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Notch-IGF1 signaling during liver regeneration drives biliary epithelial cell expansion and inhibits hepatocyte differentiation

Notch signaling promotes progenitor cell proliferation but inhibits hepatocyte differentiation during liver repair. Notching up proliferation Liver injury triggers the proliferation of biliary epithelial cells (BECs), which can give rise to cholangiocytes or hepatocytes, two major cell types in the liver. Using a model of acute, genetically driven liver injury, Minnis-Lyons et al. found that Notch signaling was required for the injury-induced proliferation of BECs because it sensitized the cells to insulin-like growth factor 1 (IGF1). Although treating mice with IGF1 after liver injury increased BEC proliferation, it did not boost hepatocyte numbers, demonstrating that additional signals are required to specify the hepatocyte fate in BECs. These findings suggest that therapeutic interventions that increase BEC proliferation may not be sufficient to stimulate tissue repair in patients with liver disease. In the adult liver, a population of facultative progenitor cells called biliary epithelial cells (BECs) proliferate and differentiate into cholangiocytes and hepatocytes after injury, thereby restoring liver function. In mammalian models of chronic liver injury, Notch signaling is essential for bile duct formation from these cells. However, the continual proliferation of BECs and differentiation of hepatocytes in these models have limited their use for determining whether Notch signaling is required for BECs to replenish hepatocytes after injury in the mammalian liver. Here, we used a temporally restricted model of hepatic repair in which large-scale hepatocyte injury and regeneration are initiated through the acute loss of Mdm2 in hepatocytes, resulting in the rapid, coordinated proliferation of BECs. We found that transient, early activation of Notch1- and Notch3-mediated signaling and entrance into the cell cycle preceded the phenotypic expansion of BECs into hepatocytes. Notch inhibition reduced BEC proliferation, which resulted in failure of BECs to differentiate into hepatocytes, indicating that Notch-dependent expansion of BECs is essential for hepatocyte regeneration. Notch signaling increased the abundance of the insulin-like growth factor 1 receptor (IGF1R) in BECs, and activating IGFR signaling increased BEC numbers but suppressed BEC differentiation into hepatocytes. These results suggest that different signaling mechanisms control BEC expansion and hepatocyte differentiation.


INTRODUCTION
After injury, the adult liver is able to regenerate from a population of biliary epithelial cells (BECs) that act as facultative hepatic stem cells by proliferating and differentiating into hepatocytes in response to injury, thereby restoring normal liver function in addition to reconstituting damaged biliary epithelia (1,2). Throughout regeneration, a cellular and acellular microenvironment surrounds BECs [also known as hepatic progenitor cells (HPCs) or oval cells] (3) and provides many developmental signals (4)(5)(6)(7). These signals are responsible for maintaining a balance between BEC proliferation and differentiation, thereby ensuring that regeneration is sufficient. In human disease, the induction of BECs and their microenvironment are collectively referred to as the ductular reaction (8). BECs are able to differentiate into two epithelial lineages: cholangiocytes that make up the bile duct, a tubular network that drains bile from the liver into the bowel, and hepatocytes, which comprise the metabolically active parenchyma of the liver. After bile duct injury, we and others have shown that Notch signaling is necessary for BECs to reconstitute the bile duct, and as such, Notch signaling is considered a master regulator of the ductular lineage (9,10). Whether Notch signaling is required for BECs to regenerate hepatocytes after hepatic injury in the mammalian liver is not clear, although data from several studies in zebrafish suggest that Notch signaling maintains BECs in a naïve state and that BECs can differentiate when the suppressive effects of Notch signaling are removed (11)(12)(13). These data from fish suggest that, contrary to evidence from mice, Notch signaling does function in the replenishment of hepatocytes after injury. Previous murine studies investigating the signals that govern the proliferation and differentiation of BECs into hepatocytes have been limited by the fact that the majority of murine models rely on establishing chronic hepatic injury. This results in continuous activation, proliferation, and differentiation of BECs. Therefore, in these models, BECs are at various stages of their life cycle, and this heterogeneity makes it difficult to resolve the signals that govern the various states of proliferation and differentiation (14)(15)(16).
To overcome this limitation, we used a model wherein large-scale, liver-wide injury is induced by the acute genetic loss of Mouse double minute 2 (Mdm2) in hepatocytes (2,17). In this model, hepatocytes become senescent (18), and, as a result, BECs undergo coordinated activation and proliferation, ultimately differentiating to reconstitute hepatocytes. Using this approach, we temporally resolved the role of Notch signaling during hepatocyte regeneration from BECs and showed that Notch signaling is activated before the overt expansion of BECs in the liver. In this model of acute hepatocyte Mdm2 loss, we found that Notch signaling promoted BEC proliferation by increasing the abundance of the insulin-like growth factor 1 receptor (IGF1R), thereby sensitizing BECs to insulin-like growth factor 1 (IGF1). Using a combination of animal models [acute hepatocyte Mdm2 loss as well as classical dietary-induced injury models in which hepatocytes arise from the differentiation of BECs or HPCs (2,19)], we found that this Notch-IGF1R pathway in vivo controlled the numbers of BECs and, thereby, the numbers of hepatocytes. Suppression of BEC proliferation resulted in a failure of BECs to differentiate into hepatocytes, indicating that a sufficient number of BECs must be generated before differentiation can occur during hepatic regeneration. Unexpectedly, we also found that increasing BEC number by stimulating IGF1 signaling inhibited differentiation, leading us to conclude that there is a fine balance between BEC generation and their differentiation into hepatocytes that must be satisfied for repair from BECs to occur.

