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Neural clocks and Neuropeptide F/Y regulate circadian gene expression in a peripheral metabolic tissue.

Erion R, King AN, Wu G, Hogenesch JB, Sehgal A - Elife (2016)

Bottom Line: Interestingly, rhythmic expression of the cytochrome P450 transcripts, sex-specific enzyme 1 (sxe1) and Cyp6a21, which cycle in the fat body independently of the local clock, depends upon clocks in neurons expressing neuropeptide F (NPF).NPF signaling itself is required to drive cycling of sxe1 and Cyp6a21 in the fat body, and its mammalian ortholog, Npy, functions similarly to regulate cycling of cytochrome P450 genes in the mouse liver.These data highlight the importance of neuronal clocks for peripheral rhythms, particularly in a specific detoxification pathway, and identify a novel and conserved role for NPF/Npy in circadian rhythms.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, United States.

ABSTRACT
Metabolic homeostasis requires coordination between circadian clocks in different tissues. Also, systemic signals appear to be required for some transcriptional rhythms in the mammalian liver and the Drosophila fat body. Here we show that free-running oscillations of the fat body clock require clock function in the PDF-positive cells of the fly brain. Interestingly, rhythmic expression of the cytochrome P450 transcripts, sex-specific enzyme 1 (sxe1) and Cyp6a21, which cycle in the fat body independently of the local clock, depends upon clocks in neurons expressing neuropeptide F (NPF). NPF signaling itself is required to drive cycling of sxe1 and Cyp6a21 in the fat body, and its mammalian ortholog, Npy, functions similarly to regulate cycling of cytochrome P450 genes in the mouse liver. These data highlight the importance of neuronal clocks for peripheral rhythms, particularly in a specific detoxification pathway, and identify a novel and conserved role for NPF/Npy in circadian rhythms.

No MeSH data available.


Related in: MedlinePlus

Oscillations of per in the fat body require an intact central clock in the absence of external cues.(A) Representative double-plotted activity records of individual control UAS-CLKΔ/CyO (left) and Pdf-GAL4/UAS-CLKΔ (right) flies over the course of 5 days in constant darkness. (B) Schematic of experimental design. Male flies, aged 7–10 days, were entrained for several days in 12 hr light: 12 hr dark cycles (LD). Male flies were dissected to obtain abdominal fat bodies (dotted red box) either on the last day in LD or on the second day of constant darkness (DD2). Graphs depict mRNA levels, normalized to α−tubulin (atub), over the course of the day in the presence of light (LD; Zeitgeber Time, ZT) or in constant darkness (DD2; Circadian Time, CT). Ablating the central clock (Pdf-GAL4/UAS-CLKΔ) (red line) does not affect per rhythms in LD (C) but abolishes per rhythms in DD2 compared to controls (blue line) (D). Each experiment was repeated independently three times. The average value for each timepoint is plotted with error bars denoting the standard error of the mean (SEM). Significant rhythmicity was determined using JTK_cycle. Asterisk (*) adjacent to genotype label indicates JTK_cycle p value <0.05. See Table 3 for JTK cycle values.DOI:http://dx.doi.org/10.7554/eLife.13552.00310.7554/eLife.13552.004Figure 1—source data 1.Data for behavioral analysis and for qPCR analysis of per in Pdf-GAL4/UAS-CLKΔ flies.DOI:http://dx.doi.org/10.7554/eLife.13552.004
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fig1: Oscillations of per in the fat body require an intact central clock in the absence of external cues.(A) Representative double-plotted activity records of individual control UAS-CLKΔ/CyO (left) and Pdf-GAL4/UAS-CLKΔ (right) flies over the course of 5 days in constant darkness. (B) Schematic of experimental design. Male flies, aged 7–10 days, were entrained for several days in 12 hr light: 12 hr dark cycles (LD). Male flies were dissected to obtain abdominal fat bodies (dotted red box) either on the last day in LD or on the second day of constant darkness (DD2). Graphs depict mRNA levels, normalized to α−tubulin (atub), over the course of the day in the presence of light (LD; Zeitgeber Time, ZT) or in constant darkness (DD2; Circadian Time, CT). Ablating the central clock (Pdf-GAL4/UAS-CLKΔ) (red line) does not affect per rhythms in LD (C) but abolishes per rhythms in DD2 compared to controls (blue line) (D). Each experiment was repeated independently three times. The average value for each timepoint is plotted with error bars denoting the standard error of the mean (SEM). Significant rhythmicity was determined using JTK_cycle. Asterisk (*) adjacent to genotype label indicates JTK_cycle p value <0.05. See Table 3 for JTK cycle values.DOI:http://dx.doi.org/10.7554/eLife.13552.00310.7554/eLife.13552.004Figure 1—source data 1.Data for behavioral analysis and for qPCR analysis of per in Pdf-GAL4/UAS-CLKΔ flies.DOI:http://dx.doi.org/10.7554/eLife.13552.004

