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The DExH box helicase domain of spindle-E is necessary for retrotransposon silencing and axial patterning during Drosophila oogenesis.

Ott KM, Nguyen T, Navarro C - G3 (Bethesda) (2014)

Bottom Line: Of the alleles that express detectable Spindle-E protein, we found that five had mutations in the DExH box domain.The phenotype of many of these alleles is as severe as the strongest spindle-E phenotype, whereas alleles with mutations in other regions of Spindle-E did not affect these processes as much.From these data we conclude that the DExH box domain of Spindle-E is necessary for its function in the piRNA pathway and retrotransposon silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Biomedical Genetics, Boston University School of Medicine, Boston, Massachusetts 02118 Graduate Program in Genetics and Genomics, Boston University School of Medicine, Boston, Massachusetts 02118.

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Dynein motor complex aggregates form in some, but not all, spn-E mutant ovaries. spn-E mutant germline clones are marked by the absence of GFP. All egg chambers were stained with α-GFP (green) to mark clones, α-Egalitarian (EGL) (red), and the DNA dye DAPI. In wild-type egg chambers, EGL is dispersed throughout the nurse cells and localizes to the oocyte (A-A′′). spn-E4-48 (B-B′′), spn-E23-17 (C-C′′), as well as spn-E9A2-17 and spn-E66-21 (not shown) show wild-type EGL localization. In spn-E155-55 DExH box mutant egg chambers (D-D′′), EGL forms aggregates throughout the egg chamber. This phenotype is present in spn-E9A9-18 mutant egg chambers (E-E′′) as well as the DExH box alleles: spn-E2A9-14, spn-E7G2-5, spn-E8D4-11, and the remainder of the spn-E alleles that do not express detectable protein (not shown). Note the small size of the oocyte in spn-E155-55 and spn-E9A9-18 egg chambers (arrow in D′ and E′). Scale bars = 20 μm.
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fig3: Dynein motor complex aggregates form in some, but not all, spn-E mutant ovaries. spn-E mutant germline clones are marked by the absence of GFP. All egg chambers were stained with α-GFP (green) to mark clones, α-Egalitarian (EGL) (red), and the DNA dye DAPI. In wild-type egg chambers, EGL is dispersed throughout the nurse cells and localizes to the oocyte (A-A′′). spn-E4-48 (B-B′′), spn-E23-17 (C-C′′), as well as spn-E9A2-17 and spn-E66-21 (not shown) show wild-type EGL localization. In spn-E155-55 DExH box mutant egg chambers (D-D′′), EGL forms aggregates throughout the egg chamber. This phenotype is present in spn-E9A9-18 mutant egg chambers (E-E′′) as well as the DExH box alleles: spn-E2A9-14, spn-E7G2-5, spn-E8D4-11, and the remainder of the spn-E alleles that do not express detectable protein (not shown). Note the small size of the oocyte in spn-E155-55 and spn-E9A9-18 egg chambers (arrow in D′ and E′). Scale bars = 20 μm.

Mentions: In wild-type egg chambers, dynein motor complex proteins are transported from the nurse cells to the oocyte, ultimately leading to a high concentration of protein in the oocyte and a more diffuse pattern in the supporting nurse cells (Figure 3A). This is in contrast to piRNA pathway mutant egg chambers where large aggregates containing components of the dynein motor machinery form in the nurse cells (Navarro et al. 2009). These aggregates contain the dynein core motor complex as well as the accessory proteins, Egalitarian (EGL) and Bicaudal-D (BIC-D), and may be sites of retrotransposon sequestration or degradation. We examined ovaries from the different spn-E mutants and determined whether dynein aggregates form by immunohistochemistry using EGL as a marker for the aggregates. Similar to what we found with the D/V patterning phenotype, those spn-E mutant flies that lay eggs with the most severe D/V patterning defects formed dynein motor complex aggregates (Figure 3, D and E), whereas the spn-E mutants that had milder D/V patterning defects did not form ovarian dynein aggregates (Figure 3, B and C). Additionally, in the mutants with the most severe dynein aggregation phenotype, the oocyte failed to grow properly, whereas in the less severe mutants the oocyte appeared to grow normally (Figure 3). The failure of oocyte growth could indicate a failure of the nurse cells to transport their contents into the oocyte, which could result in collapsed eggs. The lack of oocyte growth we found correlates well with the percentage of collapsed eggs laid by the most severe mutants. As above, we confirmed the mutant phenotypes that we saw in homozygous spn-E mutant clones in hemizygous ovaries and found the same phenotypes (Figure S3).


