Skeletal Phenotyping of Period-1-Deficient Melatonin-Proficient Mice

Abstract

In mice, variability in adult bone size and density has been observed among common inbred strains. Also, in the group of genes regulating circadian rhythmicity in mice, so called clock genes, changes in body size and skeletal parameters have been noted in knockout mice. Here, we studied the size and density of prominent bones of the axial and appendicular skeleton of clock gene Period-1-deficient (Per1^-/-) mice by means of microcomputed tomography. Our data show shorter spinal length, smaller and less dense femora and tibiae, but no significant changes in the shape of the skull and the length of the head. Together with the significantly lower total body weight of Per1^-/- mice, we conclude that Per1-deficiency in a melatonin-proficient mouse strain is associated with an altered body phenotype with smaller appendicular (hind limb) bone size, shorter spine length and lower total body weight while normal head length and brain weight. The observed changes suggest an involvement of secondary bone mineralisation with impact on long bones, but lesser impact on those of the skull. Evidence and overall physiological implications of these findings are discussed.

Keywords: bone density; clock genes; microcomputed tomography; period‐1; body size regulation; ossification.

FIGURE 1

Images of whole body microtomographical scans (μCT; dorsal view) of a WT (A) and a Per1^‐/‐ animal (B). The images are derived from videos with a 360° panoramic view, which can be found in the supplementary materials. The scale bar is 10 mm. For orientation in the µCT images, the upmost (red) line marks the beginning of the cervical part of the spine (atlas) in WT, the blue line the end of the cervical and beginning of the thoracal part, the green line the end of the thoracal and beginning of the lumbal part and the orange line the end of the lumbal part. The right part of the image shows the comparison of head (C), spinal (vertebral column; (D), femoral (E) and tibial (F) length. The statistical analysis was done by t‐test (N = 6 WT; N = 7 Per1^‐/‐; p < 0.001***; p < 0.0001****).

FIGURE 2

Figure 2 shows images of the femoral μCT of four Per1^‐/‐ (left) and four WT C3H (right side) mice. The upper images (A) display the surface view of the femora, the lower picture (C) a mid‐section view illustrating the relative cortical and spongiosal areas. The scale bar represents 5.5 mm. Statistical analysis of femoral volume (B) and femoral bone density (D) was done by t‐test (N = 6 WT; N = 7 Per1^‐/‐; p < 0.05*; p < 0.0001****).

FIGURE 3

Figure 3 shows tibial volume (A), and density as measured in g/mm³ (B) or gval (C) which differ significantly in Per1^‐/‐ in comparison to WT (p < 0.05*, t‐test). The mean difference in volume is 16.2% + 3.9% (SD). Statistical analysis was done by t‐test (N = 6 WT; N = 7 Per1^‐/‐; p < 0.05*; p < 0.0001****).

FIGURE 4

Figure 4 shows an example of the overall µCT parameters as visualised in top (red line) and side (green line) views of the whole skull as well as a sagittal (third row) and frontal (lower row) section (A). Left side of A shows WT and the right side Per1^‐/‐. Side bars indicate 10 mm for the total and sagittal views and 7 mm for the frontal view. (B) shows the statistical comparison of skull volume between WT and Per1^‐/‐ (WT N = 7; Per1^‐/‐ N = 6, p < 0.01** t‐test).

FIGURE 5

Figure 5 compares all skulls of WT and Per1^‐/‐ animals by a 1:1 analysis of every animal. The deviation in skull shape was in a low range (−1 to +2 mm) and not significantly different between WT and Per1^‐/‐ animals regarding Hausdorff distance.

FIGURE 6

Figure 6 shows the organ weights in WT and Per1^‐/‐ male mice (WT, N = 7) and (Per1^‐/‐, N = 6). Note that the brain weight did not significantly differ between Per1^‐/‐ and WT animals. However, the weights of heart (p < 0.05*), liver (p < 0.01**) and kidneys (p < 0.0001****) were significantly different between Per1^‐/‐ and WT animals.