A new mode of pancreatic islet innervation revealed by live imaging in zebrafish
- Max Planck Institute for Heart and Lung Research, Germany
- National Institute of Genetics, Japan
- SOKENDAI (The Graduate University for Advanced Studies), Japan
Figures
Figure 1
Pancreatic islet innervation in zebrafish is established early in development and maintained in juvenile and adult stages.
(A-D) Whole mount immunostaining of wild-type zebrafish at 50, 75, 100, 120 hr post fertilization (hpf) for acetylated Tubulin (nerves), Insulin (beta cells), Somatostatin (delta cells), and DAPI …
Figure 2 with 8 supplements
The sequence of cellular events preceding pancreatic islet parasympathetic innervation is revealed with single-cell resolution time-lapse confocal imaging.
(A) Tg(elavl3:Gal4-VP16); Tg(UAS:EGFP-CAAX); Tg(sst2:RFP) zebrafish mounted in 0.5% agarose containing 0.017% tricaine were imaged with laser scanning confocal microscopy at 20 min time intervals. …
Figure 2—figure supplement 1
A subset of pancreatic nerve extensions derives from neurons that were once in close contact with endocrine cells.
Whole mount immunostaining at 80 hpf for GFP (elavl3-positive cells), acetylated Tubulin (nerves), Insulin (beta cells), and RFP (delta cells). Yellow arrow points to elavl3 expressing cell …
Figure 2—video 1
Time-lapse imaging shows detachment of neurons from the pancreatic islet.
24 hpf Tg(elavl3:Gal4-VP16); Tg(UAS:EGFP-CAAX); Tg(sst2:RFP) zebrafish mounted in 0.5% agarose with tricaine was imaged with laser scanning confocal microscopy at 20 min time intervals. Movie …
Figure 2—video 2
Time-lapse imaging shows detachment of neurons from the pancreatic islet.
24 hpf Tg(elavl3:Gal4-VP16); Tg(UAS:EGFP-CAAX); Tg(sst2:RFP) zebrafish mounted in 0.5% agarose with tricaine was imaged with laser scanning confocal microscopy at 20 min time intervals.
Figure 2—video 3
Time-lapse imaging shows neurons migrating away from the pancreatic islet.
39 hpf Tg(elavl3:Gal4-VP16); Tg(UAS:EGFP-CAAX); Tg(sst2:RFP) zebrafish mounted in 0.5% agarose with tricaine was imaged with laser scanning confocal microscopy at 20 min time intervals. Movie …
Figure 2—video 4
Time-lapse imaging shows vagus nerve extending past the pancreatic islet.
59 hpf Tg(elavl3:Gal4-VP16); Tg(UAS:EGFP-CAAX); Tg(sst2:RFP) zebrafish mounted in 0.5% agarose with tricaine was imaged with laser scanning confocal microscopy at 30 min time intervals. Movie …
Figure 2—video 5
Time-lapse imaging shows neurite extending toward the pancreatic islet.
59 hpf Tg(elavl3:Gal4-VP16); Tg(UAS:EGFP-CAAX); Tg(sst2:RFP) zebrafish mounted in 0.5% agarose with tricaine was imaged with laser scanning confocal microscopy at 30 min time intervals. Movie …
Figure 2—video 6
Time-lapse imaging shows interaction of detached neurons with the vagus nerve.
59 hpf Tg(elavl3:Gal4-VP16); Tg(UAS:EGFP-CAAX); Tg(sst2:RFP) zebrafish mounted in 0.5% agarose with tricaine was imaged with laser scanning confocal microscopy at 30 min time intervals.
Figure 2—video 7
Time-lapse imaging shows detachment and caudal migration of neurons from the pancreatic islet.
83 hpf Tg(elavl3:Gal4-VP16); Tg(UAS:EGFP-CAAX); Tg(sst2:RFP) zebrafish mounted in 0.5% agarose with tricaine was imaged with laser scanning confocal microscopy at 45 min time intervals.
Figure 3
Neural crest cells are in close contact with pancreatic endocrine cells early in development.
(A) Lineage tracing of neural crest cells in Tg(sox10:CreERT2, myl7:GFP); Tg(ubb:loxP-CFP-loxP-nuc-mCherry); Tg(elavl3:GAL4-VP16); Tg(UAS:EGFP-CAAX) zebrafish following 5 µM tamoxifen treatment from …
Figure 4 with 1 supplement
Pancreatic islet parasympathetic innervation is derived from the neural crest.
(A) Lineage tracing of neural crest cells in Tg(sox10:CreERT2, myl7:GFP); Tg(ubb:loxP-CFP-loxP-nuc-mCherry) zebrafish following 5 µM tamoxifen treatment from 16 to 24 hpf and staining at 90 and 120 …
Figure 4—figure supplement 1
Neural crest cells are essential for the establishment of islet parasympathetic innervation.
