64 research outputs found

    Clonal analysis of three CTPsyn mutations in adult ovaries.

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    <p>(A–C) <i>CTPsyn<sup>d6099</sup></i>. (D–F) <i>CTPsyn<sup>e01207</sup></i>. (G–I) <i>CTPsyn<sup>d07411</sup></i> In all three mutations, no cytoophidia are detected in mutant mitotic clone (dotted lines) as indicated by lack of GFP, while cytoophidia (labelled by an antibody against CTPsyn in red) are clearly seen in adjacent wild-type germline cells (GFP positive). Scale bar, 20 µm.</p

    Expression profiles of <i>Drosophila</i> CTPsyn isoforms as revealed by qPCR.

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    <p>(A) embryos and larvae. (B) S2 cells and pupae. (C) Adult flies and tissues. WPP, white pre-pupae. P1–P4, pupal stages 1–4.</p

    Overexpression of CTPsyn isoform C induces the assembly of cytoophidia in <i>Drosophila</i> follicle cells.

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    <p>(A, B) Cytoophidia revealed by CTPsyn-GFP in protein trap line CA06746. (C, D) Long cytoophidia are induced in stage-10 follicle cells overexpressing CTPsyn isoform C. (E, F) Each follicle cell contains only one long cytoophidium in a stage-7 egg chamber. The cell boundary is outlined by membrane protein Hu-li tai shao (Hts). (G) Within the same stage 10 egg chamber, cytoophidia in follicle cells overexpressing CTPsyn isoform C (GFP+ cells) are much longer and thicker than those in neighbouring wild-type follicle cells (GFP− cells). While the average length of cytoophidia in GFP− cells is only 3.97±0.61 µm (n = 41), cytoophidia in GFP+ cells are 20.76±5.60 µm long on average (n = 32). Note that cytoophidia in GFP+ cells in E are much longer than those in C and D, which could be due to different expression of GAL4 drivers in follicle cells (actin-GAL4 in C, D and tubulin-GAL4 in E). Scale bars, 20 µm.</p

    CTPsyn mutants.

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    <p>(A) Genetic map of CTPsyn and the insertion site of <i>p-element</i> in three mutants <i>CTPsyn<sup>d6099</sup></i>, <i>CTPsyn<sup>e01207</sup></i> and <i>CTPsyn<sup>d07411</sup></i>. (B) Comparison of the expressions of CTPsyn isoforms in larvae from <i>y w</i> and three <i>CTPsyn</i> mutants (<i>CTPsyn<sup>d6099</sup></i>, <i>CTPsyn<sup>e01207</sup></i> and <i>CTPsyn<sup>d07411</sup></i>). While dramatic decrease of isofrom C expression occurs in <i>CTPsyn<sup>d6099</sup></i> and <i>CTPsyn<sup>e01207</sup></i>, isoform A expression is hugely diminished in <i>CTPsyn<sup>d07411</sup></i>.</p

    Overexpression of CTPsyn isoform C induces macro-cytoophidia in <i>Drosophila</i> embryos.

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    <p>(A, C, E) No macro-cytoophidium formation can be detected in <i>y w</i> embryos at stage 1 (A), 12 (C) and 15 (E). (B, D, F) Overexpressing CTPsyn isoform C induces macro-cytoophidia in embryos at stage 1 (B), 12 (D), and 15 (F). Segments in embryos are labeled by a transcription factor Engrailed (red). IsoC, isoform C. Scale bars, 20 µm.</p

    Overexpression of different CTPsyn isoforms in germline cells using nos-GAL4::VP16 driver.

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    <p>(A–C) Cytoophidia revealed by CTPsyn-GFP in an egg chamber from a protein trap line CA06746. (D–F) CTPsyn isoform A is localized in punctate structures in the nucleus and does not affect endogenous cytoophidia. (G–I) CTPsyn isoform B is dispersed in the cytoplasm of the egg chamber and does not affect endogenous cytoophidia. (J–L) C-terminal-tagged CTPsyn isoform C induced longer and more curved cytoophidia. (M–O) N-terminal-tagged CTPsyn isoform C affects cytoophidia morphology. Iso, Isoform. Scale bars, 20 µm.</p

    Relative expression of GFP mRNA normalised to <i>rp49</i> in Tub-GAL80<sup>TS</sup>; <i>Insc-GAL4/UAS-GFP</i> during the larval and adults experimental time courses.

