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

Version of Record: August 4, 2017
Version of Record: May 10, 2016
Accepted Manuscript: April 14, 2016

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Renske Erion

Anna N King

Gang Wu

John B Hogenesch

Amita Sehgal

(2016)
Neural clocks and Neuropeptide F/Y regulate circadian gene expression in a peripheral metabolic tissue

eLife 5:e13552.

https://doi.org/10.7554/eLife.13552
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Figures
Tables
Additional files

7 figures, 3 tables and 1 additional file

Figures

Figure 1

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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 …
https://doi.org/10.7554/eLife.13552.003

Figure 1—source data 1 Data for behavioral analysis and for qPCR analysis of per in Pdf-GAL4/UAS-CLKΔ flies.: https://doi.org/10.7554/eLife.13552.004
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Figure 2

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Rhythmic expression of genes that cycle independently of the fat body clock requires clocks in other tissues.

Daily oscillations of several fat body clock-independent genes were tested in male mutants lacking functional clocks in all tissues, *Clk^jrk* mutants, in LD. Rhythmicity of *sxe2* (A), *CG17562* (B), *sxe1* …
https://doi.org/10.7554/eLife.13552.007

Figure 2—source data 1 Data for qPCR analysis of fat body clock-independent genes in Clk^jrk mutants.: https://doi.org/10.7554/eLife.13552.008
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Figure 3

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NPF-expressing clock neurons regulate rhythmic expression of fat body genes, *sxe1* and *Cyp6a21*.

(A, B) Ablating the molecular clock by expressing CLKΔ or CYCΔ in either the LNvs (*Pdf*-GAL4) (A) or DN1s (911-GAL4) (B) does not eliminate rhythmic *sxe1* expression in the fat body. (C, D) Expressing …
https://doi.org/10.7554/eLife.13552.009

Figure 3—source data 1 Data for qPCR analysis of fat body clock-independent genes and clock genes in flies with ablated clock neurons.: https://doi.org/10.7554/eLife.13552.010
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Figure 4

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NPF is a critical circadian signal for *sxe1* and *Cyp6a21* rhythms in the fat body.

(A) Knockdown of *npf* in all NPF-positive cells does not eliminate rhythmicity but reduces expression of *sxe1* in the fat body at all times. (B) Analysis of *npf* knockdown efficiency in heads of *Npf*-GAL…
https://doi.org/10.7554/eLife.13552.011

Figure 4—source data 1 Data for qPCR analysis of fat body clock-independent genes and clock genes in flies with perturbed NPF signaling.: https://doi.org/10.7554/eLife.13552.012
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Figure 5 with 1 supplement

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Npy regulates circadian expression of cytochrome P450 genes in the murine liver.

(**A–C**) Quantitative PCR analysis in murine livers. Daily oscillations of *Cyp2b10* expression (A) are abolished in *Npy* KOs compared to their background controls (wild type), while oscillations of the …
https://doi.org/10.7554/eLife.13552.013

Figure 5—source data 1 Data for qPCR and microarray analysis in wild type and Npy knockout mouse liver.: https://doi.org/10.7554/eLife.13552.014
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Figure 5—figure supplement 1

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MetaCycle analysis of cycling liver transcripts in wild type and *NPY* KO.

(A) Microarray analysis detects 2460 and 1330 cycling transcripts (MetaCycle p<0.05) in WT and *Npy* KO liver datasets respectively. 880 cycling transcripts are common between the two datasets. (B,C) …
https://doi.org/10.7554/eLife.13552.015

Figure 6

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NPF/Npy regulate rhythmically expressed P450 enzymes in the periphery of flies and mammals.

A model of brain clock regulation of peripheral cycling. Brain clocks regulate clocks in peripheral tissues. In *Drosophila*, clocks in PDF-positive neurons (LNvs) regulate the clock in the fat body. …
https://doi.org/10.7554/eLife.13552.018

Author response image 1

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Restricted feeding between ZT9 and ZT15 alters the rhythmic expression of *sxe1* and clock gene *per* in the *Drosophila* fat body.
https://doi.org/10.7554/eLife.13552.021

Tables

Table 1

Free-running rest:activity rhythms. Clock ablation in Pdf neurons (Pdf-GAL4/UAS-CLKΔ) or in LNd neurons (Dvpdf-GAL4/UAS-CLKΔ; pdf-GAL80/+) disrupts free-running behavioral rhythms in flies. Flies …

https://doi.org/10.7554/eLife.13552.005

Genotype	n	% Rhythmic	Period (hr)	FFT
Pdf-GAL4/UAS-CLKΔ	39	36	23.51	0.04
UAS- CLKΔ/CyO	48	90	23.71	0.06
Npf-GAL4/UAS-CLKΔ	62	98	24.01	0.06
UAS-CLKΔ/+	58	100	23.70	0.05
npfr	39	95	23.66	0.05
npf/+	46	100	23.44	0.11
Dvpdf-GAL4/UAS-CLKΔ; pdf-GAL80/+	63	68	25.51	0.06
UAS-CLKΔ/+	63	100	23.91	0.08

