Zebrafish pancreatic β cell clusters undergo stepwise regeneration using Neurod1-expressing cells from different cell lineages

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.


Introduction
The pancreas is a vertebrate-specific organ with two types of functions (Cleaver and Melton 2007;Matsuda 2018).One function involves the exocrine system, which is composed of acinar cells and ductal cells and which store and deliver digestive fluids, respectively.Another is the endocrine system, which involves the islets of Langerhans (pancreatic islet).The pancreatic islets are composed of five types of endocrine cells: α cells, β cells, δ cells, ε cells, and PP cells, each of which secretes a different hormone: glucagon, insulin, somatostatin, ghrelin, and pancreatic polypeptide, respectively (Cleaver and Melton 2007;Matsuda 2018).Among these cells, β cells have been the most actively studied in various fields of research such as physiology, developmental biology, and medicine.Because pancreatic β cells secrete insulin, which is the only hormone capable of lowering blood glucose levels, they are essential for glucose homeostasis (Cleaver and Melton 2007;Matsuda 2018).
To function normally, vertebrate β cells form a cluster in the pancreatic islet.However, mammalian β cell clusters are poorly regenerative and loss of β cells leads to a reduction of insulin secretion, followed by the development of diabetes.In contrast to mammals, the teleost zebrafish can regenerate β cell clusters throughout their life (Curado et al. 2007;Pisharath et al. 2007;Moss et al. 2009;Matsuda 2018).Therefore, understanding how and why this fish can regenerate its β cell clusters could provide unique information for mammalian regeneration of these cells.
It has been reported in larval zebrafish that new β cells appear 2 days post-β cell ablation (dpa) (Curado et al. 2007;Pisharath et al. 2007).It is not clear how the β cell numbers recover and when the cluster regeneration is complete.It has also been suggested that α cells contribute to this regeneration through transdifferentiation (Ye et al. 2015); however, at most, their contribution has been reported to be 13% (Ye et al. 2015).Furthermore, there are also reports that some cell types in pancreas, which are centroacinar cells (a ductal cell type) and ductal progenitor cells, which contribute to islet neogenesis, and other endocrine progenitor cells, and δ1 cells, β/δ1hybrid cells, which are other pancreatic endocrine cells, also contribute to β cell regeneration (Delaspre et al. 2015;Ghaye et al. 2015;Carril Pardo et al. 2022;Singh et al. 2022;Wang et al. 2020), although it is unclear how much these cell types contribute to β cell cluster regeneration.Thus, the process of β cell cluster regeneration in zebrafish currently has various fundamental questions which require investigation.
In this study, we first decided to investigate the regeneration phenomenon in detail using larval zebrafish, focusing on determining how β cell clusters regenerate, when the process of β cell cluster regeneration is complete, and from what kind of cells most of regeneration for β cells arise.Several transgenic lines that are useful for analyzing cell differentiation and cell lineages were used.Our results showed that the number of β cells increased until 3 dpa, temporarily stopped increasing, then started increasing again after recovering β cell cluster functionality, and finally recovered to the normal number by 13 dpa.On the other hand, whole glucose levels were recovered by 5 dpa.This suggests that zebrafish β cell clusters regenerate functionality and morphology in a stepwise manner.In addition, we found that all regenerating β cells arise from Neurod1-expressing cells.In addition, we showed that Neurod1-expressing cells are also the primary source of β cells during development and neogenesis.This strongly suggests that all zebrafish pancreatic β cells undergo Neurod1 expression.Furthermore, our results suggest that β cells in the phase of "functional recovery" arose from Neurod1-expressing cells, which already existed in the islets, and that the Neurod1-expressing cells in the "cell number recovery" phase were newly generated after functional regeneration.This contribution of Neurod1-expressing cells is also conserved in the β cell regenerative process of adult zebrafish, suggesting that Neurod1-expressing cells of different cell lineages are used throughout their life to regenerate function and cell number in a stepwise manner.These results shed light on the fundamental cellular mechanisms underlying β cell cluster development, neogenesis, and regeneration.

