Mechanism of NOTCH2 in promoting intrahepatic bile duct development

Abstract

Background

The Notch signaling pathway plays a crucial role in intrahepatic bile duct development. Here, we aimed to investigate the effect of the Notch receptor, NOTCH2, on intrahepatic bile duct development to better understand congenital intrahepatic bile duct dysplasia.

Results

Estradiol increased NOTCH2 and its downstream proteins expression, which promoted the differentiation of hepatoblasts into intrahepatic cholangiocytes and the development of intrahepatic bile ducts by upregulating the Notch signaling pathway. NOTCH2 siRNA inhibited the above processes (P < 0.05). There was no significant difference between estradiol and estradiol + non-targeting siRNA groups (P > 0.1).

Conclusions

In conclusion, the activation of the Notch signaling pathway leads to increased NOTCH2 expression, which promotes the differentiation of hepatoblasts into intrahepatic cholangiocytes and the development of intrahepatic bile ducts during embryonic stages in C57BL/6CrSlc mice. The results of this study may provide a theoretical basis for infantile intrahepatic cholestasis treatment.

Background

The extrahepatic bile ducts and intrahepatic bile ducts are two key components of the biliary system, differing in origin. The former arises from the branches of the primitive gut diverticulum, while the latter forms in situ as the primary component of the biliary system [1].

During embryonic and early postnatal development, hepatoblasts differentiate into two major cell types: hepatocytes and intrahepatic bile duct epithelial cells, a process critically regulated by the Notch signaling pathway [2]. Our previous studies have confirmed that estradiol can promote hepatoblast differentiation into intrahepatic cholangiocytes and intrahepatic bile duct development by increasing the expression of receptors, ligands, and downstream molecules of the Notch signaling pathway [3]. Thus, the abnormal downregulation of the Notch signaling pathway is closely associated with biliary diseases.

Among diseases caused by abnormal Notch signaling, Alagille syndrome (ALGS) is particularly notable. Its hepatic pathology is mainly characterized by a reduced number of interlobular bile ducts, with clinical manifestations including cholestasis [4]. The molecular diagnostic prevalence of ALGS is approximately 1 in 70,000, making it one of the leading causes of neonatal cholestatic liver disease, accounting for about 2%−14% of cases [5, 6]. ALGS is an autosomal dominant disorder, with over 95% of cases caused by mutations in JAG1, a ligand in the Notch signaling pathway. The remaining 5% of cases are primarily attributed to mutations in NOTCH2, a receptor in the pathway [7]. Although most ALGS patients have JAG1 mutations, NOTCH2 plays a critical role in intrahepatic bile duct development, particularly in ductal plate formation [8].

It is worth noting that despite the genetic basis of ALGS, there is no complete correlation between genotype and phenotype. Even among patients with the same gene mutation, the clinical presentation including the degree of bile duct paucity and cholestasis varies. Some individuals in the same family with identical JAG1 mutations may be asymptomatic, while others may suffer from severe cholestasis [9, 10].

Currently, treatment options for ALGS are limited and focus primarily on symptomatic relief, such as reducing cholestasis and pruritus. The treatment does not effectively alter disease progression. Consequently, the prognosis for ALGS remains poor. Studies have shown that without liver transplantation, only ~ 24% of ALGS patients reach 18 years of age with their native liver [11]. Given this, novel therapeutic strategies are urgently needed. Some researchers, considering the clinical heterogeneity of ALGS, have proposed that increasing the expression of JAG1 or NOTCH2 may alleviate bile duct paucity and cholestasis in ALGS [11]. However, this hypothesis has not yet been verified.

Based on our prior studies and relevant literature, this study aims to explore whether increased expression of NOTCH2 during embryonic and early postnatal stages promotes hepatoblast differentiation into intrahepatic cholangiocytes and enhances intrahepatic bile duct development. In future research, we will investigate whether increased NOTCH2 expression can improve intrahepatic bile duct development in JAG1+/dDSL mutant mice, providing a theoretical foundation for novel ALGS treatment strategies.

