Insulin-producing β-cells regenerate ectopically from a mesodermal origin under the perturbation of hemato-endothelial specification

To investigate the role of the vasculature in pancreatic β-cell regeneration, we crossed a zebrafish β-cell ablation model into the avascular npas4l mutant (i.e. cloche). Surprisingly, β-cell regeneration increased markedly in npas4l mutants owing to the ectopic differentiation of β-cells in the mesenchyme, a phenotype not previously reported in any models. The ectopic β-cells expressed endocrine markers of pancreatic β-cells, and also responded to glucose with increased calcium influx. Through lineage tracing, we determined that the vast majority of these ectopic β-cells has a mesodermal origin. Notably, ectopic β-cells were found in npas4l mutants as well as following knockdown of the endothelial/myeloid determinant Etsrp. Together, these data indicate that under the perturbation of endothelial/myeloid specification, mesodermal cells possess a remarkable plasticity enabling them to form β-cells, which are normally endodermal in origin. Understanding the restriction of this differentiation plasticity will help exploit an alternative source for β-cell regeneration.


Introduction
The concept of embryonic development and cell fate determination was illustrated by the famous Waddington landscape model decades ago (Waddington, 1957). Waddington's model not only shows the importance of spatiotemporal precision in cell differentiation but also metaphorises cell fate determination as a sequential and irreversible event. In this hierarchical model, the endoderm follows the lineage paths downwards and progressively differentiates into multiple endodermal cell types, including pancreatic b-cells. Likewise, the mesoderm stays in the mesodermal lineage paths and differentiates into endothelial cells and other mesodermal cell types. However, in recent decades, multiple studies have suggested that committed cells are capable of differentiating across the germ layer boundaries by converting embryonic and/or adult mesodermal fibroblasts into ectodermal neuronal cells (Vierbuchen et al., 2010), multipotent neural stem cells (Ring et al., 2012), endodermal hepatocyte-like cells (Huang et al., 2011;Sekiya and Suzuki, 2011), or pancreatic blike cells (Zhu et al., 2016) in vitro. These studies highlight the feasibility of converting mesodermal cells into ectodermal or endodermal cells in vitro.
Despite the extensive studies on cell fate conversion across germ layers in vitro, the number of in vivo studies is limited. Ectopic expression of Xsox17b in Xenopus embryos relocated cells normally fated to become ectoderm to appear in the gut epithelium, suggesting a possible change in cell fate in vivo (Clements and Woodland, 2000). Similarly, ectopic expression of sox32/casanova in presumptive endothelial cells switched their cell fate to endoderm in zebrafish embryos (Kikuchi et al., 2001). Furthermore, aggregated morulae and chimeric mouse embryos of b-catenin mutants provided evidence of precardiac mesoderm formation in endodermal tissues in vivo (Lickert et al., 2002). These studies suggest that the classical in vivo germ layer boundaries may not be as clear-cut as previously thought.
In this study, we aimed to elucidate the importance of the vasculature in pancreatic b-cell regeneration, which plays a crucial role in potential therapeutic strategies against diabetes. We employed cloche zebrafish mutants as an avascular model. The mutation of npas4l, a master regulator of endothelial and hematopoietic cell fates, is responsible for the severe loss of most endothelial and blood cells in cloche mutants (Parker and Stainier, 1999;Reischauer et al., 2016;Stainier et al., 1995). Unexpectedly, the npas4l mutation induced ectopic b-cell formation in the mesenchymal region outside of the pancreas after b-cell ablation. Lineage-tracing mesodermal cells expressing draculin (drl), npas4l, and etsrp (previously named etv2) validated the mesodermal lineage of the ectopic b-cells, which are normally endodermal in origin. These findings offer novel insights into cell fate determination and an alternative source of b-cells.

