Dataset and script: "Captivity affects mitochondrial aerobic respiration and carotenoid metabolism in the house finch (<i>Haemorhous mexicanus</i>)"

This dataset includes one R script containing basic code for all major statistical analyses and figures. Five data tables (in .csv format) accompany the script, and the file names of these tables are referenced at their use in the code.All analyses were performed in R (v. 4.2.3), through RStudio (v. 2013.12.1+402). R packages used in analysis and figure creation include tidyverse (v. 2.0.0), lme4 (v. 1.1-35.1), car (v. 3.1.2), and GGally (v. 2.2.1).</p

mTOR perturbation alters the liver clock function.

<p><b>(A)</b> Bioluminescence rhythms of liver explants derived from <i>Per2</i>^<i>Luc</i> mice in the presence of DMSO or 20 nM Torin1. mTOR inhibition by Torin1 led to long period and low amplitude in liver explants cultured <i>ex vivo</i>. ** p < 0.01 vs. DMSO. <b>(B)</b> Bioluminescence rhythms of liver explants derived from <i>mTor</i>^{<i>flxflx</i>}<i>;Per2</i>^<i>Luc</i> (wt control) and <i>mTor</i>^{<i>flx/–</i>}<i>;Per2</i>^<i>Luc</i> (<i>mTor</i> heterozygous) mice. Heterozygous deletion of <i>mTor</i> reduced the rhythm amplitude in liver explants. * p < 0.05 vs. <i>mTor</i>^{<i>flxflx</i>}. <b>(C-E)</b> Western blots (C and D) and Q-PCR (E) of liver tissue samples from <i>mTor</i>^{<i>flxflx</i>} and <i>mTor</i>^{<i>flx/–</i>}mice. Mice were entrained to regular light/dark cycles and then released to constant darkness (DD), followed by tissue harvest at 4-hr intervals beginning at 52 hr in DD (CT52). While individual tissue samples were used for Q-PCR analysis, tissues from 3~5 mice were pooled at each time point for Western blotting. Relative Q-PCR values are presented for each gene and error bars represent SD of expression levels from 3 mice. Circadian time (CT): hours after animal release to constant darkness. Quantitation of the blots is shown in (D). * p < 0.05 vs. <i>mTor</i>^{<i>flxflx</i>}.</p

Genetic manipulation of mTOR pathway alters circadian clock function.

<p>RNAi knockdown of <i>mTor</i> lengthens the period length of circadian bioluminescence rhythms in MMH-D3 hepatocytes. (<b>A</b>) and 3T3-L1 adipocytes (<b>B</b>). Hepatocytes and adipocytes harboring the <i>Per2-dLuc</i> reporter were infected with lentiviral non-specific (NS) shRNA or shRNA constructs against <i>mTor</i>. Left panel: real-time bioluminescence expression was recorded in a Lumicycle luminometer on 35-mm culture dishes and the bioluminescence data are representative of at least three independent experiments. Middle panel: <i>mTor</i> knockdown efficiency was determined by Western blot analysis (middle). Right panel: period length and rhythm amplitude are mean ± standard deviation (SD) (n = 3 independent dishes). * p < 0.05 vs. NS. <b>(C)</b> Elevated mTOR via constitutively active Rheb shortens circadian bioluminescence rhythms in MMH-D3 hepatocytes. Bioluminescence rhythms from MMH-D3 hepatocytes harboring the <i>Per2-dLuc</i> reporter and overexpressing either the empty vector or constitutively active Rheb (CA-Rheb). p-S6 as a proxy of mTOR activation was hyper-phosphorylated in cells expressing CA-Rheb relative to vector control cells, whereas S6 levels were similar in the two cell lines. * p < 0.05 vs. Vector; ** p < 0.01 vs. Vector.</p

Cell Type-Specific Functions of <i>Period</i> Genes Revealed by Novel Adipocyte and Hepatocyte Circadian Clock Models

<div><p>In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of <i>Bmal1</i>, <i>Clock</i>, <i>Cry1</i>, and <i>Cry2</i> each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the <i>Period</i> and <i>Rev-Erb</i> families of clock genes. In particular, <i>Per1</i> and <i>Per2</i>, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. <i>Per3</i>, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.</p></div

Pharmacological inhibition of mTOR alters circadian clock function in MMH-D3 hepatocytes.

