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。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。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) 迁移到背根神经节 (DRG) 中的作用，并确定调节这一过程的因素。躯干 NCC 迁移缺陷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 的核糖探针，并将它们用于原位杂交 (ISH) 分析，以确定这两个基因在胚胎发育过程中的时空表达。所提出的假设是，躯干 NCC 表达 CXCR4 并迁移到高 SDF-1 浓度的中胚层区域，在那里它们分化成 DRG。鸡胚胎（汉密尔顿-汉堡 (HH) 阶段 ~7-22）用于该研究，因为它们的发育阶段已明确定义并且更易于分析和操作。鸡胚中跨越这些发育阶段的 SDF1 和 CXCR4 表达的基因表达谱已基本完成。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定抗体（NCC 的抗 HNK-1 和 DRG 的抗 Islet-1）迁移 NCC 成为 DRG 所采取的发育路径相对应。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 和趋化室的实验中，以解决上游因子（例如 TGF¿1 和 BMP4）在通过 SDF1/CXCR4 信号传导调节 NCC 迁移中的可能作用，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。 CP 是由第二腭发育缺陷引起的，这种缺陷发生在小鼠妊娠第 12-14 天 (gd) 期间。遗传和环境因素与 CP 的病因有关。环境诱导的表观遗传改变提供了一种机制，通过该机制，环境损伤可以在不改变 DNA 序列的情况下引发异常 DNA 甲基化。先前使用 Affymetrix GeneChips 对次级上颚发育过程中的 mRNA 进行分析，并结合生物信息学分析，确定 Sox4 是上颚发育过程中可能受 DNA 甲基化调节的基因，因此与口面裂的研究相关。发现 Sox4 mRNA 从 gd12 到 gd13/14 减少了两倍，表明在此期间甲基化增加。针对小鼠 gd12-14 二级腭 5' 上游区域的 Sox4，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 水平下降之间存在密切关联。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 60%-70%）被 gd13 完全甲基化。这些残基甲基化的增加与该发育时间范围内 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。