(A) Immunostaining analysis of innervation density in wild-type and sox10 mutants at 80 hpf. (B) Wild-type and sox10-/- zebrafish at 80 hpf. (C) Body length measurements at 80 hpf, mean ± SEM, n = 26…
Figure 5 with 1 supplement
The vasculature is not essential for the initial establishment of pancreatic islet parasympathetic innervation.
(A) Immunostaining analysis of innervation density in Tg(kdrl:GFP) wild-type and cloche mutants at 80 hpf. (B) Wild-type and cloche-/- larvae were imaged at 80 hpf. (C) Body length measurements at …
Figure 5—figure supplement 1
The vasculature is not essential for the initial establishment of islet parasympathetic innervation.
(A-B) Whole mount immunostaining of 4 dpf Tg(kdrl:EGFP) wild-type and cloche mutants, which lack most endothelial cells, for GFP (blood vessels), acetylated Tubulin (nerves), Insulin (beta cells), …
Figure 6
Targeted ablation studies reveal the crucial role of peri-islet neurons for the initiation of pancreatic islet parasympathetic innervation.
(A) Schematic of the two-photon ablation experiment. (B) Gross morphology at 80 hpf was comparable between control and ablated fish. (C–D) Tg(elavl3:Gal4-VP16); Tg(UAS:EGFP-CAAX); Tg(sst2:RFP) …
Figure 7 with 3 supplements
Inhibition of neural synaptic transmission diminishes islet parasympathetic innervation.
(A) Botulinum toxin (BoTx) was expressed in post-mitotic neurons to inhibit neurotransmitter release in Tg(elavl3:Gal4-VP16); Tg(UAS:BoTxBLC-GFP) zebrafish. (B) Control and BoTx-positive fish were …
Figure 7—figure supplement 1
Inhibition of synaptic transmission results in delta cell hypertrophy.
(A) Botulinum toxin (BoTx) was expressed in post-mitotic neurons to inhibit neurotransmitter release in Tg(elavl3:Gal4-VP16); Tg(UAS:BoTxBLC-GFP); Tg(sst2:RFP) zebrafish. (B) Quantification of delta …
Figure 7—figure supplement 2
Diminished nerve density and impaired neural output result in hyperglycemia.
(A) Whole larva free glucose level measurements of wild-type and sox10-/- zebrafish at 80 hpf and 5 dpf, mean ± SEM, n = 8–22 batches of five larvae per replicate. Elevated free glucose levels were …
Figure 7—figure supplement 3
Genetic ablation of neurons decreases pancreatic endocrine cell numbers.
(A) Genetic ablation in Tg(elavl3:Gal4-VP16); Tg(UAS:NTR-mCherry) zebrafish expressing nitroreductase (NTR) in post-mitotic neurons. Treatment with 2 mM metronidazole (MTZ) from 24 to 80 hpf …
Tables
Table 1
List of zebrafish transgenic and mutant lines.
https://doi.org/10.7554/eLife.34519.022| Name | Specificity/Purpose/Phenotype | Reference |
|---|---|---|
| Tissue specific promoter lines | ||
| Tg(elavl3:GAL4-VP16)zf357 | Post-mitotic neurons | (Stevenson et al., 2012) |
| Tg(sst2:RFP)gz19 | Pancreatic delta cells | (Li et al., 2009) |
| Tg(sox10:GAL4)sq9 | Neural crest cells | (Lee et al., 2013) |
| Tg(sox10:CreERT2, myl7:GFP)t007 | Neural crest cells, heart marker | (Mongera et al., 2013) |
| Tg(ubb:loxP-CFP-loxP-nuc-mCherry)jh63 | Ubiquitous | (Wang et al., 2015) |
| Tg(kdrl:EGFP)s843 | Endothelial cells | (Jin et al., 2005) |
| UAS lines | ||
| Tg(UAS:EGFP-CAAX)m1230 | Visualize neurons | (Fernandes et al., 2012) |
| Tg(UAS:BoTxBLC-GFP)icm21 | Inhibit neurotransmitter release | (Sternberg et al., 2016) |
| Tg(UAS:NTR-mCherry)c264 | Ablate neurons | (Davison et al., 2007) |
| Tg(UAS:GFP)zf82 | Visualize neural crest cells | (Asakawa et al., 2008) |
| Mutant lines | ||
| cloches5, clochem39 | Lacking endothelial cells | (Stainier et al., 1995; Liao et al., 1997) |
| sox10tw2 | Lacking neural crest cells | (Kelsh et al., 1996) |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.34519.023
