatocytes, leading to a collapsed liver, while EGFP- or nuclear mCherry-positive NRCs remained (Supporting Figs. 1D,H,2D). We examined the cellular events of hepatocyte regeneration with or without 1.5% EtOH in [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11; Tg(hand2:EGFP)pd24 23] larvae. We observed an expansion of HSCs in the MTZ-treated regenerating livers without EtOH treatment (Fig. 1B′; Supporting Fig. 3B′). In the presence of EtOH, as previously reported,24 HSCs increased in number (Fig. 1C′; Supporting Fig. 3C′) with the altered morphology from a star-like configuration to a myofibroblast-like shape with lost cytoplasmic processes (Fig. 1C′; Supporting Fig. 3C′, insets). Furthermore, production of ECM proteins, detected by laminin and fibrillar type I collagen,1, 2 was significantly augmented in the EtOH/MTZ-treated regenerating livers, suggesting that EtOH exposure triggers fibrogenic changes (Fig. 1C″,D; Supporting Fig. 3C″,D). Nevertheless, hepatocyte regeneration proceeded (Fig. 1C,C′″; Supporting Fig. 3C,C′″) at a lower efficiency (Fig. 1E; Supporting Fig. 3E). Figure 1Open in figure viewerPowerPoint Establishing a model of sustained fibrotic liver in zebrafish. (A-A′″) In [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11; Tg(hand2:EGFP)pd24] larvae treated with DMSO at 5 days-post-fertilization (dpf), quiescent HSCs showed a star-like configuration (A′, inset), whereas ECM protein laminin was almost undetectable (A″) and NRCs distributed among hepatocytes (A,A′″). (B-B′″) Hepatocytes were ablated by MTZ and allowed to regenerate for 50 hours. In the MTZ-treated regenerating livers, the number of HSCs increased (B′) with a rare deposition of laminin (B″). A population of cells coexpressing hepatocyte-specific CFP and NRC-specific nuclear mCherry was observed throughout the MTZ-treated regenerating livers (B′″, inset, white arrows) while bright-red NRCs remained CFP negative (B′″, inset, yellow arrowheads). (C-C′″) The larvae were pretreated with 1.5% EtOH from 2.5 to 3.5 dpf and then concurrently treated with MTZ from 3.5 to 4.5 dpf and allowed to regenerate for 50 hours. HSCs increased in number and lost complex cytoplasmic processes (C′, inset). Elevated laminin deposition was observed in the EtOH/MTZ-treated regenerating livers (C″, inset). A population of cells coexpressing hepatocyte-specific CFP and NRC-specific nuclear mCherry was appreciated (C′″, inset, white arrows) while bright-red NRCs remained CFP negative (C′″, inset, yellow arrowhead). (D) Representative western blot showed up-regulation of laminin protein in the EtOH/MTZ-treated regenerating livers. Numbers show relative expression of laminin protein after normalization against β-actin protein. n = 300 dissected larval livers per condition in three experiments. (E) Percentage (mean ± SD) of CFP+ cells in the population of Tg(Tp1:mCherry)-positive cells. 85.5 ± 1.6% of Tg(Tp1:mCherry)-positive cells were CFP+ in the MTZ-treated regenerating livers, while 18.7 ± 1.8% of Tg(Tp1:mCherry)-positive cells expressed CFP in the EtOH/MTZ-treated regenerating livers. Cells in five planes of confocal images from five individual larvae were counted. Asterisks indicate statistical significance: ***P < 0.001. All confocal images are single-plane images except A′, B′, and C′, which are projection images (n = 30 larvae per condition in three experiments). Scale bar, 20 μm. EtOH, ethanol; SD, standard deviation. Intriguingly, in the MTZ-treated regenerating livers at 50 hpa, we noticed a population of cells coexpressing hepatocyte-specific CFP and NRC-specific mCherry regardless of EtOH treatment (Fig. 1B′″,C′″; Supporting Fig. 3B′″,C′″, insets with arrows). The CFP and dim mCherry coexpressing NRCs are