Expression profiling of Clock mutant dorsal skin at telogen

Hair follicles undergo recurrent cycling of controlled growth (anagen), regression (catagen), and relative quiescence (telogen) with a defined periodicity. Taking a genomics approach to study gene expression during synchronized mouse hair follicle cycling, we discovered that, in addition to circadian fluctuation, CLOCK-regulated genes are also modulated in phase with the hair growth cycle. During telogen and early anagen, circadian clock genes are prominently expressed in the secondary hair germ, which contains precursor cells for the growing follicle. Analysis of Clock and Bmal1 mutant mice reveals a delay in anagen progression, and the secondary hair germ cells show decreased levels of phosphorylated Rb and lack mitotic cells, suggesting that circadian clock genes regulate anagen progression via their effect on the cell cycle. Consistent with a block at the G1 phase of the cell cycle, we show a significant upregulation of p21 in Bmal1 mutant skin. While circadian clock mechanisms have been implicated in a variety of diurnal biological processes, our findings indicate that circadian clock genes may be utilized to modulate the progression of non-diurnal cyclic processes. To gain molecular understanding of the the hair cycle delay in Clock mutant mice, we profiled the dorsal skin of Clock mutant and their wild-type littermates at P23. At P23, the skin samples are comparable because all the samples are in telogen just prior to the hair cycle delay was observed. Histological sections were used to classify each sample into specific stage of the hair growth cycle based on established morphological guidelines. RNA from each mouse dorsal skin were separately hybridized to an Affymetrix array.