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UNC79 and UNC80, putative auxiliary subunits of the NARROW ABDOMEN ion channel, are indispensable for robust circadian locomotor rhythms in Drosophila.

Lear BC, Darrah EJ, Aldrich BT, Gebre S, Scott RL, Nash HA, Allada R - PLoS ONE (2013)

Bottom Line: We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels.Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain.Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
In the fruit fly Drosophila melanogaster, a network of circadian pacemaker neurons drives daily rhythms in rest and activity. The ion channel NARROW ABDOMEN (NA), orthologous to the mammalian sodium leak channel NALCN, functions downstream of the molecular circadian clock in pacemaker neurons to promote behavioral rhythmicity. To better understand the function and regulation of the NA channel, we have characterized two putative auxiliary channel subunits in Drosophila, unc79 (aka dunc79) and unc80 (aka CG18437). We have generated novel unc79 and unc80 mutations that represent strong or complete loss-of-function alleles. These mutants display severe defects in circadian locomotor rhythmicity that are indistinguishable from na mutant phenotypes. Tissue-specific RNA interference and rescue analyses indicate that UNC79 and UNC80 likely function within pacemaker neurons, with similar anatomical requirements to NA. We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels. Yet despite this relationship, we find that the requirement for unc79 and unc80 in circadian rhythmicity cannot be bypassed by increasing NA protein expression, nor can these putative auxiliary subunits substitute for each other. These data indicate functional requirements for UNC79 and UNC80 beyond promoting channel subunit expression. Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain. Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

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RNAi knockdown of na, unc79, or unc80 in all pacemaker neurons results in anticipation defects.Normalized activity profiles from adult male populations averaged over four days of LD entrainment. White bars represent light phase; black bars indicate dark phase. Error bars represent standard error of the mean. Arrows indicate morning anticipation (black) and evening anticipation (gray). The genotypes represented in the left panels are timGAL4/ +; UAS-dcr2/ +, heterozygous for the following insertions from the Vienna Drosophila RNAi Center (VDRC): (top panel) no RNAi  =  control strain attp VIE-260B (n =  55), MAI  =  1.5+/−0.1, EAI  =  2.6+/−0.1; (second panel) na =  103754 (n  =  43), MAI  =  0.5+/−0.1, EAI  =  0.4+/−0.1; (third panel) unc79  =  108132 (n =  44), MAI  =  0.3+/−0.1, EAI  =  0.3+/−0.1; (bottom panel) unc80  =  108934 (n =  42), MAI  =  0.7+/−0.1, EAI  =  0.3+/−0.1. Genotypes represented in the right panels are pdfGAL4 UAS-dcr2/+ heterozygous for the same VDRC strains: (top panel) attp VIE-260B (n =  32), MAI  =  1.3+/−0.1, EAI  =  2.4+/−0.2; (second panel) na =  103754 (n  =  33), MAI  =  1.0+/−0.1, EAI  =  2.3+/−0.2; (third panel) unc79  =  108132 (n =  37), MAI  =  1.5+/−0.2, EAI  =  2.1+/−0.2; (bottom panel) unc80  =  108934 (n =  35), MAI  =  1.3+/−0.2, EAI  =  2.3+/−0.2. Both MAI and EAI differ significantly among the timGAL4/ +; UAS-dcr2/ + genotypes (Kruskal-Wallis one-way ANOVA, 3 degrees of freedom, P<0.001), and each RNAi genotype exhibits significantly lower MAI and EAI than the control strain (Dunn’s method, P<0.05). MAI and EAI values for each timGAL4 UAS-dcr2 RNAi strain are either lower or not significantly different from values calculated from strong mutant alleles for the corresponding gene (Kruskal-Wallis one-way ANOVA, 1-2 degrees of freedom). No significant differences in MAI or EAI are observed among pdfGAL4 UAS-dcr2/ + genotypes (Kruskal-Wallis one-way ANOVA, 3 degrees of freedom, P > =  0.146).
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pone-0078147-g002: RNAi knockdown of na, unc79, or unc80 in all pacemaker neurons results in anticipation defects.Normalized activity profiles from adult male populations averaged over four days of LD entrainment. White bars represent light phase; black bars indicate dark phase. Error bars represent standard error of the mean. Arrows indicate morning anticipation (black) and evening anticipation (gray). The genotypes represented in the left panels are timGAL4/ +; UAS-dcr2/ +, heterozygous for the following insertions from the Vienna Drosophila RNAi Center (VDRC): (top panel) no RNAi  =  control strain attp VIE-260B (n =  55), MAI  =  1.5+/−0.1, EAI  =  2.6+/−0.1; (second panel) na =  103754 (n  =  43), MAI  =  0.5+/−0.1, EAI  =  0.4+/−0.1; (third panel) unc79  =  108132 (n =  44), MAI  =  0.3+/−0.1, EAI  =  0.3+/−0.1; (bottom panel) unc80  =  108934 (n =  42), MAI  =  0.7+/−0.1, EAI  =  0.3+/−0.1. Genotypes represented in the right panels are pdfGAL4 UAS-dcr2/+ heterozygous for the same VDRC strains: (top panel) attp VIE-260B (n =  32), MAI  =  1.3+/−0.1, EAI  =  2.4+/−0.2; (second panel) na =  103754 (n  =  33), MAI  =  1.0+/−0.1, EAI  =  2.3+/−0.2; (third panel) unc79  =  108132 (n =  37), MAI  =  1.5+/−0.2, EAI  =  2.1+/−0.2; (bottom panel) unc80  =  108934 (n =  35), MAI  =  1.3+/−0.2, EAI  =  2.3+/−0.2. Both MAI and EAI differ significantly among the timGAL4/ +; UAS-dcr2/ + genotypes (Kruskal-Wallis one-way ANOVA, 3 degrees of freedom, P<0.001), and each RNAi genotype exhibits significantly lower MAI and EAI than the control strain (Dunn’s method, P<0.05). MAI and EAI values for each timGAL4 UAS-dcr2 RNAi strain are either lower or not significantly different from values calculated from strong mutant alleles for the corresponding gene (Kruskal-Wallis one-way ANOVA, 1-2 degrees of freedom). No significant differences in MAI or EAI are observed among pdfGAL4 UAS-dcr2/ + genotypes (Kruskal-Wallis one-way ANOVA, 3 degrees of freedom, P > =  0.146).