Ectopic b-cell formation in npas4l mutants
To determine the importance of vasculogenesis and vascularisation for b-cell regeneration, we examined b-cell formation in zebrafish carrying the cloche mutation (npas4l s5 mutation) after b-cell ablation, i.e., in the Tg(ins:Flag-NTR);Tg(ins:H2BGFP;ins:DsRed) model. Nitroreductase (NTR), expressed under the insulin promoter, converts the prodrug metronidazole (MTZ) to a cytotoxin to specifically ablate insulin-producing b-cells (Curado et al., 2007). The homozygous mutation of npas4l significantly increased the number of ins:H2BGFP-positive cells during the b-cell regeneration period ( Figure 1A-C). In addition, we observed a distinctive ectopic b-cell population in the mesenchymal region outside of the pancreas in the npas4l À/À group, an ectopic location that was very rarely observed in the sibling controls (including both wild-type and heterozygous siblings). This ectopic population of b-cells contributed to the major increase in the number of ins:H2BGFP-positive cells during b-cell regeneration ( Figure 1C). Moreover, the comparable and sparse numbers of ins: DsRed-positive cells in the controls and mutants indicate that the npas4l mutation did not enhance the survival of b-cells during the ablation ( Figure 1A,B) because the extended maturation time of DsRed (Baird et al., 2000) restricted the detection of DsRed to any surviving b-cells.
To visualise the location of the ectopic b-cells better, we labelled the pancreas with ptf1a:EGFP expression and observed not only a drastic reduction in the pancreas size ( Figure 1D,E, Figure 1figure supplement 1) but also the regeneration of b-cells clearly outside of the ptf1a-expressing exocrine pancreas in npas4l mutants ( Figure 1E). By labelling the mesenchyme with hand2:EGFP expression ( Figure 1F-K), we further revealed that the majority of the ectopic b-cells formed in npas4l mutants intermingled with hand2:EGFP-positive mesenchymal cells between the pronephros and the pancreas ( Figure 1J,K). In addition, we occasionally observed ectopic b-cells intermingled with hand2:EGFP-positive mesenchymal cells ventral to the pancreas ( Figure 1I,K). Although the ectopic b-cells were located among the mesenchymal cells, they did not express hand2:EGFP.
The ectopic b-cells co-express insulin and endocrine markers in npas4l mutants Next, we examined multiple pancreatic endocrine and b-cell markers, including Isl1, neurod1, pdx1, mnx1, pcsk1, and ascl1b (the functional homologue to Neurog3 in mammals), to validate the b-cell identity of the ectopic insulin-producing cells. The majority of the ectopic b-cells co-expressed insulin Representative confocal projections of the pancreas and neighbouring tissues of control siblings and npas4l À/À Tg(ins:Flag-NTR);Tg(ins:H2BGFP;ins:DsRed) zebrafish larvae at 3 dpf after b-cell ablation by MTZ at 1-2 dpf, displaying regenerated b-cells in green and older b-cells that likely survived the ablation in yellow overlap as the DsRed fluorescence driven by the insulin promoter remained in these cells, at the same time as DsRed had not had enough time to mature in the regenerated b-cells (arrowheads). The ectopic Figure 1 continued on next page as well as these markers during b-cell regeneration ( Figure 2). The high co-expression of pcsk1 ( Figure 2R,S, Table 1), which encodes an enzyme necessary for insulin biosynthesis, indicates that most of the b-cells in the ectopic population are likely functional. Consistent with previous findings in pancreatic b-cells, not all ectopic b-cells expressed ascl1b:EGFP ( Figure 2V,W, Table 1), which suggests that ascl1b works as a transient endocrine cell fate regulator (Flasse et al., 2013). In contrast with Isl1, mnx1, pcsk1, and ascl1b, we observed lower co-expression levels of neurod1 and pdx1 in ectopic b-cells compared with the pancreatic population in npas4l mutants (Table 1). In addition to the reduction in pancreas size (Figure 1-figure supplement 1), the pdx1-expressing pancreatic duct was also reduced in the npas4l mutant ( Figure 2-figure supplement 1), indicating that the pancreas and its duct did not expand to form the ectopic b-cells. These observations together suggest that the pancreatic and ectopic b-cells are similar, yet they are two distinct b-cell populations.

Ectopic b-cells can respond to glucose by displaying calcium oscillations
We further assessed the functionality and maturity of the ectopic b-cells by examining the calcium ion oscillation in vivo to determine their ability in responding to glucose to secrete insulin in the Tg (ins:GCaMP6s);Tg(ins:mCherry);Tg(ins:Flag-NTR) npas4l zebrafish mutants. After b-cell ablation by MTZ and one day of b-cell regeneration, we found comparable proportions of pancreatic (66%) and ectopic (67%) b-cells displaying mild and basal levels of calcium activity at the baseline ( Figure 3 and Video 1). Upon glucose treatment, 50% of pancreatic b-cells and 33% of ectopic b-cells elicited calcium oscillations, suggesting that a portion of the pancreatic and ectopic b-cells have the ability to respond to glucose to secrete insulin in the npas4l mutants during b-cell regeneration.
