(A) Germarium cartoon showing differentiated Terminal Filament (TF) and Cap Cells (CC), which maintain Germline Stem Cells (GSCs) and express Hh and Wg strongly. Cystoblasts (CB) and subsequent, 2, 4, 8 and 16 cell germline cysts are in black, surrounded by somatic (blue) Escort Cells (ECs). A typical single marked Follicle Stem Cell (FSC) lineage is indicated by green nuclei, including FSC daughters that move laterally (up-down) or to the posterior (right). Most FSC daughters continue to proliferate as pre-follicle cells (FC) that express Fas3 (denoted by red outlines). (B, C) FSC clones negatively marked by the loss of GFP, showing (B) a wild-type FSC (arrow) and its derivatives (no green GFP; thin white lines) and (C) a Dstat92E mutant FSC clone where the mutant FSC has been lost and replaced by GFP-positive FSCs (arrows), while some GFP-negative FSC derivatives are still detected in later egg chambers (white lines). FSCs (arrows) are the most anterior (left) cells in their lineage, contact the germarial walls and do not express the surface protein Fas3 (red). In (C) there are some “fused” egg chambers, as expected from the requirement for JAK-STAT pathway activity to specify stalk cells. (D, E) FSC clones positively marked by GFP (green) and stained with antibody to Fas3 (red) for (D) a wild-type FSC and (E) an FSC expressing excess Hop (E’ shows Fas3 staining only). Excess JAK-STAT signaling produced extra marked cells anterior to normal FSC positions (white arrows). The most anterior ectopic GFP-positive cells did not express Fas3 (yellow arrows) but Fas3 was expressed in most ectopic anterior cells, whether lining the germarial walls (no arrows) or at internal positions (arrowheads). (F, G) JAK-STAT pathway activity reported by 10xSTAT-GFP (green) in ovarioles co-stained for (F’, G’) Fas3 (red). (F) In wild-type ovarioles the JAK-STAT pathway reporter was active in FSCs (arrows), their posterior derivatives (no arrows) and in posterior Escort Cells (yellow arrowheads). (G) In ovarioles where UAS-upd RNAi was driven by neur-GAL4 in polar cells JAK-STAT reporter activity was greatly reduced in the entire germarium, including FSCs (arrows) and in egg chambers, which were sometimes fused (arrowheads) with no intervening stalks. Red scale bars represent 25µm. See also Figure S1.