In vivo imaging of β-cell function reveals glucose-mediated heterogeneity of β-cell functional development
- Peking University, China
- Institute of Basic Medical Sciences, China
- South China Normal University, China
Figures
Figure 1 with 4 supplements
Visualization and characterization of β-cell functional development in vivo using 2P3A-DSLM.
(A) An abbreviated scheme of the dual-color 2P3A-DSLM imaging system. (B) 3D-projection of all pancreatic β-cells in a live Tg (ins:Rcamp1.07) zebrafish embryo at 72 hpf. The different colors in the …
Figure 1—figure supplement 1
Rcamp1.07 was specifically expressed in pancreatic β-cells in Tg (ins:Rcamp1.07) zebrafish.
(A) An illustration showing the protocol for generating the Tg (ins:Rcamp1.07) zebrafish line. (B) Rcamp1.07 fluorescent signals were specifically localized in the islet region in Tg (ins:Rcamp1.07) …
Figure 1—figure supplement 2
Visualization of glucose-stimulated calcium transients in β-cells in live Tg (ins:Rcamp1.07) embryos under a spinning-disc confocal microscope.
(A) Representative images of Rcamp1.07-indicated calcium transients in β-cells in a live Tg (ins:Rcamp1.07) embryo at 72 hpf before and after 20 mM glucose stimulation. At the 5 min time point, 20 …
Figure 1—figure supplement 3
Reconstruction of a clear 3D structure of the islet in live zebrafish embryos with our 2P3A-DSLM setup.
(A) A detailed scheme of the in vivo imaging platform based on our self-developed dual-color 2P3A-DSLM. (B) Comparison of image qualities among single-photon selective-plane illuminative microscope …
Figure 1—figure supplement 4
Quantification of glucose-responsive β-cells, evaluation of their functional states, and observation of glucose-induced synchronized calcium transients in β-cells in live Tg (ins:Rcamp1.07) embryos under 2P3A-DSLM.
(A) Increases in the numbers of total β-cells and glucose-responsive β-cells at different stages from 28 to 74 hpf. n = 10–16 embryos per stage. (B) An illustration of the maximum amplitude (Max …
Figure 2 with 3 supplements
Sequential functional acquisition of β-cells from the mantle to the core of the islet was coordinated by islet microcirculation.
(A) A schematic illustrating the classification of β-cells into two populations: the cells in the outside (mantle, blue) and the cells in the inside (core, red) of the islet. (B) Time-dependent …
Figure 2—figure supplement 1
Categorization of β-cells based on their mantle/core localization in the islet.
(A) Representative z-stack images of β-cells in a live Tg (ins:Rcamp1.07) zebrafish embryo at 72 hpf. Red solid circles mark β-cells in the islet core determined in the current focal plane; red …
Figure 2—figure supplement 2
Islet vascularization did not affect the total β-cell number but correlated with the sequential acquisition of glucose-responsiveness of β-cells.
(A) Representative 3D-projections of β-cells (green) and blood vessels (red) at different stages in live Tg (ins:EGFP);Tg (flk1:mCherry) embryos. (B) Quantification of pancreatic β-cells located …
Figure 2—figure supplement 3
Absence of intra-islet circulation caused inefficient delivery of a low concentration of glucose to β-cells in the islet core, whereas either a high concentration of glucose or the complete establishment of islet circulation overcame this deficiency.
(A) Representative images of Rcamp1.07 (red) and 2-NBDG (green) signals in β-cells in live Tg (ins:Rcamp1.07);cloche-/- mutants at 56 hpf (top panel) and 72 hpf (bottom panel) after incubation with …
Figure 3
Optimal glucose concentrations were required to induce heterogeneous and optimal β-cell functionality at different developmental stages.
(A–B) Numbers of glucose-responsive β-cells (A) and their maximal Ca2+ responses to glucose (B) in the mantle and the core of the islets from embryos treated with 3 MPA and different concentrations …
Figure 4 with 2 supplements
Calcineurin/NFAT signaling acted as the downstream of glucose to initiate and enhance β-cell functionality.
(A) Numbers of glucose-responsive β-cells in 48 hpf embryos that had been treated with the indicated reagents. n = 4–8 embryos per condition. *p<0.05, **p<0.01. (B) Numbers of glucose-responsive …
Figure 4—figure supplement 1
Glucose activated calcineurin/NFAT to enhance and sustain the functionality of β-cells during the late hatching period.
(A) The maximum amplitudes of glucose-responsive β-cells in 48 hpf embryos that had been treated with the indicated reagents. n = 4–8 embryos per condition. *p<0.05; ns, not significant. (B) The …
Figure 4—figure supplement 2
Pharmacological treatments did not affect calcium activities of CNS neurons in living Tg (elavl3:Gcamp6s) zebrafish embryos.