distinguishable from the bright-red NRCs that are negative for CFP and positive for activated leukocyte cell adhesion molecule (Alcam), a biliary epithelial cell marker20 (Supporting Fig. 4B″,C″, insets with arrowheads). The coexpressing cells were not detected in the EtOH-only-treated larvae (Supporting Fig. 4D′″, inset). Similar to that observed in the larvae, the CFP and dim mCherry coexpressing cells were detected both in the MTZ- and EtOH/MTZ-treated regenerating livers of 12-month-old [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11] adult fish at 3 days-post-ablation (dpa) (Fig. 2E″,F″, insets with arrows) with markedly elevated deposition of type I collagen in the presence of EtOH (Fig. 2F′,G,H). Given the facts that the quiescent HPCs reside in the bile ductules4 and the proliferation of HSCs and the expansion of HPCs occur concurrently in the partial hepatectomy model with compromised hepatocyte proliferation in mammals,25 these data indicate that the CFP and dim mCherry coexpressing NRCs represent a zebrafish-counterpart of HPCs that differentiate into hepatocytes by experiencing lower levels of Notch signaling. Furthermore, these results suggest that even in the presence of sustained fibrogenic insult such as EtOH, the HPCs, responsive to Notch signaling, maintain their capacity to regenerate as hepatocytes. Figure 2Open in figure viewerPowerPoint A population of cells, responsive to Notch signaling, maintains their capacity to regenerate as hepatocytes in adult zebrafish. Liver morphology (white dashed lines) of 12-month-old [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11] adults treated with DMSO (A), MTZ (B), or 1% EtOH followed by MTZ (C). (D-F″) Confocal images of Tg(fabp10a:CFP-NTR), Tg(Tp1:mCherry), and type I collagen expression in vibratome sections of adult zebrafish livers. (D-D″) In the DMSO-treated controls, expression of Tg(fabp10a:CFP-NTR) did not overlap with that of Tg(Tp1:mCherry) (D″, inset) with almost undetectable type I collagen deposition (D′). (E-E″) Hepatocytes were ablated by 10 mM MTZ treatment for 8 hours and then allowed to regenerate for 3 days. Numerous cells coexpressing hepatocyte-specific CFP and NRC-specific nuclear mCherry were observed throughout the MTZ-treated regenerating livers (E″, inset, white arrows) with a modest increase in type I collagen deposition (E'), while bright-red NRCs remained CFP negative (E″, inset, yellow arrowhead). (F-F″) The fish were pretreated with 1% EtOH for 72 hours followed by 10 mM MTZ treatment for 8 hours and then allowed to regenerate for 3 days. Significantly elevated type I collagen deposition was observed in the EtOH/MTZ-treated regenerating livers (F'), while a population of cells coexpressing hepatocyte-specific CFP and NRC-specific nuclear mCherry was appreciated (F″, inset, white arrows). The bright-red NRCs remained CFP negative (F″, inset, yellow arrowheads). (G) qRT-PCR analysis showed up-regulation of collagen 1a1a and collagen 1a2 mRNA in the EtOH/MTZ-treated regenerating livers. n = 3 dissected adult fish livers per condition in three experiments. Asterisks indicate statistical significance: **P < 0.01 and ***P < 0.001. (H) Representative western blot showed up-regulation of type I collagen protein in the EtOH/MTZ-treated regenerating livers. Numbers show relative expression of type I collagen protein after normalization against β-actin protein. n = 3 dissected adult fish livers per condition in three experiments. A-C, Brightfield images. D-D″, E-E″, and F-F″, confocal single-plane images. For each experiment, after capturing each brightfield image, the liver from the same fish was dissected and processed for section immunostaining to get confocal images (n = 6 fish per