Functional genomic analysis of neural crest development

神经脊干细胞具有多种谱系，包括外周神经系统和黑素细胞。许多先天缺陷、疾病和包括黑色素瘤在内的癌症都是由神经脊表达基因的缺陷引起的。神经脊基因的表达受复杂的转录调控机制的调控。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脊的正常发育是必不可少的。为了确定潜在的Sox10靶基因，我们利用cDNA表达芯片分析了黑色素瘤样本中Sox10表达的变化对多个基因同时表达的影响。我们已经开发了一套工具，并正在继续开发，以发现和分析与神经脊源性黑素细胞发育和疾病有关的基因。我们使用了cDNA微阵列聚类分析，比较了来自神经脊发育不同阶段和黑色素瘤的20个细胞系的RNA。该基因芯片包括4500多个人类黑素细胞表达的EST，2000多个与已知基因相似的小鼠EST，以及在NC-M发生和黑色素瘤发生中具有重要功能的调控基因。对聚集表达谱的分析确定了两个cDNA块；每个cDNA块包含神经脊控cDNA以及尚未被归因于发育或疾病的cDNA。我们已经确认，在小鼠白化病基因巧克力中，其中一个基因发生了改变，这种基因也有眼睛缺陷。我们已经确定了其他几个基因，这些基因是神经脊发育的早期标志。我们还开发了一种有效的方法，通过在体外永生化的NC-MS和体内的黑素母细胞中过度表达这些基因来分析这些基因的功能。这包括使用逆转录病毒系统将基因过表达引导到小鼠和细胞培养中的特定谱系中。我们已经发现，这些基因中有几个具有特定的神经峰衍生物的表达模式，并在神经峰病中发生突变。我们还发现，基因的过度表达可以限制神经脊干细胞的潜力。我们已经详细介绍了WNT信号将神经脊细胞指定为黑素细胞的过程。我们已经从黑素细胞中建立了文库，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，我们已经确定了调节区。我们已经使用病毒来理解转录因子和基因产物之间的世系关系。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，并了解这种语言在黑色素瘤等疾病中是如何改变的。我们正在对黑色素瘤样本进行DNA测序，以确定发育基因的突变。

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.