(A) The schematic illustrates three different brain regions in zebrafish embryos. (B) Representative 72 hpf Tg (elavl3:Gcamp6s) embryos (top) and time courses of calcium transients (bottom) from …
Figure 5 with 1 supplement
Direct activation of calcineurin promoted the optimal maturity of neonatal mouse β-cells in isolated islets in vitro.
(A–B) Glucose-induced insulin secretion from neonatal and adult mouse islets sequentially stimulated with 3 mM glucose (A) and 20 mM glucose (B) after isolation from P0 and 8-week-old mice, and for …
Figure 5—figure supplement 1
Direct activation of calcineurin promoted the optimal maturity of neonatal mouse β-cells in isolated islets in vitro, as indicated by ex vivo islet calcium imaging.
(A) The experimental design for ex vivo islet calcium imaging used in (B–C). (B–C) Representative time courses of calcium transients under indicated glucose stimulations in islets that had been …
Author response image 1
Supplementing CGA rescued the defective β-cell function in both the core and the mantle of the islet in 72 hpf cloche mutants.
(A-B) In Tg (ins:Rcamp1.07); cloche mutants at 72 hpf, supplementing CGA significantly rescued the number (A) and the maximal Ca2+ influx (B) of glucose-responsive β-cells both in the islet core and …
Author response image 2
MafA antibody used in mouse exhibited non-specific cytoplasmic signals in β-cells of zebrafish.
https://doi.org/10.7554/eLife.41540.027
Author response image 3
MafA was not expressed in the β-cells, but was expressed in the exocrine pancreas in larval zebrafish.
https://doi.org/10.7554/eLife.41540.028
Author response image 4
ucn3 expression pattern in zebrafish islets at different developmental stages.
(A) Immunofluorescent labelling of insulin and ucn3 in zebrafish islets at indicated stages. (B) Quantification of ucn3-positive β-cells in zebrafish islets at the indicated stages in A. n = 6-12 …
Author response image 5
Blockade of calcineurin/NFAT led to reduced β-cell maturity.
(A-B) In 60 hpf Tg (ins:Rcamp1.07) embryos, not acute/short-term, but only long-term incubation with calcineurin/NFAT inhibitors significantly reduced the number (A) and the maximal Ca2+ influx (B) …
Author response image 6
α-cells in isolated mouse islets almost did not take up 2-NBDG.
Representative z-stack images of 2-NBDG (green) and EYFP (a red pseudo-color) signals in isolated 8-week-old mouse islets from the transgenic mice, whose α-cells are genetically labeled with EYFP …
Author response image 7
Genetic perturbation of calcineurin/NFAT signaling persistently prevented the glucose-responsiveness of β-cells.
(A-B) Numbers of glucose-responsive β-cells (A) and their maximal Ca2+ responses to glucose (B) in age-matched control and dn-zCnA-expressing embryos at 48 hpf, 72 hpf and 7 dpf. n = 5-7 embryos per …
Videos
Video 1
A schematic illuminating the dual-color 2P3A-DSLM and comparison of image qualities among 1P-SPIM, TPM and 2P3A-DSLM in islet 3D imaging of live zebrafish.
https://doi.org/10.7554/eLife.41540.008
Video 2
Representative time-lapse images of glucose-induced synchronized calcium transients in neighboring β-cells in a live Tg (ins:Rcamp1.07) embryo at 72 hpf under 2P3A-DSLM.
https://doi.org/10.7554/eLife.41540.009
Video 3
Representative time-lapse volumetric reconstruction of glucose-stimulated calcium transients in all β-cells in a live Tg (ins:Rcamp1.07) embryo at 72 hpf under 2P3A-DSLM.
https://doi.org/10.7554/eLife.41540.010
Video 4
Volumetric reconstruction of pancreatic β-cells and their nearby blood vessels in live Tg (ins:EGFP);Tg (flk1:mCherry) embryos from 36 to 72 hpf.
https://doi.org/10.7554/eLife.41540.015
Video 5
Representative time-lapse volumetric reconstruction of glucose-stimulated calcium transients in all β-cells in a live Tg (ins:Rcamp1.07) embryo at 72 hpf that had been treated with 3 MPA for 9 hr under 2P3A-DSLM.
https://doi.org/10.7554/eLife.41540.017
Video 6
Representative time-lapse volumetric reconstruction of glucose-stimulated calcium transients in all β-cells in a live Tg (ins:Rcamp1.07) embryo at 72 hpf that had been co-treated with 3 MPA and CGA for 9 hr under 2P3A-DSLM.
https://doi.org/10.7554/eLife.41540.021
Author response video 1
Additional files
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Supplementary file 1
Comparison of the critical events and the time windows in zebrafish and mouse pancreas development.
The table shows comparison of developmental process and critical stage in zebrafish and mouse pancreas development.
- https://doi.org/10.7554/eLife.41540.024
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Transparent reporting form
- https://doi.org/10.7554/eLife.41540.025