Changes in β cell number and whole glucose levels during β cell cluster regeneration
To study β cell cluster regeneration, we required a system that could ablate β cells easily and consistently.In this study, we used the nitroreductase/metronidazole (NTR/ Mtz) method for β cell ablation; this method was established to ablate specific cells conditionally (Curado et al. 2007;Pisharath et al. 2007).Mtz, which is a prodrug, is reduced by nitroreductase (NTR), then changes from a non-toxic to a cytotoxic active form.Therefore, by expressing NTR in target cells, we can ablate target cells in a Mtz-dependent manner.In all experiments for β cell cluster regeneration, we used the ins:NTR transgenic line (a transgenic line with NTR under a promoter from a gene specific for β cells, ins, which encodes the Insulin protein) to specifically ablate β cells (Anderson et al. 2012).
Using this system, we first observed changes in the morphology of pancreatic islets and the cell number of β cell clusters after β cell ablation, to understand how and to what extent the regeneration of β cell clusters proceeds.For this objective, we generated the triple transgenic line, ins:Switch/ gcga:GFP/ins:NTR, by crossing them with the following transgenic lines: ins:Switch (Supplement Fig. 1a; Hesselson et al. 2011), which expresses mCherry under normal conditions (Supplement Fig. 1b); gcga:GFP (Zecchin et al. 2007), which expresses GFP specifically in α cells; and ins:NTR.Then we ablated β cells from these ins:Switch/gcga:GFP/ ins:NTR lines via treatment with 24 h of Mtz between 3 and 4 days post-fertilization (dpf), followed by observation of the morphological changes of β cell clusters until 15 dpa (19 dpf) (Fig. 1a, a').We found that the first β cells appeared by 2 dpa and that almost the same size and morphology as the β cell clusters of controls were regenerated by 15 dpa (Fig. 1a').Next, we monitored changes in cell number in the β cell clusters using ins:H2BGFP/ins:NTR transgenic lines (Curado et al. 2007;Anderson et al. 2012; note that ins:H2BGFP is a transgenic line that expresses nuclear GFP in β cells and allows for easy counting of the number of β cells).Zebrafish have two types of islets, a principal islet, which is a single, huge islet, and secondary islets, which are multiple smaller islets (Fig. 1b, b', c, c').The principal islet develops during embryogenesis, followed by multiple secondary islets during postembryonic development (Parsons  The phenotype of the principal islet in animals treated with or without Mtz at 17 dpf (13 dpa).c, c' The phenotype of secondary islets (SI) in animals treated with or without Mtz at 17 dpf (13 dpa).d Changes in the number of β cells in the principal islet of animals treated with or without Mtz (mean ± STD; n = 8-11).e Changes in the number of β cells in the secondary islets of animals treated with or without Mtz (mean ± STD; n = 8-11).f Changes in the total number of β cells in the pancreas of animals treated with or without Mtz (mean ± STD; n = 8-11).Note that the difference in the number of β cells between development and β cell regeneration were no longer significant at 17 dpf (13 dpa).g Change of glucose levels in animals treated with or without Mtz (mean ± STD; n = 3).Note that the difference in whole glucose levels between development and β cell regeneration were no longer significant at 9 dpf (5 dpa).*P < 0.05 compared with control development samples of the same age by Student's t-test.Scale bars, 40 μm.dpf, days post-fertilization; dpa, days post-β ablation; PI, principal islet et al. 2009;Matsuda et al. 2013).When we first counted the β cell number in the principal islet, we found the first β cells on 2 dpa (6 dpf).Although the β cell number increased until 3 dpa (7 dpf), the increase temporarily stopped from 3 to 7 dpa (11 dpf).After 7 dpa, β cells started increasing again.Finally, the number recovered to the same level as that of the developing principal islet by 13 dpa (17 dpf) (Fig. 1d).When we next counted the β cell number in the secondary islet, we found no clear difference in the β cell number between the developing and regenerating pancreas during our period of observation (Fig. 1e).On the other hand, we found that the total number of β cells in the pancreas (sum of the number of β cells in the principal and secondary islets) showed a pattern similar to that in the principal islet (Fig. 1d, f).Together, our results indicate that zebrafish β cell clusters can recover cell numbers to a normal level by 13 dpa.In addition, under our experimental conditions, we did not find that β cell ablation affected secondary islet development.Therefore, in the remainder of our experiments, we analyzed phenotypes in only the principal islet, but not secondary islets, after β cell ablation.
It has previously been reported that whole glucose levels recovered within several days after β cell regeneration (Ye et al. 2015).To confirm these results and to estimate when β cell cluster function was recovered, we next monitored whole glucose levels in ins:H2BGFP/ins:NTR lines treated with or without Mtz, from 3 to 17 dpf (Fig. 1g).We found that whole glucose levels became high immediately after β cell ablation.However, whole glucose levels peaked at 2 dpa (6 dpf), followed by recovery by 5 dpa (9 dpa) (Fig. 1g).These results imply that the functionality of the β cell clusters was recovered by 5 dpa.Interestingly, there is a gap in the recovery timing between glucose levels and cell number after β cell ablation (Fig. 1d, f, g).This may suggest that β cell clusters undergo a two-step regeneration process, first regenerating functionality in a morphologically incomplete state by 5 dpa and then regenerating morphology by 13 dpa.