Materials and methods

Animals

C57BL/6CrSlc mice (6–8 weeks old; female: male = 2:1) of SPF grade were purchased from Hubei Ensiweier Biotechnology Co., Ltd., China. Experimental protocols related to mice surgical operations were performed according to the guidelines of the Chinese Council on Animal Care, and were approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The embryonic age was determined according to the appearance of a vaginal plug. The day that the vaginal appeared was defined as embryonic day 0.5 (E0.5).

The estradiol was administered to the pregnant mice. Since day 6 of pregnancy,mice were treated as follows:

(a) Mice in the control group received a DMSO injection diluted with isotonic saline of the same injection volume and concentration. (b) Mice in the estradiol group were given peritoneal injection with oestradiol (0.6 mg/kg) daily. (c) Mice in the estradiol + NOTCH2 siRNA group were given injection uterus with NOTCH2 siRNA adenovirus vector injection (1 × 10^9pfu) once and peritoneal injection with oestradiol (0.6 mg/kg) daily. (d) Mice in the estradiol + non-targeting siRNA group were given injection uterus with blank adenovirus injection (1 × 10^9pfu) once and peritoneal injection with oestradiol (0.6 mg/kg) daily.

The NOTCH2 siRNA adenovirus vector were obtained using AdMax adenovirus packaging system according to the instruction manual from FUBIO. (www.microbix.com) The NOTCH2 siRNA was introduced in vivo through transplacental delivery. On embryonic day 6, pregnant mice were given injection uterus with NOTCH2 siRNA adenovirus vector injection (1 × 10^9pfu).

Isolation and culture of hepatoblast

The procedure for hepatoblast isolation followed previously described protocols. Firstly, the anesthesia machine was turned on. The oxygen ventilation rate was adjusted to 1 L/min. The anesthesia concentration was adjusted to 5% vol (isoflurane). For induction anesthesia, the pregnant C57BL/6CrSlc mice were put into the anesthesia induction box, where the mice were quickly anesthetized in about 1–2 min. Then, the mice were sacrificed via cervical dislocation on embryonic day 13.5 (E13.5), and fetal livers were carefully isolated from the uterine tissues. The livers were minced and digested with type II collagenase/EDTA solution (1 g/L), filtered. After centrifugation, the cells were then resuspended in Dulbecco’s Modified Eagle Medium for adherent cultivation. The cell suspensions were collected and subjected to two rounds of adhesion cultivations. For surface antigen determination, the cells were incubated with rabbit anti-dlk1 antibodies (Abcam), washed twice in PBS, and collected for flow cytometry analysis (Becton Dickinson). The cultures were maintained in a 37 °C incubator, and the culture medium was replaced every 2–3 days. Once the cell density reached 80%, the cells were passaged. The medium was discarded, and the cells were washed once with PBS before being digested with 1–2 mL of 0.25% trypsin. After removing the trypsin, complete medium was added to create a single-cell suspension, which was passaged at a ratio of 1:3 and expanded under 37 °C, 5% CO₂, and saturated humidity conditions.

Gene transfection

Plasmids were diluted with 100 µL of serum-free Opti-MEM, gently mixed with a pipette, and left to stand at room temperature for 5 min. Lipofectamine™ 2000 was gently mixed before use, and 5 µL of Lipofectamine™ 2000 was diluted in 100 µL of Opti-MEM and left to stand at room temperature for 5 min. The diluted plasmids and Lipofectamine™ 2000 were combined (final volume: 200 µL), gently mixed, and incubated at room temperature for 20 min to form plasmid-Lipo2000 complexes.

The primary medium was removed, and the cells were washed with serum-free medium before replacing it with serum-free medium. The prepared plasmid-Lipo2000 complexes were added dropwise into the cell culture wells, gently mixed, and returned to the incubator. After 6 h, the plasmid-Lipo2000 complexes were removed, replaced with complete medium, and subcultured.

