Negative cell cycle regulation by Calcineurin is necessary for proper beta cell regeneration in zebrafish

Reviewer #1 (Public Review):

Induction of beta cell regeneration is a promising approach for the treatment of diabetes. In this study, Massoz et.al., identified calcineurin (CaN) as a new potential modulator of beta cell regeneration by using zebrafish as model. They also showed that calcineurin (CaN) works together with Notch signaling calcineurin (CaN) to promote the beta cell regeneration. Overall, the paper is well organized, and technically sound. However, some evidence seems weak to get the conclusion.

Reviewer #2 (Public Review):

This work started with transcriptomic profiling of ductal cells to identify the upregulation of calcineurin in the zebrafish after beta-cell ablation. By suppressing calcineurin with its chemical inhibitor cyclosporin A and expressing a constitutively active form of calcineurin ubiquitously or specifically in ductal cells, the authors found that inhibited calcineurin activity promoted beta-cell regeneration transiently while ectopic calcineurin activity hindered beta-cell regeneration in the pancreatic tail. They also showed similar effects in the basal state but only when it was within a particular permissive window of Notch activity. To further investigate the roles of calcineurin in the ductal cells, the authors demonstrated that calcineurin inhibition additionally induced the proliferation of the ductal cells in the regenerative context or under a limited level of Notch activity. Interestingly, the enhanced proliferation was followed by a depletion of ductal cells, suggesting that calcineurin inhibition would exhaust the ductal cells. Based on the data, the authors proposed a very attractive and intriguing model of the role of calcineurin in maintaining the balance of the progenitor proliferation and the endocrine differentiation. However, the conclusions of this paper are only partially supported by the data as some evidence from the data remains suggestive.

1. In the transcriptomic profiling, genes differentially regulated in the ablated adults could be solely due to the chemical effects of metronidazole instead of the beta-cell ablation. A control group without ins:NTR-mCherry but treated with metronidazole is necessary to exclude the side effects of metronidazole.

2. Although it has been shown that the pancreatic duct is a major source of the secondary islets in the pancreatic tail in previous studies, there is no direct evidence showing the cyclosporin A-induced cells share the source in this manuscript. Without any proper lineage tracing work, the origin of those cyclosporin A-induced cells cannot be concluded.

3. It is interesting to see an increase of beta cells in the primary islet after cyclosporin A treatment (Supplemental Fig 2B). However, it remains unclear if their formation shares the same mechanism with the newly formed beta cells in the pancreatic tail.

4. The conclusion of the effect of cyclosporin A on the endocrine progenitors (Line 175) is not convincing because the data cannot distinguish the endocrine progenitors from the insulin-expressing cells. Indeed, Figure 2E shows that neurod1+ cells are fewer than ins+ cells (Figure 2D) in the pancreatic tail at 10 dpt, suggesting that all or at least the majority of neurod1+ cells are already ins+.

5. Figure 5D shows a significant loss of nkx6.1+ cells in the combined treatment group but there is no direct evidence showing this was a result of differentiation as the authors suggested. This cell loss also outnumbered the increase in ins+ cells (Figure 4D). The cell fates of these lost cells are still undetermined, and the authors did not demonstrate if apoptosis could be a reason of the cell loss.