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

基本信息

批准号：
10457452
负责人：
SUSAN E COLE
金额：
$ 7.66万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10457452
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 gene network 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

项目摘要

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.

抽象的 Notch信号通路是一个高度保守的细胞：扮演关键的细胞通信途径在后生发展发展的许多方面的角色。该途径的紧密空间和时间调节是在许多发展决策中至关重要，并理解有助于此的分子机制优雅控制是一个广泛关注的领域。拟议的研究使用了进化变化分割时钟以探索依赖于细胞自主函数的Notch途径调节模式缺口配体。分割时钟包括一个振荡基因表达网络，该网络在时间生成的时间调节。哺乳动物时钟需要表达一个非典型配体称为无法激活Notch信号的DLL3，但能够以单元自主的方式调节途径。我们已经确定了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。这一发现突出了细分时钟作为一个发展过程这依赖于细胞自主配体功能，提供了一个模型来了解新的机制调节顺式中的缺口途径。 Notch配体之间的CIS相互作用的机制和受体或共表达配体之间的理解很少，分段时钟提供了阐述这些相互作用的杰出模型。两个目标将为单元格的中心模型提供支持自主配体活动对于调节分段时钟功能至关重要。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表达在雏鸡的胚胎发生和标记的CDLL3蛋白在细胞培养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与其他相互作用的能力 Notch途径成分及其表达如何影响Notch途径活动。第二个目标将直接检查是否需要CDLL3表达在开发雏鸡胚胎中的分割时钟功能。这里提出的工作将提供关键数据，以支持细胞自治的中心假设配体活动提供了调节分割时钟中缺口途径活动的关键机制。我们预计这项工作的结果将对我们对档次的理解产生广泛的影响途径受到调节，允许在表面上直截了当的途径有助于复杂跨后生动物的发展决策，并提供有关路径失调的新见解为人类发展的先天异常做出贡献。