Regenerating β cells arise from cells in contact with α cells
It has previously been reported that some β cells arise from α cells (gcga:GFP positive glucagon a (gcga)expressing cells) after β cell ablation, albeit at a low frequency (Ye et al. 2015).To confirm the relationship between regenerating β cells and α cells, we decided to investigate changes in α cell numbers after β cell ablation.As a result, the number of α cells itself was not significantly different from that of the control, although the size of the islet became somewhat smaller after β cell ablation (Fig. 2a, a', b).Using the gcga:GFP/ins:Switch/ ins:NTR line, we next examined the correlation between regenerating β cells and α cells after β cell ablation.

The cell lineage of N1 cells that contribute to early and late regeneration is different
There have already been abundant N1 cells in the principal islet just prior to and after β cell ablation (Supplement Fig. 2).To know if there has already been sufficient number of N1 cells to generate all β cells in the islets just after β cell ablation, we next generated a neurod1:CreERT2 transgenic line, and established neurod1:CreERT2/ins:Switch/i ns:NTR lines for lineage tracing experiments (Supplement Fig. 1b).For these experiments, neurod1:CreERT2/ ins:Switch/ins:NTR lines were treated with or without Mtz from 3 to 4 dpf and with 4-hydroxy Tamoxifen (4OHT) from 4 to 5 dpf, and then their phenotypes were analyzed (Fig. 4a).We first observed phenotypes of developing β cells in neurod1:CreERT2/ins:Switch/ins:NTR lines without Mtz.We found that most of the β cells in the principal islet expressed H2BGFP at both 9 and 17 dpf, although faint mCherry signals remained in some H2BGFP-positive cells (Fig. 4b−b'', c-c'').In contrast, in secondary islets, β cells expressed mCherry, but not H2BGFP (Fig. 4d-d'').These results indicated that N1 cells, which are necessary for β cell development in the principal islet, are already present in the pancreas by 5 dpf, but that N1 cells, which are the source of β cell development in secondary islets, develop after 5 dpf.Next, we analyzed phenotypes of these transgenic lines after β cell ablation.After β cell ablation, all regenerating β cells expressed H2BGFP at 5 dpa in the principal islet, although mCherry signals remained in some cells (Fig. 4e−e', h).However, mCherry-only expressing cells appeared by 7 dpa in the pancreas of some zebrafish.The number of mCherryonly expressing cells increased after 9 dpa (Fig. 4f-h).On the other hand, the number of H2BGFP-expressing cells did not change between 5 and 13 dpa (Fig. 4e-h).In addition, the total number of H2BGFP-and mCherry-only expressing cells (Fig. 4h) as shown to have a pattern similar to the results of Fig. 1d.These results suggest that all regenerating β cells are generated from N1 cells, which are already present in the principal islet by 5 dpa (9 dpf), and that N1 cells, which are the source of β cells after 5 dpa, are newly generated after 5 dpa.

Most new Neurod1-expressing cells generate after 7 dpa
To investigate when new N1 cells, which become the source of β cells after 5 dpa, are generated we treated neu rod1:CreERT2/ins:Switch/ins:NTR lines again with 4OHT at a time point between 4 and 11 dpa after treatment with Mtz and 4OHT, as previously investigated, and then analyzed mCherry and H2BGFP expression at 17 dpf (Fig. 5a).In these transgenic lines treated with a second treatment of 4OHT at 4 to 5 or 6 to 7 dpa, the number of H2BGFPexpressing cells and the area of mCherry-expressing cells did not significantly change compared to controls which did not receive a second 4OHT treatment (Fig. 5b, c, f, g).However, in transgenic lines treated with a second 4OHT treatment at 8 to 9 or 10 to 11 dpa, the number of H2BGFP-expressing cells increased and the area of mCherry-expressing cells was reduced significantly (Fig. 5d-g).These results suggest that most of the new N1 cells for regenerating the morphology of β cell clusters are produced after 7 dpa.

