Genetic analysis of somitogenesis in zebrafish

描述(由调查员提供)： 生物学中的一个基本问题是，在脊椎动物胚胎发生过程中，看似均匀的细胞场是如何精心构思的，以及这些过程是如何受到内在和外在信号的调控的。一个明显的例子是体细胞发生，即胚胎中线两侧的节段(体节)从发育中的脊椎动物尾芽产生的近轴前胚层依次形成。这一过程非常精确，每对体节在从前到后的方向上都有严格的周期性。在本研究中使用的模式生物斑马鱼中，每30分钟就会形成一对新的体节；而在更干燥的脊椎动物，如小鸡和老鼠，这一时间更长(分别为90分钟和120分钟)。最近这一领域的研究表明，在分裂前期细胞中，这一周期由一种内在的“分段时钟”控制，该时钟涉及到许多基因的振荡(循环)表达。在所有脊椎动物中，这种机制的核心是分裂样(HER/HES)基因的毛发/增强子(HER/HES)的循环表达，该基因编码转录抑制物，为时钟提供关键的负反馈，并可由Notch信号调节。虽然已经确定了一些影响时钟的因素，但如何管理和协调所有投入仍然是一个悬而未决的问题。时钟是由什么启动的？周期性是如何控制的？信元如何与其邻居同步？这项工作的一个目标是使用斑马鱼转基因报告系的实时成像来研究这个精致的计时器是如何在每个细胞中启动的，它如何受到邻近细胞的振荡的影响，以及振荡周期如何以及是否随着时间的变化而变化。第二个目标是确定控制时钟功能的转录后调控元件，目的是了解不同的调控输入如何影响时钟。最后一个目标是确定通过突变识别的影响振荡的新基因，并阐明它们的分子作用机制。长期目标是了解细胞如何评估它们在胚胎中的位置，对位置信号或梯度做出反应，并将位置信息传达给它们的邻居。此外，由于Hes基因的表达已被证明在非体细胞中振荡，这项工作将导致对许多细胞系中细胞计时器的深入了解。Notch途径参与了大量影响人类健康和发育的过程，包括癌症、干细胞自我更新和分化、细胞增殖、细胞和器官分化计划，以及先天性骨骼疾病，如脊柱肋骨发育不良。当今生物学家面临的关键问题之一是了解Notch信号是如何控制如此不同的发育决定的，所提出的工作将阐明一个Notch调节振荡器的分子机制，为在其他环境中如何调节Notch通路提供见解。项目简介Notch途径涉及大量影响人类健康和发育的过程，包括癌症、干细胞自我更新和分化、细胞增殖、细胞和器官分化计划，以及遗传病(包括Alagille综合征、CADASIL和脊柱肋骨发育不良)。当今生物学家面临的关键问题之一是了解Notch信号如何控制如此多样化的一组发育决定；这里提出的工作将阐明Notch调节振荡器的分子机制，以及Notch信号如何与其他信号途径整合以控制分化程序。我们预计，我们的基础研究将为Notch通路在其他环境中如何调控提供见解，并导致对人类发育、健康和疾病的重要见解。

DESCRIPTION (provided by investigator): A fundamental question in biology is how seemingly homogeneous fields of cells become elaborately patterned during vertebrate embryogenesis, and how these processes are regulated both by intrinsic and extrinsic signals. One striking example is that of somitogenesis, the process by which segments (somites) are sequentially formed on either side of the embryonic midline from the paraxial presomitic mesoderm generated by the growing vertebrate tail bud. This process is exquisitely precise, with each somite pair forming with a strict periodicity in an anterior to posterior direction. In zebrafish, the model organism used in this study, a new somite pair forms every 30 minutes; in drier vertebrates like chick and mouse, the period is longer (90 and 120 minutes, respectively). A recent explosion of work in this area has indicated that the period is controlled in presomitic cells by an intrinsic "segmentation clock" which involves the oscillating (cyclic) expression of a number of genes. Central to the mechanism in all vertebrates is the cyclic expression of hairy/Enhancer of split-like (her/hes) genes, which encode transcriptional repressors that contribute critical negative feedback to the clock and that can be regulated by Notch signals. Although a number of factors that influence the clock have been identified, it remains an open question as to how all the inputs are regulated and coordinated. What starts the clock? How is periodicity controlled? How do cells synchronize with their neighbors? One goal of this work is to use real-time imaging of a zebrafish transgenic reporter line that recapitulates her1 gene oscillation to investigate how this exquisite timer is started in each cell, how it is influenced by the oscillations of neighbor cells, and how and if the oscillation period changes over time. A second goal is to identify the post- transcriptional regulatory elements that control clock function, with the goal of understanding how different regulatory inputs influence the clock. The last goal is to characterize new genes identified by mutation that influence the oscillation and elucidate their molecular mechanism of action. The long-term goals are to understand how cells assess their position within an embryo, respond to positional signals or gradients, and communicate positional information to their neighbors. In addition, since Hes gene expression has been shown to oscillate in non-somitic cells, this work will lead to insights about cellular timers in many cell lineages. The Notch pathway has been implicated in a huge number of processes that affect human health and development, including cancers, stem cell self-renewal and differentiation, cell proliferation, and cell and organ differentiation programs, as well as in congenital skeletal disorders such as the spondylocostal dysostoses. One of the key questions facing biologists today is to understand how Notch signals control such diverse group of developmental decisions, and the work proposed will elucidate molecular mechanisms by which one Notch- regulated oscillator is controlled, providing insights into how Notch pathways may be regulated in other contexts. Project Narrative The Notch pathway has been implicated in a huge number of processes that affect human health and development, including cancers, stem cell self-renewal and differentiation, cell proliferation, and cell and organ differentiation programs, as well as genetic disease (including Alagille syndrome, CADASIL, and the spondylocostal dysostoses). One of the key questions facing biologists today is to understand how Notch signals control such a diverse group of developmental decisions; the work proposed here will elucidate molecular mechanisms by which a Notch-regulated oscillator is controlled, as well as how Notch signals integrate with other signaling pathways to control a differentiation program. We anticipate that our basic research will provide insights into how Notch pathways may be regulated in other contexts and lead to important insights into human development, health, and disease.