The DExH box helicase domain of spindle-E is necessary for retrotransposon silencing and axial patterning during Drosophila oogenesis.

Ott KM, Nguyen T, Navarro C - G3 (Bethesda) (2014)

Dynein motor complex aggregates form in some, but not all, spn-E mutant ovaries. spn-E mutant germline clones are marked by the absence of GFP. All egg chambers were stained with α-GFP (green) to mark clones, α-Egalitarian (EGL) (red), and the DNA dye DAPI. In wild-type egg chambers, EGL is dispersed throughout the nurse cells and localizes to the oocyte (A-A′′). spn-E4-48 (B-B′′), spn-E23-17 (C-C′′), as well as spn-E9A2-17 and spn-E66-21 (not shown) show wild-type EGL localization. In spn-E155-55 DExH box mutant egg chambers (D-D′′), EGL forms aggregates throughout the egg chamber. This phenotype is present in spn-E9A9-18 mutant egg chambers (E-E′′) as well as the DExH box alleles: spn-E2A9-14, spn-E7G2-5, spn-E8D4-11, and the remainder of the spn-E alleles that do not express detectable protein (not shown). Note the small size of the oocyte in spn-E155-55 and spn-E9A9-18 egg chambers (arrow in D′ and E′). Scale bars = 20 μm.
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Related In: Results  -  Collection

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Show All Figures
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fig3: Dynein motor complex aggregates form in some, but not all, spn-E mutant ovaries. spn-E mutant germline clones are marked by the absence of GFP. All egg chambers were stained with α-GFP (green) to mark clones, α-Egalitarian (EGL) (red), and the DNA dye DAPI. In wild-type egg chambers, EGL is dispersed throughout the nurse cells and localizes to the oocyte (A-A′′). spn-E4-48 (B-B′′), spn-E23-17 (C-C′′), as well as spn-E9A2-17 and spn-E66-21 (not shown) show wild-type EGL localization. In spn-E155-55 DExH box mutant egg chambers (D-D′′), EGL forms aggregates throughout the egg chamber. This phenotype is present in spn-E9A9-18 mutant egg chambers (E-E′′) as well as the DExH box alleles: spn-E2A9-14, spn-E7G2-5, spn-E8D4-11, and the remainder of the spn-E alleles that do not express detectable protein (not shown). Note the small size of the oocyte in spn-E155-55 and spn-E9A9-18 egg chambers (arrow in D′ and E′). Scale bars = 20 μm.
Mentions: In wild-type egg chambers, dynein motor complex proteins are transported from the nurse cells to the oocyte, ultimately leading to a high concentration of protein in the oocyte and a more diffuse pattern in the supporting nurse cells (Figure 3A). This is in contrast to piRNA pathway mutant egg chambers where large aggregates containing components of the dynein motor machinery form in the nurse cells (Navarro et al. 2009). These aggregates contain the dynein core motor complex as well as the accessory proteins, Egalitarian (EGL) and Bicaudal-D (BIC-D), and may be sites of retrotransposon sequestration or degradation. We examined ovaries from the different spn-E mutants and determined whether dynein aggregates form by immunohistochemistry using EGL as a marker for the aggregates. Similar to what we found with the D/V patterning phenotype, those spn-E mutant flies that lay eggs with the most severe D/V patterning defects formed dynein motor complex aggregates (Figure 3, D and E), whereas the spn-E mutants that had milder D/V patterning defects did not form ovarian dynein aggregates (Figure 3, B and C). Additionally, in the mutants with the most severe dynein aggregation phenotype, the oocyte failed to grow properly, whereas in the less severe mutants the oocyte appeared to grow normally (Figure 3). The failure of oocyte growth could indicate a failure of the nurse cells to transport their contents into the oocyte, which could result in collapsed eggs. The lack of oocyte growth we found correlates well with the percentage of collapsed eggs laid by the most severe mutants. As above, we confirmed the mutant phenotypes that we saw in homozygous spn-E mutant clones in hemizygous ovaries and found the same phenotypes (Figure S3).

Bottom Line: Of the alleles that express detectable Spindle-E protein, we found that five had mutations in the DExH box domain.The phenotype of many of these alleles is as severe as the strongest spindle-E phenotype, whereas alleles with mutations in other regions of Spindle-E did not affect these processes as much.From these data we conclude that the DExH box domain of Spindle-E is necessary for its function in the piRNA pathway and retrotransposon silencing.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Biomedical Genetics, Boston University School of Medicine, Boston, Massachusetts 02118 Graduate Program in Genetics and Genomics, Boston University School of Medicine, Boston, Massachusetts 02118.

Show MeSH
Related in: MedlinePlus