REGULATION OF NEURAL CREST CELL MIGRATION BY SDF1-CXCR4 SIGNALING

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 项目一：该项目的总体目标是研究SDF 1/CXCR 4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SDF 1和CXCR 4的核糖核酸探针，并利用它们在原位杂交（ISH）分析，以确定这两个基因在胚胎发育过程中的时空表达。提出的假设是，躯干NCC表达CXCR 4并向高SDF-1浓度的中胚层区域迁移，在那里它们分化成DRG。鸡胚胎（Hamilton-Hamburger（HH）阶段~7-22）用于研究，因为它们的发育阶段已经明确定义，更适合分析和操作。跨越鸡胚这些发育阶段的SDF 1和CXCR 4表达的基因表达谱已经在很大程度上完成。正在进行的研究正在确定CXCR 4和SDF-1的表达模式是否与使用特异性抗体--抗HNK-1用于NCC和抗Islet-1用于DRG--迁移NCC成为DRG所进行的发育路径相对应。在一项平行研究中，PC 12细胞（来源于肾上腺;神经嵴起源）被用于涉及transwells和趋化性室的实验中，以解决上游因子（例如，转化生长因子1和BMP 4）在通过SDF 1/CXCR 4信号调节NCC迁移中的作用，以及该信号对推定的下游靶点如IP 3激酶的作用。 项目#2：（新）： 口面裂，如腭裂（CP）和唇裂伴或不伴腭裂（CL/P），是美国最常见的出生缺陷之一。CP是由次级腭发育缺陷引起的，在小鼠中，这发生在妊娠期（gd）12-14天。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，通过这种机制，环境损伤可以在不改变DNA序列的情况下触发异常DNA甲基化。先前使用AffysseGeneChips对次生腭发育过程中的mRNA谱进行的分析，加上生物信息学分析，将Sox 4确定为在腭发育过程中可能受DNA甲基化调控的基因，因此与口面裂的研究相关。发现Sox 4 mRNA从gd 12至gd 13/14降低两倍，表明在此期间甲基化增加。在gd 12 -14次级腭中的5'上游区域中开发了小鼠Sox 4的广泛CpG甲基化谱。结果分析表明，5'侧翼区的DNA甲基化与gd 12 -14期间观察到的mRNA水平降低之间存在强相关性。具体而言，位于差异甲基化区域（DMR）中的两个CpG残基在gd 12处被60%-70%甲基化，在gd 13处被完全甲基化。这些残基的甲基化增加与Sox 4 mRNA水平在此发育时间范围内的降低相关。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残基的功能性。

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.