The ectopic b-cells are of mesodermal origin in npas4l mutants and etsrp morphants We have previously shown that npas4l expression is first initiated in the lateral plate mesoderm at the tailbud stage by in situ hybridisation (Reischauer et al., 2016). In this study, we examined npas4l expression at 20 hr postfertilisation (hpf) and found that npas4l was severely reduced in the lateral plate mesoderm in the npas4l mutants (  (C) Quantification of the pancreatic or ectopic b-cells per larva at 3 dpf. ****p<0.0001 (Š idá k's multiple comparisons test); n = 24 (control) and 19 (npas4l À/À ). Quantification data are represented as the mean ± SEM. (D, E) Representative image projections of the pancreas and neighbouring tissues in control siblings and npas4l -/-Tg(ins:Flag-NTR);Tg(ptf1a: EGFP) larvae at 3 dpf after b-cell ablation by MTZ at 1-2 dpf, displaying b-cells in red with immunostaining for insulin and ptf1a:EGFP + exocrine pancreas in green. Pancreata are outlined by solid white lines. Dashed line outlines ectopic b-cells in the mesenchyme (E). (F-K) Representative images and projections of the pancreas and neighbouring mesenchyme of control siblings and npas4l -/-Tg(ins:Flag-NTR);Tg(hand2:EGFP) zebrafish larvae at 3 dpf after b-cell ablation by MTZ at 1-2 dpf, displaying b-cells in red with immunostaining for insulin and hand2:EGFP + mesenchyme in green. White arrowheads point to b-cells in the pancreas (F, I). Dashed rectangles indicate the ectopic b-cells intermingling with the mesenchyme between the pronephros and the pancreas, without co-expressing insulin and hand2:EGFP (J, K). Selected area in dashed ovals shows other ectopic b-cells intermingling with the mesenchyme ventral to the pancreas (I, K). Scale bars = 20 mm. Anatomical axes: D (dorsal), V (ventral), A (anterior), and P (posterior). The online version of this article includes the following figure supplement(s) for figure 1: . $ -; . $  Figure 2. The ectopic b-cells co-express insulin and endocrine markers in npas4l mutants. Representative confocal images of the tissues adjacent to the pancreas of control siblings and npas4l À/À Tg(ins:Flag-NTR) zebrafish larvae at 3 dpf after b-cell ablation by MTZ at 1-2 dpf, displaying cells expressing pancreatic endocrine cell markers Isl1 (A-B'), neurod1 (E-F'), pdx1 (I-J'), mnx1 (M-N'), pcsk1 (Q-R'), and ascl1b (U-V') in green, and ectopic b-cells in red with immunostaining for insulin (except Q-R' with the mCherry fluorescence driven by the insulin promoter). Arrowheads point to ectopic b-cells Figure 2 continued on next page affect the hematopoietic and endothelial lineages (Parker and Stainier, 1999), we hypothesised that the ectopic b-cells originated from a mesodermal lineage.
To determine whether the mesoderm was the origin of the ectopic b-cells, we genetically traced the mesodermal cells using drl:CreER T2 , a tamoxifen-inducible Cre transgene driven by a drl promoter (Mosimann et al., 2015). The spatial expression pattern of drl in the npas4l mutants resembled that in the sibling controls (Figure 4-figure supplement 2), suggesting that npas4l mutation did not induce any ectopic expression of drl to disrupt the lineage-tracing approach. In combination with a À3.5ubb:LOXP-EGFP-LOXP-mCherry (ubi:Switch) reporter (Mosimann et al., 2011), the drlexpressing mesodermal cells were labelled in red in Tg(drl:CreER T2 );Tg(ubi:Switch);Tg(ins:Flag-NTR) (drl-tracing) zebrafish larvae after 4-hydroxytamoxifen (4-OHT) induction ( Figure 4A, Figure 4-figure supplement 3). We treated the transgenic embryos with 4-OHT at 10-12 hpf. We chose to label the mesodermal cells during this period as neither endothelial/hematopoietic cells nor b-cells have developed at that stage, i.e., to exclude confounding effects of endothelial/hematopoietic cells or possible ectopic expression of the lineage tracer in the b-cells of the npas4l mutant. To ablate the bcells, we incubated the 4-OHT-treated transgenic embryos in MTZ at 1-2 days postfertilisation (dpf). We allowed the b-cells to regenerate for 30 hr before we fixed the larvae at 3 dpf for immunostaining ( Figure 4B).