Notch signaling is activated early in BECs after hepatic injury
The Notch signaling pathway is essential for the formation of bile ducts from embryonic hepatoblasts (20,21) and is reactivated in adult bile duct regeneration, during which it directs BECs into a cholangiocyte lineage (9,22,23). We sought to address whether Notch also regulates hepatocyte regeneration from BECs. We have previously described a mouse model of hepatic regeneration in which large numbers of hepatocytes can be generated from BECs (2,17). Known as the Ah-Cre::Mdm2 fl/fl mouse, this model relies on the specific expression of Cre recombinase in hepatocytes after administration of -naphthoflavone, which activates the Cyp1a1 promoter to drive Cre expression. In the liver, the administration of -naphthoflavone activates Cre expression only in hepatocytes and not in BECs. In these mice, both alleles of Mdm2, which encodes an E3 ubiquitin ligase that promotes the degradation of the tumor suppressor TRP53, are also flanked by LoxP sites. Cre-dependent deletion of Mdm2 stabilizes TRP53, leading to the induction of p21 and cell cycle arrest in hepatocytes. In this model, large-scale p21-triggered hepatocyte senescence results in liver injury (fig. S1A), hepatocyte apoptosis (fig. S1B), and BEC proliferation and differentiation to reconstitute the liver parenchyma (2,17). Because liver regeneration is initiated at a single time point (when -naphthoflavone is administered), it is possible to track the temporal dynamics of BECs as they proliferate before their differentiation into hepatocytes.
After Mdm2 loss in Ah-Cre::Mdm2 fl/fl mice, the number of BECs labeled by a pan-specific antibody against basic cytokeratins (panCK), which are present only in BECs in the liver, began to increase throughout the liver within 5 to 7 days after induction (Fig. 1A). By 14 days after induction, there were significantly higher numbers of panCK + BECs invading the parenchyma compared with mice that still had intact Mdm2 (Fig. 1A). These cells were also positive for the BEC marker SOX9 (fig. S1C).
Given the rapid increase in BEC numbers after hepatic injury, we hypothesized that they would continuously proliferate throughout regeneration to produce large numbers of daughter cells to replenish lost hepatocytes. Therefore, we stained Mdm2-deleted livers for Ki67, a marker of proliferation, and for the epithelial marker panCK. Contrary to what we expected, the proportion of proliferating BECs was high in the early phase of regeneration (days 3 to 5 after initiation of injury), when we began to see the emergence of BECs; however, this high proliferative rate was not maintained throughout regeneration, and after 5 days, the proportion of actively cycling BECs decreased (Fig. 1, A and B, and fig. S5A). Because Notch signaling is mitogenic in other progenitor cell systems (24,25) and is required for bile duct regeneration (9, 10), we evaluated whether components of the Notch signaling pathway were expressed during hepatocyte regeneration. After initiation of injury, there was a rapid increase in the expression of Notch1 and Notch3 (Fig. 1C) and the Notch ligands, Jagged1 and Jagged2 (Fig. 1D), between days 3 and 5 of regeneration. Notch2 expression did not increase, nor did expression of Dll1, the only other detectable Notch ligand ( fig. S1D). To verify that the Notch signaling pathway was activated in proliferating BECs, we used a transgenic mouse line, CBF::H2B Venus , that reports active Notch signaling with single-cell resolution (26) and crossed this into our Ah-Cre::Mdm2 fl/fl line to generate Ah-Cre::Mdm2 fl/fl ::CBF H2BVenus mice, in which we could identify cells that have active Notch signaling during hepatocyte regeneration by the expression of the enhanced yellow fluorescent protein (eYFP) variant, Venus. Proliferating BECs were strongly positive for Venus in the early phase (day 5) after hepatocyte Mdm2 loss ( Fig. 1E and fig. S5B), suggesting that, in agreement with the expression of Notch genes, this signaling pathway was active in these cells. The expression of transcripts encoding Notch pathway components was reduced at later time points, having peaked at 3 to 5 days after hepatocyte Mdm2 loss ( Fig. 1, C and D). To validate independently that the Notch pathway is activated in BECs, we stained liver tissue from mice with hepatocyte Mdm2 deletion for Notch pathway components and found that Notch1 and Notch3 proteins were present in BECs at days 3 and 5, respectively (Fig. 1F). Furthermore, the Notch1 intracellular domain localized to the nucleus in BECs, demonstrating that the Notch1 is cleaved and the pathway is activated ( Fig. 1F and fig. S5C). Previous work in ductular regeneration has shown that Jagged1 is produced by myofibroblasts surrounding proliferating ducts (9); however, during hepatocyte regeneration from BECs, we found little evidence indicating Notch ligands were present on fibroblasts or macrophages (Fig. 1G). Instead, Jagged1 was produced by cells that were intercalated in the duct and are likely the BECs themselves (Fig. 1G).

