Title: Mechanism of NOTCH2 in Promoting Intrahepatic Bile Duct Development
Version: 1 Date: 26 May 2025
Reviewer's report:
This manuscript addresses a clinically relevant question, providing novel insights into the role
of Notch2 in hepatoblast differentiation and intrahepatic bile duct development, with potential
implications for understanding the pathogenesis and clinical heterogeneity of Alagille Syndrome
(ALGS). The findings are intriguing and may have translational relevance. However, to improve
the clarity, scientific rigor, and overall impact of the manuscript, several critical points need to
be addressed:
1. All figures must include full legends describing sample sizes (n), statistical tests used, and
meaning of error bars. Additionally, figures should be revised to ensure they are of publication-
quality resolution and include clearly labeled axes.
2. In the Results section (3.2.1), the authors describe immunofluorescence staining of Notch2,
Hes-1, and CK19 in different treatment groups. However, all markers appear to have been
visualized using single-color (red) staining, with no co-localization analysis performed. Since
Notch2 and Hes-1 are functionally linked to biliary differentiation, it is critical to demonstrate
that their expression co-localizes with CK19-positive intrahepatic bile duct cells. Without such
analysis, it remains uncertain whether these signals are derived from bile duct epithelium
or surrounding non-ductal cells.The authors are strongly encouraged to perform double
immunofluorescence staining (e.g., Notch2/CK19 or Hes-1/CK19) and provide merged images
to clarify the cellular context of these findings.
3. While the study demonstrates that estradiol promotes Notch2 expression and downstream
signaling, the mechanism underlying this regulation remains unexplored. Given the potential
involvement of estrogen receptor-mediated transcriptional pathways, the authors should either
provide additional mechanistic discussion or propose experimental strategies to validate
estradiol's regulation of Notch2 expression.
4. The manuscript frequently discusses the potential compensatory relationship between Notch2
and JAG1 as a basis for clinical heterogeneity in ALGS. While this is an interesting hypothesis,
it is important to acknowledge that the current study does not use JAG1-deficient models or
patient-derived cells to support this claim. Therefore, any suggestion that Notch2 upregulation
compensates for JAG1 mutations and mitigates bile duct paucity in ALGS should be presented
as speculative, with a clear recommendation for future experimental validation using JAG1+/
# or JAG1+/dDSL mutant models.
Title: Mechanism of NOTCH2 in Promoting Intrahepatic Bile Duct Development
Version: 1 Date: 26 May 2025
Reviewer's report:
This manuscript addresses a clinically relevant question, providing novel insights into the role
of Notch2 in hepatoblast differentiation and intrahepatic bile duct development, with potential
implications for understanding the pathogenesis and clinical heterogeneity of Alagille Syndrome
(ALGS). The findings are intriguing and may have translational relevance. However, to improve
the clarity, scientific rigor, and overall impact of the manuscript, several critical points need to
be addressed:
1. All figures must include full legends describing sample sizes (n), statistical tests used, and
meaning of error bars. Additionally, figures should be revised to ensure they are of publication-
quality resolution and include clearly labeled axes.
2. In the Results section (3.2.1), the authors describe immunofluorescence staining of Notch2,
Hes-1, and CK19 in different treatment groups. However, all markers appear to have been
visualized using single-color (red) staining, with no co-localization analysis performed. Since
Notch2 and Hes-1 are functionally linked to biliary differentiation, it is critical to demonstrate
that their expression co-localizes with CK19-positive intrahepatic bile duct cells. Without such
analysis, it remains uncertain whether these signals are derived from bile duct epithelium
or surrounding non-ductal cells.The authors are strongly encouraged to perform double
immunofluorescence staining (e.g., Notch2/CK19 or Hes-1/CK19) and provide merged images
to clarify the cellular context of these findings.
3. While the study demonstrates that estradiol promotes Notch2 expression and downstream
signaling, the mechanism underlying this regulation remains unexplored. Given the potential
involvement of estrogen receptor-mediated transcriptional pathways, the authors should either
provide additional mechanistic discussion or propose experimental strategies to validate
estradiol's regulation of Notch2 expression.
4. The manuscript frequently discusses the potential compensatory relationship between Notch2
and JAG1 as a basis for clinical heterogeneity in ALGS. While this is an interesting hypothesis,
it is important to acknowledge that the current study does not use JAG1-deficient models or
patient-derived cells to support this claim. Therefore, any suggestion that Notch2 upregulation
compensates for JAG1 mutations and mitigates bile duct paucity in ALGS should be presented
as speculative, with a clear recommendation for future experimental validation using JAG1+/
# or JAG1+/dDSL mutant models.
Please review this paper.
user
Title: Mechanism of NOTCH2 in Promoting Intrahepatic Bile Duct Development
Version: 1 Date: 26 May 2025
Reviewer's report:
This manuscript addresses a clinically relevant question, providing novel insights into the role
of Notch2 in hepatoblast differentiation and intrahepatic bile duct development, with potential
implications for understanding the pathogenesis and clinical heterogeneity of Alagille Syndrome
(ALGS). The findings are intriguing and may have translational relevance. However, to improve
the clarity, scientific rigor, and overall impact of the manuscript, several critical points need to
be addressed:
1. All figures must include full legends describing sample sizes (n), statistical tests used, and
meaning of error bars. Additionally, figures should be revised to ensure they are of publication-
quality resolution and include clearly labeled axes.
2. In the Results section (3.2.1), the authors describe immunofluorescence staining of Notch2,
Hes-1, and CK19 in different treatment groups. However, all markers appear to have been
visualized using single-color (red) staining, with no co-localization analysis performed. Since
Notch2 and Hes-1 are functionally linked to biliary differentiation, it is critical to demonstrate
that their expression co-localizes with CK19-positive intrahepatic bile duct cells. Without such
analysis, it remains uncertain whether these signals are derived from bile duct epithelium
or surrounding non-ductal cells.The authors are strongly encouraged to perform double
immunofluorescence staining (e.g., Notch2/CK19 or Hes-1/CK19) and provide merged images
to clarify the cellular context of these findings.
3. While the study demonstrates that estradiol promotes Notch2 expression and downstream
signaling, the mechanism underlying this regulation remains unexplored. Given the potential
involvement of estrogen receptor-mediated transcriptional pathways, the authors should either
provide additional mechanistic discussion or propose experimental strategies to validate
estradiol's regulation of Notch2 expression.
4. The manuscript frequently discusses the potential compensatory relationship between Notch2
and JAG1 as a basis for clinical heterogeneity in ALGS. While this is an interesting hypothesis,
it is important to acknowledge that the current study does not use JAG1-deficient models or
patient-derived cells to support this claim. Therefore, any suggestion that Notch2 upregulation
compensates for JAG1 mutations and mitigates bile duct paucity in ALGS should be presented
as speculative, with a clear recommendation for future experimental validation using JAG1+/
# or JAG1+/dDSL mutant models.
assistant