Mentions: To determine the anatomical requirements for unc79 and unc80 in circadian behavior, we utilized tissue-specific RNA interference (RNAi). We find that pan-neuronal knockdown of na, unc79, or unc80 using elavGAL4 driven expression of UAS-RNAi constructs results in a strong decrease in protein expression for the targeted gene, particularly when these constructs are co-expressed with the RNAi component dicer2 (UAS-dcr2; Figure S2A). To address whether unc79 and unc80 function is required in pacemaker neurons, we crossed these UAS-RNAi lines to circadian GAL4 strains. Expression of unc79, unc80, or na RNAi using the broad circadian driver timelessGAL4 (timGAL4) plus UAS-dcr2 produces strong LD and DD rhythmicity defects (Figure 2, left panels, P<0.05; Table 2, P<0.001), which are comparable to the strongest phenotypes observed in the corresponding mutant strains (Figure 1; Table 1, P > =  0.385). Restricting RNAi expression to the PDF+ pacemaker neurons using pdfGAL4 UAS-dcr2 produces strong defects in DD rhythmicity compared to control strains (Table 2, P<0.001), but does not clearly alter LD behavior (Figure 2, right panels, P > =  0.146). These data suggest that both unc79 and unc80 are required broadly among pacemaker neurons to promote locomotor rhythmicity. Moreover, both genes appear to have similar anatomical requirements as na itself.


UNC79 and UNC80, putative auxiliary subunits of the NARROW ABDOMEN ion channel, are indispensable for robust circadian locomotor rhythms in Drosophila.