Mentions: While some peripheral clocks in Drosophila are completely autonomous, e.g. malphigian tubules (Hege et al., 1997), others rely upon cell-extrinsic factors, in particular the clock in the brain. For example, PDF-positive LNvs are required for rhythmic expression of clock components in the prothoracic gland, a peripheral tissue that gates rhythmic eclosion (Myers et al., 2003). In addition, PDF released by neurons in the abdominal ganglion is necessary to set the phase of the clock in oenocytes (Krupp et al., 2013), which regulate sex pheromone production and mating behavior (Krupp et al., 2008). We investigated whether clocks in PDF-positive LNvs were necessary for clock function in the abdominal fat body. The molecular clock in Drosophila consists of an autoregulatory loop in which the transcription factors, CLOCK (CLK) and CYCLE (CYC), activate expression of the genes period (per) and timeless (tim) and PER and TIM proteins feedback to inhibit the activity of CLK-CYC (Zheng and Sehgal, 2012). To disrupt the molecular clock exclusively in PDF-positive cells, we used the GAL4/UAS system to express a dominant-negative version of the CLK transcription factor, CLKΔ. CLKΔ lacks regions of its DNA-binding domain, preventing it from binding DNA and activating transcription of genes, including components of the molecular clock. However, CLKΔ can still heterodimerize with its partner, CYC, through its protein interaction domain (Tanoue et al., 2004). Behavioral assays of Pdf-GAL4/UAS-CLKΔ flies showed that a majority of the flies had arrhythmic locomotor activity in constant darkness (DD) (Figure 1A and Table 1), confirming that CLKΔ expression in the LNvs disrupts circadian rhythms.10.7554/eLife.13552.003Figure 1.Oscillations of per in the fat body require an intact central clock in the absence of external cues.


Neural clocks and Neuropeptide F/Y regulate circadian gene expression in a peripheral metabolic tissue.

Erion R, King AN, Wu G, Hogenesch JB, Sehgal A - Elife (2016)