Immunostaining against insulin displayed a normal set of b-cells in the pancreas of the drl-tracing larvae with or without npas4l mutation after 30 hr of regeneration ( Figure 4C,E, and F). In line with the findings shown in Figure 1, the npas4l mutation induced the formation of ectopic b-cells in the mesenchymal region ( Figure 4D,G, and H). Furthermore, 98.9% of the ectopic b-cells in the mesenchymal region were mCherry-positive ( Figure 4H'-H'''), indicating that they derived from the drlexpressing mesodermal cells.

The mesodermal cells lose npas4l expression after differentiating into ectopic b-cells
We then further examined the autonomous role of the progenitors of endothelial and hematopoietic cells in ectopic b-cell emergence in npas4l mutants by tracing the npas4l cell lineage with a zebrafish knock-in line, npas4l Pt(+36-npas4l-p2a-Gal4-VP16)bns423 . This knock-in line not only carries a +36 bp mutation in exon 2 of npas4l (npas4l bns423 mutation) but also expresses the transcription activator Gal4 under control of the endogenous npas4l promoter (npas4l:Gal4), allowing us to trace the npas4l lineage after crossing it into Tg(UAS:Cre);Tg(ubi:Switch) ( Figure 5A). We obtained a transheterozygous npas4l mutant (npas4l s5/bns423 ) by crossing this npas4l tracer carrying the npas4l bns423 mutation into the npas4l s5 mutant used in the studies above, and observed similar ectopic b-cell formation ( Figure 5B-E''') as found in the homozygous npas4l s5 mutant (Figure 1). Some of these ectopic b-cells (32.6%) could be traced back to the npas4l lineage as they were labelled by ubb:mCherry after the Cre-recombination driven by npas4l:Gal4 and UAS:Cre ( Figure 5C and E-E'''), which further supports a mesodermal origin of the ectopic b-cells.
By crossing Tg(npas4l:Gal4) into Tg(UAS:EGFP), the endogenous promoter activity of npas4l could be revealed by EGFP ( Figure 5F). The ectopic b-cells emerged in close proximity to mesodermal cells with active npas4l expression in the transheterozygous npas4l s5/bns423 mutants ( Figure 5G-G'''). However, we did not observe any ectopic b-cells with EGFP expression, suggesting that these mesodermal cells have lost the identity of the lateral plate mesoderm with npas4l expression and started to express insulin as in the endodermal pancreatic b-cells instead.

The ectopic b-cells derive from the etsrp-expressing mesodermal lineage in etsrp morphants
To confirm the origin of the ectopic b-cell using a different lineage-tracing approach, we generated Tg(etsrp:Cre) zebrafish, based on the promoter of À2.3estrp:GFP that has been demonstrated to closely recapitulate the endogenous etsrp expression in the lateral plate mesoderm and vasculature (Veldman and Lin, 2012). We then crossed Tg(etsrp:Cre) into Tg(ubi:Switch);Tg(ins: Flag-NTR), and thereby labelling descendants of the etsrp lineage in red ( Figure 6A). We validated the efficiency of the etsrp-lineage tracer and revealed that the majority of kdrl-expressing endothelial cells were being traced in the intersegmental vessels (86.6%) and other vasculature (  Together, we used three different lineage-tracing models as well as three different loss of function models, that is using the promoter of drl, npas4l, or etsrp to drive Cre in npas4l s5/s5 mutants, npas4l s5/bns423 mutants, or etsrp morphants. This suggests that the ectopic b-cell formation is not restricted to the loss of a specific gene, but rather due to the perturbation of endothelial/myeloid specification.