<p>Top panel: representative records of bioluminescence rhythms of MMH-D3 hepatocytes harboring the <i>Per2-dLuc</i> reporter in the presence of mTOR inhibitors: 50 nM rapamycin <b>(A)</b>, 20 nM Torin1 <b>(B)</b>, or 10 uM PP242 <b>(C)</b>. Real-time bioluminescence expression was recorded in a Synergy microplate luminometer on 96-well plates. Bottom panel: period length and amplitude are mean ± SD (n = 8 independent wells) for each treatment. All three inhibitors caused significantly longer period length and lower rhythm amplitude. ***p < 0.001 vs. DMSO.</p

Knockdowns of <i>Bmal1</i>, <i>Clock</i>, <i>Cry1</i>, <i>Cry2</i>, and <i>Fbxl3</i> lead to cell type-ubiquitous circadian phenotypes.

<p>Bioluminescence expression patterns upon KD of <i>Bmal1</i> or <i>Clock</i> (A), <i>Cry1</i> or <i>Cry2</i> (B), and <i>Fbxl3</i> (C) in all three cell types. For clock phenotyping, both reporters were used for each cell line and phenotypes were independent of the reporter used. For phenotyping, we selected 3T3 cells expressing the <i>Bmal1</i>-d<i>Luc</i> reporter, and 3T3-L1 and MMH-D3 cells expressing the <i>Per2</i>-d<i>Luc</i> reporter. Cells were infected with specific lentiviral shRNAs as indicated. Real-time bioluminescence expression was recorded by Synergy microplate reader as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004244#pgen-1004244-g001" target="_blank">Figure 1</a>. Out of the 6 shRNAs tested, two validated shRNAs (orange and green) are shown. NS, non-specific shRNA as control (black). While KD of <i>Bmal1</i> or <i>Clock</i> resulted in low amplitude, rapid damping or arrhythmicity, <i>Fbxl3</i> KD led to long period and low amplitude in 3T3, long period in 3T3-L1, and low amplitude and rapid damping in MMH-D3 cells. <i>Cry1</i> KD caused rapid damping or low amplitude, and <i>Cry2</i> KD lengthened period and increased rhythm amplitude. Bioluminescence data are representative of four independent experiments for 3T3 and 3T3-L1 cells, and three independent experiments for MMH-D3 cells. Knockdown of endogenous mRNA expression in non-synchronized cells was determined by qPCR (insert). Values for each gene are expressed as percentage of gene expression in NS control cells. qPCR data are mean ± SD (two samples/wells from one experiment).</p

shRNA-mediated knockdowns of <i>Per1</i>, <i>Per2</i> and <i>Per3</i> lead to cell type-specific circadian phenotypes.

<p>Bioluminescence expression patterns upon KD of <i>Per1</i> (A), <i>Per2</i> (B), and <i>Per3</i> (C) in all three cell types. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004244#pgen-1004244-g002" target="_blank">Figure 2</a> for details. Whereas <i>Per3</i> KD led to short periods in all three cell types, <i>Per1</i> and <i>Per2</i> KDs caused different clock phenotypes depending on cell type. (D) Summary of period length phenotypes. Data are mean ± SD (n = 4 independent experiments for 3T3 cells; n = 3 samples/wells of one experiment for 3T3-L1 and MMH-D3 cells). NS, non-specific shRNA. Compared to NS controls, significant difference in period length was detected in the following KDs: NS vs. <i>Per1</i> KD in MMH-D3, t-test, p<0.001; NS vs. <i>Per2</i> KD in 3T3, t-test, p = 0.013; NS vs. <i>Per2</i> KD in MMH-D3, t-test, p<0.001; NS vs. <i>Per3</i> KD in 3T3, t-test, p<0.001; NS vs. <i>Per3</i> KD in 3T3-L1, t-test, p = 0.003; NS vs. <i>Per3</i> KD in MMH-D3, t-test, p<0.001). *p<0.01; ** p<0.001. (E) <i>Per3</i> deletion led to short period length defects in SCN explants (left) and even stronger defects in dissociated SCN neurons (right). <i>Per3^−/−</i> SCN explants show a slightly shorter period than WT (mean ± SEM: WT, 24.4 hr±0.17, n = 5; <i>Per3^−/−</i>, 23.78 hr±0.18, n = 5). The mean period of rhythms in <i>Per3^−/−</i> neurons was substantially shorter than in WT cells (mean ± SEM: WT, 27.23 hr±0.24, n = 106; <i>Per3^−/−</i>, 25.58 hr±0.12, n = 157; t-test, p<10E-10; ** p<0.001).</p

shRNA-mediated single and composite knockdown effects of <i>Per1</i>, <i>Per2</i> and <i>Per3</i> in MMH-D3 hepatocytes.