Adult zebrafishes undergo two-step regeneration using Neurod1-expressing cells from different cell lineages
Neurod1:eGFP-expressing cells are present in adult zebrafish islets both before and after β cell ablation (Fig. 6a, a').We wondered whether these N1 cells contribute to β cell regeneration in adults.To investigate it, we observed β cell regeneration until 17 dpa using the neurod1:Cre/ins:Switch/ ins:NTR triple transgenic line with or without β cell ablation (Fig. 6b−b""").The number of H2BGFP-expressing β cells recovered to the number prior to β cell ablation by 17 dpa in this transgenic line (Fig. 6c).On the other hand, blood glucose levels recovered to normal by 5 dpa (Fig. 6d).Thus, there is a gap of recovery timing between blood glucose levels and β cell numbers during β cell regeneration in adults as well.This suggests that in adults, β cell clusters regenerate through a two-step process involving functional recovery followed by β cell number recovery.We next investigated the contribution of N1 cells present in the pancreas prior to β cell ablation, using the neurod1:CreERT2/ins:Switch/ ins:NTR triple transgenic line.First, this transgenic line was treated with 4OHT for 24 h.After 1 day of rest, these fishes were treated with Mtz for 24 h and then observed to determine whether the regenerating β cells expressed H2BGFP or mCherry (Fig. 6e-l).At 5 dpa, we found only H2BGFPexpressing β cells or mCherry-and H2BGFP-co-expressing cells but not cells that expressed only mCherry (Fig. 6h, k,  l).However, at 17 dpa, a very large number of mCherryexpressing β cells appeared (Fig. 6i).In contrast, H2BGFPexpressing cells showed no significant difference compared to 5 dpa (Fig. 6 h, i, k).When this triple transgenic line was treated with 4OHT between 11 and 12 dpa again, mCherryexpressing β cells decreased and H2BGFP-expressing β cells increased at the same levels as those prior to β cell ablation (-1 dpa) (Fig. 6f, j-l).These findings suggest that, in adults as well as larvae, N1 cells in pancreatic islets first recover the function of the β cell cluster by regenerating a small number of β cells, followed by recovering the number of β cells using N1 cells newly generated after β cell ablation.