Table 1—source data 1 Data for circadian analysis of fly behavior in constant darkness.: https://doi.org/10.7554/eLife.13552.006
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Table 2

MetaCycle statistics for cycling of liver clock-independent genes in wild type and Npy knockout mice. Our Npy KO data compared to the previously reported set of liver transcripts whose expression …

https://doi.org/10.7554/eLife.13552.016

Liver clock-independent genes with disrupted cycling of expression in Npy KO
Affymetrix transcript ID	Gene	WT MetaCycle P-value	WT Phase	WT Median Exp	WT Relative Amp	KO MetaCycle P-value	KO Phase	KO Median Exp	KO Relative Amp
17503756	Rbl2	0.0003	8.94	367.0	0.217	0.0753	9.20	338.7	0.132
17287733	Ddx46	0.0007	7.77	264.2	0.229	0.1756	11.46	262.8	0.158
17235227	Cirbp	0.0037	6.28	58.1	0.188	0.0924	7.83	54.3	0.056
17311807	Sqle	0.0039	21.00	193.4	0.790	0.7685	9.43	229.9	0.310
17365314	Ldb1	0.0067	10.38	234.1	0.164	0.1503	12.05	216.2	0.171
17475360	Cyp2b10	0.0102	17.75	155.9	0.869	0.0782	0.00	85.7	0.486
17331429	Actg1	0.0153	18.56	155.7	0.470	0.3570	1.52	124.5	0.512
17290173	Hmgcs1	0.0230	0.50	751.5	0.687	0.1625	4.83	695.2	0.083
17239493	Heca	0.0265	9.30	255.6	0.132	0.1921	10.93	258.6	0.086
17232235	Ctgf	0.0331	13.18	37.9	0.364	0.9984	11.52	42.8	0.152

Liver clock-independent gene with altered phase of expression in Npy KO
Affymetrix transcript ID	Gene	WT MetaCycle P-value	WT Phase	WT Median Exp	WT Relative Amp	KO MetaCycle P-value	KO Phase	KO Median Exp	KO Relative Amp
17268729	Fbxl20	0.0001	7.66	72.8	0.338	0.0287	3.35	74.6	0.024

Exp, expression level; Amp, amplitude.

Table 2—source data 1 Microarray data for MetaCycle analysis.: https://doi.org/10.7554/eLife.13552.017
Download elife-13552-table2-data1-v3.xlsx

Table 3

JTK_Cycle Statistics and Two-factor ANOVA Results. All qPCR data were tested for circadian rhythmicity with JTK_cycle test and two-way ANOVA for repeated measures and a Tukey’s post hoc test. …

https://doi.org/10.7554/eLife.13552.019

Figure	Genotype	Gene	Tissue	JTK_cycle P-value	Time P-value	Genotype P-value	Time X Genotype P-value
1C	[ZT] UAS-CLK∆/+	per	Fat Body (FB)	0.0194	< 0.0001	0.7826	0.4679
1C	[ZT] Pdf-GAL4/UAS-CLK∆	per	FB	0.0094
1D	[CT] UAS-CLK∆/+	per	FB	0.0041	0.0313	0.6650	0.0281
1D	[CT] Pdf-GAL4/UAS-CLK∆	per	FB	1

2A	Iso31	sxe2	FB	0.0014	0.0131	0.1852	0.0843
2A	Clk^jrk	sxe2	FB	1
2B	Iso31	CG17562	FB	0.0014	0.0223	0.6409	0.1746
2B	Clk^jrk	CG17562	FB	0.6945
2C	Iso31	sxe1	FB	0.0020	0.0019	<0.0001	0.0020
2C	Clk^jrk	sxe1	FB	1
2D	Iso31	CG14934	FB	0.0820	0.1146	<0.0001	0.0266
2D	Clk^jrk	CG14934	FB	0.5429

