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. 2019 Jan 16;8:e40806. doi: 10.7554/eLife.40806

Figure 1. Ova is a niche factor for GSCs and ovary development in Drosophila.

(a) Phase contrast images of dissected ovaries from flies of indicated genotypes. Scale bar, 500 μm. (b) A graph shows the total offspring number of indicated females (n = 20, 20, 16, 16, 20 respectively). (c) Representative image of germaria from indicated genotypes labeled by α-Spectrin (red), Vasa (green), and DAPI (blue). ova1/1 and ova1/4 ovaries have numerous spherical-shaped spectrosome-containing cells (tumorous) or are empty of germline cells (germless), indicated by lack of germline cell marker Vasa (green). A wild-type (WT) germarium is usually 2 GSCs localized to the anterior tip. Scale bar, 10 μm. (d) A graph shows the percentage of normal, germless, and tumorous germaria of indicated genotypes (n = 14, 20, 30, 117, 57 respectively). (e) c587ts > ova RNAi germarium accumulated GSC-like cells after shift to 29°C for 7 and 14 days. Scale bars, 10 μm. (f) Escort cell-specific expression of ova rescued oogenesis and GSC differentiation defect of ova1/4 females. Red, α-Spectrin; Green, Vasa (g) Quantification of GSC-like cell number in germaria of indicated genotypes (n = 25, 20, 30, 117, 57, 30, 21, 31, 23 respectively). (h) Confocal sections of germaria stained by indicated antibodies or reporter. Scale bars, 10 µm. (i) Quantitative results of pMad and Dad-lacZ positive cell numbers from germaria of indicated genotypes. Values are mean ± SEM.; n > 20. P values by two-tailed Student t-test.

Figure 1.

Figure 1—figure supplement 1. Ova is allelic to CG5694.

Figure 1—figure supplement 1.

(a) Top, a schematic drawing of mapping results of ova1 allele using deficiency kit. Two small deficiencies, Df(2L)ED737 and Df(2L)BSC144, both failed to complement ova1 (Figure 1—figure supplement 1a). Bottom, analysis of the overlapping regions of these deficiencies implicated five candidate genes. Gray box, ova genomic rescue fragment. (b) DNA sequencing revealed a nucleotic deletion in the coding region of CG5694. (c) Schematic drawings of Ova, Drosophila Ewg-PA, and mouse NRF-1 PA proteins. Black box, Nrf1 DNA-binding domain. Grey box, Nrf1 activator-binding domain. (d) Multiple sequence alignments of Nrf1-binding domain of Ova, Ewg, and NRF-1 by Clustal Omega. (e) Schematic drawings of two CG5694/ova alleles, ova1, and ova4. ova1 is a deletion allele generated using CRISPR-Cas9.

Figure 1—figure supplement 2. Ova is not cell- autonomously required for GSC differentiation.

Figure 1—figure supplement 2.

(a–d) Clonal analysis of wild-type and ova mutant GSCs. Anti-α-Spectrin staining was shown in red. The control clones marked by the absence of lacZ (anti-β-galactosidase, green) at 4 days or 14 days after clone induction (ACI) (a,b). The ova1 mutant clones at 4 days or 14 days ACI (c, d). Scale bar, 10 μm. (e) A ova1 germline clone with properly specified oocyte (anti-Orb, red). Scale bar, 20 μm. (f) A time course analysis of GSC maintenance rate in wild-type and ova mutant GSC clones. (g) Phase contrast images of germline-specific knock-down ova (ova GLKD) ovary. Scale bar, 500 μm. (h) Quantification of GSC-like cell number in ova GLKD germaria. Values are mean ± SEM.; n = 24.