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. 2015 Jul 1;403(1):57-68.
doi: 10.1016/j.ydbio.2015.03.024. Epub 2015 Apr 14.

The insulator protein Suppressor of Hairy wing is required for proper ring canal development during oogenesis in Drosophila

Shih-Jui Hsu  1 , Maria P Plata  1 , Ben Ernest  2 , Saghi Asgarifar  1 , Mariano Labrador  3
Affiliations

The insulator protein Suppressor of Hairy wing is required for proper ring canal development during oogenesis in Drosophila

Shih-Jui Hsu et al. Dev Biol. .

Abstract

Chromatin insulators orchestrate gene transcription during embryo development and cell differentiation by stabilizing interactions between distant genomic sites. Mutations in genes encoding insulator proteins are generally lethal, making in vivo functional analyses of insulator proteins difficult. In Drosophila, however, mutations in the gene encoding the Suppressor of Hairy wing insulator protein [Su(Hw)] are viable and female sterile, providing an opportunity to study insulator function during oocyte development. Whereas previous reports suggest that the function of Su(Hw) in oogenesis is independent of its insulator activity, many aspects of the role of Su(Hw) in Drosophila oogenesis remain unexplored. Here we show that mutations in su(Hw) result in smaller ring canal lumens and smaller outer ring diameters, which likely obstruct molecular and vesicle passage from nurse cells to the oocyte. Fluorescence microscopy reveals that lack of Su(Hw) leads to excess accumulation of Kelch (Kel) and Filament-actin (F-actin) proteins in the ring canal structures of developing egg chambers. Furthermore, we found that misexpression of the Src oncogene at 64B (Src64B) may cause ring canal development defects as microarray analysis and real-time RT-PCR revealed there is a three fold decrease in Src64B expression in su(Hw) mutant ovaries. Restoration of Src64B expression in su(Hw) mutant female germ cells rescued the ring phenotype but did not restore fertility. We conclude that loss of su(Hw) affects expression of many oogenesis related genes and down-regulates Src64B, resulting in ring canal defects potentially contributing to obstruction of molecular flow and an eventual failure of egg chamber organization.

Keywords: Chromatin insulators; Drosophila; Oogenesis; Ring canals; Src64b; Su(Hw); Suppressor of Hairy wing.

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Figures

Figure 1
Figure 1
su(Hw) mutant ovaries show oocyte and ring canal developmental defects. F-actin staining using Phalloidin on wildtype and su(Hw) mutant ovaries appears red and DAPI staining of DNA appears blue. The oocyte was observed at different stages of each genotype and the scale bar is 50 μm in each image. The dash lines highlight oocytes at stage 8 and 9.
Figure 2
Figure 2
Orb mislocalizes to the cytoplasm of nurse cells in stage 8 egg chambers from su(Hw) mutant ovaries. Egg chambers were stained with Orb antibody (green) and DAPI (blue). High concentration of Orb is observed in oocytes through oogenesis in wildtype (A). Oocyte localization of Orb is significantly reduced at stage 8 in su(Hw)v/TM6B heterozygotes (B), and is totally absent and limited to nurse cell cytoplasm in su(Hw)v/e04061 mutant (C). Arrowheads point to stage 8 oocytes. Scale bar is 50 μm.
Figure 3
Figure 3
Su(Hw) regulates expression of many genes involved in oogenesis. Heatmap of eighty-two genes related to ovary development with a significant change in expression greater than three-fold with up-regulation showed in red and down-regulation showed in blue. Corresponding fold changes of gene symbols are shown in supplementary table 1. Color key is shown at bottom with numbers indicating fold (A). Table in B shows a group of selected genes with their corresponding change in expression. qRT-PCR shows that expression of Src64B is reduced 30% in ovaries from su(Hw) mutants (C).
Figure 4
Figure 4
Rings from su(Hw) mutant egg chambers show significant morphological differences compared to wildtype. Ring canals in wildtype and mutant egg chambers are stained with antibody anti-Kelch in green and Phalloidin in red. Stage 8 egg chambers stained with Kelch are shown in wildtype (A) and su(Hw) mutant (B). Zoom in images of wildtype individual rings from dashed squares in A are shown in C and E. Zoom in images of su(Hw) mutant individual rings from dashed squares in B are shown in D and F. Isosurface images of individual rings in wildtype (G and H) and su(Hw) mutant (I and J) rings were generated using Leica Deblur software, and show the accumulation of actin in rings. The scale bars in egg chamber images represent 10 µm, and in individual ring images represent 1µm.
Figure 5
Figure 5
Ring canals in su(Hw) mutant egg chambers show excess accumulation of structural proteins. A cartoon ring image illustrating the structure and organization of a ring canal (A). Staining of rings at stage 6 using antibodies against ring structural proteins Kelch (B and C), Ovhts-RC (D and E) and F-actin (B-E). Kelch and Ovhts-RC show accumulation at the inner rim in su(Hw) mutants (C and E) but not in wildtype (B and D). Scale bars represent 1µm.
Figure 6
Figure 6
Ring canal development is defective in su(Hw) mutants. From stage 4 to 9, ring canals were detected using F-actin staining (A), and the sizes of rings at each stage were quantified using the measurement tool in ImageJ. The measurements of ring outer diameter in each genotype at stage 5 and 8 are shown in histograms (B and C). Scale bars represent 1µm.
Figure 7
Figure 7
Fusome development in su(Hw) mutants show no differences with wildtype. Ovary staining was performed using 1B1 antibody (green) to detect fusomes. F-actin is shown in red and DAPI in blue. Early germarium stage egg chambers in wildtype (A) and su(Hw) mutants (D) are shown. Dashed areas are shown in detail for wildtype (B and C) and mutant (E and F), showing no major differences in fusome organization between su(Hw) mutants and wildtype.
Figure 8
Figure 8
Restoration of Src64B expression rescues ring phenotype in su(Hw) mutants. Ring morphology was imaged using F-actin staining in wildtype (A), su(Hw)v/e04061 mutant (B), and nosGal4>> Src64B rescued su(Hw)v/e04061 mutant egg chambers (C). Scale bars represent 20 µm.
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