Genetic analysis of somitogenesis in zebrafish

描述（由研究者提供）： 生物学中的一个基本问题是，在脊椎动物胚胎发生过程中，看似均匀的细胞场如何变得精心图案化，以及这些过程如何受到内在和外在信号的调节。一个突出的例子是体节发生，通过该过程，节（体节）从近轴前体中胚层在胚胎中线的两侧依次形成，所述近轴前体中胚层由生长的脊椎动物尾芽产生。这个过程是非常精确的，每一对体节的形成都有严格的周期性。在本研究中使用的模式生物斑马鱼中，每30分钟形成一对新的体节;在鸡和小鼠等干燥脊椎动物中，时间更长（分别为90和120分钟）。最近在这一领域的工作爆炸表明，该时期是控制在preomitic细胞由一个内在的“分割时钟”，其中涉及到振荡（循环）表达的一些基因。在所有脊椎动物中，该机制的核心是分裂样（her/hes）基因的毛状/增强子的循环表达，其编码对生物钟贡献关键负反馈的转录抑制因子，并且可以由Notch信号调节。虽然已经确定了一些影响时钟的因素，但如何调节和协调所有输入仍然是一个悬而未决的问题。时钟是怎么开始的？如何控制周期性？细胞如何与它们的邻居同步？这项工作的一个目标是使用重现her 1基因振荡的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如何在每个细胞中启动，它如何受到邻近细胞振荡的影响，以及如何以及是否振荡周期随着时间的推移而变化。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。最后一个目标是表征通过突变识别的影响振荡的新基因，并阐明其作用的分子机制。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谱系中细胞计时器的见解。Notch通路涉及影响人类健康和发育的大量过程，包括癌症、干细胞自我更新和分化、细胞增殖和细胞及器官分化程序，以及先天性骨骼疾病如脊椎肋骨发育不全。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发育决定，并且所提出的工作将阐明一个Notch调节振荡器被控制的分子机制，从而提供对Notch途径如何在其他环境中进行调节的见解。Notch通路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、细胞和器官分化程序，以及遗传疾病（包括Alagille综合征、CADASIL和脊椎肋骨发育不全）。今天生物学家面临的关键问题之一是了解Notch信号如何控制这样一组不同的发育决定;这里提出的工作将阐明Notch调节振荡器控制的分子机制，以及Notch信号如何与其他信号通路整合以控制分化程序。我们预计，我们的基础研究将为Notch途径在其他背景下如何调节提供见解，并为人类发育、健康和疾病提供重要见解。

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.