Research ArticleCIRCADIAN RHYTHM

Timing of expression of the core clock gene Bmal1 influences its effects on aging and survival

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Science Translational Medicine  03 Feb 2016:
Vol. 8, Issue 324, pp. 324ra16
DOI: 10.1126/scitranslmed.aad3305
  • Fig. 1. Loss of circadian rhythms in iKO mice.

    (A) Representative double-plotted actograms of wheel-running activity of 3-month-old Bmal1f/f and Bmal1f/f-EsrCre mice (red dots, tamoxifen treatment). Similar results were obtained in n = 8 to 9 mice per group. (B) Counts of wheel revolutions per hour from control mice (Ctrls) and iKO mice under conditions of DD (n = 8 to 9, Student’s t test; ns, no significant difference). (C) Representative double-plotted actograms of wheel-running activity from 18-month-old Ctrl and iKO mice under DD. Similar results were obtained in n = 6 to 7 mice per group. (D) Counts of wheel revolutions from 18-month-old Ctrls and iKOs under DD (n = 6 to 7; Student’s t test). (E) Representative radiotelemetry results of locomotor activity, systolic BP (SBP), and HR in Bmal1f/f and Bmal1f/f-EsrCre mice (red inverted triangles, tamoxifen treatment). Similar results were obtained in n = 3 mice per group. (F) Hepatic mRNA levels of canonical clock genes and clock-controlled gene Dbp were determined by qRT-PCR [n = 4 per genotype per time point; x axis, circadian time (CT); y axis, relative mRNA levels; *P < 0.05; **P < 0.01; ***P < 0.001, two-way analysis of variance (ANOVA)].

  • Fig. 2. General status of iKO mice.

    (A) Life span of Ctrls and iKOs with medians of 745 and 765 days, respectively (P = 0.9852, log-rank test). TAM, tamoxifen. (B) Body weight of Ctrls and iKOs for both genders (Student’s t test). (C) Ratio of organ to body weight in 5- and 11-month-old Ctrls and iKOs (**P < 0.01, multiple t tests with Holm-Sidak correction). (D) Fertility analysis in both male and female Ctrl, iKO, and untreated Cre+ mice (**P < 0.01; ***P < 0.001, χ2 test). (E) Blood glucose concentrations of Ctrls and iKOs that underwent GTT and ITT (n = 6 to 7; two-way ANOVA; no significant difference between Ctrls and iKOs at any time point).

  • Fig. 3. Hair cycling and arthropathy.

    (A) Six-week-old Bmal1f/f and Bmal1f/f-EsrCre mice were treated with tamoxifen. Meanwhile, dorsal hair was shaved. Pictures were taken 7 weeks after hair shaving. Mice with obvious hair regrowth are highlighted with orange boxes. (B and C) Contingency bar graphs show frequency distribution of hair regrowth in both cKO and iKO strains. The hair regrowth was observed 7 weeks (B) or 12 weeks (C) after hair shaving. WT, wild type. (D) Hair regrowth assay in 6-month-old and 1.5-year-old mice. The hair regrowth was observed 7 weeks after hair shaving. (E) Representative photographs of Alizarin Red–stained ribcages and hindlimbs from both cKO and iKO strains. Blue circles indicate calcification.

  • Fig. 4. HFD-induced atherosclerosis.

    (A and B) Both cKO (A) and iKO (B) mice and their littermate controls were in an LDLR−/− background and fed an HFD (42% kcal from fat) from 12 weeks of age. Sixteen weeks after HFD treatment, mice were euthanized and whole aortas were dissected and stained with oil red O. Plaque areas (shown in red) were quantified with Image-Pro. The percentage of plaque area relative to the entire area was calculated (*P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test).

  • Fig. 5. Ocular abnormalities and astrogliosis in iKO mice.

    (A to D) Representative gross images (left) and hematoxylin and eosin (H&E)–stained sections of eyes (middle and right) from Ctrl (A) and iKOs (B to D). (A) Unremarkable globe from a Ctrl mouse. AC, anterior chamber; Co, cornea; Ir, iris; Le, lens; Re, retina. (B) Pathologic changes in a male mouse eye. Grossly, there is a leukoplakic plaque on the cornea (left, arrow). Histologically, the cornea appears thickened with keratinization of the epithelium (right, arrow) and chronic inflammation and neovascularization of the stroma (right, arrowhead). (C) In the contralateral eye of the same mouse, the corneal surface appears irregular with a flattened chamber. Histologically, the cornea appears thickened. The retina is adherent to the lens. The corneal epithelium is attenuated (right, arrow) with chronic inflammation and neovascularization in the stroma. A subcapsular anterior cataract is present (right, arrowhead). (D) A female mouse eye shows an irregular corneal surface with leukoplakia (left, arrow) and corneal neovascularization (right, arrow). (E) Immunofluorescence staining of 4′,6-diamidino-2-phenylindole (DAPI) (all nuclei), NeuN (neuronal nuclei), BMAL1, and GFAP (activated astrocytes) in the motor cortex from cKO, iKO, and nKO mouse strains. Representative images show loss of Bmal1 immunoreactivity in all KOs, which is accompanied by severe astrogliosis (GFAP panels). Similar results were obtained in n = 5 mice per group. Scale bar, 150 μm.