Discussion
In this study, we analyzed the β cell cluster regeneration process using several larval zebrafish transgenic lines to understand which cells were utilized during β cell cluster regeneration and how zebrafish β cell clusters are regenerated.Our results showed that newly formed β cells appeared by 2 dpa and that the number of β cells increased until 3 dpa at which time they stopped increasing.On the other hand, whole glucose levels increased  c-c'', d-d'', and g-g'').Note that all developing β cells in the principal islet (PI), but not secondary islets (SI), arose from Neurod1-expressing cells from 4 to 5 dpf.h Quantifica-tion of changes in the number of regenerating β cells with H2BGFP and/or mCherry expression (mean ± STD; n = 5-10).Note that the number of H2BGFP-expressing cells did not change, but the number of mCherry-expressing cells without H2BGFP expression was significantly increased during β cell regeneration after 11 dpa.**P < 0.01 compared with samples at 5 dpa by Tukey's honestly significant difference test after ANOVA.Scale bars, 40 μm.PI, principal islet cells; SI, secondary islet cells 1 3 sion (mean ± STD; n = 9).g Quantification of the area of regenerating β cells with mCherry expression (mean ± STD; n = 9).Note that the number of H2BGFP-expressing cells was elevated after 7 dpf and that the area of mCherry-expressing cells was reduced after 7 dpf.*P < 0.05, **P < 0.01 compared with samples at 5 dpa by Tukey's honestly significant difference test after ANOVA.Scale bars, 40 μm immediately after β cell ablation, peaked at 2 dpa, then decreased and recovered to normal levels by 5 dpa.Thus, the timing of the decrease of whole glucose levels coincided with that of the increase in β cell number.This suggests that the reduction of whole glucose levels after 2 dpa was due to the regeneration of functional β cells in the islets.Interestingly, after whole glucose levels became normal, β cell numbers increased again and eventually recovered to normal levels by 13 dpf.These findings suggest that zebrafish β cell clusters undergo a two-step regeneration process: first, their β cell clusters regenerate functionally with the conditions of a small number of β cells; second, after blood glucose levels become normal, cell numbers in β cell clusters are recovered by creating new N1 cells to compensate the missing cells (Fig. 7).Furthermore, this two-step regeneration process was conserved in the adult zebrafish pancreas (Fig. 7).Taken together, these findings suggest that throughout their lifetime, zebrafish pancreatic islets can regenerate β cell clusters through two phases of "functional recovery" and "cell number recovery" using N1 cells of different cell lineages (Fig. 7).
In this study, we found that all zebrafish β cells regenerate via Neurod1-expressing cells (N1 cells).The zebrafish islets already had sufficient N1 cells, which may be used during development, to regenerate functional β cell clusters immediately after β cell ablation (Fig. 7).Furthermore, cell number recovery was also performed by recruiting the missing N1 cells after functional recovery (Fig. 7).Thus, zebrafish regenerate β cell clusters in a very rational way, first using a minimal number of β cells to restore the function of the cluster, and then supplementing the missing cells to complete the regeneration.Likely the gap between the timing of the consumption of N1 cells for "functional recovery" and neogenesis of N1 cells for "cell number recovery" is the reason why the increase of the β cell number stops transiently during larval β cell regeneration.
Most previous zebrafish studies have focused on the phase of functional recovery, which we identified in this study.They reported that α cells, δ1 cells, β/δ1 hybrid cells, and some endocrine progenitor cells contribute to β cell regeneration ( Ye et al. 2015;Lu et al. 2016;Wang et al. 2020;Carril Pardo et al. 2022;Singh et al. 2022).Our current results suggest that these cells already express Neurod1 or express Neurod1 within 24 h after β cell ablation and that Neurod1 expression is a common characteristic of cells which give rise to the regeneration of β cells.On the other hand, a large number of N1 cells are present in the islets even before β cell ablation.Nevertheless, only a small number of β cells could be regenerated during functional recovery.These results indicate that N1 cells are a heterogeneous population, some of which can give rise to regenerating β cells but not others.Interestingly, in our results, all regenerating β cells, including cells co-expressed with glucagon, were adjacent to α cells (glucagon-expressing cells) at 3 dpa.Given that all β cells arise from N1 cells, this suggests that the regenerating β cells during functional recovery arise from N1 cells adjacent to α cells (Fig. 7).In addition, α cells are likely also a heterogeneous population, some of which are special α cells (glucagon-expressing cells) that are able to participate in β cell cluster regeneration and regulate differentiation of special N1 cells to β cells.
We found that N1 cells for cell number recovery are newly generated after 7 dpa.On the other hand, the question remained regarding the cells that give rise to N1 cells for cell number recovery.Some groups have reported that centroacinar cells and ductal progenitor cells contribute to pancreatic endocrine neogenesis and β cell regeneration ( Parsons et al. 2009;Wang et al. 2011;Matsuda et al. 2013;Delaspre et al. 2015;Ghaye et al. 2015).Interestingly, N1 cells also arise from centroacinar cells and ductal progenitor cells during endocrine neogenesis ( Kimmel et al. 2011;Ninov et al. 2012;Flasse et al. 2013;Matsuda et al. 2013).These may suggest that for the cell number recovery phase, N1 cells also arise from centroacinar and ductal progenitor cells.On the other hand, it is unclear whether all N1 cells arise from centroacinar cells or ductal progenitor cells during endocrine neogenesis.Hence, the possibility that some N1 cells may arise from other unknown cell populations cannot currently be ruled out.To accurately understand the process of cell number recovery, we need to clarify which cells give rise to N1 cells.If we identify these cell types, it will be a great advantage to understanding the fundamental questions related to the phase of cell number recovery, how Fig. 6 Neurod1-expressing cells contribute to β cell regeneration in adult zebrafish.a, a' neurod1:EGFP-expressing cells in the principal islet of adult zebrafish before and after β cell ablation.b-b""" Morphological changes of the principal islets (neurod1:CreERT2/ ins:loxp-mCherry-loxp-H2BGFP/ins:NTR) during β cell regeneration in adults.c Changes in the number of β cells (H2BGFPexpressing cells) in 45000μm 2 of a region, where a principal islet was located, before and after Mtz treatment (mean ± STD; n = 5-8).d Changes in blood glucose levels in animals treated with or without Mtz (mean ± STD; n = 5).Note that the recovery timing was different between blood glucose levels and β cell number after β cell ablation.e-l neurod1:CreERT2/ins:loxp-mCherry-loxp-H2BGFP/ins:NTR lines were treated with or without Mtz after 24 h 4OHT treatment (from -3 to -2 dpa) and then sacrificed for analysis at -1 (f), 0 (g), 5 (h), and 17 dpf with (j) or without (i) the second 4OHT treatment between 11 and 12 dpa.k Quantification of changes in the number of β cells with H2BGFP expression in 45,000 um 2 of a region, where a principal islet was located (mean ± STD; n = 6-9).l Quantification of the area of regenerating β cells with mCherry expression in 45,000 um 2 of a region, where a principal islet was located (mean ± STD; n = 6-9).Note that the number of H2BGFP-expressing cells did not change between 5 and 17 dpa, but the mCherry-expressing cells was significantly increased during β cell regeneration at 17 dpa.*P < 0.05, **P < 0.01 compared with samples at -1 dpa by Tukey's honestly significant difference test after ANOVA.Scale bars, 40 μm ◂ pancreatic islets and the source cells of N1 cells sense the lack of N1 cells, how new N1 cells are generated, and how N1 cells are recruited into β cell clusters.
Interestingly, Neurod1 is expressed in the pancreatic islets of adult mammals (Gu et al. 2010).However, it is unclear whether these N1 cells have the potential to contribute to β cell cluster regeneration in a similar way to zebrafish N1 cells.Further characterization of zebrafish N1 cells will reveal whether mammals also have N1 cells which can contribute to β cell cluster regeneration.We will then be able to propose critical methods to make completely functional β cell clusters in the mammalian pancreas for developing new diabetic therapies.Thus, through the results of this study, we have succeeded in making a valuable discovery that is a significant first step toward the regeneration of mammalian β cell clusters.

