Functional genomic analysis of neural crest development

神经嵴发育的功能基因组分析

• 批准号: 8349981
• 负责人: William J Pavan
• 金额: $ 69.38万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349981
• 关键词: Albinism; Cell Culture Techniques; Cell Line; Chocolate; Cleft Lip; Cluster Analysis; Complementary DNA; Complex; Congenital Abnormality; Congenital Megacolon; DNA; DNA Sequence; Defect; Development; Developmental Gene; Diagnosis; Disease; Expressed Sequence Tags; Eye; Functional RNA; Gene Expression; Gene Targeting; Genes; Goals; Human; In Situ; In Vitro; Language; Lead; Libraries; Malignant Neoplasms; Methods; Molecular Profiling; Mus; Mutate; Mutation; Neural Crest; Neural Crest Cell; Nucleic Acid Regulatory Sequences; Pathway interactions; Patients; Pattern; Peripheral Nervous System; Process; Proteins; RNA; Retroviridae; Sampling; Sequence Analysis; Specific qualifier value; Staging; Stem cells; System; Transcription factor genes; Transcriptional Regulation; Virus; WNT Signaling Pathway; Waardenburg syndrome; Work; cDNA Arrays; cDNA Expression; comparative; deafness; functional genomics; human disease; in vivo; melanoblast; melanocyte; melanoma; mouse genome; overexpression; therapy development; tool; transcription factor

Neural crest stem cells give rise to multiple lineages including the peripheral nervous system and melanocytes. Many birth defects, diseases and cancers including melanoma are caused by defects in neural crest expressed genes. The expression of neural crest genes is controlled by a complex transcriptional regulation. One of the transcription factors needed for appropriate expression is SOX10. The target genes of SOX10 may themselves be essential for proper neural crest development. To identify potential SOX10 target genes, we have analyzed the effects of altered SOX10 expression occurring in melanoma samples on the expression of multiple genes simultaneously using cDNA expression microarrays. We have developed and are continuing to develop sets of tools to discover and analyze genes that are involved in neural crest-derived melanocyte development and disease. We used cDNA microarray cluster analysis comparing RNA from twenty cell lines from varying stages of neural crest development and melanoma. The cDNA microarray contained over 4500 human melanocyte expressed ESTs; over 2000 mouse ESTs with similarity to known genes; and control genes with important functions in NC-M development and melanoma. Analysis of the clustered expression profiles identified two blocks of cDNAs; each containing neural crest control cDNAs as well as cDNAs that have not been attributed to development or disease. We have identifed that one of the genes is altered in a mouse albinism gene chocolate which also has eye defects. We have identifed several other genes that are early markers of neural crest development. We have also developed an efficient method for functional analysis of these genes by over-expression in immortalized NC-Ms in vitro and melanoblasts in vivo. This involves using a retrovirus system to direct overexpression of genes into defined lineages in mice and in cell culture. We have found that several of these genes have expression patterns of specific neural crest derivatives and are mutated in neural crest diseases. We have also found that overexpression of genes can limit the potential of the neural crest stem cells. We have detailed the process by which WNT signaling specifies neural crest cells to melanocytes. We have generated libraries from melanocytes and used them to annotate the genome of the mouse. This will help in our identification of human disease loci that have melanocyte defects.This involves analysis of non-coding DNA. Utilizing comparative sequence analysis and trangenesis we have identified regulatory regions. We have used viruses to understand heirarchies between transcription factors and gene products. Using gene expression and in situ comparaison and comparative sequence analysis we are defining the transcriptional language needed for melanocyte development and understanding how this is altered in diseases such as melanoma. We are sequencing DNA from melanoma samples to identify mutations in developmental genes.

神经嵴干细胞产生多种谱系，包括外周神经系统和黑素细胞。许多出生缺陷、疾病和癌症包括黑色素瘤是由神经嵴表达基因缺陷引起的。神经嵴基因的表达受复杂的转录调控。适当表达所需的转录因子之一是SOX 10。SOX 10的靶基因本身可能对神经嵴的正常发育至关重要。为了确定潜在的SOX 10靶基因，我们分析了改变SOX 10表达的影响发生在黑色素瘤样本上的多个基因的表达，同时使用cDNA表达微阵列。我们已经开发并将继续开发一套工具来发现和分析与神经嵴源性黑素细胞发育和疾病有关的基因。我们使用cDNA微阵列聚类分析比较来自神经嵴发育和黑色素瘤不同阶段的20个细胞系的RNA。cDNA微阵列包含超过4500个人类黑素细胞表达的EST;超过2000个与已知基因相似的小鼠EST;以及在NC-M发展和黑色素瘤中具有重要功能的对照基因。聚类的表达谱的分析确定了两个区块的cDNA，每个区块包含神经嵴控制cDNA以及未归因于发育或疾病的cDNA。我们已经确定，在一种老鼠白化基因巧克力中，其中一个基因被改变了，它也有眼睛缺陷。我们已经确定了其他几个基因是神经嵴发育的早期标志物。我们还开发了一种有效的方法，这些基因的功能分析过表达的永生化NC-MS在体外和黑素细胞在体内。这涉及到使用逆转录病毒系统来指导基因在小鼠和细胞培养物中的过度表达到确定的谱系中。我们已经发现，这些基因中的几个具有特定的神经嵴衍生物的表达模式，并在神经嵴疾病中发生突变。我们还发现基因的过度表达会限制神经嵴干细胞的潜力。 我们已经详细介绍了WNT信号将神经嵴细胞指定为黑素细胞的过程。 我们已经从黑素细胞中产生了文库，并用它们来注释小鼠的基因组。 这将有助于我们识别出有黑素细胞缺陷的人类疾病位点，这涉及到非编码DNA的分析。利用比较序列分析和转基因，我们已经确定了调控区域。 我们利用病毒来了解转录因子和基因产物之间的等级关系。 使用基因表达和原位比较和比较序列分析，我们正在定义黑素细胞发育所需的转录语言，并了解这在疾病如黑色素瘤中是如何改变的。 我们正在对黑色素瘤样本的DNA进行测序，以确定发育基因的突变。