  • Fig. 6. Hepatic transcriptome.

    RNA samples from iKOs and their littermates under DD condition were used for RNA-Seq. (A) Of the 37,681 transcripts, 5457 exhibited circadian variability in Ctrl mice. By contrast, only one gene in iKOs exhibited a circadian pattern. Heat map rendering of the temporal gene expression pattern of these 5457 circadian genes is shown with the average gene expression level measured for each time point. (B) Erh is the only hepatic gene that shows a circadian expression pattern in iKOs, and the q value (JTK_CYCLE analysis) and fold change (ratio of peak/trough) are shown [x axis, circadian time; y axis, fragments per kilobase of transcript per million mapped reads (FPKM)]. (C) Venn diagram (sizes not to scale) depicting the number of differentially expressed genes in cKO and iKO strains. (D) Mouse Genome Informatics Mammalian Phenotype Level 3 enrichment analysis for cKO differentially expressed genes. The top 17 (adjusted P < 0.05) phenotypes related to these genes are given. Overlap, number of appearing gene/number of background genes. (E) Mup3, Serpina3k, and Apoa1 are the only differentially expressed genes in the iKO strain.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/324/324ra16/DC1

    Fig. S1. Validation of Bmal1 deletion and dampening effect of other clock genes.

    Fig. S2. Wheel-running activity, body weight, and fertility in cKO mice.

    Fig. S3. Life span of iKO, cKO, and nKO mice.

    Fig. S4. mRNA levels of Ccnd1 and Mki67 in skin.

    Fig. S5. Other parameters in HFD-fed mice.

    Fig. S6. iKO mice display similar Gfap induction in the brain as cKO.

    Fig. S7. RNA-Seq results revealed dampening effect in core clock genes in iKO mice.

    Table S1. Top 20 oscillating hepatic genes (JTK_CYCLE q value) in Ctrl mice show no circadian pattern in iKO mice.

    Table S2. The ratio of differentially expressed gene numbers in cKO strain to the numbers in iKO strain.

    Table S3. Summary of phenotypes of cKO and iKO mice.

    Data file S1. Circadian transcriptome.

    Data file S2. Differentially expressed genes irrespective of time points.

    Data file S3. Phenotype enrichment analysis of differentially expressed genes.

  • Supplementary Material for:

    Timing of expression of the core clock gene Bmal1 influences its effects on aging and survival

    Guangrui Yang, Lihong Chen, Gregory R. Grant, Georgios Paschos, Wen-Liang Song, Erik S. Musiek, Vivian Lee, Sarah C. McLoughlin, Tilo Grosser, George Cotsarelis, Garret A. FitzGerald*

    *Corresponding author. E-mail: garret{at}upenn.edu

    Published 3 February 2016, Sci. Transl. Med. 8, 324ra16 (2016)
    DOI: 10.1126/scitranslmed.aad3305

    This PDF file includes:

    • Fig. S1. Validation of Bmal1 deletion and dampening effect of other clock genes.
    • Fig. S2. Wheel-running activity, body weight, and fertility in cKO mice.
    • Fig. S3. Life span of iKO, cKO, and nKO mice.
    • Fig. S4. mRNA levels of Ccnd1 and Mki67 in skin.
    • Fig. S5. Other parameters in HFD-fed mice.
    • Fig. S6. iKO mice display similar Gfap induction in the brain as cKO.
    • Fig. S7. RNA-Seq results revealed dampening effect in core clock genes in iKO mice.
    • Table S1. Top 20 oscillating hepatic genes (JTK_CYCLE q value) in Ctrl mice show no circadian pattern in iKO mice.
    • Table S2. The ratio of differentially expressed gene numbers in cKO strain to the numbers in iKO strain.
    • Table S3. Summary of phenotypes of cKO and iKO mice.
    • Legends for data files S1 to S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Circadian transcriptome.
    • Data file S2 (Microsoft Excel format). Differentially expressed genes irrespective of time points.
    • Data file S3 (Microsoft Excel format). Phenotype enrichment analysis of differentially expressed genes.

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