Figure 2: PS11 hub cells migrate anteriorly along the periphery.

Representative stills (time in minutes) from live imaging of six4-nlsGFP marking all SGPs (green) and Prd-GAL4>tdTomato to mark SGPs originating from PS11 (red). Scalebar is 10 microns. A) Merge, and A’) Prd>tdTomato channel alone. At Time 0 before anterior movement of pro-niche cells, tdTomato is expressed only in a central column of SGPs (originating from PS11) and the posterior msSGPs (from PS13, bracket), and not SGPs from even-numbered parasegments. The stills in this time series were from Z-slices at mid-depth within the gonad (13-16 um in from the periphery of the gonad (26.5 um represents full depth in this gonad)). At Time 0 a PS11 hub cell can be seen extending a protrusion towards the gonad periphery, and moving on to it. This same cell also has a trailing extension that is likely wrapped around a germ cell (arrow). As this cell moves to the lower edge of the gonad, it is moving from within the internal milieu of the gonad to the periphery. This cell is followed from time 0 through 139 min, and the boxed region is magnified in the inset, where the cell body is outlined. A trailing extension visible at Time 0 and 15 min, is retracted by 32 min (asterisk), while the anterior extension persists as the cell moves along the gonad periphery (arrow). By 124min, this PS11 cell has joined the anterior assembly of pro-niche cells, and retracted its anterior extension.B) Two PS11 pro-niche cells (inset) that were already located on the gonad periphery at the start of imaging. Both cells migrated anteriorly and ended in assembly (72 min) with a third PS11 pro-niche cell, and two nonPS11 SGPs (expressing six4-nlsGFP, but not Prd>tdTomato). Both PS11 cells intially had cytoplasmic protrusions surrounding a germ cell (0min; arrows) that were subsequently retracted (asterisks, 15 and 30min). (C) The inset initially focuses on one peripheraly located PS11 pro-niche cell, with cytoplasmic protrusions surrounding a germ cell (arrows, 0min) that were retracted (asterisk, 11min) or remodeled (0 – 26min, asterisk) as the cell moved anteriorly. Pro-niche cells tracked in (B) and (C) were visible in the first z-slice containing cells from their region of the gonad; i.e., within 1 micron of the gonad periphery. (D) Quantification of total niche cells and PS11 niche cells in each gonad imaged (n = 22; hashmark, mean). Connectors pair data collected from each gonad. A larger contribution from PS 11 did not always correlate with a larger niche. E) Basement membrane is desposited during hub assembly. Live-imaging of SGPs (six4-nlsGFP) simultaneously with Collagen::GFP to monitor depositon of ECM. A PS11 pro-hub cell moves out to and then along the periphery arrow). ECM was detectable by the time the PS11 cell reached the periphery (t=10m and onward, arrowhead; and insets).