Fig 1.
Experimental design of the iTRAQ 4-plex labeling.
(A) Diagram of a wild-type germarium. The arrangement of somatic and germline-derived cell types of the germarium and the three regions in which germaria are subdivided are shown. Region 2 is further subdivided into 2a and 2b, depending on the shape of the 16-cell cysts (in 2b they stretch across the width of the germarium). (B) iTRAQ experiment workflow. (C) Table of the ECM components identified in the iTRAQ analysis. Only the two Collagen IV α chains are significantly underrepresented in the experimental tissue. TFCs: terminal filament cells; CpCs: cap cells; ECs: escort cells; FSC: follicle stem cell; FCs: follicle cells; GSCs: germline stem cells; Diff. cysts: differentiating cystoblasts, 4- and 16-cell cysts; BM: basement membrane. See also S1 and S2 Figs, S1 and S2 Tables.
Fig 2.
In vivo zymography reveals increased collagenase activity in timp mutant ovaries.
Control and timp mutant ovaries were incubated in culture medium with human Collagen IV-FITC for two hours then fixed and stained to show filamentous actin (F-actin). Green fluorescence corresponds to Col IV-FITC molecules cleaved by zymogen activity in the ovaries. (A) Fluorescence along each ovariole was measured at multiple positions within the following regions: the TF (terminal filament, 12 measurements/ovariole), germarial regions 1-2a (15 measurements/ovariole), and 2b-3 (9 measurements/ovariole), interfollicular stalks (12 measurements/ovariole) and successive egg chamber s (ECh, 12 measurements/ovariole). 5 control and 3 experimental ovarioles were measured. (B) Control ovariole. Note the Col IV-FITC staining decorating the basement membrane and the slightly increased signal in the stalks. (C) timp mutant ovariole. Note that the Col IV-FITC staining, particularly in the anterior tip of the germarium and in the stalks, is stronger than the control. (D) Graph showing the average fluorescence intensity in arbitrary units along the anterior-posterior axis of the ovariole. Images are composites of several focal planes. P-values were obtained using a Student’s t-test. P values <0.05 were considered statistically significant (*:P<0.05, **:P<0.005, ***:P<0.0005). Unless otherwise stated, the genotypes used in this and the remaining figures are: control w1118;; timp28/TM3 and timp mutant w1118;; timp28/Df(3R) ED5472. See also S2 and S3 Figs.
Fig 3.
timp over-expression impairs cyst encapsulation without affecting cell fate acquisition.
(A, B) Ovarioles from control (no heat-shock) and experimental (daily heat-shock regime for 2 weeks post-eclosion) animals carrying UAS-timp and hs-Gal4. Arrows compare a normal egg chamber containing 16 germ cells with an abnormal, fused egg chamber encasing 32 germ cells. (C, D) Control (c587-Gal4) and experimental (c587-Gal4; UAS-timp) ovarioles grown at 18°C and then switched to 25°C for 9 days. Arrowheads indicate Lamin C-positive stalk cells. (E, F) Control (c587-Gal4) and experimental (c587-Gal4; UAS-timp) ovarioles grown at 18°C and aged 4 weeks at 25°C. Large, empty arrowheads show Mmp-1 accumulation from region 2. The ring of strong Mmp1 staining that coincides with the region contracting to pinch off a new egg chamber in controls is absent in timp-overexpressing germaria (small, empty arrowheads). Occasionally, the TF of experimental germaria show a prominent accumulation of Mmp1 staining, indicating that timp overexpression may affect Mmp1 localization in this region. Images can be composites of several focal planes.
Fig 4.
timp mutant ovarioles have altered physical properties.
(A) Subdivision of regions for AFM-based analysis of ovariole tissue stiffness. (B) Comparison of the apparent elastic modulus K at discrete points along 3–4 week old wild-type (n = 7) and timp null mutant ovarioles (n = 12) ex-vivo. Mutant ovarioles exhibited significantly lower levels of tissue stiffness throughout the regions tested. Differences were most severe in the germarium, early egg chambers and their associated interfollicular stalks. Results shown refer to an indentation depth of 0.2 μm. The image is a composite of several focal planes. ** = p values of two-tailed t-tests <0.01. See also S6 Fig.
Fig 5.
Ovaries from timp mutant females are smaller and lack integrity.
(A) Ovaries dissected from 2-week old control females. (B) Small disorganized ovaries dissected from a 2-week old timp null mutant. (C) Normal ovary morphology restored in ovaries dissected from 2-week old females lacking the endogenous timp gene but carrying a UASt-timp transgene. (D) Germarium from a 2-week old control ovary stained with Rhodamine-Phalloidin to visualize F-actin (red), Vasa to label de germline cells (green) and Hoescht to mark DNA (blue). (E) Germarium from a 2-week old mutant female stained as before. Note the marked decrease in the number of developing cysts within the germarium. (F) Ovary from a 4-week old mutant female stained to visualize the outline of the tissue and DNA. Empty arrowheads point to depleted ovarioles containing very few or no follicles. Arrows: terminal filament. Arrowheads: first interfollicular stalk. Images can be composites of several focal planes.
Fig 6.
timp is required to maintain germarium organization and shape.
Distribution of the stalk-cell marker Lamin C in control (A, A’) and timp mutant ovarioles (B, B’). (C) Normal germarium morphology in ovaries dissected from 2-week old control females. FasIII (green) is expressed by pre-follicle cells in the germarium. Phalloidin-labeled actin (red) and DNA (blue) are shown. (D) Abnormal rounded shape of germaria commonly seen in ovaries dissected from 2-week old mutant females. In most cases, despite the altered shape, overall organization of the terminal filament and FasIII expressing cells seem broadly similar to wild-type germaria. (E) Graph quantifying the changing shape of timp mutant germaria 1–7 and 14 days post-eclosion. Differences in length-to-width ratios between control and mutants are statistically significant (p value of two-tailed t-tests <0.001 for 14-day old germaria). (F) Abnormal organization of distinct domains of a timp mutant germarium. In extreme cases, the terminal filament is located to the posterior of region 3, adjacent to egg chambers already pinched off from the germarium. This highly unusual phenotype has not been described previously and is never observed in wild-type germaria. (G, H) The distribution of Escort Cells (labeled in green with the Tj protein) seems normal in aberrantly shaped germaria. The position of GSCs, as determined by their spectrosomes (anti-Hts staining, red) indicates that these cells are still associated to the terminal filament and cap cells, albeit in the example in (H) they are adjacent to follicle cells. Arrows: terminal filament. Arrowheads: first interfollicular stalk. Asterisks: GSC spectrosomes. Images can be composites of several focal planes. See also S2 Table.
Fig 7.
Mmp1 and Mmp2 are abnormally distributed in timp mutant ovaries.
(A, A’) Pattern of Mmp1 accumulation in control ovaries. (B, B’) timp null mutant ovaries (timp28/Df ED5472) displayed a diffused Mmp1 staining, particularly in the stalks. (C, C’) Mmp2::EGFP is localized to the anterior half of the germarium in control ovaries. (D, D’) Mmp2::EGFP localization at the anterior tip of timp mutant germaria was not detected. Arrows: terminal filament. Arrowheads: interfollicular stalk. (E, E’) Double in situ hybridization and antibody staining to visualize the pattern of expression of timp mRNA in a wild-type germarium (E and red signal in E’) and the spectrosome marker Hts (green in E’). DNA was counterstained in blue. timp was strongly expressed at the tip of region 1, were GSCs and cystoblasts reside, and in region 2. Images can be composites of several focal planes.