Function genomic analysis of neural crest development

神经嵴干细胞可分化为多系，包括周围神经系统和黑色素细胞。许多出生缺陷、疾病和癌症，包括黑色素瘤，都是由神经嵴表达基因的缺陷引起的。神经嵴基因的表达受复杂的转录调控控制。适当表达所需的转录因子之一是SOX10。SOX10的靶基因本身可能对神经嵴的正常发育至关重要。为了鉴定潜在的SOX10靶基因，我们利用cDNA表达微阵列分析了黑色素瘤样本中SOX10表达改变对多个基因同时表达的影响。我们已经并将继续开发一系列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了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4500多个人类黑素细胞表达的ESTs；超过2000个与已知基因相似的小鼠ESTs；并控制在NC-M发展和黑色素瘤中起重要作用的基因。集群表达谱分析鉴定了两个片段的cdna；每一种都含有神经嵴控制cdna以及未归因于发育或疾病的cdna。我们已经确认，一只白化病基因巧克力的一个基因被改变了，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。我们还开发了一种有效的方法，通过在体外永生化NC-Ms和体内黑色素母细胞中过表达来分析这些基因的功能。这包括在小鼠和细胞培养中使用逆转录病毒系统将基因过度表达引导到确定的谱系中。我们发现其中一些基因具有特定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表达会限制神经嵴干细胞的潜能。我们详细介绍了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生成了文库，并用它们来注释小鼠的基因组。这将有助于我们识别有黑素细胞缺陷的人类疾病基因座。这包括对非编码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。我们已经用病毒来了解转录因子和基因产物之间的遗传关系。利用基因表达、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，并了解这在黑色素瘤等疾病中是如何改变的。

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 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 lineaegs in mice and in cell culture. We have found that several of these genes have expression pattersn 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 facotrs and gene products. Using gene expression and in situ comparasin and comparative sequence analysis we are defining the transcriptional language needed for melanocyte development and undersatnding how this is altered in diseases such as melanoma.