3A	UAS-CLK∆/+	sxe1	FB	0.0007	0.0003	0.0049	0.7061
3A	Pdf-GAL4/UAS-CLK∆	sxe1	FB	9.00E-07
3B	UAS-CYC∆/+	sxe1	FB	0.0363	0.0006	0.7995	0.9592
3B	911-GAL4/UAS-CYC∆	sxe1	FB	0.0044
3C	UAS-CLK∆/+	sxe1	FB	0.0014	0.0060	<0.0001	0.0749
3C	Npf-GAL4/UAS-CLK∆	sxe1	FB	0.1969
3D	UAS-CYC∆/+	sxe1	FB	0.0029	<0.0001	<0.0001	<0.0001
3D	Npf-GAL4/UAS-CYC∆	sxe1	FB	1
3E	UAS-CLK∆/+	sxe1	FB	0.0196	0.0038	0.0017	0.5326
3E	Dvpdf-GAL4/UAS-CLK∆;Pdf-GAL80/+	sxe1	FB	0.0001
3F	UAS-CLK∆/+	Cyp6a21	FB	0.0009	<0.0001	<0.0001	0.0574
3F	Npf-GAL4/UAS-CLK∆	Cyp6a21	FB	0.0259
3G	UAS-CLK∆/+	per	FB	0.0568	0.0057	0.8767	0.9902
3G	Npf-GAL4/UAS-CLK∆	per	FB	0.0441

4A	UAS-Npf RNAi/+	sxe1	FB	0.0020	0.0005	<0.0001	0.1421
4A	Npf-GAL4/UAS-Npf RNAi	sxe1	FB	0.0441
4B	Not analyzed with JTK	npf	Head	-
4C	npfr/+	sxe1	FB	0.0128	0.0008	<0.0001	0.0038
4C	npfr	sxe1	FB	0.1969
4D	npfr/+	Cyp6a21	FB	0.0916	0.0319	<0.0001	0.4182
4D	npfr	Cyp6a21	FB	0.1150
4E	UAS-CLK∆/+	npf	Head	1	0.7588	<0.0001	0.846
4E	Npf-GAL4/UAS-CLK∆	npf	Head	1
4F	UAS-CLK∆/+	per	Head	0.0001	<0.0001	0.1410	0.0420
4F	Npf-GAL4/UAS-CLK∆	per	Head	1.19E-05

5A	Wild type	Cyp2b10	Liver	0.0010	0.0209	0.0034	0.1640
5A	Npy Knockout (KO)	Cyp2b10	Liver	0.0510
5B	Wild type	Reverbα	Liver	7.46E-12	<0.0001	0.4979	0.0661
5B	Npy KO	Reverbα	Liver	7.17E-09
5C	Wild type	Alas1	Liver	9.81E-06	0.0007	0.8476	0.8202
5C	Npy KO	Alas1	Liver	0.0018

Additional files

Supplementary file 1 MetaCycle statistics of microarray data generated in this study. MetaCycle p-value, phase, baseline expression (baselineEXP), and relative amplitude (relativeAMP) are calculated for each gene in wild type and NPY KO animals.: https://doi.org/10.7554/eLife.13552.020
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Figures

Oscillations of per in the fat body require an intact central clock in the absence of external cues.

Figure 1—source data 1

Rhythmic expression of genes that cycle independently of the fat body clock requires clocks in other tissues.

Figure 2—source data 1

NPF-expressing clock neurons regulate rhythmic expression of fat body genes, sxe1 and Cyp6a21.

Figure 3—source data 1

NPF is a critical circadian signal for sxe1 and Cyp6a21 rhythms in the fat body.

Figure 4—source data 1

Npy regulates circadian expression of cytochrome P450 genes in the murine liver.

Figure 5—source data 1

MetaCycle analysis of cycling liver transcripts in wild type and NPY KO.

NPF/Npy regulate rhythmically expressed P450 enzymes in the periphery of flies and mammals.

Restricted feeding between ZT9 and ZT15 alters the rhythmic expression of sxe1 and clock gene per in the Drosophila fat body.

Tables

Table 1—source data 1

Table 2—source data 1

Additional files

Supplementary file 1

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Oscillations of per in the fat body require an intact central clock in the absence of external cues.

Figure 1—source data 1

Rhythmic expression of genes that cycle independently of the fat body clock requires clocks in other tissues.

Figure 2—source data 1

NPF-expressing clock neurons regulate rhythmic expression of fat body genes, sxe1 and Cyp6a21.

Figure 3—source data 1

NPF is a critical circadian signal for sxe1 and Cyp6a21 rhythms in the fat body.

Figure 4—source data 1

Npy regulates circadian expression of cytochrome P450 genes in the murine liver.

Figure 5—source data 1

MetaCycle analysis of cycling liver transcripts in wild type and NPY KO.

NPF/Npy regulate rhythmically expressed P450 enzymes in the periphery of flies and mammals.

Restricted feeding between ZT9 and ZT15 alters the rhythmic expression of sxe1 and clock gene per in the Drosophila fat body.

Table 1—source data 1

Table 2—source data 1

Supplementary file 1