Inhibition of Notch signaling impairs BEC proliferation and hepatocyte differentiation
Although the expression of Notch pathway components correlates with an increased number of BECs entering the cell cycle, it is not clear whether Notch signaling drives proliferation of BECs per se. To determine whether this is the case, we induced Mdm2 loss in hepatocytes to stimulate BEC proliferation and treated the mice with DAPT {N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester}, a pharmacological inhibitor of Notch signaling that prevents the activity of -secretase, thereby blocking Notch cleavage (hereafter referred to as Notch-i). Because our model relies on a single genetic insult to initiate injury and hepatocyte regeneration, we were able to inhibit Notch signaling either early during regeneration or later in this process ( Fig. 2A). When we administered Notch-i early during regeneration (between days 2 and 4 after injury), when Notch signaling is highly activated, the number of BECs that were proliferative and the total number of panCK + BECs were significantly reduced (Fig. 2B). In contrast, administering Notch-i late, at a point where Notch1 and Notch3 transcripts are less abundant (at 7 to 10 days after injury), did not affect the number of proliferative BECs, demonstrating that Notch signaling only drives BEC proliferation in the early phases of hepatocyte repair (Fig. 2, B and C).
Given that inhibition of -secretase can influence the cleavage of other cell surface proteins in addition to Notch, we sought to verify that Notch1 and Notch3 specifically promoted BEC proliferation in the early stages. After hepatocyte Mdm2 loss and the activation of BECs, we inhibited Notch1 and Notch2 using receptor-specific inhibitory antibodies (administered 1 day after Mdm2 loss) or removed Notch3 genetically using a Notch3 −/− mutant mouse line. Inhibition of Notch1 or deletion of Notch3 significantly reduced the number of proliferative BECs and the total number of panCK + BECs after injury in vivo (Fig. 2, D and E) and was associated with a significant reduction in BEC proliferation and expression of Notch pathway target genes in vitro ( fig. S2, A to F). Inhibition of the Notch signaling pathway (either with DAPT, blocking antibodies, or through receptor deletion) and reduction in BEC numbers did not appear to make liver injury worse [as determined by alanine transaminase (ALT) levels; fig. S2, G to J]. However, in the context of Notch1 inhibition or genetic Notch3 deletion, wherein BEC proliferation is reduced, we found that the levels of serum bilirubin decreased significantly, suggesting that the formation of a ductular reaction, in these models at least, promotes cholestasis (fig. S2, K and L).
Prior studies have suggested that BECs differentiate into hepatocytes and that these differentiated cells then undergo proliferation to reconstitute the parenchyma (27). To determine whether inhibition of Notch signaling and reduced BEC numbers affected hepatocyte differentiation, we used a lineage tracing system in which the tamoxifen-inducible expression of CreERT 2 from the Osteopontin (Opn) promoter irreversibly labels BECs and their progeny with eYFP (OPN-iCRE ERT2 ::R26R eYFP ) to monitor the differentiation of BECs into hepatocytes (19). Whereas OPN-iCRE ERT2 labels only BECs in the healthy adult mouse liver, Opn is induced in hepatocytes after injury (28). To ensure we did not capture any other cells and that we only labeled BECs, we dosed animals with tamoxifen and then left them for a 2-week "washout" period before initiating injury (28). Hepatocyte injury was induced by feeding the tamoxifen-treated OPN-iCRE ERT2 ::R26R eYFP mice a choline-deficient, ethioninesupplemented (CDE) diet for 3 weeks and then allowing the animals to recover for 2 weeks with or without Notch-i treatment (Fig. 2F).
In the absence of Notch-i, the proportion of hepatocytes labeled with eYFP ( Fig. 2G) was significantly higher than in uninjured livers ( Fig. 2H), as previously reported (19). After Notch-i treatment in these OPN-iCRE ERT2 ::R26R eYFP , CDE-fed mice, however, the number of proliferative eYFP + BECs was reduced ( fig. S3A) as was the number of lineage-traced hepatocytes (Fig. 2, G and H). We also confirmed that in a second dietary model of hepatocyte injury, the methionine and choline-deficient (MCD) model (2), Notch-i consistently reduced the number of proliferating BECs ( fig. S3A), demonstrating that BEC proliferation driven by Notch signaling was not restricted to the CDE model. Collectively, our data indicate that Notch-dependent BEC proliferation is essential for normal hepatocyte reconstitution from a BEC origin.

