Figure 4. Absence of gone early causes an excessive PGC differentiation in LL3 ovaries.

(A) Average numbers of total germ cells, PGCs, and differentiating germ cells in LL3 wild type (white bars, y w), goe^5–11/331 (black bars), goe^5–11/331 with nos-gal4 alone (gray bars, goe^5–11/331; nos>), goe^5–11/331 carrying nos-gal4 and UAS-goe (pale yellow bars, goe^5–11/331; nos>goe), or UAS-goeFLAG (yellow bars, goe^5–11/331; nos>goeFLAG). Excessive PGC differentiation in the goe mutant was rescued when goe was expressed in germline by nos-gal4 (compare goe^5–11/331; nos> control with goe^5–11/331; nos>goe and goe^5–11/331; nos>goe-FLAG), indicating that the phenotype is due to loss of goe function. No significant difference in the number of PGCs was observed between these ovaries (p>0.2, U-test). Between 9 and 23 ovaries were examined for each data point. (**p<0.001, *p<0.04; U-test). (B–D′) All confocal images depict LL3 ovaries triple-stained for Vasa (green, B–D), Hts (magenta, B–D), and GFP (bam-GFP) (white, B′–D′). (B, B′) A y w control ovary. (C, C′) A goe^5–11/331 ovary. (D, D′) A rescued ovary (goe^5–11/331; nos>goe). Inset in C shows a 4-cell cyst harboring a U-shaped fusome (white arrowhead). White and magenta dashed lines in B′–D′ outline whole ovaries and GC/IC regions, respectively. Anterior is up. (E–F′) Dedifferentiation of germline cysts can be identified by the presence of a dot-like fusome in the ring canal remnant and bam-GFP expression (see Materials and Methods and Figure S4) [25]. Comparison of a dividing PGC (E, E′) and a dedifferentiating 2-cell cyst (F, F′) at LL3. Anti-Vasa (green) and anti-Hts (magenta) mark the germ cell cytoplasm and fusome, respectively. Both the PGC undergoing cytokinesis (E) and the 2-cell cyst undergoing dedifferentiation (F) had a dot-like fusome (white arrowheads) in the ring canal remnants (Figure S2), but only the 2-cell cyst expressed bam-GFP (E′, F′). (G) Average numbers of dedifferentiating 2-cell cysts in LL3 ovaries. There were 2-fold more dedifferentiating 2-cell cysts in goe^5–11/331 ovaries than in y w control ovaries (*p = 0.002, U-test). At LL3, dedifferentiation was observed mainly in 2-cell cysts, as observed in adult testis [49]. (H) The average number of PH3-positive PGCs in LL3 ovaries. No significant difference in the number of dividing PGCs was observed between goe^5–11/331 and y w control ovaries (p = 0.58, U-test). (I) The average number of TUNEL-positive germ cells in LL3 ovaries. No significant difference in the number of apoptotic germ cells was found between goe^5–11/331 and y w control ovaries (p = 0.18, U-test). The total number of ovaries examined is indicated at the bottom of each bar (G, H) or over each bar (I). Error bars in G–I indicate SEM. Scale bar: 20 µm.