Figure 1.
Panoistic ovariole of B. germanica and dynamics of follicular cell proliferation.
(A) Panoistic ovariole from a 0-day old B. germanica adult female. Only the basal ovarian follicle (BOF) grows and matures in any one gonadotrophic cycle. Germinal vesicles are indicated by the arrowheads. Pe: pedicel, OM: oocyte membrane, FE: follicular epithelium; sBOF: sub-basal ovarian follicle, G: germaria, TF: terminal filament. LOv: lateral oviduct, s: stalk. Scale bar: 50 µm. (B) Follicular cell number and length of the basal ovarian follicle (BFL) during the sixth nymphal instar and previtellogenic period in the adult. Data are expressed as mean ± s.e.m. C: Progression of follicular cell cycle in the basal ovarian follicle from the first day of the sixth instar nymph (N6D0) to chorion formation in the adult (Ad7c), compared to the progress of the follicular cell cycle through the different eggs chambers (stages, S) in one ovariole of D. melanogaster.
Figure 2.
Expression pattern of the Hippo pathway components in ovaries of B. germanica.
mRNA expression pattern of the components of the Hippo pathway in ovaries in sixth nymphal instar and adult females during the first gonadotrophic cycle. The dashed line indicates the moult to adult. Data represent copies of mRNA per 1000 copies of BgActin-5c (relative expression) and are expressed as the mean ± s.e.m. (n = 3).
Figure 3.
BgHpo controls follicular cell proliferation in developing follicles.
Ovarioles from 5-day-old adult females treated with dsBgHpo (A), or dsMock (B). The posterior end of the ovarian follicles is towards the left. Scale bar: 100 µm. (C) Expression of BgHpo, BgWts and BgYki in ovaries from 5-day-old adult dsMock and dsBgHpo-treated females. BgHpo is significantly depleted in treated ovaries [P(H1) = 0.004]. (D) Expression of BgVg in fat body from 5-day-old adult dsMock and dsBgHpo-treated females (no significant difference [P(H1) = 0.098]. Data represent normalized values against the control (reference value = 1) (n = 3). (E, F, F′) Follicular epithelium from 0-day-old adult females in which cells are dividing actively in dsMock- (E and Figure S1) and dsBgHpo- (F, F′) treated females. Scale bar: 10 µm. (G, H, H′) Follicular cells from 5-day-old adult females. In the dsMock (G and Figure S1) females, mitosis is arrested and patency is already apparent. In the dsBgHpo (H, H′) females, the cells are still dividing and the cytoskeleton is disorganized Scale bar: 10 µm. (I, J, K) Follicular cells in adult ovarioles labelled with an anti-phospho-histone 3 antibody (PH3). (I) Follicular cells from 0-day-old and (J) 5-day-old dsMock-treated females. (K) Follicular cells from a 5-day-old dsBgHpo-treated female. Scale bar: 20 µm. (L) Optical section of (K), the arrow indicates the layer of endosymbiont bacteriocytes. Oo: ooplasm. Scale bar: 10 µm.
Figure 4.
Loss of BgHpo generates long-stalks activating BgN.
(A) Stalk from a 5-day-old dsMock adult female. F-actin labelling in the apical tips of the cells. Scale bar: 20 µm. (B, B′, B″) Long-stalk phenotype from a 5-day-old dsBgHpo adult female. (B) F-actin labelling is concentrated in the central axis of the stalk. (B′) The cells are smaller but greater in number. (B″) Merging of (B) and (B′). Scale bar: 20 µm. (C) Ovariole from 0-day-old adult female showing NICD localization. (C′) Merge image of NICD and F-Actin labelling. The arrow indicates the labelling in the germarium. Scale bar: 50 µm. (D) Follicular cells from 0-day-old dsMock adult female; the dividing cells show strong NICD labelling. Scale bar: 20 µm. (E) Stalk from 0-day-old dsMock adult female showing NICD labelling. Scale bar: 10 µm. (F) follicular cells from 5-day-old dsMock adult female. Scale bar: 20 µm (the optical section of 5-day-old dsMock adult female is showed in Figure 5D). (G, H, I) Optical section of a basal ovarian follicle showing the different localization of NICD between the apical (Ap) and basal pole (Ba) of the follicular cells. (G) 0-day-old dsMock adult female. (H) 0-day-old and (I) 5-day-old dsBgHpo adult female. Arrowheads in (I) indicate NICD accumulation in the ooplasm. Scale bar: 20 µm. (J) Stalk in a 5-day-old dsBgHpo adult female ovariole. Oo: ooplasm, FC: follicular cells. Scale bar: 10 µm. (K) Basal ovarian follicle from a 5-day-old dsBgHpo adult female. Ov: oviduct. Scale bar: 50 µm. (L) mRNA expression of different components of the Notch pathway in ovaries of 5-day-old dsBgHpo adult females. BgSer, BgHnt, BgCut and BgEya were upregulated (P(H1) = 0.0001, 0.010, 0.005 and 0.001 respectively); BgDl and BgN were not significantly affected. Data represent normalized values against dsMock controls (reference value = 1, dashed line) (n = 3). In all images the posterior pole of the basal follicle is towards the left.
Figure 5.
Notch depletion suppresses the stalk and reduces the long stalk phenotype in dsBgHpo knockdowns.
Ovariole from a 5-day-old adult female treated with dsBgN (A), and a basal ovarian follicle from a dsMock 5-day-old adult female (B). Scale bar: 100 µm. (C, D) Localization of Notch in ovarioles of 5-day-old adult dsBgN (C) and dsMock (D) females showing the position of the stalk (white box). In (C) there is no stalk. Notch was detected with an anti-NICD antibody. Scale bar: 20 µm. (E) mRNA relative expression of BgN, BgHnt, BgCut and BgEya in ovaries from 5-day-old adult dsMock- and dsBgN-treated females. BgN, BgCut and BgEya were significantly down-regulated (P(H1) = 0.0001) in dsBgN-treated ovaries. (F) mRNA relative expression of BgHpo, BgWts, BgYki and BgN in ovaries from 5-day-old adult dsMock and dsBgHpo + dsBgN-treated females. BgHpo and BgN were significantly reduced (P(H1) = 0.047 and 0.028 respectively). Data represent normalized values against the control (reference value = 1) (n = 3). (G) Ovariole from a 5-day-old, adult, double knockdown female (dsBgHpo + dsBgN). Scale bar: 100 µm. (H) Follicular epithelium where no mitoses were detected in opposite to dsBgHpo treatment (see Figure 3K). Scale bar: 50 µm. (I–J) Different range of stalk phenotypes resulting from the double knockdown. Scale bar in I and K: 50 µm, in J: 100 µm. In all images the posterior end of the basal ovarian follicle is towards the left, except in (C) in which it is towards the bottom.