Notch signaling negatively regulates the abundance of IGF1R in BECs
Notch signaling is involved in the expansion of various progenitor cell pools throughout development and in adult tissues (29)(30)(31). It is widely accepted that Notch does this by suppressing differentiation and maintaining progenitor cells in a state that is receptive to mitogenic signals rather than by directly stimulating proliferation (32,33). In a mouse model of Alagille syndrome, the loss of Notch signaling due to mutations in Jagged1 reduces IGF1 signaling (34). Therefore, we investigated whether Notch signaling regulates IGF1 signaling in adult BECs and whether IGF1 stimulates BECs to proliferate.   After deletion of Mdm2 in hepatocytes and initiation of hepatic regeneration from BECs, both IGF1 and its activated receptor, pIGF1R Y1161 , were detected on Keratin-19 + BECs in vivo ( Fig. 3A  and fig. S5D), and, throughout the time course after Mdm2 inactivation, Igf1r expression correlated with the expression of the Notch pathway target gene HeyL ( fig. S3B). In vitro, BMOL (bipotential mouse oval cell line) cells, a mouse BEC cell line, produced their own IGF1, which they secreted into the media (Fig. 3B). Treatment with Notch-i had no effect on the expression of Igf1 ( fig. S3C) but reduced both the expression of Igf1r (Fig. 3C) and the abundance of the IGF1R protein ( Fig. 3D and fig. S3D), suggesting that Notch inhibition may reduce signaling through the IGF1 pathway in vitro by negatively regulating the amounts of the IGF1 receptor. We also measured the amounts of both Igf1 and Igf1r transcripts in mice that had received Notch-i for 3 days during early hepatocyte regeneration after Mdm2 deletion in the Ah-Cre::Mdm2 fl/fl line, the time period during which Notch1 and Notch3 are most highly expressed ( Fig. 2A). Consistent with the in vitro findings, expression of Igf1r mRNA, but not that of Igf1, was significantly reduced in vivo by Notch-i (Fig. 3E). Furthermore, in these Ah-Cre::Mdm2 fl/fl mice  undergoing hepatocyte regeneration, treatment with Notch-i reduced the abundance of pIGF1R Y1161 , whereas pIGF1R Y1161 was abundant and localized to the plasma membrane of BECs in vehicle-treated animals (Fig. 3F).

Signaling through the IGF1R positively regulates BEC proliferation
Having determined that Notch signaling can reduce the abundance of IGF1R by inhibiting Igf1r expression in BECs and given that IGF1-IGF1R signaling is potently mitogenic in other cell types, we sought to address whether signaling through IGF1R promoted the proliferation of BECs. In vitro, BECs treated with the IGF1R inhibitor AG1024 (hereafter referred to as IGF1R-i) reduced their proliferation in a dose-dependent manner ( fig. S4A), indicating that IGF1-IGF1R signaling is mitogenic in BECs. Because IGF1R is produced by many different cell types in vivo (35) and to better understand the role of IGF1 signaling in hepatic regeneration, we conditionally deleted Igf1r (using Igf1r flox mice) in BECs using the OPN-CreER T2 transgenic mouse line, while concurrently labeling BECs with eYFP (designated as OPN-CreER T2 ::IGF1 fl/fl ::R26R eYFP mice) (Fig. 3G) and inducing liver injury by feeding the mice a hepatotoxic CDE diet. After CDE-induced hepatic injury, OPN-CreER T2 ::IGF1 fl/fl ::R26R eYFP mice, which lack Igf1r in their BECs, failed to mount a proliferative BEC response, as determined by reduced numbers of eYFP + BECs and fewer eYFP + BECs entering the cell cycle (Fig. 3, H and I). Given that IGF1-IGF1R signaling is a potent activator of the AKT and mechanistic target of rapamycin (mTOR) signaling pathway, we verified that mTOR was activated (phosphorylated) in proliferating BECs in Ah-Cre::Mdm2 fl/fl mice after Mdm2 deletion ( fig. S4B). Treating Ah-Cre::Mdm2 fl/fl mice undergoing BEC-mediated hepatocyte regeneration with the mTOR inhibitor rapamycin resulted in fewer proliferating BECs in vivo compared with vehicle-treated animals ( fig. S4, C and D), indicating that mTOR can promote BEC proliferation and may mediate proliferative IGF1-IGF1R signaling in BECs.

