Fig. S4

High Glucose Increases Endocrine Differentiation; TOR Is Required for the Expansion of the Progenitor Pool in the IPD, Related to Figure 4

(A–D) Tg(TP1:H2BmCherry);Tg(neuroD:GFP) animals were incubated in egg water (A), 3% D -glucose solution (B), DMSO (C) or 10 μM Prednisolone (Glucocorticoid) (D) from 3.5 to 7dpf. Whereas each control animal (A, C) exhibits only one Tg(neuroD:GFP)⁺ SI (arrowheads), the glucose- and prednisolone-treated animals have formed several SIs (arrowheads). The high magnification projections with separate channels show that all ectopic Tg(neuroD:GFP)⁺ cells are also Tg(TP1:H2BmCherry)⁺ and thus have recently 10 differentiated from NRCs. Yellow arrowheads point to Tg(neuroD:GFP)⁺ cells that also exhibit Insulin immunoreactivity.
(E) Quantification of the average number of Tg(neuroD:GFP)⁺ SIs for animals incubated in egg water (n = 22 animals), 3% D-Glucose (n = 18 animals), DMSO (n = 20 animals) or 10 μM Prednisolone (n=20 animals). Statistical significance was calculated using an unpaired one-tailed t-test. (*p < 0.05).
(F and G) Tg(TP1:H2BmCherry) animals were fed regularly until 17dpf (F) or fasted from 15 to 17dpf (G) and then stained with an antibody against phosphorylated RPS6 (p-RPS6) (green). p-RPS6 levels appeared to be reduced in the pancreata of fasted animals compared to fed ones. Note that in the fed animals, the pancreas (outlined by a white dashed line) appears to exhibits higher levels of p-RPS6 immunoreactivity compared to liver and gut.
(H and I) High magnification single planes showing p-RPS6 immunoreactivity in the NRCs (arrowheads) of fed animals (H) and a strong downregulation in the NRCs (arrowheads) after fasting (I). The yellow arrowhead in I points to a single NRC that exhibits low levels of p-RPS6 immunoreactivity.
(J and K) Epifluorescence images of WT (J) and mtor^xu015Gt homozygous mutants (K). In WT, by 5dpf, the pancreatic NRCs have expanded in number and form the IPD (arrowheads). mtor^xu015Gt homozygous mutant animals display a strong defect in NRC expansion resulting in a reduced pool of NRCs, as well as a failure of IPD branching by 5dpf (arrowhead). The PI (arrow) appears unaffected compared to WT. Note that there is very limited proliferation of β-cells during the embryonic stages [2]. In addition, the NRCs in the liver appear less severely affected (the liver is outlined by a green dashed line in F and G). A-I show lateral views, anterior to the top, dorsal to the left. J,K show anterior to the left, dorsal to the top. Error bars = s.e.m.. Scale bars, 20 μm.