PMC

A–H) CB and cyst number in control animals and animals with compromised ecdysone signaling. A, B) CB and 2-, 4- and 8-cell cyst number are unaffected whereas the number of 16-cell cysts is reduced (C–H). C–G) 16-cell cysts outlined, no outline indicates an absence of 16-cell cysts. C) c587 alone 29^oC day 8; D) c587::USP RNAi 29^oC day 8; E) c587::EcR RNAi 29^oC day 8; F) ecd¹ 18^oC control; G) ecd¹ 29°C day 4. Green: somatic cells (anti-Tj), magenta: cell membranes and spectrosome/fusome (anti-Hts and anti-FasIII). I) No change in TUNEL positive 16-cell cyst number was seen when ecdysteroid signaling was limited. J-N) Germ cells and cysts within germaria from control flies and flies where ecdysone signaling was compromised were stained for C(3)G protein to reveal synaptonemal complex-containing cells. Region 2a cysts (dashed outline), region 2b follicles (solid outline) and region 3 follicle (solid outline) indicated. No outline indicates absence of C(3)G positive cysts or follicles. Green, synaptonemal complex (anti-C(3)G), magenta, cell membranes and spectrosome/fusome (anti-Hts and anti-FasIII). J) c587 alone 29^oC day 8; K) c587::USP RNAi 29^oC day 8; L) c587::EcR RNAi 29^oC day 8; M) ecd¹ 18^oC control; N) ecd¹ 29^oC day 4. Scale bar: 10 µm. Error bars indicate s.d.

A) The number of male GSCs was counted after the indicated periods following a shift to 29°C to compromise ecdysone signaling as indicated. Error bars indicate s.d. B) c587 alone 29°C day 8; C) c587::USP RNAi 29°C day 8; D) c587::EcR RNAi 29°C day 8; E) c587::E75 RNAi 29°C day 8; F) ecd¹ 29°C day 4. (B′–F′: enlarged regions). GSCs outlined in B′, C′, D′, E′ and F′ and asterisk position of hub cells. Green: somatic cells (anti-Tj) magenta: cell membranes and fusome (anti-hts and anti-FasIII). Scale bar: 10 µm. G, H) EM analysis of ecd¹ cysts from males kept G) at 18°C, or H) 29°C day 8. Magenta: pseudocolour (germ cells within a single cyst), green: pseudocolour (cyst cells in contact with the pseudocoloured cyst). Scale bar: 2 µm.

A–J) Germaria from control flies and animals in which ecdysone signaling was compromised following temperature shift for the indicated periods, were analyzed to determine the number of region 2a (dashed outline), region 2b (solid outline) and region 3 (solid outline) 16-cell cysts. Asterisk  =  missing follicle(s). Germaria from ecd¹ animals (B) and animals in which ecdysone signaling components were knocked-down (F) were scored as to whether follicle formation was defective by analysing the number of cysts present (at least one cyst is normally present in each of regions 2a, 2b and 3) and whether the location and morphology of these cysts appeared normal. A) ecd¹ 18°C control; C–E) ecd¹ 29°C day 4; G) c587 29^oC day 8; H–I) c587::USP RNAi 29C day 8; J) c587::EcR.B1 dominant °negative 29°C day 8. Green: somatic cells (anti-Tj), magenta: cell membranes and fusome (anti-hts and anti-FasIII). Error bars indicate s.d. Scale bar: 10 µm.

A–C) Escort and early follicle cell processes (labeled with anti-Fax) entirely surround each germline cyst and early follicle in control germaria (A, red arrows) but are completely (B) or partially (C, red arrow indicates intact process) retracted when ecd¹ flies are shifted to 29^oC. A) ecd¹ 18^oC control; B/C) ecd¹ 29^oC day 8. Scale bar: 10 µm. D–E”) EM analysis of somatic process retraction, germ cells in a single cyst pseudocoloured magenta and escort cells green. D′ is an enlargement of outlined region in D and E′ and E′′ are enlargements of outlined region within E. D/D′) ecd¹ 18^oC control; E/E′/E′′) ecd¹ 29^oC day 4 Scale bar: 2 µm. G) Knock down of usp in a sub-population of escort cells (green, single cell outlined) causes cell shape changes (compare to control, F). H) Knock down of EcR expression in a single escort cell (outlined) does not change the cell shape. I) Over expression of EcR.B1 dominant negative in a sub-population of escort cells (green, single cell outlined) does cause shape changes. Green: GFP and RNAi expression, magenta: cell membranes and fusome (anti-Hts). F) Flipout::GFP 29° day 7; G) Flipout::GFP USP RNAi 29°C day 7; H) Flipout::GFP EcR RNAi 29°C day 7; I) Flipout:: EcR.B1 dominant negative 29°C day 5. Scale bar: 10 µm.

A) Diagram of the Drosophila germarium: terminal filament cells (light blue), cap cells (yellow), GSCs (magenta), cystoblasts (pink), cysts (beige), escort cells (dark blue), follicle cells (green). Subregions 1–3 of the germarium are indicated. B) anti-USP staining of somatic escort cell nuclei (arrows), follicle cell nuclei (arrowhead) and germ cells within forming follicles (asterisks). C–H) Regions 1 and 2a of the germaria containing GSCs and cysts (dashed outline) appear smaller when ecdysteroid signaling is reduced. C) ecd¹ 18^oC control; D) ecd¹ 29^oC day 4; E) c587 alone 29^oC day 8; F) c587::USP RNAi 29^oC day 8; G) c587::EcR RNAi 29^oC day 8; H) c587::E75 RNAi 29^oC day 8. Green, somatic cells (anti-Tj), magenta, cell membranes and spectrosome/fusome (anti-Hts and anti-FasIII). I) Time course showing the number of GSCs present in germaria from controls and flies in which ecdysone signaling was reduced for the duration shown as described. J-K) Control germaria usual have two to three GSCs (J, dashed outline), whereas c587:USP RNAi flies after 8 days at 29°C usually have only one (K, dashed outline), white: cell membranes and spectrosome/fusome (anti-Hts and anti-FasIII). Asterisk marks the position of the cap cells. L) The number of cap cells was counted in germaria from controls and flies in which ecdysone signaling was reduced for the duration shown. Cap cell number is not affected by reducing ecdysteroid signaling. Error bars indicate s.d. Scale bar: 10 µm.