Fig 1.
Neuronal (elav-GAL4) RNAi knockdown screen of Drosophila genes encoding voltage gated potassium channels.
A) Cumulative percent paralyzed flies over time, with dotted line indicating 50% level. B) Direct comparison of time at which 50% of flies are paralyzed, using the same data as in A. n = 12 vials/genotype, 10 flies per vial, placed in 41–42°C water. ANOVA (p < 0.0001) followed by Dunnett’s post hoc test was used to determine groups significantly different than the Luciferase control. Data are presented as mean ±SEM, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2.
sei mutants display heat hypersensitivity.
A-B) Acute heat assay paralysis behavior of homozygous and heterozygous seiP mutants and controls. n = 4 vials/genotype, 10 flies per vial, placed in 41–42°C water. Letters above bars represent significantly different groups by ANOVA (p < 0.001) followed by Tukey’s post hoc analysis (p < 0.01). C-D) Gradual heat assay paralysis behavior of seiP mutant flies and controls. n = 12 vials, 10 flies per vial. E) Larval behavioral responses when placed on an agar surface heated to 37 °C, in seiP mutants and controls. n = 9. F) Response time of seiP mutant larvae and controls to 50°C thermal nociception assay. G-H) seiP mutant and wildtype larvae distance travelled in five minute trials at room temperature (25°; n = 6) or cold temperature (13°; n = 12). I-J) Survival of adult male seiP mutants and controls exposed to sucrose alone or with H2O2 (n = 60 per group). Significance determined via log-rank (Mantel-Cox) test. Data are presented as mean ±SEM, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test unless otherwise stated).
Fig 3.
sei expression is required in neurons and glia for the homeostatic response to acute heat stress.
A cell-type specific RNAi-dependent sei knockdown screen. A-B) Neurons (elav-GAL4); C-D) glia (repo-GAL4); E-F) heart (hand-GAL4); G-H) muscle (BG57-GAL4). n = 12 vials/genotype, 10 flies per vial, placed in 41–42°C water. Data are presented as mean ±SEM, **p < 0.01, ***p < 0.001 (Student’s t-test).
Fig 4.
sei promoter drives expression in neurons and glia.
A-B) Representative confocal z-stack images of an adult brain (A) and ventral nerve cord (B) from sei-LexA>LexAOp-GFPnls (green) flies. C-E) Overlap of sei-LexA>LexAOp-GFPnls (green) and glial marker RepoGAL4>UAS-RedStinger (magenta). Arrows point to examples of co-labeled cells. Scalebars are 20μm.
Fig 5.
sei expression is specifically required in cholinergic, glutamatergic and octopaminergic neurons for response to acute heat stress.
Neuronal subtype specific RNAi-dependent sei knockdown screen. A-B) Cholinergic neurons (ChAT-GAL4) n = 12 vials/genotype; C-D) Glutamatergic neurons (VGlut-GAL4) n = 6; E-F) GABAergic neurons (Gad1-GAL4) n = 12; G-H) Octopaminergic neurons (Tbh-GAL4) n = 12; I-J) Dopaminergic neurons (ple-GAL4) n = 12; K-L) Serotonergic neurons (Trh-GAL4) n = 12; M-N) Peptidergic neurons (C929-GAL4) n = 6; O-P) Sensory neurons (PO163-GAL4) n = 6. All experiments conducted with 10 flies per vial, placed in 41–42°C water. Data are presented as mean ±SEM, **p < 0.01, ***p < 0.001 (Student’s t-test).
Fig 6.
Organismal response to acute heat stress depends on sei action in neuropile ensheathing and perineurial glia.
Glia subtype specific RNAi-dependent sei knockdown screen. A-B) Neuropile ensheathing glia (R56F03-GAL4) n = 6 vials/genotype; C-D) Perineurial glia (R85G01-GAL4) n = 12; E-F) Astrocyte-like glia (alrm-GAL4) n = 12; G-H) Subperineurial glia (R54C07-GAL4) n = 12; I-J) Cortex glia (R54H02-GAL4) n = 12; K-L) Tract ensheathing glia (R75H03-GAL4) n = 12. All experiments conducted with 10 flies per vial, placed in 41–42°C water. Data are presented as mean ±SEM, *p < 0.05, **p < 0.01 (Student’s t-test).
Fig 7.
Effects of sei mutation on NMJ synaptic morphology in larvae and adults.
A) SeiP mutants display increased branching at the larval neuromuscular junction (NMJ). n = 11–12. B) No difference in the number of boutons was observed between seiP mutants and controls at the larval NMJ. n = 11-12/genotype. C-D) Representative 63x confocal z-stack images of larval NMJs stained with anti-HRP antibody labeling neuronal processes. E-F) No differences were observed in number of branches or bouton number at the NMJ of adult ventral abdominal muscles. n = 7/genotype. G-H) Representative 63x confocal z-stack images of adult abdominal NMJs stained with anti-HRP antibody labeling neuronal processes. Data are presented as mean ±SEM, **p < 0.01 (Student’s t-test). Scale bars are 20μm.
Fig 8.
Nervous system expression of seiGFP in adults and larvae is primarily localized to neuronal axons.
A) Strategy for the generation of the seiGFP allele by using CRISPR/Cas9-dependent DNA editing. Stars represent single nucleotide substitutions in the PAMs of sgRNA sites. B-C) Behavior of seiGFP and wildtype flies in the acute heat assay. n = 12. Data was analyzed using Student’s t-test and presented as mean ±SEM. D-G) Representative 20x confocal z-stack images of the adult brain (D-E) and adult ventral nerve cord (F-G) following immunostaining for seiGFP (green) and nc82 (magenta). H-I) 20x confocal z-stack images of the larval brain and ventral nerve cord of seiGFP (green) and DAPI (blue). Note that low levels of green autofluorescence are observed in the w1118 larval brain. J-M) 40x confocal z-stack images of the adult antennal lobe (J), optic lobe (K) and ventral nerve cord (L) with seiGFP (green), nc82 (magenta) and DAPI (blue). M) 40x confocal z-stack images of the larval ventral nerve cord of seiGFP (green) and DAPI (blue). Scalebars are 20μm. White arrows point to neuronal tracks with enriched seiGFP localization.