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Fig. 2
Increasing the level of Notch signaling downregulation leads to an increase in endocrine differentiation of NRCs. (A-D2) Tg(Tp1:H2BmCherry); Tg(neurod:EGFP) larvae were used to label NRCs (red) and follow endocrine differentiation (green). Larvae were treated with (A) DMSO, (B) 100 μM DAPT, (C) 10 μM LY411575 and (D) 50 μM LY411575 from 3.5 to 6.5 dpf and analyzed at 7.5 dpf. 2F11 staining marks the IPD. (A,A2) In control larvae, the pancreatic NRCs form the IPD, as assessed by 2F11 immunoreactivity (arrowhead). White arrows indicate endocrine cells along the IPD. (B,B2) In the DAPT-treated larvae, some NRCs posterior to the PI differentiate into the endocrine lineage, as assessed by Tg(neurod:EGFP) expression (arrows). The majority of NRCs retained IPD cell identity, as assessed by 2F11 immunoreactivity (arrowheads), and did not undergo endocrine differentiation (arrowheads). (C,C2) In the larvae treated with 10 μM LY411575, a smaller number of NRCs retained IPD cell identity (compared with B), as assessed by 2F11 immunoreactivity (arrowheads). Note that the majority of Tg(Tp1:H2BmCherry)+ cells differentiated into endocrine cells, as assessed by Tg(neurod:EGFP) expression. Arrows indicate endocrine cells. (D,D2) In the larvae treated with 50 μM LY411575, the vast majority of NRCs converted into endocrine cells, turned on Tg(neurod:EGFP) expression and aggregated into secondary islets (arrows). The IPD network was absent, as assessed by 2F11 immunoreactivity. (E-G2) Larvae obtained from a cross of Tg(hsp:Gal4); Tg(UAS:myc-Notch1a-intra) hemizygous and Tg(Tp1:H2BmCherry); Tg(neurod:EGFP) fish were heat shocked at 3.5 dpf to induce myc-Notch1a-intra expression and incubated in DMSO or 10 μM LY411575 until 6.5 dpf. (E,E2) A DMSO-treated (myc-Notch1a-intra)– larva. Upper arrowhead, PI; lower arrowhead, IPD. (F,F2) A LY411575-treated (myc-Notch1a-intra)– larva with ectopic secondary islets (lower arrowheads). Note the increased size of the PI (upper arrowhead; compare with E). [Tg(neurod:EGFP) expression is also increased in the brain (yellow arrowhead).] (G,G2) A (myc-Notch1a-intra)+ larva treated with LY411575. No secondary islets were present along the IPD (lower arrowhead) and no increase in the size of the PI was observed (upper arrowhead). Note the dramatic increase in Tg(Tp1:H2BmCherry) expression. (H-I32) Tg(Tp1:H2BmCherry) larvae treated with (H,H2) DMSO and (I-I32) 50 μM LY411575 from 3.5 to 6.5 dpf, and stained for glucagon and insulin at 7.5 dpf. In the LY411575-treated larvae, NRCs have formed five islets posterior to the PI (arrows). These islets contain NRC-derived α- and β-cells. (I3,I32) High-magnification projections of several planes for two of the islets (for the full stack, see supplementary material Movie 4). Yellow and white arrowheads in I3 point to α- and β-cells, respectively. All images are lateral views, anterior towards the top, ventral towards the right. 2F11+, Tg(Tp1:H2BmCherry)+ and Tg(neurod:EGFP)+ cells ventral to the IPD are gut cells. Scale bars: 20 μm in A-D2,H-I2 100 μm in E-G.