Expression profiling of Bmal mutant dorsal skin at telogen of hair follicle cycling

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 Bmal mutant mice, we profiled the dorsal skin of Bmal knockout (-/-) and their heterozygous (+/-) littermates at P22. At P22, 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 Mouse Gene 1.0 ST array.毛囊会以明确的周期性，经历受控的循环往复：生长期（anagen）、退行期（catagen）以及相对静止期（telogen）。我们采用基因组学方法研究同步化小鼠毛囊循环过程中的基因表达，发现除昼夜波动外，受CLOCK调控的基因也会与毛发生长周期同步发生调控变化。在静止期（telogen）与早期生长期（anagen）阶段，生物钟核心基因在次级毛芽（secondary hair germ）中显著表达，而次级毛芽包含了毛囊生长所需的前体细胞。对Clock与Bmal1突变小鼠的分析显示，其生长期进程出现延迟，且次级毛芽细胞内磷酸化Rb的水平降低，同时缺乏有丝分裂细胞，这表明生物钟核心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期阻滞的现象一致，我们在Bmal1突变小鼠的皮肤中观察到p21的显著上调。尽管生物钟调控机制已被证实参与多种昼夜生物过程，但本研究结果表明，生物钟核心基因或可用于调控非昼夜性的循环进程。为从分子层面解析Bmal1突变小鼠的毛发周期延迟现象，我们在P22日龄时对Bmal1基因敲除（-/-）小鼠及其杂合子（+/-）同窝小鼠的背部皮肤进行了转录组分析：在P22日龄时，所有样本均处于即将出现毛发周期延迟的静止期（telogen）阶段，因此各组皮肤样本具有可比性。研究人员依据已确立的形态学标准，通过组织切片将每一份样本归类至毛发生长周期的特定阶段。将每只小鼠背部皮肤提取的RNA分别与Affymetrix小鼠基因1.0 ST芯片（Affymetrix Mouse Gene 1.0 ST array）进行杂交实验。