Flow cytometry

On day 13.5 of pregnancy, the liver of the fetal mice was isolated. Mouse embryonic hepatoblasts were made into single-cell suspensions using hepatoblast culture medium and evenly seeded into 6-well plates. The cells were cultured overnight at 37 °C under 5% CO₂ and saturated humidity. The cells were incubated and transfected according to the experimental groups (estradiol 10 nM, estradiol + non-targeting siRNA, estradiol + Si-NOTCH2, and blank control). For each well, 200 µL of the transfection mixture was added, and the culture plates were gently shaken to mix the solution. The cells were incubated in a 37 °C, 5% CO₂ incubator for 4–6 h, after which the transfection solution was removed and replaced with regular culture medium. The cells were harvested using trypsin/EDTA, washed with PBS and flow cytometry staining buffer, and incubated with rabbit anti-CK19 antibody at 4 °C in the dark for 30 min. After incubation, the cells were washed twice with permeabilization buffer and resuspended in 200 µL of flow cytometry staining buffer for analysis using a flow cytometer.

Immunofluorescence analysis

Immunofluorescence analysis of the NOTCH2 and cholangiocyte lineage-associated proteins in the cells was performed. Slides with adherent cells were washed with PBS for 3 times, fixed with 4% paraformaldehyde for 15 min, and washed with PBS for another 3 times. After being blocked with goat serum for 30 min, the primary antibody was added, and the slides were incubated overnight at 4 °C. The next day, the slides were washed with PBS and incubated with the diluted fluorescent secondary antibody at 37 °C for 1 h. DAPI was added for nuclear staining under light-protected conditions for 5 min. Excess DAPI was washed off with PBST, and images were captured using a fluorescence microscope.

For in vivo experiments, embryonic day 18 fetal livers were examined. Tissue samples were dehydrated in a graded ethanol series, cleared, and embedded in paraffin at 60 °C. Tissue blocks were trimmed and embedded with paraffin wax at slightly higher temperature to ensure complete integration. The samples were sectioned, deparaffinized, and subjected to antigen retrieval using an electric ceramic stove. Slides were dried with absorbent paper, circled with an immunohistochemical pen, and blocked with normal goat serum at room temperature for 30 min to reduce nonspecific staining. Subsequent steps were similar to those used for in vitro experiments.

RNA extraction and real-time PCR

RNA was extracted from in vivo and in vitro samples using the Trizol method. Cells were lysed in 1 mL of Trizol reagent, pipetted to mix thoroughly, and transferred to RNase-free 1.5 mL EP tubes. After a 10-min lysis, 200 µL of chloroform was added, mixed, and left at room temperature for 5 min. RNA was separated from the aqueous phase using isopropanol precipitation. Total RNA (2 µg) was reverse transcribed into cDNA under the following conditions: 15 min at 50 °C, 5 s at 85 °C, and 10 min at 4 °C. The resulting cDNA was diluted 1:8, and the reaction mixture was prepared for real-time polymerase chain reaction (PCR) analysis. The qPCR reaction conditions were as follows: pre-denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s.

Data analysis

In vivo portion of the study, there were three independent experiments. Every time, three pregnant mice were included in each group (Control, Estradiol, Estradiol + NOTCH2 siRNA and Estradiol + non-targeting siRNA). Three litters from different pregnant mice were analyzed per condition. Therefore, nine embryos were examined in each group in total. In vitro portion, there were also three independent experiments. Every time, three pregnant mice were included in each group (Control, Estradiol, Estradiol + NOTCH2 siRNA and Estradiol + non-targeting siRNA). Three cultures from embryos of different pregnant mice were analyzed per condition. Therefore, nine cultures of embryos were examined in each group in total.

