REGULATION OF NEURAL CREST CELL MIGRATION BY SDF1-CXCR4 SIGNALING

SDF1-CXCR4 信号传导对神经嵴细胞迁移的调节

• 批准号: 8167655
• 负责人: Ratnam Sathiagana Seelan
• 金额: $ 23.8万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167655
• 关键词: 5' Flanking Region; Address; Adrenal Glands; Affect; Antibodies; BMP4; Bioinformatics; CXCL12 gene; CXCR4 gene; Chemotaxis; Chick Embryo; Chickens; Cleaved cell; Cleft Palate; Cleft lip with or without cleft palate; Computer Retrieval of Information on Scientific Projects Database; Congenital Abnormality; Coupled; DNA Methylation; DNA Sequence; Defect; Development; Embryo; Embryonic Development; Environmental Risk Factor; Epigenetic Process; Etiology; Funding; Gene Expression Profiling; Genes; Genetic; Goals; Grant; Immigration; In Situ Hybridization; Institution; Luciferases; Messenger RNA; Methylation; Mus; Neural Crest; Neural Crest Cell; PC12 Cells; Palate; Pattern; Perinatal; Peripheral Nervous System; Phosphotransferases; Process; Regulation; Research; Research Personnel; Resources; Role; Secondary Palate; Signal Transduction; Source; Spinal Ganglia; Staging; Stromal Cell-Derived Factor 1; Time; United States; United States National Institutes of Health; cell motility; expression vector; islet; migration; orofacial; research study; spatiotemporal

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Project #1: The overall goal of the project is to examine the role of SDF1/CXCR4 signaling in the migration of trunk neural crest cells (NCCs) to the Dorsal Root Ganglion (DRG) and to identify factors that regulate this process. Defective migration of trunk NCCs can cause perinatal lethality and affect the development and formation of the peripheral nervous system. Towards this end, we constructed riboprobes for chicken SDF1 and CXCR4, and utilized them in in situ hybridization (ISH) analyses to determine the spatiotemporal expression of the two genes during embryonic development. The hypothesis being addressed is that trunk NCCs express CXCR4 and migrate towards mesodermal regions of high SDF-1 concentration where they differentiate into the DRG. Chicken embryos (Hamilton-Hamburger (HH) stages ~7-22) were used for the study as their developmental stages have been well defined and are more amenable to analysis and manipulation. Gene expression profiling of SDF1 and CXCR4 expression spanning these developmental stages in the chick embryo has largely been accomplished. Ongoing studies are determining if the expression patterns of CXCR4 and SDF-1 correspond to the developmental paths undertaken by migrating NCCs to become DRG using specific antibodies -- anti-HNK-1 for NCCs and anti-Islet-1 for the DRG. In a parallel study, PC12 cells (derived from the adrenal gland; neural crest origin) are being used in experiments involving transwells and chemotaxis chambers to address possible roles for upstream factors (e.g., TGF¿1 and BMP4) in regulating NCC migration via SDF1/ CXCR4 signaling, and the effect of this signaling on putative downstream targets, such as IP3 kinase. Project #2: (new): Orofacial clefts such as cleft palate (CP), and cleft lip with or without cleft palate (CL/P), are amongst the most prevalent birth defects in the United States. CP is caused by defects in the development of the secondary palate, which in mice occurs during gestational days (gd) 12-14. Genetic and environmental factors have been implicated in the etiology of CP. Environmentally-induced epigenetic alterations provide a mechanism by which environmental insults could trigger abnormal DNA methylation without a change in DNA sequence. Previous analyses of mRNA profiling during secondary palate development, using Affymetrix GeneChips, coupled with bioinformatic analyses, identified Sox4 as a gene that is potentially regulated by DNA methylation during palate development, and therefore, relevant to the study of orofacial clefting. Sox4 mRNA was found to decrease two-fold from gd12 to gd13/14, indicative of increased methylation during this period. An extensive CpG methylation profile was developed for mouse Sox4 in the 5' upstream region in gd12-14 secondary palates. The resulting analyses indicated a strong association between DNA methylation in the 5' flanking region and decreased mRNA levels seen during gd12-14. Specifically, two CpG residues residing in a Differentially Methylated Region (DMR), which are 60%-70% methylated at gd12, become fully methylated by gd13. Increased methylation of these residues correlates with the decrease in Sox4 mRNA levels during this developmental time-frame. Current studies seek to validate the functionality of these CpG residues using methylation-dependent luciferase expression vectors.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 项目#1：该项目的总体目标是检查 SDF1/CXCR4 信号在躯干神经嵴细胞 (NCC) 向背根神经节 (DRG) 迁移中的作用，并确定调节此过程缺陷迁移的因素。躯干 NCC 的缺失可导致围产期死亡并影响周围神经系统的发育和形成为此，我们构建了鸡 SDF1 和 CXCR4 的核糖核酸探针，并在原位使用它们。杂交（ISH）分析以确定胚胎发育过程中这两个基因的时空表达，所讨论的假设是躯干NCC表达CXCR4并迁移到高SDF-1浓度的中胚层区域，在那里它们分化为DRG鸡胚胎。 -汉堡 (HH) 阶段 ~7-22) 用于研究，因为它们的发育阶段已明确定义，并且更适合 SDF1 和基因表达谱的分析和操作。鸡胚中跨这些发育阶段的 CXCR4 表达已基本完成，正在进行的研究正在确定 CXCR4 和 SDF-1 的表达模式是否与使用特异性抗体（抗 HNK）迁移 NCC 成为 DRG 所采取的发育路径相对应。 1 用于 NCC，抗 Islet-1 用于 DRG 在一项平行研究中，PC12 细胞（源自肾上腺；神经嵴起源）被用于涉及 Transwell 和趋化性的实验。室来解决上游因素（例如 TGF¿ 1 和 BMP4）通过 SDF1/CXCR4 信号传导调节 NCC 迁移，以及该信号传导对假定的下游靶标（例如 IP3 激酶）的影响。 项目#2：（新）： 口面部裂，如腭裂 (CP) 和伴或不伴腭裂的唇裂 (CL/P) 是美国最常见的出生缺陷之一，是由次级腭发育缺陷引起的。小鼠的妊娠期 (gd) 12-14 周发生 CP 的病因与遗传和环境因素有关。环境诱导的表观遗传改变提供了环境损伤可能的机制。先前使用 Affymetrix GeneChips 结合生物信息学分析对次级上颚发育过程中的 mRNA 分析进行了分析，发现 Sox4 是上颚发育过程中可能受 DNA 甲基化调节的基因，因此具有相关性。在口面裂研究中，发现 Sox4 mRNA 从 gd12 到 gd13/14 减少了两倍，这是在此期间甲基化增加的指标。小鼠 Sox4 在 gd12-14 二级腭的 5' 上游区域开发了广泛的 CpG 甲基化谱。结果分析表明 5' 侧翼区域的 DNA 甲基化与 gd12-14 期间观察到的 mRNA 水平降低之间存在密切关联。具体来说，位于差异甲基化区域 (DMR) 的两个 CpG 残基（在 gd12 处甲基化程度为 60%-70%）变成这些残基的甲基化程度增加与发育期间 Sox4 mRNA 水平的降低相关，目前的研究试图使用甲基化依赖性荧光素酶表达载体来验证这些 CpG 残基的功能。