Molecular analysis for the novel two-hour cycle biological clocks Hes1 and Hes7

调节胚胎发育的生物钟的分子机制直到最近才开始被阐明。我们最近发现，bHLH抑制物Hes1和Hes7具有生物钟的功能，周期为两小时。HES1和Hes7自主地表现出两小时周期振荡表达。这种振荡可以用以下equations.dp(t)/dt=am(t-T_p)-bp(t)dm(t)/dt=k/{1+{p(t-T_m}^2/po^2]-cm(t)These方程来模拟，这些方程是基于HES蛋白质二聚体的负反馈环。这些方程表明，HES蛋白的不稳定性是稳定振荡所必需的。HES蛋白质的半衰期约为20min，但该模拟模型预测，如果半衰期变为30min，振荡将被抑制。为了确定Hes蛋白不稳定的意义，我们用转基因小鼠代替野生型Hes7，表达稳定的Hes7蛋白(半衰期约为30min)。在这些小鼠中，Hes7振荡在三到四个周期后受到抑制，体节融合。这一结果与模拟模型的预测很好地吻合。为了了解HES振荡在除体节分割之外的其他系统中的作用，我们搜索了其表达以两小时为周期振荡的基因。在血清处理后的不同时间点，从培养的成纤维细胞中提取RNA并进行微阵列分析。从这个分析中，我们识别了大约10个基因，它们的表达像Hes1一样以两个小时的周期振荡。一些基因的振荡与Hes1处于同一阶段，而另一些基因的振荡则不同。我们正计划通过过度表达和敲除实验来阐明它们的功能。这些实验将有助于理解HES振荡在许多生物系统中的意义。

The molecular mechanism for biological clocks that regulate embryogenesis has been only recently beginning to be elucidated. We have recently shown that the bHLH repressors Hes1 and Hes7 function as biological clocks with a periodicity of two hours. Hes1 and Hes7 autonomously exhibit two-hour cycle oscillatory expression. This oscillation could be simulated by the following equations.dp(t)/dt=am(t-T_p)-bp(t)dm(t)/dt=k/{1+{p(t-T_m}^2/po^2]-cm(t)These equations are based on the negative feedback loop by a dimer of Hes proteins. These equations indicate that the instability of Hes proteins is essential for stable oscillation. The half-life of Hes proteins is about 20 min, but this simulation model predicts that if the half-life becomes 30 min, the oscillation could be damped. To determine the significance of the instability of Hes proteins, we generated transgenic mice, which express stabilized Hes7 protein (the half-life is about 30 min) instead of the wild-type Hes7. In these mice, Hes7 oscillation becomes damped after three or four cycles, and somites are fused. This result agrees well with the prediction of the simulation model.To understand the roles of Hes oscillation in other systems than the somite segmentation, we searched for genes whose expression oscillates with a periodicity of two hours. RNA was extracted from cultured fibroblast cells at several time points after serum treatment and subjected to micro-array analysis. From this analysis, we identified about ten genes whose expression oscillates with a periodicity of two hours like Hes1. Oscillation of some genes is in the same phase as Hes1 while that of others is different. We are planning to elucidate their functions by over-expression and knock-down experiments. These experiments will lead to understanding of the significance of Hes oscillation in many biological systems.