Discussion
In this study, we first examined the role of blood vessels in b-cell regeneration in the cloche zebrafish mutant, which carries a homozygous npas4l mutation (Reischauer et al., 2016). We then unexpectedly revealed b-cells regenerating ectopically in the mesenchymal area. The ectopic b-cells were likely functional because they expressed several endocrine and b-cell markers, including Isl1, mnx1, and pcsk1, and were capable of responding to glucose to induce calcium oscillations during b-cell regeneration, although we do not know if they possess all the features of bona fide b-cells. By combining in situ hybridisation, lineage tracing, and confocal microscopy, we successfully traced the origin of the ectopic b-cells to the mesodermal lineage. A recent study has reported the conversion of Etsrp-deficient vascular progenitors into skeletal muscle cells and highlighted the plasticity of mesodermal cell fate determination within the same germ layer (Chestnut et al., 2020). Our data demonstrated the plasticity of b-cell differentiation across the committed germ layers in vivo, i.e., switching from a mesodermal to an endodermal fate in a regenerative setting, while gastrulation and cell fate commitment in the germ layers are considered to be irreversible in development. Ectopic pancreata have been observed before, e.g., in Hes1 mutant mice (Fukuda, 2006;Sumazaki et al., 2004), although that has been shown to be through an expansion of the pancreas rather than through changes in cell fate determination across organs or germ layers (Jørgensen et al., 2018). Our discovery is, to our knowledge, the first demonstration of ectopic b-cells with a mesodermal origin in vivo.
The recent genome-wide study with zebrafish embryos has confirmed the crucial role of npas4l in the early specification of endothelium and blood as the expressions of some endothelial and hematopoietic genes like tal1, etsrp, lmo2, gfi1aa, and gata1a were downregulated in homozygous npas4l mutants (Marass et al., 2019), suggesting that some populations of mesodermal cells may not have acquired their designated cell fates and remained in a more plastic state. In line with the important role of etsrp in the endothelial cell fate determination, Chestnut et al., 2020 have reported a significant reduction in the cell clusters of endothelial cells and endothelial progenitor cells in homozygous etsrp mutants in a single-cell RNA-seq analysis. Interestingly, they have also shown a remarkable increase in the cluster of lateral plate mesoderm, which points to the possibility    However, it is difficult to deduce why those mesodermal cells with perturbed cell fates would be competent to differentiate into ectopic insulin-expressing b-cells from these transcriptomic data; because the population of these competent mesodermal cells might be very small according to the number of ectopic b-cells that we observed and their single-cell RNA-seq was not performed after b-cell ablation.
Since we could trace the origin of ectopic b-cells back to drl, npas4l, and etsrp lineages, we believe that the versatile lateral plate mesoderm close to the pancreas could contribute to the b-cell regeneration in npas4l mutants or etsrp morphants. Tracing the lineages with early mesodermal, endodermal, and endocrine markers, and sorting these lineage-traced cells could be necessary for a single-cell transcriptomic study to fully understand the cellular status of this small population of versatile cells and further elucidate the underlying molecular mechanisms. Before deciphering these mechanisms, we cannot formally rule out the possibility that the mechanisms of ectopic b-cell formation in npas4l mutants and etsrp morphants could be different. The mutated gene in the cloche mutant was named npas4l because its encoded protein shares homology with human NPAS4 (Reischauer et al., 2016). Although injecting human NPAS4 mRNA or zebrafish npas4l mRNA into zebrafish cloche mutant embryos at the one-cell stage could rescue the mutants, Npas4 knockout mice are unlikely to share the same severe vascular and hematopoietic defects as zebrafish npas4l mutants because Npas4 knockout mice survive to adulthood (Lin et al., 2008). This discrepancy suggests that other members of the mammalian NPAS protein family or other proteins may be functionally redundant with NPAS4 in vascular and hematopoietic development. Mammalian NPAS4 has been shown to have important cell-autonomous functions in b-cells (Sabatini et al., 2018;Speckmann et al., 2016). In zebrafish, npas4a is the main npas4 paralogue expressed in b-cells (Tarifeño-Saldivia et al., 2017), meaning that it is unlikely the phenotype we identified early in development in npas4l mutants is related to the functions of Npas4 in b-cells. Further studies on NPAS4, related bHLH transcription factors, and ETV2 in mammals should elucidate whether inactivating such factors promotes b-cell formation with or without significantly perturbing the development of blood cells and vessels.
In summary, we have shown that the npas4l mutation and etsrp knockdown each induces ectopic regeneration of b-cells from the mesoderm. Our findings suggest a plasticity potential of mesodermal cells to differentiate into endodermal cells including b-cells (Figure 7). Future studies on the restriction of this plasticity would not only increase our understanding of the gating role of Npas4l and Etsrp in cell fate determination but also help to exploit an alternative source for b-cell regeneration.
