Figure 1.
Runx2 gene and protein expression is detectable in neurons of the adult mouse brain.
A) Qualitative PCR analysis of Runx2 mRNA (289 bp) expression in the suprachiasmatic nucleus (SCN) of adult mice. Lane 1: 100 bp molecular weight standard ladder (black arrowhead: 300 bp; grey arrowhead: 500 bp). Lane 2–4: Runx2 (289 bp) PCR product from reactions performed using E15 mouse calvarial bone mRNA extracts and Runx2-specific primers. Lane 5–7: Runx2 (289 bp) PCR product from reactions performed using adult mouse SCN mRNA extracts and Runx2-specific primers. Sequencing confirmed the identity of all positive PCR amplification products. B) Western blot analysis of Runx2 protein (∼60 kDa; black arrowhead) expression in the SCN of adult mice. Lane 1: Molecular weight standard ladder. Lane 2–3: protein isolated from E15 mouse calvarial bone. Lane 4–5: protein isolated from SCN of adult mice. GAPDH (∼37 kDa; grey arrowhead) protein detection served as a loading control. C) Immunolabelling of Runx2 (green) and NeuN (red) in the frontal cortex. Overlay image showing co-localization of Runx2 and NeuN (yellow). Scale bar: 50 µm.
Figure 2.
Examples of RUNX2 immunolabeling in the adult mouse brain.
RUNX2 immunlabelling in the cortex (A), ventral striatum (B), hippocampus (C), thalamus (D), amygdala (E), and hypothalamus (F) in a representative adult male mouse at CT9. Scale bar indicates 250 µm. Abbreviations: aco: anterior commissure; AM: anteromedial nucleus of the thalamus; ARC: arcuate nucleus; BLA: basolateral nucleus of amygdala; cc: corpus callosum; CeA: central nucleus of amygdala; DB: diagonal band of Broca; DG: dentate gyrus; lv: lateral ventricle; M: motor cortex; MeA: medial nucleus of amygdala; MS: medial septum, NAc: nucleus accumbens; ot: optic tract; PVA: paraventricular thalamic nucleus, anterior; Re: reuniens thalamic nucleus; RS: retrosplenial cortex, S1: somatosensory cortex; sm: stria medularis; Tu: olfactory tubercle; VMH: ventromedial nucleus of hypothalamus.
Figure 3.
Example of RUNX2 immunolabeling in the suprachiasmatic nucleus (SCN) and paraventricular nucleus (PVN) in a representative adult male mouse at CT9.
The dotted lines outline the area of interest, the SCN in A and the PVN in B. Scale bar depicts 125 µm in A and 250 µm in B. Abbreviations: 3V: third ventricle; och: optic chiasm; PVN: paraventricular nucleus; SCN: suprachiasmatic nucleus.
Figure 4.
Runx2 gene expression is rhythmic in the SCN of wild type mice.
Fold changes in levels of gene expression (± SEM) in the SCN for Runx2 (red circle), Per2 (green triangle; A and B), Bmal1 (blue square; A and B) and Runx1 (black diamond; C and D) and OC (blue circle; C and D) determined by real-time qRT-PCR under 12 h∶ 12 h LD (A and B) and 24 hr DD (C and D). Values represent the level of normalized gene expression relative to the mean overall normalized gene expression across the day. In 12∶12 h LD conditions, time of day is represented by white and black horizontal bars indicating periods of day (light) and dark (night), respectively, with dark onset marking ZT12. In 24 h DD conditions, black horizontal bars represent darkness during subjective day and night with running wheel activity onset marking CT12. Runx2 gene expression data for 12∶12 h LD and 24 h DD were plotted in both panels to allow for comparison with multiple gene profiles. Significant peaks levels of expression (*) compared to nadir points (#) for given gene presented in respective color (p<0.05).
Figure 5.
Runx2 gene expression is rhythmic in the brain regions outside of the SCN.
The paraventricular nucleus, the olfactory bulb and the hippocampus tissues were assayed fewer than 12∶12 h LD (A, C and E) and 24 h DD (B, D and F) conditions. Fold changes in levels of gene expression (± SEM) for Runx2 (red circle), Per2 (green triangle) and Bmal1 (blue square) determined by real-time qRT-PCR. In 12 h∶12 h LD conditions, time of day is represented by white and black horizontal bars indicating periods of day and night, respectively, with dark onset marking ZT12. In 24 h DD conditions, grey and black horizontal bars represent subjective day and night, respectively, with running wheel activity onset marking CT12. Significant peaks levels of expression (*) compared to nadir points (#) for given gene presented in respective color (p<0.05).
Figure 6.
Runx2 protein immunoreactivity is rhythmic in the SCN and PVN of wild type mice.
Fold change (mean ± SEM) in Runx2 protein levels measured by immunodensity analyses in the SCN (A) and PVN (D) of mice housed in 24 h DD. Data are expressed as the fold change (mean ± SEM) relative to the overall mean across times of day for all animals for each brain region. . Significant peaks levels of expression (*) compared to nadir points (#; p<0.05). Representative images of sections through the SCN (B and C) and PVN (E and F) at peak (B and E) and nadir (C and F) time points. Traces represent area of analyses. 3V: third ventricle; och: optic chiasm. Scale bar: 100 µm.
Figure 7.
Diurnal changes in Runx2 gene expression are absent in Bmal1−/− but not Bmal1+/− mouse SCN.
Fold changes in mRNA levels (± SEM) of Per1, Per2, Runx2 and Runx1 determined by real-time qRT-PCR under DD conditions. Values represent the level of normalized gene expression relative to the mean overall normalized gene expression at observed times of day. *: significant difference in levels of gene expression between times of day; p<0.05.
Figure 8.
Mean period of mPer2Luc-mediated bioluminescence rhythms is lengthened in the SCN of Runx2 deficient mice.
A) Mean period (± SEM) of mPer2Luc rhythms in SCN cultures of Runx2+/+ (black), Runx2+/− (grey) and Runx2−/− (white) determined from real-time monitoring of bioluminescence from isolated E19/20 mice. Inlayed number represents n value of experimental group. B) Representative trace of mPer2Luc-mediated bioluminescence observed over several cycles from Runx2+/+ (black), Runx2+/− (grey) and Runx2−/− (dashed) SCN cultures. *: significant difference in levels of gene expression between times of day; p<0.05.
Figure 9.
Free-running period of running wheel activity is lengthened in Runx2+/− mice.
A) Mean (± SEM) free-running period of Runx2+/+ (grey) and Runx2+/− (white) mice. Inlayed number represents n value of experimental group. B) Mean ± SEM wheel revolutions of male Runx2+/+ (grey) and male Runx2+/− (white) entrained to a 12∶12 h LD schedule. Inlayed number represents n value of experimental group. C) Representative running wheel activity records (upper panels) for Runx2+/+ (left panels) and Runx2+/− (right panels) mice. Activity records are in single-plotted format with each horizontal line represents a 24 h period. In 12 h∶12 h LD conditions, time of day is represented by white and black horizontal bars indicating periods of day and night, respectively, with dark onset marking ZT12. Black arrow with white asterisk: transition into 24 h DD. *: significant difference between genotypes; p<0.05.
Table 1.
Runx2+/− mice display responses to phase shifting light pulses indistinguishable from Runx2+/+ mice.