Data are presented as mean ± standard deviation. Statistical analysis was performed using SPSS (v13.0, SPSS Inc.). One-way analysis of variance (ANOVA) was used for comparisons of quantitative data. A P-value < 0.05 was considered statistically significant.

Results

Increased level of NOTCH2 enhances hepatoblast differentiation into intrahepatic cholangiocytes during embryonic period in vitro

Flow cytometry analysis confirmed that increased level of NOTCH2 due to upregulation of the Notch signaling pathway enhanced hepatoblast differentiation into intrahepatic cholangiocytes in vitro (Fig. 1).

Fig. 1.

Differentiation of hepatoblasts into intrahepatic cholangiocytes in different experimental groups in vitro (intrahepatic cholangiocytes are defined as CK19 + cells). (A-D) The proportions of hepatoblasts differentiating into intrahepatic cholangiocytes in the control group, estradiol group, estradiol + non-targeting siRNA group, and estradiol + NOTCH2 siRNA group were 2%, 41%, 41%, and 27%, respectively. P2 in A-D is the gating of CK19⁺ cells. (E) * p < 0.05 vs. Control, # p < 0.05 vs. Estradiol. (A–D: flow cytometry dot plots for each group; E: quantification of CK19⁺cell percentages) (Three independent experiments have been completed. Every time, three pregnant mice were included in each group. Three cultures from embryos of different pregnant mice were analyzed per condition. Therefore, nine cultures of embryos were examined in each group in total.)

Flow cytometry was used to measure the differentiation of hepatoblast, with intrahepatic cholangiocytes defined as CK19 + cells. P2 in Fig. 1 is the flow cytometry gating, based on unstained controls. As shown in Figs. 1A-D, CK19⁺ cholangiocytes comprised 2%, 41%, 41%, and 27% of the total cells in the control, estradiol, estradiol + non-targeting siRNA, and estradiol + NOTCH2 siRNA groups, respectively. Figure 1E indicates that estradiol significantly increased the proportion of intrahepatic cholangiocytes, compared to the control group (p < 0.5). NOTCH2 siRNA significantly reduced this promotion (p < 0.05), while non-targeting siRNA had no significant effect on estradiol-induced promotion.

Immunofluorescence analysis and real-time PCR confirmed that upregulation of the Notch signaling pathway enhances NOTCH2 expression, promoting downstream protein expression and gene transcription in vitro (Fig. 2).

Fig. 2.

Protein expression levels of NOTCH2 and downstream Hes1 of the Notch signaling pathway in different experimental groups in vitro. (A, C) Hepatoblasts were incubated with anti-Hes1 and anti-NOTCH2 antibodies, and cell nuclei were stained with DAPI (blue). Scale bar, 10 μm. (B, D) Quantitative fluorescence analysis comparing the IOD/pixel values of proteins in hepatoblasts between the estradiol-treated group and the control group, as well as between the estradiol-treated group and the estradiol + NOTCH2 siRNA group (* p < 0.05). (A: Hes1 immunofluorescence; B: quantification of Hes1 staining; C: NOTCH2 immunofluorescence; D: quantification of NOTCH2 staining in vitro) (Three independent experiments have been completed. Every time, three pregnant mice were included in each group. Three cultures from embryos of different pregnant mice were analyzed per condition. Therefore, nine cultures of embryos were examined in each group in total.)

As shown in Fig. 2A and C, immunofluorescence staining intensity for NOTCH2 and its target gene Hes1 was significantly increased in the estradiol group compared to control (p < 0.05). Hes1 is one of the most important downstream target genes of Notch signaling pathway, promoting hepatoblast differentiation into intrahepatic cholangiocytes. Both the staining intensity was significantly reduced compared to the estradiol group when NOTCH2 siRNA was added (p < 0.05). 42% reduction in staining intensity of NOTCH2 was achieved relative to estradiol treatment without siRNA. The estradiol + blank siRNA group showed no significant difference compared to the estradiol group.