The Tg(UAS:Cre, cryaa:Cerulean) bns382 line, abbreviated as Tg(UAS:Cre), was generated via Tol2mediated transgenesis by injecting a construct that placed the Cre recombinase coding sequence downstream of five tandem copies of the upstream activation sequence (UAS). The eye-marker cryaa:Cerulean cassette (Hesselson et al., 2009) was inserted downstream of Cre in the reverse orientation using 0.56 kb of the cryaa promoter (Kurita et al., 2003).
Males and females ranging in age from 3 months to 2 years were used for breeding to obtain new offspring for experiments. Individuals were sorted into the control sibling group (npas4l +/+ , npas4l s5/+ , or npas4l bns423/+ ) and the npas4l mutant group (npas4l s5/s5 or npas4l s5/bns423 ) based on the characteristic pericardial oedema and blood-cell deficit. Zebrafish larvae were allocated into different experimental groups based on their phenotypes and genotypes in experiments involving cloche mutants. In morpholino knockdown experiments, zebrafish embryos were randomly assigned to each experimental condition for injection. Experimental procedures were performed on the zebrafish from 10 hpf to 3 dpf before the completion of sex determination and gonad differentiation. All zebrafish, except npas4l mutants and etsrp morphants, appeared healthy and survived to adulthood. The npas4l mutants exhibited pericardial oedema, bell-shaped hearts, and blood deficits, as previously reported (Stainier et al., 1995). The etsrp morphants had similar phenotypes. All studies involving zebrafish were performed in accordance with local guidelines and regulations and approved by regional authorities.

Lineage tracing by tamoxifen-inducible cre recombinase
To genetically trace the mesodermal lineage, we treated Tg(ins:Flag-NTR);Tg(ubi:Switch);Tg(drl: CreER T2 ) zebrafish embryos with 10 mM 4-OHT (Sigma-Aldrich) in E3 medium in 90 mm Petri dishes, with approximately 60 individuals per dish, from 10 to 12 hpf. Upon induction by 4-OHT, cytoplasmic CreER T2 would be translocated to the nucleus to excise the loxP-flanked EGFP to enable mCherry expression in drl-expressing cells and their descendants, indicating a mesodermal lineage.

Sample fixation for immunostaining
Before fixing the zebrafish larvae, we confirmed the presence of the transgenes by determining the corresponding fluorescent markers and subsequently examined them under a widefield fluorescence microscope LEICA M165 FC (Leica Microsystems). We then euthanised the zebrafish larvae with 250 mg/L tricaine (Sigma-Aldrich) in E3 medium followed by washing in distilled water three times. We fixed the samples in 4% formaldehyde (Sigma-Aldrich) in PBS (ThermoFisher Scientific) at 4˚C overnight. After washing away the fixative with PBS three times, we removed the skin and crystallised yolk of the zebrafish larvae by forceps under the microscope to expose the pancreas and mesenchyme for immunostaining.
Before confocal imaging, we mounted the stained samples in VECTASHIELD Antifade Mounting Medium (Vector Laboratories) on microscope slides with the pancreas facing the cover slips. We imaged the pancreas and neighbouring mesenchyme of every zebrafish sample that we had mounted with the confocal laser scanning microscopy platform Leica TCS SP8 and LAS X (Leica Microsystems). We analysed the images by Fiji (Schindelin et al., 2012) and classified a b-cell as pancreatic when it was located in the pancreas, as delineated by pan-cadherin, DAPI, or ptf1a:EGFP labelling. The insulin-positive cells outside of the pancreas were defined as ectopic b-cells.

Live calcium imaging of zebrafish b-cells
Imaging of pancreatic and ectopic b-cells was performed on 3 dpf Tg(ins:GCaMP6s);Tg(ins: mCherry);Tg(ins:Flag-NTR) npas4l mutants on a ZEISS LSM 980 confocal microscope equipped with a W Plan-Apochromat Â20/1 NA water correction lens and operated by ZEN. The GCaMP6s and mCherry signals from b-cells were simultaneously acquired using the 488 nm and 587 nm laser lines. The GCaMP6s signal was rendered in green, and the mCherry signal was rendered in red. Time series recordings were taken with an in-plane resolution of 1024 Â 1024 pixels and a fully open pinhole to maximise light capture. The videos were recorded for 400 cycles, with approximately 2 s acquisition time per frame. Videos were analysed in ImageJ (Schneider et al., 2012).