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

调节胚胎发生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阻遏蛋白Hes 1和Hes 7的功能就像生物钟一样，周期为两个小时。Hes 1和Hes 7自主地呈现两小时周期振荡表达。这种振荡可以用下面的方程来模拟：dp（t）/dt=am（t-T_p）-bp（t）dm（t）/dt=k/{1+{p（t-T_m}^2/po^2]-cm（t）这些方程是基于Hes蛋白二聚体的负反馈回路。这些方程表明，Hes蛋白的不稳定性是稳定振荡所必需的。Hes蛋白的半衰期约为20分钟，但该模拟模型预测，如果半衰期变为30分钟，则振荡可以被阻尼。为了确定Hes蛋白不稳定性的意义，我们产生了转基因小鼠，其表达稳定的Hes 7蛋白（半衰期约为30分钟）而不是野生型Hes 7。在这些小鼠中，Hes 7振荡在三或四个周期后变得阻尼，体节融合。为了理解Hes振荡在体节分割以外的其他系统中的作用，我们寻找了表达以两小时为周期振荡的基因。在血清处理后的几个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，我们确定了大约10个基因，其表达以两个小时的周期振荡，如Hes 1。有些基因的振荡与Hes 1处于同一阶段，而有些基因的振荡与Hes 1不同。我们计划通过过量表达和敲低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.