Identification and analysis of chicken DLL3 as a segmentation clock component

摘要 Notch信号通路是一种高度保守的细胞：起关键作用的细胞通讯通路 在后生动物发展的许多方面发挥作用。对这一途径的严格时空调节是 在许多发育决策中至关重要，并了解导致这一现象的分子机制 优雅的控制是一个广受关注的领域。这项拟议的研究使用了生物进化过程中的 分段时钟探索依赖于细胞自主功能的Notch通路调节模式 缺口配体。 分段时钟包括一个振荡的基因表达网络，该网络在 体细胞发生的时间调控。哺乳动物的时钟需要表达一种非典型的配体 DLL3不能激活Notch信号，但能够以细胞自主的方式调节该途径。我们 已经确定了DLL3的一个鸡同源基因，并发现它的预测结构表明它也将是 无法激活Notch信令。这一发现强调了分段时钟是一个发育过程 这依赖于细胞自主配体的功能，为理解新的机制提供了一个模型 调控Cis中的Notch通路。Notch配体与Cis相互作用的机制 受体，或共表达的配体之间的了解很少，分段时钟提供了一个 剖析这些互动的杰出模型。两个目标将为中心模型提供支持，即细胞 自主性配体活动对节段性时钟功能的调节至关重要。首先，我们将评估 鸡DLL3蛋白的表达及功能研究RNA原位杂交将评估cDll3的表达 在鸡胚胎发生过程中，标记的cDLL3蛋白在细胞培养中的表达将被用于鉴定 蛋白质的亚细胞定位。功能分析将评估cDLL3与其他基因相互作用的能力 Notch途径的组成，以及其表达如何影响Notch途径的活性。第二个目标将直接 研究鸡胚胎发育过程中是否需要cDLL3的表达来实现分段时钟功能。 这里提出的工作将提供关键数据来支持细胞自主的中心假说 配基的活性在分段时钟中为调节Notch途径的活性提供了一个关键机制。我们 预计这项工作的结果将对我们理解凹槽如何产生广泛的影响 途径是受调控的，允许表面上看起来直接的途径有助于形成复合体 后生动物的发育决策，并提供了对途径失调如何 导致人类发育中的先天异常。

Abstract The Notch signaling pathway is a highly conserved cell:cell communication pathway that plays critical roles in many aspects of metazoan development. Tight spatial and temporal regulation of this pathway is crucial in many developmental decisions, and understanding the molecular mechanisms contributing to this elegant control is an area of broad interest. The proposed research uses evolutionary changes in the segmentation clock to explore modes of Notch pathway regulation that rely on cell-autonomous functions of Notch ligands. The segmentation clock comprises a network of oscillatory gene expression that functions in the temporal regulation of somitogenesis. The mammalian clock requires expression of an atypical ligand called DLL3 that can not activate Notch signaling, but is able to modulate the pathway in a cell autonomous way. We have identified a chicken homolog of DLL3, and find that its predicted structure suggests that it too will be unable to activate Notch signaling. This finding highlights the segmentation clock as a developmental process that relies on cell autonomous ligand functions, providing a model to understand the novel mechanisms that regulate the Notch pathway in cis. The mechanisms that underlie cis interactions between Notch ligands and receptors, or between co-expressed ligands are poorly understood, and the segmentation clock provides an outstanding model to dissect these interactions. Two aims will provide support for the central model that cell autonomous ligand activities are critical for regulation of segmentation clock function. First, we will assess the expression and function of the chicken DLL3 protein. RNA in situ hybridization will assess cDll3 expression during chick embryogenesis and expression of tagged cDLL3 protein in cell culture will be used to identify the subcellular localization of the protein. Functional assays will asses the ability of cDLL3 to interact with other Notch pathway components, and how its expression affects Notch pathway activity. A second aim will directly examine whether cDLL3 expression is required for segmentation clock function in developing chick embryos. The work proposed here will provide critical data to support the central hypothesis that cell-autonomous ligand activity provides a critical mechanism to regulate Notch pathway activity in the segmentation clock. We anticipate that the results from this work will have broad implications for our understanding of how the Notch pathway is regulated, allowing a pathway that appears straightforward on the surface to contribute to complex developmental decisions across metazoans, and providing new insights into how pathway dysregulation can contribute to congenital anomalies in human development.