Quantitative live imaging of Venus::BMAL1 in a mouse model reveals complex dynamics of the master circadian clock regulator
Fig 1
Generation and validation of Venus::BMAL1 mouse.
A) sgRNA targeting design across Bmal1 start codon (ATG in bold, shading indicates gene coding sequence). HDR targeting cassette design, cut site indicated by scissors, corresponding homology arms by red boxes and Venus-linker-linker in yellow. Black arrows show post-HDR allele primers for genotyping. B) WB in mouse lung with anti-BMAL1 antiserum, showing increased molecular weight of Venus::BMAL1 (V/V). C) There was no difference in the pattern of BMAL1 expression (shown by IHC) between Venus and non-Venus mouse hip. “No antibody control” confirms the specificity of the antiserum. D) Top: Representative confocal images of SCN from brain sections taken from Venus::BMAL1 animals. Venus::BMAL1 showed nearly 100% registration with endogenous BMAL1 in the SCN from heterozygous animals. Expression of Venus::BMAL1 (yellow) was revealed by confocal fluorescence imaging and IF with an anti-BMAL1 antibody (red). Bottom: Venus::BMAL1-positive localisation was compared with nuclear staining of DAPI. Venus::BMAL1 was predominantly expressed in the nuclei of cells in the SCN. Scale bars as written in Figure. E) Representative confocal micrographs showing close-up images of cells in fixed SCN sections where Venus::BMAL1 (green) is colocalised with SCN neuropeptides (VIP, AVP and GRP shown in red) by IHC and co-stained with DAPI (blue). Colocalised cells are indicated by arrows shown in orange. Red arrows show gaps in Venus::BMAL1 signal which align with GRP cells. F) Percentage neuropeptide-immunostained cells that co-localise with Venus::BMAL1-positive cells (n = 4 animals). G) Representative Venus::BMAL1 signals in femoral head articular cartilage and intervertebral disc tissues (top) and in primary chondrocyte, fibroblast and IVD cultures (bottom). The IVD preparation was co-stained with DAPI, shown below the fluorescence image. Scale bars as written in Figure. H) Representative visualisation of Venus::BMAL1 fluorescence in MEF and chondrocyte nuclei generated in Imaris from Z-stack spectral imaging. Spectral linear unmixing revealed nuclear localization of Venus::BMAL1, with most of the cytoplasmic fluorescence attributed to auto-fluorescence. Yellow: Venus::BMAL1; Red: CellMask Deep Red Plasma membrane stain; blue: auto-fluorescence.