Real-time PCR further confirmed the results (Fig. 3A and B). Compared to the control group, NOTCH2 and Hes1 mRNA levels increased by 3.10-fold and 2.15-fold in the estradiol group. Estradiol + NOTCH2 siRNA showed a smaller increase (~ 1.50- and 1.22-fold; significantly less than estradiol alone, p < 0.05). 52% reduction in NOTCH2 mRNA level was achieved relative to estradiol treatment without siRNA. The estradiol + non-targeting siRNA group was similar to the estradiol group (~ 2.99- and 2.24-fold; no significant difference vs. estradiol). (The fold-change is relative to the control group set as 1.0.) The knockdown was known to be partial according to the results, which might explain why estradiol + NOTCH2 siRNA still showed slight increases in some markers.

Fig. 3.

Gene transcription levels of NOTCH2 and downstream Hes1 of the Notch signaling pathway in different experimental groups in vitro. RT-PCR was used to measure the relative mRNA expression levels of Hes1 and NOTCH2 normalized to β-actin in the control group, estradiol-treated group, estradiol + blank siRNA group, and estradiol + NOTCH2 siRNA group (3 A for Hes1, 3B for Notch2). Gene expression was normalized to β-actin and then to control = 1. The y-axis indicated that fold change in mRNA was relative to control. Comparisons were made between the estradiol-treated group and the control group, as well as between the estradiol-treated group and the estradiol + NOTCH2 siRNA group. * p < 0.05 vs. Control, # p < 0.05 vs. Estradiol. (Three independent experiments have been completed. Every time, three pregnant mice were included in each group. Three cultures from embryos of different pregnant mice were analyzed per condition. Therefore, nine cultures of embryos were examined in each group in total.)

Increased NOTCH2 level due to the activation of the Notch signaling pathway improves the intrahepatic bile duct development in vivo

Immunofluorescence analysis confirmed that increased NOTCH2 expression due to the activation of the Notch signaling pathway enhanced its target protein expression in vivo

As shown in Fig. 4, immunofluorescence analysis was performed on different groups to detect NOTCH2, Hes1, and CK19. CK19 is one of the most important cholangiocyte differentiation markers. Therefore, CK19 staining in vivo has been used to assess the intrahepatic bile duct development during our experiment. Estradiol significantly increased the staining intensity of NOTCH2, as well as its target Hes1 and bile duct marker CK19 compared to control (p < 0.05). When NOTCH2 siRNA was used to knock down NOTCH2 expression, the staining intensities of NOTCH2, Hes1, and CK19 were significantly reduced, compared to the estradiol group (p < 0.05). 36% reduction in staining intensity of NOTCH2 was achieved relative to estradiol treatment without siRNA. No significant difference was observed between the estradiol + non-targeting siRNA group and the estradiol group.

Fig. 4.

Protein expression of NOTCH2, downstream Hes1 of the Notch signaling pathway, and biliary lineage marker CK19 in different experimental groups in vivo. (A, C, E) Liver progenitor cells were incubated with anti-CK19, anti-Hes1, and anti-NOTCH2 antibodies, respectively, and nuclei were stained with DAPI (blue). Scale bar: 50 μm. (B, D) Quantitative fluorescence analysis showing the IOD/pixel values of protein expression in liver progenitor cells, comparing the estradiol-treated group with the control group and the estradiol-treated group with the estradiol + NOTCH2 siRNA group. * p < 0.05 vs. Control, # p < 0.05 vs. Estradiol. Fluorescence intensity measured through average area positive percentage per group, n = 3 experiments. (A: CK19 immunofluorescence; B: quantification of CK19 staining; C: Hes1 immunofluorescence; D: quantification of Hes1 staining; E: NOTCH2 immunofluorescence; F: quantification of NOTCH2 staining in vivo) (Three independent experiments have been completed. Every time, three pregnant mice were included in each group. Three litters from different pregnant mice were analyzed per condition. Therefore, nine embryos were examined in each group in total.)