condition in three experiments). Scale bars: A-C, 2 mm; D-D″, E-E″, and F-F″, 20 μm. EtOH, ethanol; SD, standard deviation. Heterogeneity of Notch Signaling Activity in the Regenerating Livers We further investigated the relationship between heterogeneity of Notch signaling and hepatocyte regeneration by performing time-course analyses in [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11] larvae. Compared with the dimethyl sulfoxide (DMSO)-treated controls, MTZ treatment led to the destruction of layers of hepatocytes, resulting in NRCs in closer proximity to each other (Fig. 3A). The number of mCherry-expressing NRCs increased at 5 hpa with two discrete populations of dimming red NRCs (Fig. 3B, inset with arrows) and bright-red NRCs (Fig. 3B, inset with arrowheads). At 13-15 hpa, hepatocyte-specific CFP began to appear and was coexpressed in a few dimming mCherry-positive NRCs throughout the regenerating livers (Fig. 3C, inset with arrows). The remaining bright-red NRCs (Fig. 3C, inset with arrowhead) suggested an NRC-to-hepatocyte conversion in a subset of NRCs. These CFP and mCherry coexpressing NRCs continued to increase in number, resulting in two clearly distinct heterogeneous populations of NRCs: dim-red NRCs coexpressing CFP (Fig. 3D′,D″, insets with arrows) and the bright-red NRCs without CFP markers (Fig. 3D′,D″, insets with arrowheads). The former gradually became CFP-only-positive cells with the loss of red, suggesting that they differentiated into hepatocytes and may no longer be subject to Notch signaling (Fig. 3E′,E″, insets with arrows). The percentage of dim-red NRCs increased during regeneration with all of the CFP-positive hepatocytes being derived from these cells (Fig. 3G). By 70 hpa, the conversion was nearly complete (Fig. 3F), resulting in the repopulation of the entire liver with newly generated functional hepatocytes, which processed PED6, a fluorescent fatty acid reporter26 at 96 hpa (Supporting Fig. 5C, arrow). The larvae continued to express Alcam and restored the expression of Abcb11, a bile transport pump27 located in the bile canaliculi of hepatocytes at 96 hpa (Supporting Fig. 5F,J) and 1 month-post-ablation (mpa) (Supporting Fig. 5G,K). This indicated that the liver function was recovered and maintained after near-complete eradication of hepatocytes. Similar regeneration events occurred in the EtOH/MTZ-treated regenerating livers but at a lower efficiency (Supporting Fig. 6) and a decreased recovery of liver function (Supporting Fig. 5D,H,L). Furthermore, regardless of EtOH treatment, we detected comparable occurrences in [Tg(fabp10a:mCherry-NTR)gt2; Tg(Tp1:EGFP)um14]larvae (Supporting Fig. 7, insets), while other differentiated liver cells including endothelial cells and HSCs had minimum capacity to convert into hepatocytes (Supporting Fig. 8B,D, insets). These data indicate that NRCs experience different levels of Notch signaling during hepatocyte regeneration; the HPCs with continuously lower levels of Notch activity differentiate into hepatocytes, while those with higher levels remain as cholangiocytes. Figure 3Open in figure viewerPowerPoint Heterogeneity of Notch signaling activity in the regenerating livers. (A-F) MTZ-treated larvae were collected at the indicated timepoints during liver regeneration. (A) NRCs marked by Tg(Tp1:mCherry) clustered together in close proximity at 0 hpa. (B) The number of mCherry-expressing NRCs expanded at 5 hpa. Yellow arrowheads indicate bright-red NRCs (inset), while white arrows indicate NRCs with dim-red colors (inset). (C) At 13-15 hpa, hepatocyte-specific CFP started to coexpress in a few dimming mCherry-positive NRCs throughout the regenerating livers (inset, white arrows), while the bright-red NRCs