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. 2015 Dec 9;4:e08497. doi: 10.7554/eLife.08497

Figure 5. Myc acts downstream of Insulin and Notch pathways during AMP reactivation.

(A, A”) A single cluster of AMPs from the tPGH third-instar larvae stained for GFP to reveal activation of PI3K/Insulin pathway and for phospho-histone H3 (PH3) to identify AMPs that undergo proliferation. Note that PH-GFP localizes to the cell membranes, indicating the activity of PI3K/Insulin signaling in AMPs that proliferate. (B–D”) Single clusters of third-instar larva lateral AMPs stained for dMyc and NICD (B–C”) and for dMyc and Lamin (D–D’’). (B, B”) Control m6-GAL4 larva. (C, C”) m6-GAL4-driven expression of Inr-CAAX in AMPs upregulates dMyc and NICD expression. (D, D”) Targeted expression of NICD in AMPs results in an increased dMyc signal in AMPs. (E–I) Double transgenic mutant contexts and their effects on number of lateral AMPs. Attenuations of Notch (F) and dMyc (G) rescue the InRCA-induced overproliferation phenotype. Similarly, attenuating dMyc in AMPs expressing NICD dramatically reduces AMP numbers (I) compared to NICD context (H). (J) Mean fluorescence intensity of the dMyc signal detected in loss- and gain-of-function contexts for Insulin and Notch pathway components. (K) Mean fluorescence intensity of the NICD signal detected in InRCA and PTEN contexts. (L) Mean number of lateral AMPs counted in different genetic contexts shown in (E–I). (***) and (**) indicate P≤ 0.001 and P≤ 0.01, respectively. Scale bars are (A, A”): 9 microns; (B–D”): 15 microns; (E–I): 45 microns. (M) Schematic illustration of genetic hierarchy between Insulin, Notch and Myc during AMP reactivation.

DOI: http://dx.doi.org/10.7554/eLife.08497.018

Figure 5—source data 1. Table showing mean number of dorsal, lateral and ventral AMPs in the abdominal segments from the genotypes shown in Figure 5E–I,L and Figure 5—figure supplement 1.
For each genotype, the average number of cells ± standard error mean is shown. Sample size (n) is indicated in brackets. Below a table with mean signal intensity measurements for Myc and NICD in lateral AMP cluster from the genotypes shown in Figure 5B–D'',J,K. For each genotype, the average signal intensity value ± standard error mean is shown. Sample size (n) is indicated in brackets.
DOI: http://dx.doi.org/10.7554/eLife.08497.019
elife-08497-fig5-data1.docx (50KB, docx)
DOI: 10.7554/eLife.08497.019

Figure 5.

Figure 5—figure supplement 1. Proliferation of AMPs is positively regulated by Insulin, Notch and their downstream target Myc.

Figure 5—figure supplement 1.

(A) A graph representing the mean number of AMPs from the dorsal and ventral abdominal groups of mid-third-instar larvae from the genotypes shown in Figure 5E–I. Notice that ventral AMPs reactivation appears independent of Insulin and Notch but dependent on Myc. (***) and (**) indicate P≤0.001 and P≤0.01, respectively. P-values were computed using t test and Prism software with Gal4 as a control sample. (B-D) A single cluster of AMPs from the lateral group of M6-gapGFP early third-instar larvae stained for GFP (green) visualizing the AMPs and for proliferation marker PH3 (magenta). (E-E”) Cluster of lateral AMPs from early third-instar t-PGH larvae stained for GFP, gray (E) to reveal subcellular PGH localisation and (E’) for PH3 (magenta). (E”) merged view. Arrow indicates membrane localized PGH in cell with a high level of PH3 and arrowhead points to a cell with faint PH3 staining and with ubiquitously distributed PGH. Scale bar in (B): 15 microns; in (E): 9 microns.
DOI: http://dx.doi.org/10.7554/eLife.08497.020