Lear BC, Darrah EJ, Aldrich BT, Gebre S, Scott RL, Nash HA, Allada R - PLoS ONE (2013)

RNAi knockdown of na, unc79, or unc80 in all pacemaker neurons results in anticipation defects.Normalized activity profiles from adult male populations averaged over four days of LD entrainment. White bars represent light phase; black bars indicate dark phase. Error bars represent standard error of the mean. Arrows indicate morning anticipation (black) and evening anticipation (gray). The genotypes represented in the left panels are timGAL4/ +; UAS-dcr2/ +, heterozygous for the following insertions from the Vienna Drosophila RNAi Center (VDRC): (top panel) no RNAi  =  control strain attp VIE-260B (n =  55), MAI  =  1.5+/−0.1, EAI  =  2.6+/−0.1; (second panel) na =  103754 (n  =  43), MAI  =  0.5+/−0.1, EAI  =  0.4+/−0.1; (third panel) unc79  =  108132 (n =  44), MAI  =  0.3+/−0.1, EAI  =  0.3+/−0.1; (bottom panel) unc80  =  108934 (n =  42), MAI  =  0.7+/−0.1, EAI  =  0.3+/−0.1. Genotypes represented in the right panels are pdfGAL4 UAS-dcr2/+ heterozygous for the same VDRC strains: (top panel) attp VIE-260B (n =  32), MAI  =  1.3+/−0.1, EAI  =  2.4+/−0.2; (second panel) na =  103754 (n  =  33), MAI  =  1.0+/−0.1, EAI  =  2.3+/−0.2; (third panel) unc79  =  108132 (n =  37), MAI  =  1.5+/−0.2, EAI  =  2.1+/−0.2; (bottom panel) unc80  =  108934 (n =  35), MAI  =  1.3+/−0.2, EAI  =  2.3+/−0.2. Both MAI and EAI differ significantly among the timGAL4/ +; UAS-dcr2/ + genotypes (Kruskal-Wallis one-way ANOVA, 3 degrees of freedom, P<0.001), and each RNAi genotype exhibits significantly lower MAI and EAI than the control strain (Dunn’s method, P<0.05). MAI and EAI values for each timGAL4 UAS-dcr2 RNAi strain are either lower or not significantly different from values calculated from strong mutant alleles for the corresponding gene (Kruskal-Wallis one-way ANOVA, 1-2 degrees of freedom). No significant differences in MAI or EAI are observed among pdfGAL4 UAS-dcr2/ + genotypes (Kruskal-Wallis one-way ANOVA, 3 degrees of freedom, P > =  0.146).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818319&req=5

