Figure 2. 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. Maximum intensity projections of selected timeframes are presented (A, anterior; D, dorsal). Yellow arrows point to the detachment of neurons from the pancreatic islet. (B) Whole mount immunostaining at 34 hpf for GFP (neurons), RFP (delta cells), Insulin (beta cells), and Glucagon (alpha cells) after 10 hr time-lapse imaging. The detached neurons are not positive for endocrine cell markers. (C-E) Confocal imaging of Tg(elavl3:Gal4-VP16); Tg(UAS:EGFP-CAAX); Tg(sst2:RFP) zebrafish at 20–30 min time intervals. Maximum intensity projections of selected timeframes are presented. Yellow arrows point to the cellular events of interest. (F) Quantification of the time when the indicated cellular events were observed for individual fish (mean ± SEM). (G) Schematic of the sequence of cellular events preceding parasympathetic innervation of the pancreatic islet. V, vagus nerve; E, enteric nerve.

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 projecting neural extensions toward the pancreatic islet.

Figure 2—video 1. Time-lapse imaging shows detachment of neurons from the pancreatic islet.

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DOI: 10.7554/eLife.34519.005

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 corresponds to Figure 2A.

Figure 2—video 2. Time-lapse imaging shows detachment of neurons from the pancreatic islet.

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DOI: 10.7554/eLife.34519.006

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.

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DOI: 10.7554/eLife.34519.007

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 corresponds to Figure 2C.

Figure 2—video 4. Time-lapse imaging shows vagus nerve extending past the pancreatic islet.

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DOI: 10.7554/eLife.34519.008

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 corresponds to Figure 2D.

Figure 2—video 5. Time-lapse imaging shows neurite extending toward the pancreatic islet.

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DOI: 10.7554/eLife.34519.009

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 corresponds to Figure 2E.

Figure 2—video 6. Time-lapse imaging shows interaction of detached neurons with the vagus nerve.

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DOI: 10.7554/eLife.34519.010

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.

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DOI: 10.7554/eLife.34519.011

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.