Figure S5.
Conditional knockout of tra-2 leads to a neuron-specific degradation of TRA-1. (A) Expression of endogenous GFP::TRA-1 in hermaphrodites, males, and hermaphrodites with pan-neuronal tra-2 knockout. (B–D) Expression of endogenous GFP::TRA-1 in hermaphrodites with tra-2 specific knockout in cholinergic neurons (B and C) and GABAergic neurons (D). GFP::TRA-1 (green), cholinergic neurons (magenta in B and C, labeled by endogenous UNC-17::mkate2), and GABAergic neurons (magenta in D, labeled by RFP::RAB-3 driven by unc-25 promoter) are shown. White triangles and white dotted circles indicate cholinergic neurons in B and C and GABAergic neurons in D. Yellow arrows indicate the other neurons. GFP::TRA-1 in muscle cells is marked by asterisks (*). Scale bar, 10 μm. (E) Locomotion behavior analysis of hermaphrodites with tra-2 knockout in all neurons or specifically in cholinergic neurons. The minigene encoding Cre recombinase was driven by the rgef-1 and unc-17 promoters to knock out tra-2 gene in all neurons and cholinergic motor neurons, respectively. For control groups, we used transgenic animals with two loxP sites insertion but without Cre recombinase expression as control 1, and transgenic animals with pan-neuronal Cre recombinase expression, but without two loxP sites insertion as control 2. The averaged and individual crawling locomotion velocities were plotted. (F) Time course analysis of 1.4 mM aldicarb-induced paralysis in hermaphrodites expressing FEM-3 in all neurons (under rab-3 promoter), cholinergic neurons (under unc-17 promoter), GABAergic neurons (unc-25 promoter), and muscles (myo-3 promoters). (G) Time course analysis of 1.4 mM aldicarb-induced paralysis in males expressing TRA-2ic in all neurons (under rab-3 promoter), cholinergic neurons (under unc-17 promoter), GABAergic neurons (unc-25 promoter), and muscles (under myo-3 promoter). In E, data are presented as mean values ± SEM. Ns represent the number of animals tested. One-way ANOVA with post-hoc Bonferroni’s multiple comparisons. In F and G, for each of the group, n = 3 biologically independent samples; each sample contains ≥25 animals. Data are presented as mean values ± SEM. Two-way ANOVA comparing all of the time points. ***P < 0.001, n.s. not significant.

Conditional knockout of tra-2 leads to a neuron-specific degradation of TRA-1. (A) Expression of endogenous GFP::TRA-1 in hermaphrodites, males, and hermaphrodites with pan-neuronal tra-2 knockout. (B–D) Expression of endogenous GFP::TRA-1 in hermaphrodites with tra-2 specific knockout in cholinergic neurons (B and C) and GABAergic neurons (D). GFP::TRA-1 (green), cholinergic neurons (magenta in B and C, labeled by endogenous UNC-17::mkate2), and GABAergic neurons (magenta in D, labeled by RFP::RAB-3 driven by unc-25 promoter) are shown. White triangles and white dotted circles indicate cholinergic neurons in B and C and GABAergic neurons in D. Yellow arrows indicate the other neurons. GFP::TRA-1 in muscle cells is marked by asterisks (*). Scale bar, 10 μm. (E) Locomotion behavior analysis of hermaphrodites with tra-2 knockout in all neurons or specifically in cholinergic neurons. The minigene encoding Cre recombinase was driven by the rgef-1 and unc-17 promoters to knock out tra-2 gene in all neurons and cholinergic motor neurons, respectively. For control groups, we used transgenic animals with two loxP sites insertion but without Cre recombinase expression as control 1, and transgenic animals with pan-neuronal Cre recombinase expression, but without two loxP sites insertion as control 2. The averaged and individual crawling locomotion velocities were plotted. (F) Time course analysis of 1.4 mM aldicarb-induced paralysis in hermaphrodites expressing FEM-3 in all neurons (under rab-3 promoter), cholinergic neurons (under unc-17 promoter), GABAergic neurons (unc-25 promoter), and muscles (myo-3 promoters). (G) Time course analysis of 1.4 mM aldicarb-induced paralysis in males expressing TRA-2ic in all neurons (under rab-3 promoter), cholinergic neurons (under unc-17 promoter), GABAergic neurons (unc-25 promoter), and muscles (under myo-3 promoter). In E, data are presented as mean values ± SEM. Ns represent the number of animals tested. One-way ANOVA with post-hoc Bonferroni’s multiple comparisons. In F and G, for each of the group, n = 3 biologically independent samples; each sample contains ≥25 animals. Data are presented as mean values ± SEM. Two-way ANOVA comparing all of the time points. ***P < 0.001, n.s. not significant.

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