Role of TGF-β signaling for coupling of circadian oscillators in peripheral tissues

人们早就知道，视交叉上核中昼夜节律振荡器细胞之间的耦合是内聚、稳健和高振幅节律所必需的。然而，外周细胞时钟之间是否也存在耦合是有争议的，可能的机制尚不清楚。最近，Schibler实验室能够明确地表明外周振荡细胞（肝细胞）在活小鼠中也偶联。同时，我们已经确定了典型的TGF-β信号作为外周细胞生物钟正常耦合所需的途径。在这里，我们假设典型的TGF-β信号有助于体内肝细胞的细胞间偶联，从而在昼夜生理中发挥重要作用。为了验证这一假设，我们提出了以下两个研究目标：目的1：表征典型TGF-β信号在体内肝细胞偶联中的作用。外周组织内偶联的损害被预测会影响内聚性、振幅、相以及对授时细胞的反应。我们将创建典型TGF-β信号的组织特异性遗传功能丧失小鼠模型，并测试(i)时钟基因和时钟控制基因的昼夜节律振幅和相位是否受到影响；（ii）进料反转时相移动力学是否改变。由于外周组织的基因表达节律既依赖于局部昼夜节律振荡器，也依赖于有节奏的全身授时因子，耦合的扰动可能会影响基因表达的昼夜动力学，因为对全身输入的反应可能发生改变。我们将分析肝细胞中典型TGF-β信号受到干扰的小鼠肝脏的昼夜基因表达节律，并确定可能受影响的生理途径。

It has long been known that coupling among circadian oscillator cells in the suprachiasmatic nucleus is required for cohesive, robust and high amplitude rhythms. However, whether coupling also exists between cellular clocks in the periphery was controversial, and a possible mechanism was unknown. Recently, the Schibler laboratory was able to show unequivocally that peripheral oscillator cells (hepatocytes) are also coupled in living mice. At the same time, we have identified canonical TGF-β signaling as a pathway required for normal coupling of cellular circadian clocks in peripheral cells. Here, we hypothesize that canonical TGF-β signaling contributes to intercellular coupling of hepatocytes in vivo thereby playing an important role for circadian physiology. To test this hypothesis, we propose to study the following two objectives: Objective 1: Characterize the role of canonical TGF-β signaling for coupling of hepatocytes in vivo Impairment of coupling within peripheral tissues is predicted to affect cohesiveness, amplitudes, phases as well as responses to zeitgebers. We will create tissue-specific genetic loss-of-function mouse models of canonical TGF-β signaling and test (i) whether circadian amplitudes and phases of clock genes and clock-controlled genes are affected; (ii) whether the kinetics of phase shifting upon feeding reversal is altered. Objective 2: Study the role of canonical TGF-β signaling for liver transcriptome rhythms As gene expression rhythms in peripheral tissues depend on both the local circadian oscillator and rhythmic systemic zeitgebers, a perturbation of coupling likely affects circadian dynamics of gene expression due to a potentially altered response to systemic inputs. We will analyze circadian gene expression rhythms in murine liver with perturbed canonical TGF-β signalling in hepatocytes and identify potentially affected physiological pathways.