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. 2017 Jul 27;8:15990. doi: 10.1038/ncomms15990

Figure 6. Neuronal activity promotes Actβ expression which drives hemocyte expansion.

Figure 6

(a–d) Carbachol induces dose-dependent transcriptional induction of an Actβ reporter. (a) Quantification of luciferase expression in larval fillets of Actβ-GAL4, UAS-CD4-tdGFP/UAS-luciferase animals, stimulated by increasing doses of carbachol as intact larvae (2.5 mgml−1; 10 mgml−1). Pools of 2-3 larval fillets per sample in biological replicates; carbachol-stimulated conditions were normalized to unstimulated condition (total n=4–6 per condition); statistics show average of four such normalized independent experiments. (b) Carbachol-induced increase of GFP expression in fillets of Actβ-GAL4, UAS-CD4-tdGFP larvae, signal quantification over three independent sets of images, normalized to unstimulated condition and averaged (n=3). (c,d) Sample set of images as in b. (c) Control, (d) 4 h stimulation with carbachol as intact larva, 2.5 mg ml−1. (e–g) Transient PNS silencing with UAS-Kir2.1 reduces GFP expression; induction for 22 h at 27 °C. (e) Fraction of larvae showing visible GFP expression in PNS neurons, genotype tubGAL80ts/+; Actβ-GAL4, UAS-CD4-tdGFP/+ (control) and tubGAL80ts/+; Actβ-GAL4, UAS-CD4-tdGFP/UAS-Kir 2.1 (experiment); n=20 to 22, average of 2 independent biological replicates. (f) Sample images of control and (g) Kir2.1 expressing larva. Note that Kir2.1 coexpression reduces GFP expression in PNS and CNS; CNS on left side (anterior). (h) Comparison of hemocyte expansion in ActβED80 mutant larvae and controls, genotypes HmlΔ-DsRed/+; ActβED80/ED80 and control HmlΔ-DsRed/+. Total hemocyte counts per larva of carbachol-treated cohorts (0.7 mg ml−1) and control food cohorts; n=6–8 per genotype and condition. (i,j) Comparison of control and ActβED80 mutant in the recruitment of hemocytes to HPs after short-term carbachol exposure (10 mg ml−1, 15 min), genotypes as in h, dorsal view. (k) Model. Actβ in sensory neuron-induced blood cell adaptation. PNS neuronal activity triggered by carbachol elevates Actβ expression in neurons, resulting in enhanced hemocyte proliferation and localization to HP. Neuronal silencing has opposite effects. Scale bars, c,d, 100 μm; f,g, 0.5 mm; i,j, 0.2 mm. Error bars represent s.d., and two-tailed t-test values correspond to NS (not significant) P>0.05; *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001.