Real-time PCR confirmed that increased NOTCH2 expression due to the activation of the Notch signaling pathway promotes its target gene transcription in the Notch signaling pathway in vivo

Real-time PCR results corroborated the immunofluorescence findings (Fig. 5). Compared to control, estradiol increased NOTCH2, Hes1, and CK19 mRNA by 2.95-, 2.54-, and 2.09-fold (p < 0.05). 56% reduction in NOTCH2 mRNA level was achieved relative to estradiol treatment without siRNA. Estradiol + NOTCH2 siRNA showed a smaller increase (~ 1.72-, 1.53-, and 1.32-fold; significantly less than estradiol alone, p < 0.05), while the estradiol + non-targeting siRNA group was similar to the estradiol group (~ 3.00-, 2.40-, and 2.22-fold; no significant difference vs. estradiol). (The fold-change is relative to the control group set as 1.0.)

Fig. 5.

Gene transcription of NOTCH2, downstream Hes1 of the Notch signaling pathway, and biliary lineage marker CK19 in different experimental groups in vivo. The expression levels of mRNA relative to β-Actin were measured by RT-PCR in the control group, estradiol-treated group, estradiol + blank siRNA group, and estradiol + NOTCH2 siRNA group. (3 A for CK19, 3B for Hes1, 3 C for Notch2). Gene expression was normalized to β-actin and then to control = 1. The y-axis indicated that fold change in mRNA was relative to control. Comparisons were made between the estradiol group and the control group, as well as between the estradiol group and the estradiol + NOTCH2 siRNA group. * p < 0.05 vs. Control, # p < 0.05 vs. Estradiol. (Three independent experiments have been completed. Every time, three pregnant mice were included in each group. Three litters from different pregnant mice were analyzed per condition. Therefore, nine embryos were examined in each group in total.)

These findings further confirm that estradiol promotes intrahepatic bile duct development during embryonic period by upregulating NOTCH2 transcription and expression via activation of the Notch signaling pathway.

Discussion

In recent years, significant progress has been made in understanding the molecular mechanisms underlying infantile (including neonatal) cholestatic liver diseases. Genetic testing has provided reliable diagnosis evidence for the clinic. However, treatment options for infantile cholestatic diseases remain limited, most of which are symptomatic therapies. It is important to seek targeted treatment strategies for each type of cholestatic disease.

Even with the advent of genetic testing, infantile cholestatic diseases remain a highly complex group of conditions. Identical genetic mutations might manifest in vastly different clinical phenotypes across patients. This complexity complicates diagnosis; however, it may also provide an opportunity for new breakthroughs in understanding and treating infantile cholestatic diseases.

Clinical heterogeneity is particularly pronounced in Alagille syndrome (ALGS), an autosomal dominant disorder. Both literature and our clinical observations support the significant variability in ALGS manifestations [12]. Estradiol is an important inducer of cell differentiation and growth, mainly mediated by estrogen receptors (ERs). ERα and ERβ are the most characterized receptors [13]. In liver, cholangiocytes express both ERα and ERβ, while hepatocytes mainly express ERα. First, estradiol binds to the receptor, activating the estrogen responsive elements (ERE) [3]. In C57BL/6CrSlc mice, estradiol promotes the differentiation of hepatoblast into intrahepatic cholangiocytes and facilitates intrahepatic bile duct development during embryonic period. The promotion has been demonstrated through in vitro and in vivo studies building on our earlier research. The study has also demonstrated that the promotion is achieved by upregulating the Notch signaling pathway [3].

The Notch signaling pathway plays a critical role in intrahepatic bile duct development [14]. It is a highly conserved pathway, where receptor-ligand interactions (Notch1, NOTCH2, Notch3, Notch4 and JAG1, JAG2, Delta-like 1/3/4) activate intracellular cascades, promoting downstream target gene transcription and cellular differentiation [15]. Hes1, one of the detection indexes in this study, is a downstream target gene of NOTCH2 and also a transcription factor. Despite a shared downstream mechanism, the expression and activity of Notch receptors and ligands vary significantly across tissues and developmental stages [16, 17]. In the context of intrahepatic bile duct development, the primary receptor and ligand involved are NOTCH2 and JAG1 [18, 19].

