Expression profiling of mouse dorsal skin during hair follicle cycling. Mus musculus

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. Overall design: To investigate the molecular control of hair follicle cycling, we profiled mRNA expression in mouse dorsal skin at multiple representative time points in the synchronized second postnatal hair growth cycle and in a depilation-induced hair growth cycle. For profiling of second synchronized and depilation-induced hair growth cycle, the same upper-mid region of dorsal skin was excised from C57BL/6 mice at representative postnatal days (P). The time points for second hair growth cycle are classified into different phases of the hair growth cycle based on established morphological guidelines as follow: early anagen (P23, P25), mid anagen (P27), late anagen (P29, P34), early catagen (P37, P39), mid catagen (P41), and telogen (P44). Depilation-induced hair growth cycle by applying wax/rosin mixture on the dorsal skin of seven-week old mice (all follicles in telogen) was performed on mice. The corresponding phases of the hair growth cycle at number of days following depilation (D) is as follow: early anagen (D3), mid anagen (D5), late anagen (D8, D12), and early catagen (D17). For each time point, multiple biological replicates were profiled, with each mouse dorsal skin separately hybridized to an Affymetrix array.

毛囊以固定周期反复经历受控的生长（anagen，生长期）、退行（catagen，退行期）与相对静止（telogen，休止期）的循环过程。本研究采用基因组学方法探究同步化小鼠毛囊周期中的基因表达情况，发现除昼夜波动外，受CLOCK调控的基因同样会随毛发生长周期同步发生表达调控变化。
在休止期与早期生长期阶段，生物钟基因（circadian clock genes）在次级毛芽（secondary hair germ）中显著表达，该结构包含发育中毛囊的前体细胞。对CLOCK与Bmal1突变小鼠的分析显示，其毛囊生长期进程出现延迟，且次级毛芽细胞内的磷酸化Rb（phosphorylated Rb）水平降低，同时未检测到有丝分裂细胞，这提示生物钟基因可通过影响细胞周期来调控生长期进程。与细胞周期G1期（G1 phase）阻滞的现象一致，我们在Bmal1突变小鼠的皮肤中观察到p21蛋白（p21）出现显著上调。
尽管生物钟调控机制已被证实参与多种昼夜生物学过程，本研究结果表明，生物钟基因或可用于调控非昼夜循环过程的进程。

总体实验设计：
为探究毛囊周期的分子调控机制，我们对同步化的出生后第二次毛发生长周期以及脱毛诱导的毛发生长周期中的多个代表性时间点的小鼠背部皮肤进行了mRNA表达谱分析。
对于同步化第二次毛发生长周期与脱毛诱导毛发生长周期的样本采集，我们均从C57BL/6小鼠背部皮肤的同一中上区域取材，取材时间为对应的代表性出生后天数（P）。第二次毛发生长周期的时间点依据已确立的形态学标准划分为不同毛发生长阶段：早期生长期（P23、P25）、中期生长期（P27）、晚期生长期（P29、P34）、早期退行期（P37、P39）、中期退行期（P41）以及休止期（P44）。
我们通过在7周龄小鼠（此时所有毛囊均处于休止期）的背部皮肤涂抹蜡/松香混合物来构建脱毛诱导的毛发生长模型。脱毛后天数（D）对应的毛发生长周期阶段如下：早期生长期（D3）、中期生长期（D5）、晚期生长期（D8、D12）以及早期退行期（D17）。每个时间点均设置多个生物学重复，每只小鼠的背部皮肤样本分别与Affymetrix基因芯片（Affymetrix array）进行杂交。