puastattb vectors  (Addgene inc)

Journal: iScience

Article Title: Tet controls axon guidance in early brain development through glutamatergic signaling

doi: 10.1016/j.isci.2024.109634

Figure Lengend Snippet: Tet regulates β axon guidance via insulin-producing cells (IPCs) and the Tet and Fmr1 mutant phenotypes can be rescued by overexpressing Gs2 in the IPCs or by MPEP treatment (A) 201Y-GAL4 driven membrane-tagged CD8::GFP expression pattern in 24 h APF brain, the driver expresses in MB neurons and midline cells (MLCs). vL: vertical lobe, mL: medial lobe. (B–G) 201Y-GAL4 driven CD8::GFP cells (green) and Gs2 expressing cells (magenta, anti-Gs2 staining). Gs2 is not expressed in MB neurons (B–D) but instead is expressed in MLCs (E–G, asterisks marks midline cells expressing Gs2). (H–J) MB α/β lobes stained with anti-Fas2 antibody in the Dilp2-GAL4 driver control (H), Dilp2-GAL4 driven Tet RNAi (I), or Dilp2-GAL4 driven Gs2 RNAi (J). (K) Quantification of β fusion brain in (H–J); n = 41–45. (L––N) Schematic representation of the experimental design for rescuing Tet or Fmr1 mutant phenotypes by expressing Gs2 under the control of Dilp2-GAL4. (O) Percentage of brains showing MB β axon midline crossing from wild type, Tet AXXC mutants, and Gs2 rescued Tet AXXC mutants; n = 37–49. (P) Percentage of brains from wild type, Fmr1 3 mutants, and Gs2 rescued Fmr1 3 mutants showing MB β axon midline crossing; n = 30–33. (Q) MPEP and glutamate treatment scheme for wild-type and Tet AXXC mutant flies. Third-instar larvae were reared on food containing MPEP or glutamate and changed to normal food after eclosion. (R) Percentage of wild type or Tet AXXC mutant brains showing MB β axon midline crossing after MPEP or glutamate treatment; n = 31–48. Chi-square test with ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. Scale bar: 50 μm. See also Figures S2 I‒S2L, and E.

Article Snippet: The pUASTattB-Gs2 vector was injected to y 1 w 67c23 ; PCaryPattP40 stock by the BestGene company to insert the UAS-Gs2 construct to attP40 landing site on the second chromosome.

Techniques: Mutagenesis, Membrane, Expressing, Staining

Journal: iScience

Article Title: Tet controls axon guidance in early brain development through glutamatergic signaling

doi: 10.1016/j.isci.2024.109634

Figure Lengend Snippet: Tet regulates β axon guidance by controlling glutamate synthetase 2 levels in the brain (A) Volcano plot of RNA sequencing results, showing differentially expressed RNAs in Tet AXXC and wild-type brains. Significant differences are defined by p -value <0.01 and fold change >2. (B) Representation of the glutamatergic and GABAergic pathways showing that Gs2 and Gat are the main enzyme and transporter, respectively, of the pathway. (C) Comparison of Gs2 and Gat transcript levels in wild-type or Tet AXXC and Fmr1 3 mutant brains assessed by RT-qPCR. RQ: relative quantification, error bars represent standard deviation (SD). (D) Western blot showing Gs2 protein levels in the brains of control (Act5C-Cas9) or CRISPR/Cas9 mutated Gs2 (Act5C-Cas9> Gs2 TKO) blotted with anti-Gs2 antibody. (E and F) MB α/β lobes stained with anti-Fas2 antibody showing Gs2 mutated brain exhibiting MB β lobe midline crossing (F, Act5C-Cas9> Gs2 TKO) not observed in control brains (E, Act5C-Cas9 Control). (G) Quantification of phenotype seen in (E andF); n = 30–38. (H–M) Gs2 RNAi KD driven by 201Y-GAL4 (K–M) or control (H–J). All brains express 201Y-GAL4 driven CD8::GFP. Brains were stained with anti-Fas2 and anti-GFP antibodies to visualize whole α/β lobes (Fas2, magenta) or core α/β (GFP, green). Gs2 KD using MB driver 201Y-GAL4 recapitulates the Tet AXXC midline crossing phenotype not observed in control. (N) Quantification of midline crossing phenotype; n = 35–39. Chi-square test with ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001. Scale bars: 50 μm. See also Figures S3 and A‒S4D, and Table S4 and .

Article Snippet: The pUASTattB-Gs2 vector was injected to y 1 w 67c23 ; PCaryPattP40 stock by the BestGene company to insert the UAS-Gs2 construct to attP40 landing site on the second chromosome.

Techniques: RNA Sequencing Assay, Comparison, Mutagenesis, Quantitative RT-PCR, Standard Deviation, Western Blot, CRISPR, Staining

Journal: iScience

Article Title: Tet controls axon guidance in early brain development through glutamatergic signaling

doi: 10.1016/j.isci.2024.109634

Figure Lengend Snippet:

Article Snippet: The pUASTattB-Gs2 vector was injected to y 1 w 67c23 ; PCaryPattP40 stock by the BestGene company to insert the UAS-Gs2 construct to attP40 landing site on the second chromosome.

Techniques: Recombinant, Plasmid Preparation, Protease Inhibitor, SYBR Green Assay, Clone Assay, CRISPR, Mutagenesis, Software, Imaging