<p>(A) Representative bioluminescence expression patterns recorded on LumiCycle upon knockdowns of <i>Per1</i>, <i>Per2</i>, <i>Per3</i> (single KD); <i>Per1</i>/<i>Per2</i>, <i>Per1</i>/<i>Per3</i>, <i>Per2</i>/<i>Per3</i> (double KD); and <i>Per1</i>/<i>Per2</i>/<i>Per3</i> (triple KD) in MMH-D3 hepatocytes. sh62, sh67 and sh74 were used to knock down <i>Per1</i>, <i>Per2</i> and <i>Per3</i>, respectively. All single KDs led to short periods in all three cell types, consistent with Synergy assays. <i>Per1</i>/<i>Per2</i> double and <i>Per1</i>/<i>Per2</i>/<i>Per3</i> triple KDs caused arrhythmicity. All other double composite KDs caused short period phenotype. (B) Summary of period length phenotypes. Data are mean ± SD (n = 3 independent samples/wells from one experiment). NS, non-specific shRNA. Compared to NS controls, significant difference in period length was detected (NS vs. <i>Per1</i> KD, t-test, p<0.001; NS vs. <i>Per2</i> KD, t-test, p<0.001; NS vs. <i>Per3</i> KD, t-test, p<0.001; NS vs. <i>Per1</i>/<i>Per3</i> KD, t-test, p<0.001; NS vs. <i>Per2</i>/<i>Per3</i> KD, t-test, p<0.001). (C) qPCR analysis of clock gene expression upon <i>Per</i> KD in non-synchronized MMH-D3 cells. Values for each gene are expressed as percentage of gene expression in NS control cells. Data represent two samples/wells from one experiment.</p

<i>mTor</i>^{<i>flx/–</i>}mice have long period length of circadian locomotor activity rhythms.

<p><b>(A)</b> Representative double-plotted actograms of wheel-running activity rhythms in <i>mTor</i>^{<i>flxflx</i>} and <i>mTor</i>^{<i>flx/–</i>}mice. X axis: zeitgeber time (ZT) of the 12 h/12 h light/dark cycle (LD) indicated by the bar (top). Y axis: number of days during the experiment (left). Mice were first entrained to a regular LD cycle for 10 days and then released to constant darkness (DD) for 20 days. On the 31^st day, mice were released into constant light (LL) for 40 days. Grey and white areas indicate dark and light periods. <b>(B)</b> Circadian free-running period lengths of mice in DD and LL. Data are mean ± SEM (red) of individual values that are shown in black dots (n = 6 for <i>mTor</i>^{<i>flxflx</i>} mice and n = 9 for <i>mTor</i>^{<i>flx/–</i>}mice. <b>(C)</b> Average wheel-running activity of mice expressed in wheel revolutions per 10 min across 24 h in LD, DD and LL.</p

mTOR perturbation alters the central SCN clock function.

<p><b>(A)</b> SCN explants from <i>Per2</i>^<i>Luc</i> reporter mice were treated with either DMSO or 50 nM rapamycin as indicated by an arrow. Representative bioluminescence records are shown (left). Period and amplitude data (right) are mean ± SD (n = 4 mouse SCN slices). ** p < 0.01 vs. rapamycin pre-treatment. Rapamycin treatment altered tissue-autonomous circadian rhythms of SCN explants cultured <i>ex vivo</i>. <b>(B)</b> Bioluminescence record from one representative SCN explant treated with PP242. Period and amplitude data (right) are mean ± SD (n = 4 mouse SCN slices). ** p < 0.01 vs. PP242 pre-treatment. <b>(C)</b> Bioluminescence rhythms of SCN explants derived from <i>mTor</i>^{<i>flxflx</i>}<i>;Per2</i>^<i>Luc</i> and <i>mTor</i>^{<i>flx/–</i>}<i>;Per2</i>^<i>Luc</i> mice. Heterozygous deletion of <i>mTor</i> reduced tissue-autonomous circadian rhythm amplitude and lengthened the period length of SCN explants. Period length and amplitude data are mean ± SD (n = 5 mouse SCN slices). ** p < 0.01 vs. <i>mTor</i>^{<i>flxflx</i>}.</p