Fig. 1.

PI3K and Tor activity are observed during CySC differentiation. (A) Simplified model of the PI3K/Tor pathway. Here, PI3K is activated by the insulin receptor (InR) tyrosine kinase following binding of a Drosophila insulin-like peptide (Dilp). InR phosphorylates PI3K, which in turn phosphorylates PIP₂ lipids to create PIP₃. Pten is a lipid phosphatase that catalyzes the reverse reaction. Subsequently, PIP₃ activates Akt, which inactivates the Tor inhibitor Tsc1/2. The TORC1 complex is then activated and leads to the phosphorylation of S6K and 4E-BP. The secreted Dilp antagonist ImpL2 blocks Dilps from binding to InR. Phosphorylation events are indicated by the yellow symbols. (B) The PH-GFP reporter of PIP₃ consists of GFP fused to the pleckstrin homology (PH) domain of Grp1, which binds PIP₃. This GFP fusion is cytoplasmic, but when PIP₃ levels are high it translocates to the plasma membrane. (C) The PI3K reporter PH-GFP (green) was predominantly cytoplasmic in CySCs around the hub, indicating low PI3K activity. Arrow in C′,C‴ marks a CySC with low membrane PH-GFP. Membrane-associated PH-GFP was observed as cyst cells begin to differentiate, one further cell diameter away from the hub. Arrowheads in C′,C‴ mark differentiating cyst cells that have upregulated membrane PH-GFP. Tj (blue and C‴) labels CySCs and early cyst cells, Dlg (red and C″) marks somatic cell membranes. (D) The Tor activity reporter p4E-BP (green) was detected at high levels in somatic cells adjacent to stem cells. Arrow in D-D″ marks a Zfh1-positive CySC next to the niche that has low p4E-BP. Arrowhead in D-D″ labels a Zfh1-positive early CySC daughter cell primed for differentiation that is one cell diameter away from the CySCs and that has high levels of p4E-BP. Arrowhead in D‴ marks an Eya-positive differentiating cyst cell. Fas3 (blue and D‴) marks the hub; CySCs are the Zfh1-positive cells (red and D″) that are closest to the hub. Dotted red line outlines the hub.