Therapeutically stimulating BEC proliferation with IGF1 does not increase hepatocyte regeneration
One of the ultimate aims of regenerative medicine is to identify small molecules that can alter the regenerative response and improve tissue repair. A core tenet of many therapies is that increasing the pool of endogenous stem or progenitor cells will ultimately improve the regenerative output of the tissue. Whether this is actually true in BEC-mediated liver regeneration, however, has not been determined.
Our data indicate that therapeutic modulation of Notch-IGF1 signaling could affect BEC number and ultimately alter how many hepatocytes are replenished after injury. Because BEC number undergoes rapid expansion from day 5 after hepatocyte Mdm2 loss, and that this is the point at which Igf1r mRNA is most highly expressed, we treated Ah-Cre::Mdm2 fl/fl mice with either IGF1R-i to reduce signaling through IGF1R or recombinant IGF1 (rIGF1) to stimulate IGF1R activation on days 6 to 9 after hepatocyte Mdm2 loss. After treatment with IGF1R-i, the number of proliferating BECs was reduced, as was the total number of BECs throughout the liver (Fig. 4, A and B). Conversely, treatment with rIGF1 significantly increased the number of BECs that entered the cell cycle compared with animals that received vehicle treatment, and the overall number of BECs within the liver increased (although this did not reach statistical significance) (Fig. 4, C and D). Together, these results indicate that IGF1-IGF1R signaling promotes BEC proliferation during hepatic regeneration.
Having determined that modulation of IGF1R through either therapeutic inhibition or activation can directly affect the number of BECs that are proliferative in the Ah-Cre::Mdm2 fl/fl model after hepatic injury, we sought to determine whether increasing the pool of BECs in the liver would increase the number of BEC-derived hepatocytes. To do this, we used a previously published model of regeneration wherein hepatocyte senescence is induced in the livers of Krt19Cre ERT ::R26 tdTomato transgenic mice by expressing p21 in hepatocytes (using AAV8:TBG-p21, an adeno-associated virus that is highly specific for hepatocytes) and injuring the liver using the hepatotoxic MCD diet (2). In the context of liver injury, the Krt19Cre ERT mouse does not have ectopic activation of the Cre transgene in hepatocytes (28) and therefore is a robust tool for genetically labeling and lineage tracing BECs to assess BEC-to-hepatocyte differentiation, particularly in contexts in which the number of hepatocytes arising from BECs could be limited. Because previous studies have shown that hepatocytes can be lineage traced from Krt19 + BECs (by staining for tdTomato) after the withdrawal of the MCD diet in this mouse line, we administered rIGF1 or vehicle alone in this model to determine whether stimulating IGF1R results in the formation of more lineage-traced hepatocytes (Fig. 4E). After rIGF1 treatment, the number of proliferating BECs and total number of BECs within the livers of rIGF1-treated mice were increased at the end of the 52-day study compared with vehicle-treated controls (Fig. 4F), reiterating that exogenous IGF1 increases BEC number in a range of murine models of hepatocyte regeneration. Last, we stained and quantified tissue for lineage-traced (tdTomato + ) hepatocytes that were also positive for hepatocyte nuclear factor 4 (HNF4), a marker of hepatocytes. There was a significant reduction in tdTomato + hepatocytes derived from BECs (Fig. 4, G and H). Because the rapid increase in cell size as BECs differentiate into hepatocytes dilutes the amounts of tdTomato in the hepatocyte cytoplasm, making them appear fainter compared with the BECs, we confirmed that lineage-traced hepatocytes were present by immunohistochemical staining (Fig. 4G). These results indicate that increasing BEC number alone is not sufficient to increase BEC-to-hepatocyte differentiation (Fig. 4I). Together, our data show that the Notch signaling pathway is necessary for the proliferation of BECs destined to become hepatocytes and that Notch does this by sensitizing BECs to IGF1, but other factors are necessary to promote the differentiation of these cells into hepatocytes. Our study affirms the central role of Notch signaling in the maintenance of progenitor cells, and although this role for the Notch signal has been previously identified, we have uncovered its central importance in expanding BEC number after injury and before hepatocyte transdifferentiation.