pone-0078147-g002: RNAi knockdown of na, unc79, or unc80 in all pacemaker neurons results in anticipation defects.Normalized activity profiles from adult male populations averaged over four days of LD entrainment. White bars represent light phase; black bars indicate dark phase. Error bars represent standard error of the mean. Arrows indicate morning anticipation (black) and evening anticipation (gray). The genotypes represented in the left panels are timGAL4/ +; UAS-dcr2/ +, heterozygous for the following insertions from the Vienna Drosophila RNAi Center (VDRC): (top panel) no RNAi  =  control strain attp VIE-260B (n =  55), MAI  =  1.5+/−0.1, EAI  =  2.6+/−0.1; (second panel) na =  103754 (n  =  43), MAI  =  0.5+/−0.1, EAI  =  0.4+/−0.1; (third panel) unc79  =  108132 (n =  44), MAI  =  0.3+/−0.1, EAI  =  0.3+/−0.1; (bottom panel) unc80  =  108934 (n =  42), MAI  =  0.7+/−0.1, EAI  =  0.3+/−0.1. Genotypes represented in the right panels are pdfGAL4 UAS-dcr2/+ heterozygous for the same VDRC strains: (top panel) attp VIE-260B (n =  32), MAI  =  1.3+/−0.1, EAI  =  2.4+/−0.2; (second panel) na =  103754 (n  =  33), MAI  =  1.0+/−0.1, EAI  =  2.3+/−0.2; (third panel) unc79  =  108132 (n =  37), MAI  =  1.5+/−0.2, EAI  =  2.1+/−0.2; (bottom panel) unc80  =  108934 (n =  35), MAI  =  1.3+/−0.2, EAI  =  2.3+/−0.2. Both MAI and EAI differ significantly among the timGAL4/ +; UAS-dcr2/ + genotypes (Kruskal-Wallis one-way ANOVA, 3 degrees of freedom, P<0.001), and each RNAi genotype exhibits significantly lower MAI and EAI than the control strain (Dunn’s method, P<0.05). MAI and EAI values for each timGAL4 UAS-dcr2 RNAi strain are either lower or not significantly different from values calculated from strong mutant alleles for the corresponding gene (Kruskal-Wallis one-way ANOVA, 1-2 degrees of freedom). No significant differences in MAI or EAI are observed among pdfGAL4 UAS-dcr2/ + genotypes (Kruskal-Wallis one-way ANOVA, 3 degrees of freedom, P > =  0.146).
Mentions: To determine the anatomical requirements for unc79 and unc80 in circadian behavior, we utilized tissue-specific RNA interference (RNAi). We find that pan-neuronal knockdown of na, unc79, or unc80 using elavGAL4 driven expression of UAS-RNAi constructs results in a strong decrease in protein expression for the targeted gene, particularly when these constructs are co-expressed with the RNAi component dicer2 (UAS-dcr2; Figure S2A). To address whether unc79 and unc80 function is required in pacemaker neurons, we crossed these UAS-RNAi lines to circadian GAL4 strains. Expression of unc79, unc80, or na RNAi using the broad circadian driver timelessGAL4 (timGAL4) plus UAS-dcr2 produces strong LD and DD rhythmicity defects (Figure 2, left panels, P<0.05; Table 2, P<0.001), which are comparable to the strongest phenotypes observed in the corresponding mutant strains (Figure 1; Table 1, P > =  0.385). Restricting RNAi expression to the PDF+ pacemaker neurons using pdfGAL4 UAS-dcr2 produces strong defects in DD rhythmicity compared to control strains (Table 2, P<0.001), but does not clearly alter LD behavior (Figure 2, right panels, P > =  0.146). These data suggest that both unc79 and unc80 are required broadly among pacemaker neurons to promote locomotor rhythmicity. Moreover, both genes appear to have similar anatomical requirements as na itself.

Bottom Line: We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels.Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain.Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
In the fruit fly Drosophila melanogaster, a network of circadian pacemaker neurons drives daily rhythms in rest and activity. The ion channel NARROW ABDOMEN (NA), orthologous to the mammalian sodium leak channel NALCN, functions downstream of the molecular circadian clock in pacemaker neurons to promote behavioral rhythmicity. To better understand the function and regulation of the NA channel, we have characterized two putative auxiliary channel subunits in Drosophila, unc79 (aka dunc79) and unc80 (aka CG18437). We have generated novel unc79 and unc80 mutations that represent strong or complete loss-of-function alleles. These mutants display severe defects in circadian locomotor rhythmicity that are indistinguishable from na mutant phenotypes. Tissue-specific RNA interference and rescue analyses indicate that UNC79 and UNC80 likely function within pacemaker neurons, with similar anatomical requirements to NA. We observe an interdependent, post-transcriptional regulatory relationship among the three gene products, as loss of na, unc79, or unc80 gene function leads to decreased expression of all three proteins, with minimal effect on transcript levels. Yet despite this relationship, we find that the requirement for unc79 and unc80 in circadian rhythmicity cannot be bypassed by increasing NA protein expression, nor can these putative auxiliary subunits substitute for each other. These data indicate functional requirements for UNC79 and UNC80 beyond promoting channel subunit expression. Immunoprecipitation experiments also confirm that UNC79 and UNC80 form a complex with NA in the Drosophila brain. Taken together, these data suggest that Drosophila NA, UNC79, and UNC80 function together in circadian clock neurons to promote rhythmic behavior.

Show MeSH
Related in: MedlinePlus