remained (inset, yellow arrowhead). (D-D″) At 30 hpa, NRCs were clearly segregated into two heterogeneous populations, dim-red NRCs coexpressing CFP (D′, D″, insets, white arrows) and bright-red NRCs without CFP markers (D′, D″, insets, yellow arrowheads). (E-E″) CFP-positive NRCs became CFP only-positive cells with the loss of red (E′, E″, insets, white arrows), continuing to increase in numbers at 50 hpa. Bright-red NRCs remained as CFP-negative (E′, E″, insets, yellow arrowheads). (F) By 70 hpa, the conversion was nearly complete, resulting in the repopulation of the entire liver with newly generated hepatocytes. (G) Percentages (mean ± SD) of dim-red NRCs that contributed to newly formed CFP-positive hepatocytes in the first 50 hours of regeneration. At 15 hpa, hepatocyte-specific CFP began to emerge from the population of dim-red NRCs (13.7 ± 0.5% of dim-red NRCs were CFP-positive). The percentage of dim-red NRCs increased (0 hpa, 7.1 ± 1.9%; 5 hpa, 15.9 ± 2.6%; 10 hpa, 22.3 ± 3.5%; 15 hpa, 37.7 ± 4.6%; 25 hpa, 69.8 ± 5.4%; 50 hpa, 87 ± 0.7%) during the regeneration with all of the CFP-positive hepatocytes being derived from these cells (15 hpa, 13.7 ± 0.5%; 25 hpa, 37.7 ± 1.3%; 50 hpa, 87 ± 0.7%). Cells in five planes of confocal images from five individual larvae were counted at each timepoint. All images are confocal single-plane images except A, D, E, and F, which are projections images (n = 30 larvae per each timepoint in three experiments). Scale bars, 20 μm. To further examine the contribution of the HPCs to regenerating hepatocytes, we used the [Tg(Tp1:CreERT2)jh12; Tg(actb2:loxP-STOP-loxP-hmgb1-mCherry)jh15]28 lines, in which nuclear mCherry expression is genetically induced in NRCs. Treating tamoxifen either prior to (24-48 hpf) or after (0-24 hpa) hepatocyte ablation, we detected a subset of mCherry-expressing NRCs which coexpressed hepatocyte-specific CFP at 35 hpa (Supporting Fig. 9B,B′,D,D′, arrows) and 1 mpa (Supporting Fig. 9E,F, arrows). In the control livers without MTZ treatment, mCherry-expressing NRCs were only detected in the Alcam-positive biliary epithelial cells but not in the hepatocytes (Supporting Fig. 9A,C, yellow arrowheads). Altogether, these data suggest that NRCs portray two populations of cells: the cholangiocytes and the HPCs, which represent a progenitor population with the potential to regenerate as hepatocytes. Distinct Levels of Notch Signaling Are Essential for the Proliferation and Differentiation of the HPCs To analyze how the discrete levels of Notch signaling regulate the fate of HPCs, we first examined the dynamics of Notch signaling in the MTZ-treated regenerating livers. We used transgenic lines expressing a destabilized fluorescent protein with a short half-life (VenusPEST) and a fluorescent protein with enhanced stability (Histone2BmCherry) under the Notch responsive TP1 element, Tg(Tp1:VenusPEST)s939 and Tg(Tp1:H2BmCherry)s940, respectively.16 Thus, the Tg(Tp1:H2BmCherry); Tg(Tp1:VenusPEST)-double-positive cells are presently experiencing Notch signaling, whereas the Tg(Tp1:H2BmCherry)-positive; Tg(Tp1:VenusPEST)-negative cells were positive for Notch signaling in the recent past but have since switched it off. In the control livers, there was a clear overlap between Tg(Tp1:H2BmCherry) and Tg(Tp1:VenusPEST) expression with a small number of Tg(Tp1:H2BmCherry)-single-positive NRCs (Fig. 4A, white arrowheads). Alcam is expressed in all of the Tg(Tp1:H2BmCherry)-positive NRCs. At 0 hpa in the MTZ-treated livers, we observed heterogeneity in the expression of Venus, segregating Tg(Tp1:H2BmCherry)-positive NRCs into three groups based on Venus intensity and Alcam expression: Tg(Tp1:VenusPEST)high /Alcam+, Tg(Tp1:VenusPEST)–/Alcam–, and Tg(