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    (A) The GAL80TS system was used to eliminate any adult GAL4 expression. For larval experiments, a temperature sensitive GAL80 (GAL80TS) represses GAL4 at 19°C but becomes inactive at 29°C was used. Embryos were reared for 24 h at 29°C, during which GAL4 is expressed, then switched to 19°C to eliminate expression. (B) GFP RNA was measured in whole embryos and larval CNS over the time course analogous to that used in the locomotor and pupation assays. GFP expression was seen to diminish by 0 hrs. We detected no further GFP expression throughout the course of the experimental period. (C) For adult studies, larvae were reared at 29°C (GAL80TS is inactive; GAL4 is active) and then switched to 19°C (GAL80TS is active; GAL4 is repressed) at the start of pupation. (D) Relative expression of GFP mRNA normalised to rp49 in Tub-GAL80TS; Insc-GAL4/UAS-GFP larvae, pupae and adults. GFP RNA was measured in larval, pupae and adults over the time course analogous to that used in the adult activity and flight assays. The Larvae were switched from 29 to 19°C at the late L3 stage. GFP expression was seen to diminished during larval growth and maturation. We detected no GFP expression throughout the pupal and adult periods studied. (L2, 2nd Instar Larvae; L3, 3rd Instar Larvae). (TIF)</p

    Dual knockdown of <i>survival motor neuron</i> (SMN) in neuroblasts and differentiated neurons synergistically enhances the SMA model phenotypes.

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    (A) GAL4 nervous system expression patterns detailing the driver type and single and double-driver combinations used to knock down Smn. For negative controls Elav-GAL4 + Insc-GAL4 and nSyb-GAL4 + Insc-GAL4 were used. Elav-GAL4, Pros-GAL4 and Insc-GAL4 driven UAS-SMN-RNAiN4 were used as positive controls and compared with Elav-GAL4 + Insc-GAL4 and nSyb-GAL4 + Insc-GAL4 driven UAS-SMN-RNAiN4; (B) the genotypes were assessed for locomotor dysfunction at 72 h after hatching. Elav-GAL4 + Insc-GAL4 and nSyb-GAL4 + Insc-GAL4 driven UAS-SMN-RNAiN4 underwent reduced peristaltic contractions compared with negative and positive controls (***P n >20, Kruskal–Wallis test with Dunn’s multiple comparisons); (C) fly hatching number was then assessed to analyse survival to adulthood. Elav-GAL4 + Insc-GAL4 and nSyb-GAL4 + Insc-GAL4 driven UAS-SMN-RNAiN4 survived compared with negative and positive controls (***P n > 50; Kruskal–Wallis test with Dunn’s multiple comparisons). All error bars [SEM].</p

    The <i>Drosophila melanogaster</i> CG6854/CTPsyn gene locus encodes three isoforms.

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    <p>(A) Genome Browser view of the CG6854/CTPsyn gene locus (<a href="http://www.flybase.org" target="_blank">www.flybase.org</a>). In two protein trap lines (CA06746 and CA07332), GFP was trapped between the first and second exons of the CTPsyn isoform. (B) The protein map of three isoforms of CTPsyn. UTP binding sites are indicated by red triangles.</p

    CTPsyn mutants survive until larval stages.

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    <p><i>CTPsyn<sup>d6099</sup></i> homozygous mutants (−/− in A–E) and <i>CTPsyn<sup>e01207</sup></i> homozygous mutants (−/− in F–J) survive until 7 days after egg deposition (DAE) with slower growth compared to heterozygous mutants (+/−) or y w (+/+). (K–M) <i>CTPsyn<sup>d07411</sup></i> homozygous mutants (−/−) show severely delayed development, in comparison with heterozygous mutants (+/−and <i>y w</i> (+/+). <i>CTPsyn<sup>d07411</sup></i> (−/−) mutant larvae only survive until 5 days after egg deposition (DAE) with very little growth. Heterozygous mutants were balanced with TM6B Tb (+/− in all panels).</p
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