Oscillations of per in the fat body require an intact central clock in the absence of external cues.(A) Representative double-plotted activity records of individual control UAS-CLKΔ/CyO (left) and Pdf-GAL4/UAS-CLKΔ (right) flies over the course of 5 days in constant darkness. (B) Schematic of experimental design. Male flies, aged 7–10 days, were entrained for several days in 12 hr light: 12 hr dark cycles (LD). Male flies were dissected to obtain abdominal fat bodies (dotted red box) either on the last day in LD or on the second day of constant darkness (DD2). Graphs depict mRNA levels, normalized to α−tubulin (atub), over the course of the day in the presence of light (LD; Zeitgeber Time, ZT) or in constant darkness (DD2; Circadian Time, CT). Ablating the central clock (Pdf-GAL4/UAS-CLKΔ) (red line) does not affect per rhythms in LD (C) but abolishes per rhythms in DD2 compared to controls (blue line) (D). Each experiment was repeated independently three times. The average value for each timepoint is plotted with error bars denoting the standard error of the mean (SEM). Significant rhythmicity was determined using JTK_cycle. Asterisk (*) adjacent to genotype label indicates JTK_cycle p value <0.05. See Table 3 for JTK cycle values.DOI:http://dx.doi.org/10.7554/eLife.13552.00310.7554/eLife.13552.004Figure 1—source data 1.Data for behavioral analysis and for qPCR analysis of per in Pdf-GAL4/UAS-CLKΔ flies.DOI:http://dx.doi.org/10.7554/eLife.13552.004
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fig1: Oscillations of per in the fat body require an intact central clock in the absence of external cues.(A) Representative double-plotted activity records of individual control UAS-CLKΔ/CyO (left) and Pdf-GAL4/UAS-CLKΔ (right) flies over the course of 5 days in constant darkness. (B) Schematic of experimental design. Male flies, aged 7–10 days, were entrained for several days in 12 hr light: 12 hr dark cycles (LD). Male flies were dissected to obtain abdominal fat bodies (dotted red box) either on the last day in LD or on the second day of constant darkness (DD2). Graphs depict mRNA levels, normalized to α−tubulin (atub), over the course of the day in the presence of light (LD; Zeitgeber Time, ZT) or in constant darkness (DD2; Circadian Time, CT). Ablating the central clock (Pdf-GAL4/UAS-CLKΔ) (red line) does not affect per rhythms in LD (C) but abolishes per rhythms in DD2 compared to controls (blue line) (D). Each experiment was repeated independently three times. The average value for each timepoint is plotted with error bars denoting the standard error of the mean (SEM). Significant rhythmicity was determined using JTK_cycle. Asterisk (*) adjacent to genotype label indicates JTK_cycle p value <0.05. See Table 3 for JTK cycle values.DOI:http://dx.doi.org/10.7554/eLife.13552.00310.7554/eLife.13552.004Figure 1—source data 1.Data for behavioral analysis and for qPCR analysis of per in Pdf-GAL4/UAS-CLKΔ flies.DOI:http://dx.doi.org/10.7554/eLife.13552.004
Mentions: While some peripheral clocks in Drosophila are completely autonomous, e.g. malphigian tubules (Hege et al., 1997), others rely upon cell-extrinsic factors, in particular the clock in the brain. For example, PDF-positive LNvs are required for rhythmic expression of clock components in the prothoracic gland, a peripheral tissue that gates rhythmic eclosion (Myers et al., 2003). In addition, PDF released by neurons in the abdominal ganglion is necessary to set the phase of the clock in oenocytes (Krupp et al., 2013), which regulate sex pheromone production and mating behavior (Krupp et al., 2008). We investigated whether clocks in PDF-positive LNvs were necessary for clock function in the abdominal fat body. The molecular clock in Drosophila consists of an autoregulatory loop in which the transcription factors, CLOCK (CLK) and CYCLE (CYC), activate expression of the genes period (per) and timeless (tim) and PER and TIM proteins feedback to inhibit the activity of CLK-CYC (Zheng and Sehgal, 2012). To disrupt the molecular clock exclusively in PDF-positive cells, we used the GAL4/UAS system to express a dominant-negative version of the CLK transcription factor, CLKΔ. CLKΔ lacks regions of its DNA-binding domain, preventing it from binding DNA and activating transcription of genes, including components of the molecular clock. However, CLKΔ can still heterodimerize with its partner, CYC, through its protein interaction domain (Tanoue et al., 2004). Behavioral assays of Pdf-GAL4/UAS-CLKΔ flies showed that a majority of the flies had arrhythmic locomotor activity in constant darkness (DD) (Figure 1A and Table 1), confirming that CLKΔ expression in the LNvs disrupts circadian rhythms.10.7554/eLife.13552.003Figure 1.Oscillations of per in the fat body require an intact central clock in the absence of external cues.

Bottom Line: Interestingly, rhythmic expression of the cytochrome P450 transcripts, sex-specific enzyme 1 (sxe1) and Cyp6a21, which cycle in the fat body independently of the local clock, depends upon clocks in neurons expressing neuropeptide F (NPF).NPF signaling itself is required to drive cycling of sxe1 and Cyp6a21 in the fat body, and its mammalian ortholog, Npy, functions similarly to regulate cycling of cytochrome P450 genes in the mouse liver.These data highlight the importance of neuronal clocks for peripheral rhythms, particularly in a specific detoxification pathway, and identify a novel and conserved role for NPF/Npy in circadian rhythms.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, United States.

ABSTRACT
Metabolic homeostasis requires coordination between circadian clocks in different tissues. Also, systemic signals appear to be required for some transcriptional rhythms in the mammalian liver and the Drosophila fat body. Here we show that free-running oscillations of the fat body clock require clock function in the PDF-positive cells of the fly brain. Interestingly, rhythmic expression of the cytochrome P450 transcripts, sex-specific enzyme 1 (sxe1) and Cyp6a21, which cycle in the fat body independently of the local clock, depends upon clocks in neurons expressing neuropeptide F (NPF). NPF signaling itself is required to drive cycling of sxe1 and Cyp6a21 in the fat body, and its mammalian ortholog, Npy, functions similarly to regulate cycling of cytochrome P450 genes in the mouse liver. These data highlight the importance of neuronal clocks for peripheral rhythms, particularly in a specific detoxification pathway, and identify a novel and conserved role for NPF/Npy in circadian rhythms.

No MeSH data available.


Related in: MedlinePlus