Identification and analysis of chicken DLL3 as a segmentation clock component

作为分段时钟组件的小鸡DLL3的识别与分析

• 批准号: 10303586
• 负责人: SUSAN E COLE
• 金额: $ 7.67万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303586
• 关键词: Address; Affect; Anterior; Area; Binding; Biological; Biological Assay; Birds; Cell Communication; Cell Culture Techniques; Cell Nucleus; Cell model; Cell surface; Cells; Chick; Chick Embryo; Chickens; Complex; Congenital Abnormality; Data; Defect; Deformity; Development; Developmental Process; Disease; EGF gene; Embryo; Embryonic Development; Evolution; Exhibits; Future; Gene Expression; Genes; Genetic; Homologous Gene; Human; Human Development; In Situ Hybridization; Ligands; Light; Link; Mammals; Mesoderm; Modeling; Molecular; Nature; Notch Signaling Pathway; Orthologous Gene; Pathway interactions; Pattern; Play; Positioning Attribute; Post-Transcriptional Regulation; Process; Proteins; RNA; Regulation; Research; Role; Segmentation Clock Pathway; Signal Transduction; Skeletal Development; Skeleton; Somites; Structure; Surface; Time; Variant; Work; Zebrafish; congenital anomaly; extracellular; gene function; in vivo; insight; interest; loss of function mutation; notch protein; novel; protein function; receptor; rib bone structure; scoliosis; skeletal; somitogenesis; spine bone structure; vertebrate embryos

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.

摘要 Notch信号通路是一种高度保守的细胞：细胞通讯通路， 在后生动物发育的许多方面发挥作用。该途径的紧密空间和时间调节是 在许多发展决定中至关重要，并了解有助于这一点的分子机制 优雅控制是一个广泛关注的领域。这项拟议中的研究利用了 分段时钟，以探索依赖于细胞自主功能的Notch途径调节模式， Notch配体。 分段时钟包括振荡基因表达的网络，其在细胞中起作用。 体节发生的时间调节。哺乳动物的生物钟需要表达一种非典型配体， DLL3不能激活Notch信号传导，但能够以细胞自主的方式调节该途径。我们 我已经鉴定了DLL3的鸡同源物，并发现其预测的结构表明它也将是 无法激活Notch信号。这一发现强调了分割时钟作为一个发展过程 它依赖于细胞自主配体功能，提供了一个模型来理解新的机制， 顺式调节Notch通路。Notch配体和Notch受体之间的顺式相互作用的机制是： 受体之间，或共表达配体之间的相互作用知之甚少，而分段时钟提供了一个 这是一个很好的模型来剖析这些相互作用。两个目标将为细胞的中心模型提供支持， 自主配体活性对于调节分段时钟功能是关键的。首先，我们将评估 鸡DLL3蛋白的表达和功能。RNA原位杂交将评估cDll3表达 在鸡胚胎发生过程中，标记的cDLL3蛋白在细胞培养物中的表达将用于鉴定 蛋白质的亚细胞定位。功能测定将评估cDLL3与其他细胞相互作用的能力。 Notch途径组分，以及其表达如何影响Notch途径活性。第二个目标将直接 检查cDLL3表达是否是发育中的鸡胚中的分割时钟功能所必需的。 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设， 配体活性提供了在分割时钟中调节Notch途径活性的关键机制。我们 预计这项工作的结果将对我们理解Notch如何产生广泛的影响。 途径受到调节，允许表面上看起来简单的途径有助于复杂的 发育决策的后生动物，并提供新的见解，如何途径失调， 导致人类发育的先天性异常