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Fig. 4
Progenitor amplification and differentiation can be uncoupled by modulating the duration and/or extent of Notch signaling downregulation. (A-B2) Tg(Tp1:H2BmCherry) larvae were used to label NRCs (red). Larvae were treated with DMSO or 10 μM LY411575 from 4 to 4.5 dpf. At 4.5 dpf, the chemicals were washed away and EdU was added from 4.5 to 5.5 dpf for cell cycle analysis. The positions of the EdU+ NRCs are indicated by white outlines. This brief period of LY411575 treatment was sufficient to induce cell cycle entry in the majority of NRCs compared with controls. (C) Average percentage of EdU+ NRCs in A and B (±s.d.). Forty Tg(Tp1:H2BmCherry)+ cells per larva were counted, starting from the posterior part of the pancreas (n=12 larvae for DMSO and n=12 larvae for LY411575). In DMSO controls, on average 23% of the NRCs were EdU+, whereas in LY411575-treated larvae an average of 69% were EdU+ (P<0.001). (D-F) Tg(Tp1:H2BmCherry); Tg(neurod:EGFP) larvae were treated with (D) DMSO or (E,F) 10 μM LY411575. (E) Larvae treated with LY411575 from 4 to 4.5 dpf. At 4.5 dpf, LY411575 was washed away and the animals were allowed to develop until 7 dpf. On average, 12 endocrine cells were present posterior to the PI (s.d.=4 cells, n=8 larvae), which translates into <14% of the NRCs in the pancreatic tail differentiating into the endocrine lineage. DMSO controls showed on average 1 endocrine cell (s.d.=1 cell, n=7 larvae). Arrowheads indicate NRC-derived endocrine cells. Arrows indicate Tg(neurod:EGFP)+ cells in the gut. (F) Larvae treated with LY411575 from 4 to 7 dpf. White arrowheads indicate three new islets composed of numerous endocrine cells. Only two NRCs did not undergo endocrine differentiation (yellow arrowheads). (G-H2) To induce moderate Notch signaling downregulation, Tg(Tp1:H2BmCherry); Tg(neurod:EGFP) larvae were treated with (G) DMSO or (H,H2) 1 μM LY411575 from 4 to 7 dpf. In each case, the chemicals were replaced every 24 hours. Larvae treated with 1 μM LY411575 (H,H2) exhibited a significant increase in the total number of Tg(Tp1:H2BmCherry)+ cells posterior to the PI (yellow arrowheads) (118 cells, s.d.=21 cells, n=20 larvae) compared with DMSO controls (71 cells, s.d.=19 cells, n=18 larvae) (P<0.0001). NRC-derived endocrine cells represented, on average, 17% (s.d.=7.26%, n=19 larvae) of the total number of Tg(Tp1:H2BmCherry)+ cells posterior to the PI (white arrowheads) (see also supplementary material Fig. S5A,B,D for low-magnification views and data quantitation). All images are lateral views, anterior towards the top, ventral towards the right. Scale bars: 20 μm. (I) NRCs experience different levels of Notch signaling regulating their quiescent, proliferative or differentiation states. (J) Upon downregulation of Notch signaling, NRCs (red) enter the cell cycle. Under sustained downregulation of Notch signaling, they differentiate into endocrine cells and exit from the cell cycle. Some NRCs can differentiate directly without entering the cell cycle; some NRCs differentiate into endocrine cells even after a brief and/or a moderate level of Notch signaling downregulation.