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Essential role of Notch/Hes1 signaling in postnatal pancreatic exocrine development

Abstract

Background

Notch/Hes1 signaling has been shown to play a role in determining the fate of pancreatic progenitor cells. However, its function in postnatal pancreatic maturation is not fully elucidated.

Methods

We generated conditional Hes1 knockout and/or Notch intracellular domain (NICD) overexpression mice in Ptf1a- or Pdx1-positive pancreatic progenitor cells and analyzed pancreatic tissues.

Results

Both Ptf1acre/+; Hes1f/f and Ptf1acre/+; Rosa26NICD mice showed normal pancreatic development at P0. However, exocrine tissue of the pancreatic tail in Ptf1acre/+; Hes1f/f mice atrophied and was replaced by fat tissue by 4 weeks of age, with increased apoptotic cells and fewer centroacinar cells. This impaired exocrine development was completely rescued by NICD overexpression in Ptf1acre/+; Hes1f/f; Rosa26NICD mice, suggesting compensation by a Notch signaling pathway other than Hes1. Conversely, Pdx1-Cre; Hes1f/f mice showed impaired postnatal exocrine development in both the pancreatic head and tail, revealing that the timing and distribution of embryonic Hes1 expression affects postnatal exocrine tissue development.

Conclusions

Notch signaling has an essential role in pancreatic progenitor cells for the postnatal maturation of exocrine tissue, partly through the formation of centroacinar cells.

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Acknowledgements

We thank Y Kawaguchi (Kyoto University, Kyoto, Japan) for supplying Ptf1acre/+ mice. We thank D Melton (Harvard University, MA, USA) for supplying Rosa26NICD mice. We thank Yuta Kawamata and Taichi Ito for the excellent technical support.

Funding

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP16K09395.

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Correspondence to Yuzo Kodama.

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535_2021_1779_MOESM1_ESM.pdf

Supplementary Figure S1 Mouse strains. a The protein-coding region of Ptf1a was precisely replaced with that of Cre, encoding the recombinase Cre. b Hes1 is floxed by loxp and conditionally knocked out in the Cre recombinase promoter site. c Rosa-26, Lox-STOP-Lox, and LacZ construct. LacZ expresses in the Cre recombinase promoter site. d Rosa-26, Lox-STOP-Lox, and NICD construct. NICD expresses in the Cre recombinase promoter site. e Cre recombinase is under the transcriptional control of the mouse Pdx1 promoter. Supplementary Figure S2. Hes1 immunohistochemistry Hes1 expression in pancreatic tissue was analyzed by immunohistochemistry in Hes1 cKO (Ptf1acre/+;Hes1f/f ;Rosa26LacZ) and control (Ctrl, Ptf1acre/+;Hes1f/f ;Rosa26LacZ ) mice at P0. Hes1 expression was deleted in Hes1 cKO mice (b), while it was positive in the ducts and centroacinar cells of control mice (a). Supplementary Figure S3 Changes in body weight and blood glucose level Changes in body weight (a) and blood glucose level (b) after birth in Hes1 cKO (Ptf1acre/+;Hes1f/f ;Rosa26LacZ) and control (Ctrl, Ptf1a+/+;Hes1f/f;Rosa26LacZ ) mice. The median and the standard error of mean are shown. Supplementary Figure S4 Hes1 knockout in Ptf1a-positive progenitor cells. X-gal staining of Hes1 conditional knockout mice (Ptf1acre/+;Hes1f/f;Rosa26LacZ) and control mice (Ptf1acre/+;Hes1f/+;Rosa26LacZ) at 4 weeks of age. b and d are a magnification of the broken line in a and c. Arrow head: islet, arrow: duct. Scale bars: 50 μm (b, d). Supplementary Figure S5 a Western blot analysis for LC3-I and its lapidated form LC3-II. We found expression of LC3-I but not its lipidated form LC3-II in both pancreatic head and tail of Hes1 cKO mice and control mice at P4. Supplementary Figure S6 a Immunofluorescence images of Hes1 and Aldh1a1 in Hes1 cKO (Ptf1acre/+;Hes1f/f) and control (Ctrl, Ptf1a+/+;Hes1f/f) mice at P3. Scale bars: 50 μm. b Microarray analysis of pancreatic head and tail tissues of Hes1 cKO and control mice at P7. A heatmap of genes related to pancreatic development is shown. Supplementary Figure S7 a FACS isolation of Aldh-positive cells with Aldefluor reagent. FACS gating was established by negative control. The number of Aldh-positive cells were decreased in Hes1 cKO (Ptf1acre/+;Hes1f/f) mice compared with that in control mice at P7 (b). The number of organoid formation from pancreatic cells (c, d). *P < 0.05. Supplementary Figure S8 a H&E staining and immunohistochemical staining for Hes5 and Hey1 of control mice, Ptf1acre/+;Hes1f/f mice (cKO), Ptf1acre/+;Rosa26NICD mice, and Ptf1acre/+;Hes1f/f;Rosa26NICD mice at P0. Marked increase of Hes5 expression was observed in Ptf1acre/+;Hes1f/f;Rosa26NICD mice. Scale bars: 50 μm. Supplementary Table 1. Primary antibodies used in this study. (PDF 4987 KB)

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Kuriyama, K., Kodama, Y., Shiokawa, M. et al. Essential role of Notch/Hes1 signaling in postnatal pancreatic exocrine development. J Gastroenterol 56, 673–687 (2021). https://doi.org/10.1007/s00535-021-01779-y

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  • DOI: https://doi.org/10.1007/s00535-021-01779-y

Keywords

  • Hes1
  • Notch signaling
  • Pancreas
  • Postnatal development
  • Centroacinar cell