Ed by cap cell loss (Fig. 1L).One of the most fundamental events of early oogenesis is entry into meiosis, a process that commences in newly formed 16-cell cysts. The signals triggering entry into meiosis remain poorly understood, especially in metazoans, but in yeast involve nutrient limitation (reviewed in [26]). Because the onset of meiosis (premeiotic S phase) occurs shortly after 16-cell cyst formation, we investigated whether ecdysone signaling affects meiotic induction by immunostaining for C(3)G, a AN 3199 web component of the synaptonemal complex [27]. In controls, two to three C(3)G positive 16-cell cysts were found in each germarium, as expected (Fig. 2J and M dashed line outlines region 2a cysts and solid line region 2b or 3 follicles). However, the reduction in 16-cell cyst production caused by knock down of ecdysone signaling components resulted in an absence of C(3)G staining in region 2a/b of many CP21 site germaria (Fig. 2K ). A few older cysts that probably entered meiosis before RNAi became effective stained positively (Fig. 2L, solid line). 16-cell cysts present in ecd mutant germaria, which likely formed before the temperature shift and were unable to continue oogenesis due to follicle formation defects (see below) stained positively for C(3)G (Fig. 2N, dashed line). Thus, compromised ecdysone pathway function severely impairs new 16-cell cyst formation and entry into meiosis.Follicle Formation Requires Steroid Hormone SignalingAfter 16-cell cysts progress through meiosis I in region 2a of the germarium, their covering of escort cells is replaced by follicle cells that proliferate and eventually coat each new follicle. The apparent retention of already formed 16-cell cysts in ecd1 germaria following temperature shift suggests that follicle formation might also require ecdysone signaling. Control germaria contained at least one `lens-shaped’ region 2b follicle and one spherical, region 3 follicle (Fig. 3A, solid lines). However, 70 of ecd1 germaria already showed morphological defects in region 2b and/or 3 follicles within two days at the restrictive temperature (Fig. 3B). Defects included the absence of a region 2b or a region 3 follicle (Fig. 3C, position of missing 2b and 3 follicle marked by an asterisk) and/or fusions between adjacent late follicles (Fig. 3D, outlined). Additionally, the normal perpendicular alignment of 16cell cysts relative to the A/P axis in region 2a (Fig. 3A, dashed lines), was often disrupted (Fig. 4E, dashed lines), which may impede follicle formation. These data provide evidence that follicle formation requires ecdysone signaling. Unlike in ecd mutants, knock down of ecdysone signaling pathway components does not 
lead to the retention of 16-cell cysts due to a rapid block in follicle formation after temperature shift (Fig. 2H, Fig. 3B). However, follicle formation defects could occur at later time points after the pre-formed 16-cell cysts have formed follicles. Examination of germaria in which USP had been knocked down showed this to be the case. After 4 days at 29oC few germaria contained abnormal or missing region 2b or 3 follicles, but by day 8 defects were seen in 96618 of germaria (Fig. 3F). Either no follicles of these stages were present (Fig. 3H, I, asterisk marks missing region 2b and 3 follicles), or the 2a cystsEcdysone Mediated Signaling in Somatic Cells is Required to Form Meiotic 16-cell CystsCompromised ecdysone signaling might also reduce the size and germ cell content of germaria by affecti.Ed by cap cell loss (Fig. 1L).One of the most fundamental events of early oogenesis is entry into meiosis, a process that commences in newly formed 16-cell cysts. The signals triggering entry into meiosis remain poorly understood, especially in metazoans, but in yeast involve nutrient limitation (reviewed in [26]). Because the onset of meiosis (premeiotic S phase) occurs shortly after 16-cell cyst formation, we investigated whether ecdysone signaling affects meiotic induction by immunostaining for C(3)G, a component of the synaptonemal complex [27]. In controls, two to three C(3)G positive 16-cell cysts were found in each germarium, as expected (Fig. 2J and M dashed line outlines region 2a cysts and solid line region 2b or 3 follicles). However, the reduction in 16-cell cyst production caused by knock down of ecdysone signaling components resulted in an absence of C(3)G staining in region 2a/b of many germaria (Fig. 2K ). A few older cysts that probably entered meiosis before RNAi became effective stained positively (Fig. 2L, solid line). 16-cell cysts present in ecd mutant germaria, which likely formed before the temperature shift and were unable to continue oogenesis due to follicle formation defects (see below) stained positively for C(3)G (Fig. 2N, dashed line). Thus, compromised ecdysone pathway function severely impairs new 16-cell cyst formation and entry into meiosis.Follicle Formation Requires Steroid Hormone SignalingAfter 16-cell cysts progress through meiosis I in region 2a of the germarium, their covering of escort cells is replaced by follicle cells that proliferate and eventually coat each new follicle. The apparent retention of already formed 16-cell cysts in ecd1 germaria following temperature shift suggests that follicle formation might also require ecdysone signaling. Control 
germaria contained at least one `lens-shaped’ region 2b follicle and one spherical, region 3 follicle (Fig. 3A, solid lines). However, 70 of ecd1 germaria already showed morphological defects in region 2b and/or 3 follicles within two days at the restrictive temperature (Fig. 3B). Defects included the absence of a region 2b or a region 3 follicle (Fig. 3C, position of missing 2b and 3 follicle marked by an asterisk) and/or fusions between adjacent late follicles (Fig. 3D, outlined). Additionally, the normal perpendicular alignment of 16cell cysts relative to the A/P axis in region 2a (Fig. 3A, dashed lines), was often disrupted (Fig. 4E, dashed lines), which may impede follicle formation. These data provide evidence that follicle formation requires ecdysone signaling. Unlike in ecd mutants, knock down of ecdysone signaling pathway components does not lead to the retention of 16-cell cysts due to a rapid block in follicle formation after temperature shift (Fig. 2H, Fig. 3B). However, follicle formation defects could occur at later time points after the pre-formed 16-cell cysts have formed follicles. Examination of germaria in which USP had been knocked down showed this to be the case. After 4 days at 29oC few germaria contained abnormal or missing region 2b or 3 follicles, but by day 8 defects were seen in 96618 of germaria (Fig. 3F). Either no follicles of these stages were present (Fig. 3H, I, asterisk marks missing region 2b and 3 follicles), or the 2a cystsEcdysone Mediated Signaling in Somatic Cells is Required to Form Meiotic 16-cell CystsCompromised ecdysone signaling might also reduce the size and germ cell content of germaria by affecti.
