Abstract
Pancreatic β cell clusters produce insulin and play a central role in glucose homeostasis. The regenerative capacity of mammalian β cells is limited and the loss of β cells causes diabetes. In contrast, zebrafish β cell clusters have a high regenerative capacity, making them an attractive model to study β cell cluster regeneration. How zebrafish β cell clusters regenerate, when the regeneration process is complete, and the identification of the cellular source of regeneration are fundamental questions that require investigation. Here, using larval and adult zebrafish, we demonstrate that pancreatic β cell clusters undergo a two-step regeneration process, regenerating functionality and then β cell numbers. Additionally, we found that all regenerating pancreatic β cells arose from Neurod1-expressing cells and that cells from different lineages contribute to both functional and β cell number recovery throughout their life. Furthermore, we found that during development and neogenesis, as well as regeneration, all β cells undergo Neurod1expression in zebrafish. Together, these results shed light on the fundamental cellular mechanisms underlying β cell cluster development, neogenesis, and regeneration.
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All data generated or analyzed during this study are included in this published article.
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Acknowledgements
The authors thank Dr. Didier Stainier (Max Planck Institute for Heart and Lung Research) for the gcga:GFP, ins:Switch, ins:NTR, ins:H2BGFP, and neurod1:EGFP lines, and Dr. David Raible (University of Washington) for plasmids of the neurod1 promoter. RIKEN WT was provided by National Bio Resource Project Zebrafish.
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This work was supported by JSPS KAKENHI Grant Number JP20K06660, the Naito Foundation, and funds from the Ritsumeikan University to HM.
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HM conceived, designed, and performed the experiments and analyzed the data. HM and YK contributed reagents, materials, analysis, and wrote the manuscript.
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Supplement Fig. 1
ins:Switch (ins:loxp-mCherry-loxp-H2BGFP) systems for expression analysis and lineage tracing. a. Schematic representation of construct of ins:Switch (ins:loxp-mCherry-loxp-H2BGFP). b. Changes in phenotypes of ins:Switch transgenic line with or without Cre or CreERT2. Only mCherryis expressed inthepancreatic β cells of the ins:Switch transgenic line without Cre expression. However, H2BGFP begins to be expressed in β cells after Cre recombination. We always confirmed that there were no leaks of H2BGFP expression without Cre expression using siblings of neurod1:Cre transgenic lines. In the neurod1:CreERT2 transgenic lines, we always confirmed that there were no leaks of H2BGFP expression without 4OHT using siblings of neurod1:CreERT2 transgenic lines used for 4OHT treatment. Scale bars, 40 μm (TIF 16668 KB)
Supplement Fig. 2
Serial z-stack images in gcga:GFP/ins:Switch/ins:NTR. Here, we showed serial z-stack images, which have 6 μm intervals, in a gcga:GFP/ins:Switch/ins:NTR transgenic line at 7 dpa (3 dpa). We found that regenerating β cells (red cells) were always located adjacent to α cells (green cells). Scale bars, 40 μm (TIF 16671 KB)
Supplement Fig. 3
Neurod1 expression pattern in the principal islet in larvae during development and β cell regeneration, using neurod1:GFP/ins:Switch/ins:NTRtreated with or without Mtz. We can see that many neurod1-expressing cells (green cells) existed within the islets during the pancreatic developmental and β cell regenerative processes. Scale bars, 40 μm (TIF 16667 KB)
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Matsuda, H., Kubota, Y. Zebrafish pancreatic β cell clusters undergo stepwise regeneration using Neurod1-expressing cells from different cell lineages. Cell Tissue Res 394, 131–144 (2023). https://doi.org/10.1007/s00441-023-03805-2
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DOI: https://doi.org/10.1007/s00441-023-03805-2