Specification of embryonic C. elegans mesoderm by a T-box factor

T 盒因子对胚胎秀丽隐杆线虫中胚层的规范

• 批准号: 7185646
• 负责人: MORRIS F MADURO
• 金额: $ 7.5万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-15 至 2009-03-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7185646
• 关键词: Adopted; Animal Model; Animals; Blood; Boxing; Caenorhabditis elegans; Cells; Congenital Abnormality; Coupled; DNA Binding; DNA Sequence Rearrangement; Development; Ectoderm; Embryo; Endoderm; Evaluation; Family; Gene Targeting; Genes; Genetic; Genomics; Germ Layers; Goals; Green Fluorescent Proteins; Heat-Shock Response; Histocompatibility Testing; Human; In Situ Hybridization; In Vitro; Lead; Light; Malignant Neoplasms; Mammals; Mesoderm; Mesoderm Cell; Muscle; Mutation; Myocardium; Nematoda; Nuclear; Pathway interactions; Pharyngeal structure; Phenotype; Phytohemagglutinins; Process; Production; Protein Overexpression; Proteins; RNA Interference; Regulator Genes; Reporter; Repression; Research Personnel; Role; Signal Transduction; Sister; Site; Skeleton; Specific qualifier value; Testing; Time; Tissues; To specify; base; blastomere structure; cell type; cleft lip and palate; mutant; programs; promoter

DESCRIPTION (provided by applicant): The broad goal of this application is to understand how embryonic cells are directed to follow particular paths of development. The conserved T-box family of transcriptional regulators specifies the development of the middle germ layer, mesoderm, in triploblastic animals. In mammals, mesoderm includes muscle, heart, skeleton, and blood. Mutations of T-box genes can result in birth defects such as cleft lip and palate. In the model animal C. elegans, many mesodermal cell types, including much of the body muscle and part of the pharynx, are made by descendants of a single embryonic cell called MS. The investigator has identified a T-box gene, tbx-35, that is required to specify the fate of MS. In embryos lacking tbx-35, MS descendants are not made. The conserved Wnt pathway, important in human cancer, transduces a signal that blocks activation of tbx-35 in the sister cell of MS, called E. Surprisingly, this repression does not involve the classical C. elegans Wnt effector TCF/POP-1. Specific Aim 1 will fully characterize the phenotype of tbx-35(-) embryos by investigating the expression of pharynx and muscle genes using tissue-specific GFP reporters and in situ hybridization to understand in which tissues are the activities absent and identify the aberrant fate MS descendants ultimately adopt. Specific Aim 2 will assess the ability of the TBX-35 protein to specify mesoderm fates when expressed outside of its normal context by using a heat-shock promoter fusion to overexpress tbx-35 throughout the embryo and assess production of ectopic muscle and pharynx fates by reporter and in situ hybridization. Specific Aim 3 will test direct interaction of TBX-35 with the promoters of known pharynx (pha-4/FoxA) and muscle (hlh-1/MyoD) regulators, and to identify other putative TBX-35 targets using an in vitro genomic selection strategy to amplify fragments of C. elegans DNA that bind TBX-35. Specific Aim 4 will characterize the Wnt-dependent repression of tbx-35 that occurs in E by using mutations and RNA interference to deplete function of Wnt/MAPK pathway genes that are known to participate in regulatory changes that make E different from MS, and assess their effect on tbx-35 repression. The investigator will also identify the cis-regulatory sites in tbx-35 that are responsive to this repression. As T-box genes and the Wnt pathway have similar roles in development of all animals, an understanding of how tbx-35 functions in C. elegans may shed light on how development goes awry, a process which can lead to birth defects or cancer.

描述（由申请人提供）：本申请的主要目标是了解胚胎细胞如何被引导遵循特定的发育路径。 转录调节因子的保守 T 盒家族指定三叶细胞动物中胚层（中胚层）的发育。 在哺乳动物中，中胚层包括肌肉、心脏、骨骼和血液。 T-box基因突变可导致出生缺陷，例如唇裂和腭裂。 在模型动物秀丽隐杆线虫中，许多中胚层细胞类型，包括大部分身体肌肉和部分咽部，都是由称为 MS 的单个胚胎细胞的后代产生的。 研究人员已经鉴定出 T 盒基因 tbx-35，它是确定 MS 命运所必需的。 在缺乏 tbx-35 的胚胎中，不会产生 MS 后代。 保守的 Wnt 通路在人类癌症中很重要，它转导一个信号，阻止 MS 姐妹细胞（称为 E）中 tbx-35 的激活。令人惊讶的是，这种抑制并不涉及经典的线虫 Wnt 效应子 TCF/POP-1。 具体目标 1 将通过使用组织特异性 GFP 报告基因和原位杂交研究咽部和肌肉基因的表达来全面表征 tbx-35(-) 胚胎的表型，以了解哪些组织缺乏活动并确定 MS 后代最终采用的异常命运。 具体目标 2 将评估 TBX-35 蛋白在正常环境之外表达时指定中胚层命运的能力，方法是使用热休克启动子融合在整个胚胎中过度表达 tbx-35，并通过报告基因和原位杂交评估异位肌肉和咽命运的产生。 具体目标 3 将测试 TBX-35 与已知咽 (pha-4/FoxA) 和肌肉 (hlh-1/MyoD) 调节因子的启动子的直接相互作用，并使用体外基因组选择策略来扩增结合 TBX-35 的线虫 DNA 片段，以确定其他假定的 TBX-35 靶点。 具体目标 4 将通过使用突变和 RNA 干扰来消除已知参与使 E 不同于 MS 的调节变化的 Wnt/MAPK 途径基因的功能，来表征 E 中发生的 tbx-35 的 Wnt 依赖性抑制，并评估它们对 tbx-35 抑制的影响。 研究人员还将确定 tbx-35 中对这种抑制有反应的顺式调控位点。 由于 T-box 基因和 Wnt 通路在所有动物的发育中都具有相似的作用，因此了解 tbx-35 在秀丽隐杆线虫中的功能可能有助于揭示发育如何出错，这一过程可能导致出生缺陷或癌症。