DISCUSSION
The role of Notch signaling in bile duct development is well described (20,34,36), and developmental signaling pathways are reactivated in the adult when bile ducts are injured (9,10). However, early data from us and others indicated that there was no role for Notch signaling in hepatic regeneration, because genes encoding Notch pathway components are transcriptionally repressed in BECs destined for hepatocyte differentiation, and Notch function is suppressed in BECs through the E3 ubiquitin ligase Numb (9,12). Previous mammalian models of hepatic regeneration promoted continuous BEC proliferation and differentiation (16). Thus, progenitor cells underwent proliferation and differentiation in an unsynchronized manner in these models, making the identification of pathways that regulate BEC proliferation and differentiation in the mammalian liver difficult. Furthermore, data from zebrafish have suggested that Notch signaling is required for BEC-to-hepatocyte differentiation (12,13). Recent advances in mammalian models of liver regeneration, however, have provided new tools to answer fundamental questions about BEC biology (2). These types of models in which a single, timed insult results in the proliferation and differentiation of BECs into hepatocytes have been discussed elsewhere (37,38), and they allow for temporal resolution of the signaling pathways involved in hepatocyte regeneration from a progenitor cell. Single-cell RNA sequencing from regenerating adult livers has found that the Yap signaling pathway is critically important for BEC proliferation (6, 7). These studies have indicated that Yap signaling, through the promotion of Notch signaling, could promote the proliferation of BECs (39). Whether this is a direct action of Notch acting as a mitogen or whether Notch signaling sensitizes BECs to other mitogenic signals has not been investigated (6,7).
We hypothesized that Notch signaling sits at the center of a signaling network (potentially regulated by Yap signaling) that maintains BECs in a state permissive to proliferation. Here, we show that expression of two Notch-encoding genes (Notch1 and Notch3) increased in BECs in a temporally restricted manner after liver injury. Expression of Notch2, which has an essential role in bile duct development during embryogenesis, did not change over time, and Notch4 was undetectable ( fig. S1D). In our model, activation of Notch1 or Notch3 signaling promotes the proliferation of BECs. However, specific  inhibition or genetic deletion of either receptor was sufficient to reduce the number of actively dividing BECs, suggesting that signaling through Notch1 and Notch3 is not redundant and indicating that Notch1 and Notch3 might play different roles in the activation and subsequent proliferation of BECs. We found in our model of largescale hepatocyte regeneration (using the Ah-Cre::Mdm2 fl/fl line) that the proliferation of BECs preceded hepatocyte apoptosis. The proliferation of BECs has previously been associated with the formation of an inflammatory microenvironment (3,40,41), and indeed, signaling from immune cells to BECs has been shown to initiate the formation of the ductular reaction (42,43). At a tissue level, this temporal separation of BEC proliferation and differentiation from the apoptosis of hepatocytes makes sense because the number of BECderived hepatocytes needs to be sufficient to maintain liver function before the clearance of injured hepatocytes. However, how this balance is maintained during regeneration is poorly defined. What is clear from our studies is that Notch signaling increases the sensitivity of BECs to stimulation with IGF1 through positivity regulating IGF1R signaling in BECs. Inhibiting Notch signaling or inhibiting signaling through the IGF1R axis was sufficient to reduce the number of actively cycling BECs. This has interesting implications for both liver cancer, which appears to rely on the activation of developmental signaling pathways, including Notch (44,45), and regenerative medicine, in which relatively few therapeutically tractable signaling pathways have been identified that can increase the number of progenitor cells within a regenerating system. In this study, we identify IGF1 as one such factor that increases the number of proliferating BECs and has the potential to improve progenitormediated regeneration. In our work in animal models of hepatocyte regeneration from BECs, however, we found that merely increasing the expansion of BECs was not sufficient to regenerate more hepatocytes. Instead, increasing the number of BECs suppressed hepatocyte differentiation compared to control animals. The relationship between proliferation and differentiation in stem and progenitor cell systems is complex and requires the integration of the actions of various proproliferative factors (such as cyclin-dependent kinases) with those of prolineage regulators and fate determinants (such as transcription factors). How these two states are reconciled in hepatic regeneration is not known; however, work investigating the epigenetic landscape in BEC-mediated liver regeneration could start to clarify the relationship between these two fundamental cellular states (46,47). We cannot discount an alternative model in which IGF1R signaling directly suppresses BEC-to-hepatocyte differentiation, and further work is needed to understand this, particularly given the biphasic role of Notch in BEC differentiation in zebrafish models of hepatocyte regeneration (11,12).
This work highlights the importance of Notch-IGF1 signaling in hepatocyte regeneration and, rather than being a strict hepatocyte fate-promoting signal or a signal that limits BECs committed into the cholangiocyte lineage, Notch acts as a common signal to expand progenitor cells before their differentiation. Furthermore, these data suggest that increasing the number of BECs in patients with hepatic injury is likely to be insufficient to promote regeneration and that additional therapies that drive expansion and differentiation would be required.
The Keratin19-CreERT::R26R tdTomato mice were induced by three individual intraperitoneal injections of tamoxifen (20 mg/ml; Sigma-Aldrich) at a dose of 4 mg. Mice received 2 weeks of normal diet after the last tamoxifen injection. Viral AAV8.TBG.p21 (Addgene) particles (5 × 10 11 ) were reconstituted in 100 l and intravenously injected through the tail vein with Ultra-Fine insulin syringes (BD). Mice received one more week of normal diet before starting the MCD diet (MP Biomedicals) for 14 days followed by 4 days of normal diet. Then, the mice received two intraperitoneal daily doses of recombinant murine-IGF1 (PeproTech, prepared at 0.05 mg per dose in 100 l of sterile saline) or saline control during four consecutive days followed by four additional days of recovery on normal diet. In our lineage tracing study, one animal of the saline group did not have a tdTomato expression and was therefore excluded from the analysis of the tdTomato + hepatocytes.
All animal experiments were conducted in accordance with the UK Home Office PPL awarded to S.J.F. All mice were genotyped by Transnetyx (Memphis, TN, USA) and were maintained in 12-hour light/dark cycles on a predominantly C67BL6/J genetic background.