Fluorescence imaging
Fluorescence images were acquired with a FV10i-DOC Laser Scanning Microscope (OLYMPUS, Shinjuku, Japan).Cell numbers were counted manually for experiments using ins:H2BGFP, gcga:GFP with DAPI, or ins:Switch with DAPI.The area of mCherry in ins:Switch was calculated using FLOVIEW software.

Fig. 1
Fig.1Zebrafish can regenerate pancreatic β cell functionally and morphologically.a, a' Morphological changes of the pancreatic islet (gcga:GFP, α cell; ins:Switch, β cell) during development and β cell regeneration.b, b' The phenotype of the principal islet in animals treated with or without Mtz at 17 dpf (13 dpa).c, c' The phenotype of secondary islets (SI) in animals treated with or without Mtz at 17 dpf (13 dpa).d Changes in the number of β cells in the principal islet of animals treated with or without Mtz (mean ± STD; n = 8-11).e Changes in the number of β cells in the secondary islets of animals treated with or without Mtz (mean ± STD; n = 8-11).f Changes in the total number of β cells in the pancreas of animals treated with or without Mtz (mean ± STD; n = 8-11).Note that the difference in the number of β cells between development and β cell regeneration were no longer significant at 17 dpf (13 dpa).g Change of glucose levels in animals treated with or without Mtz (mean ± STD; n = 3).Note that the difference in whole glucose levels between development and β cell regeneration were no longer significant at 9 dpf (5 dpa).*P < 0.05 compared with control development samples of the same age by Student's t-test.Scale bars, 40 μm.dpf, days post-fertilization; dpa, days post-β ablation; PI, principal islet

Fig. 2 Fig. 3
Fig. 2 Regenerating pancreatic β cells in direct contact with α cells.a, a' Morphology of pancreatic α cells (gcga:GFPexpressing cells) in animals treated with or without Mtz at 5 dpf (1 dpa).b Changes in number of α cells in the principal islet in animals treated with or without Mtz (mean ± STD; n = 9-10).c−c''and d−d" Relationship of the localization between α cells and regenerating β cells (ins:Switchexpressing cells) at 3 dpa.e Quantification of the relationship of localization between α cells and regenerating β cells (mean ± STD; n = 9-10).Note that 15% of regenerating β cells were gcga:GFP-expressing cells at 3 dpa (c-c'' and e) and that 85% of regenerating β cells were juxtaposed to gcga:GFPexpressing cells at 3 dpa (d-d" and e).Scale bars, 40 μm

Fig. 5
Fig. 5 Most new Neurod1-expressing cells rapidly generate after 7 dpa.a−f neurod1:CreERT2/ins:loxp-mCherry-loxp-H2BGFP/ ins:NTR lines were treated with 4OHT (red arrows in a) between 4 to 5 (b), 6 to 7 (c), 8 to 9 (d), or 10 to 11 dpa (e) after treatment with Mtz from 3 to 4 dpf (green arrows in a) and 4OHT from 4 to 5 dpf, and then sacrificed for analysis at 17 dpf.f Quantification of changes in the number of regenerating β cells with H2BGFP expres-

Fig. 7
Fig. 7 A model for pancreatic β cell regeneration in zebrafish.Zebrafish islets regenerate the functionality and cell number of β cell clusters in a stepwise manner.First, Neurod1-expressing cells (N1