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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 项目＃1：该项目的总体目标是检查SDF1/CXCR4信号传导在躯干神经元细胞（NCCS）迁移到背根神经节（DRG）中的作用，并确定调节该过程的因素。躯干NCC的有缺陷的迁移会导致围产期致死性，并影响周围神经系统的发展和形成。为此，我们为鸡SDF1和CXCR4构建了带状探针，并利用了原位杂交（ISH）分析来确定胚胎发育过程中两个基因的空间时间表达。要解决的假设是，躯干NCCS表达CXCR4并迁移到高SDF-1浓度的中胚层区域，它们分化为DRG。鸡肉胚（Hamilton-Hamburger（HH）阶段〜7-22）被用于研究，因为它们的发育阶段已经很好地定义，并且更适合分析和操纵。在很大程度上完成了跨越这些发育阶段的SDF1和CXCR4表达的基因表达分析。正在进行的研究正在确定CXCR4和SDF-1的表达模式是否对应于使用特定抗体迁移NCC成为DRG所采取的发育路径-NCCS的抗HNK-1和DRG的抗ISLET-1。在一项并行的研究中，在涉及Transwells和趋化室的实验中，使用PC12细胞（源自肾上腺；神经crest起源）来解决上游因素（例如TGF¿1和BMP4）的可能作用（例如，TGF。1和BMP4）在通过sdf1/ cxcr4信号的NCC迁移中进行调节，并在此信号下效应效应。 项目＃2 ：（新）： 在美国最普遍的出生缺陷之一，包括口腔裂（CP）和带有或不带有裂口的裂口等口腔裂口。 CP是由次生口感发育的缺陷引起的，次生口感的发育发生在小鼠中发生在妊娠日（GD）12-14。 CP的病因中隐含了遗传和环境因素。环境诱导的表观遗传改变提供了一种机制，环境损伤可以触发异常的DNA甲基化而不会改变DNA序列。使用Affymetrix Genechips对MRNA分析的先前分析，再加上生物信息学分析，将SOX4鉴定为一种基因，是一种可能受到da甲基发育过程中DNA甲基化的基因，因此与大地裂裂的研究有关。发现Sox4 mRNA从GD12到GD13/14降低了两倍，表明在此期间甲基化增加。在GD12-14次生味的5'上游区域中为小鼠Sox4开发了广泛的CpG甲基化谱。结果分析表明，在5'侧翼区域的DNA甲基化与GD12-14期间看到的mRNA水平降低之间存在很强的关联。具体而言，在GD12时为60％-70％甲基化的60％-70％甲基化的两个CPG残差被GD13完全甲基化。这些残留物的甲基化增加与此发育时间框架中Sox4 mRNA水平的降低相关。当前的研究试图使用甲基化依赖性的荧光素酶表达向量验证这些CpG的功能。