Cell culture
The BEC cell line BMOL was maintained in Williams' E medium containing 2% fetal calf serum (Life Technologies). Cells were tested for the presence of mycoplasma in-house at the beginning of the study and were found to be negative. For in vitro cell viability assays, cells were plated at 2 × 10 5 /cm 2 density and treated for 48 hours with DAPT or AG1024 or the relevant vehicle at the concentrations detailed in the figures. Anti-Notch antibodies or control was used at 10 g/ml. MTT assay was conducted using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/ml; Sigma-Aldrich) diluted in phosphate-buffered saline (PBS) (Sigma-Aldrich). Cells were treated for 12 hours, and MTT crystals were dissolved in dimethyl sulfoxide (DMSO) and read at 570/690 nm using a FLUOstar Omega (BMG LABTECH). EdU incorporation assay (Thermo Fisher Scientific) was conducted as described in the manufacturer's instructions after 24-hour treatment of cells with DAPT (10 M) or volume-matched DMSO.

Immunohistochemistry
At euthanasia, livers were perfused with 5 ml of sterile PBS into the inferior vena cava (IVC) before fixation overnight in 10% buffered formaldehyde. After paraffin embedding, tissue sections were cut at 4 m. After dewaxing, sections were subject to heat-mediated antigen retrieval followed by blocks to peroxide (Vector), avidin/biotin (Life Technologies) where appropriate before protein block (Vector). Sections were incubated overnight with primary antibodies at 4°C. After washing, sections were stained with species-appropriate secondary antibodies conjugated to Alexa 488, Alexa 555 (Life Technologies), or Biotin (Vector). For colorimetric stains, sections were incubated with ABC reagent (Vector) and developed using DAB (Vector) before counterstaining with Harris hematoxylin and Scott's tap water. For a complete list of primary antibodies, see table S1.