In this study, we further confirmed that the increased expression of NOTCH2 following activation of the Notch signaling pathway is the primary mechanism driving these effects.

The incremental advance in the current work further proposed that NOTCH2 played the key role in both intrahepatic bile duct development inferred from molecular markers and the process of hepatoblasts differentiation into intrahepatic cholangiocytes. The central new finding here is the specific requirement of NOTCH2 in mediating that effect. The new finding provided a more detailed explanation of the clinical heterogeneity observed in patients with JAG1 mutations. Despite the same JAG1 mutation, increased NOTCH2 level may improve the interlobular bile ducts development and cholestasis of some patients. It is also in accordance with that NOTCH2 mutation is another common mutation site in ALGS.

Studies have shown a compensatory effect among Notch ligands, where different ligands binding to the same receptor produce similar biological effects [18, 20]. Additionally, JAG2 and DLL1, which are also expressed in bile duct tissues, can interact with NOTCH2 [21]. Given that NOTCH2 is a critical gene regulating intrahepatic bile duct development [22], we hypothesize that increased NOTCH2 expression in some ALGS patients with JAG1 mutations may enhance binding with alternative ligands (JAG2 and DLL1) to activate the Notch signaling pathway, compensating for the JAG1 deficiency and improving bile duct development.

Our new findings suggest more explicitly that targeting NOTCH2 could be a therapeutic strategy for ALGS. Some researchers have proposed that increasing the expression of JAG1 or NOTCH2 may alleviate bile duct paucity and cholestasis in ALGS [11]. However, the hypothesis has not been verified. The mechanistic insight proposed by our present study advance understanding of ALGS.

Of course, there are deficiencies in our research work. Though our new findings show that NOTCH2 is necessary for the estradiol-induced enhancement of cholangiocyte differentiation since knocking it down impairs the effect. However, it remains somewhat unclear if increased NOTCH2 alone is sufficient to drive bile duct development. The enhancement of bile duct development in vivo is inferred from molecular marker. Our future research will supplement additional evidence whether overexpression of NOTCH2 in hepatoblasts leads to similar pro-differentiation effects as estradiol and intrahepatic bile duct development through direct morphological counts.

Conclusions

Our study demonstrated that increased NOTCH2 due to upregulation of the Notch signaling pathway promoted intrahepatic bile duct development during the embryonic stage in C57BL/6CrSlc mice. Of course, potential compensatory relationship between NOTCH2 and JAG1 in ALGS is only a hypothesis. Our future experimental using JAG1+/dDSL mutant models will be finished to validate the hypothesis. We will focus on verifying whether NOTCH2 is a major determinant of clinical heterogeneity in JAG1+/dDSL mutant mice. ALGS models and human ALGS tissues will also be collected to verify the central hypothesis of NOTCH2 compensating for JAG1 mutations, which may provide more information about the disease.

Abbreviations

ALGS

Alagille syndrome

ANOVA

One-way analysis of variance

siRNA

Small interfering RNA

E0.5

Ebryonic day 0.5

PCR

Polymerase chain reaction

Authors’ contributions

All authors contributed to the study conception and design. Material preparation and data collection were performed by Benping Zhang, Sainan Shu, Biao Zou and Shengxuan Liu. Data analysis was mainly finished by Chen Dong. The first draft of the manuscript was written by Chen Dong. All authors commented on previous versions of the manuscript. Benping Zhang revised the article critically. All authors read and approved the final manuscript.

Funding

This study did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Experimental protocols related to mice surgical operations were performed according to the guidelines of the Chinese Council on Animal Care, and were approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.