Microscopy and cell counting
Images were obtained on a Nikon Eclipse E600 microscope. Cell counts were performed manually using Fiji ImageJ (EMBL) on blinded slides and more than 20 consecutive nonoverlapping fields at ×200 (colorimetric) or ×400 (immunofluorescence) magnification. Interlobular bile ducts were excluded from quantification. Picrosirius red quantification was performed on 30 consecutive nonoverlapping ×200 fields by pixel analysis using Adobe Photoshop CS6.
For analysis of proliferating cell nuclear antigen (PCNA) positivity in red fluorescent protein (RFP)-or YFP-expressing cells after performing immunofluorescence staining for the fluorescent reporter, PCNA and 4′,6-diamidino-2-phenylindole (DAPI), slides were scanned using the Operetta high-content analysis system (PerkinElmer). A minimum of thirty 10× long working distance objective fields (0.4 cm 2 ) liver tissue was analyzed using the Columbus image data storage and analysis system. Briefly, a sliding parabola was applied to the DAPI channel to remove variation in background level across and between samples. All nuclei were identified using method M standard settings, and from this population, cells positive for the reporter were identified on the basis of a common threshold. From this second population, those with nuclear PCNA staining were again identified using a common threshold. Results are reported as % RFP or YFP + cells also PCNA + . Fields containing artefacts that affected image analysis were excluded manually.
Quantification of the percentage of biliary-derived hepatocytes (Fig. 4) was performed in a semi-automated manner. Thirty-five nonoverlapping images (magnification, ×20) were acquired per mouse using Operetta High-Content Imaging System (PerkinElmer). Total cell count (DAPI + nuclei) and total hepatocyte count (HNF4 + nuclei) were quantified in an automated manner using a segmentation strategy and Columbus image analysis software (PerkinElmer). Biliary-derived hepatocytes were quantified manually, identified by the colocalization of DAPI and HNF4 in the nucleus and tdTomato + cytoplasm.

Real-time polymerase chain reaction and gene expression analysis
Cells and tissues were lysed into Tri-Reagent (Ambion) and homogenized. Lysates were mixed with 1:5 chloroform/Tri-Reagent, and the aqueous phase containing RNA was collected. Cells were lysed directly into RLT lysis buffer (Qiagen). RNA was precipitated with isopropanol. The precipitate was applied to an RNeasy Mini column (Qiagen) according to the manufacturer's protocol. Isolated RNA was quantified using a NanoDrop 1000, and 1 g was reverse transcribed using the Quantitect RT kit (Qiagen) as per the manufacturer's instructions. Quantitative real-time polymerase chain reaction was performed using SYBR green master mix (Qiagen) and QuantiTect primers (Qiagen). Primers are listed in table S2. Data were analyzed using the LightCycler system and normalized to the housekeeping gene Gapdh. All samples were run in triplicate.

Enyzme-linked immunosorbent assay detection of IGF1
Conditioned medium from BMOL cells for 48 hours was tested for IGF1 concentration using an IGF1 mouse enzyme-linked immunosorbent assay (ELISA) kit (Abcam ab100695) following the manufacturer's protocol. A standard curve was generated using protein standards within the kit, and absorbance of this and test samples was read using a spectrophotometer (SPECTROstar Omega). This was normalized to protein content of secreting cells [determined using a bovine serum albumin (BSA) standard and Pierce reagent] as a surrogate for cell number.

Serum analysis
Serum analysis used commercial kits according to the manufacturer's instructions: serum albumin (Alb), bilirubin, and ALT (Alpha Laboratories); aspartate aminotransferase and alkaline phosphatase (Randox Laboratories); and cholesterol (Wako Chemicals GmbH). All kits were adapted for use on a Cobas Fara centrifugal analyzer (Roche Diagnostics).

Immunoblotting
Cell extracts were prepared from BMOL cells treated with DAPT (10 M) or vehicle for 48 hours. Protein content was quantified using a BSA standard and Pierce reagent before being resolved using SDS-polyacrylamide gel electrophoresis. Immunoblots were incubated at 4°C overnight with primary antibody rabbit anti-Igf1R Y1161 (Abcam) or anti--actin clone AC-74 (Sigma-Aldrich). Appropriate horseradish peroxidase-labeled secondary antibodies were used (Cell Signaling Technologies), and signal was detected with ECL reagent (Thermo Fisher Scientific).

Statistical analysis
Prism software (GraphPad Software version 8) was used for all statistical analysis. Data are presented as means ± standard error of the mean (SEM). n refers to biological replicates. Normal distribution of data was determined using the D'Agostino and Pearson omnibus normality test. For parametric data, data significance was analyzed using a two-tailed unpaired Student's t test. In cases where more than two groups were being compared, a one-way analysis of variance (ANOVA) was used. In instances where the n was too small to determine normal distribution or the data were nonparametric, a two-tailed Mann-Whitney U test was used. Kruskall-Wallis test was used when comparing multiple nonparametric data.
Normal distribution was determined by the Shapiro-Wilk test. The data were normally distributed, and statistical significance was determined using unpaired t test. Statistical significance was assumed at P < 0.05. Data are presented as means ± SEM.

Study approval
All studies involving human tissue were approved by the University of Edinburgh and NHS Lothian Academic and Clinical Central Office for Research Development (ACCORD) tissue governance unit.