Using RNA-seq to identify Hox transcriptional targets in neuronal migration

描述（申请人提供）：神经元迁移是形成大脑和中枢神经系统的关键发育事件。在秀丽隐杆线虫中，双侧对称的Q神经母细胞产生依赖于Wnt信号向前（右侧）和向后（左侧）迁移的后代。虽然已经了解了导致转录变化的典型Wnt途径，但尚不清楚Wnt诱导的这些转录变化如何导致特定的细胞和生长锥指导决策。在这个提议中，我们试图了解wnt诱导的转录变化在秀丽隐杆线虫中引导神经母细胞迁移的机制。在没有Wnt信号的情况下，Q神经母细胞后代迁移到前侧。Hox转录因子MAB-5在Wnt应答中介导这种后向引导，并可能调节其他指定后向与前向迁移的基因。我们将利用这一相对简单的指导决策来确定由单克隆抗体-5调控的控制后向和前向迁移的基因。我们将利用Illumina Genome AnalyzerII仪器上新兴的转录本测序技术（RNA-seq）分析野生型和单克隆抗体-5功能丧失和功能获得突变体的转录组，以确定在单克隆抗体-5活性响应中上调或下调的基因。然后，我们将使用高通量RNAi来确定哪些候选基因控制Q后代迁移。通过鉴定受转录因子调控的下游分化因子，这将代表对分化转录调控的独特进展和见解。这些研究还将测试新兴的RNA-seq技术的有效性，该技术通过突变体分析来识别转录因子的转录靶点，这是一个通常用微阵列来解决的问题。虽然有技术可以从模式生物的特定细胞群中分离RNA池，但由于细胞特异性启动子的可用性限制或RNA回收率低，它们可能不适用于所有细胞类型。在这个提议中，我们将测试RNA-seq的敏感性，以检测从整个生物体分离的RNA群体中基因表达的变化。这一建议具有重要意义，因为它将在引导细胞迁移中识别MAB-5 Hox靶点，并且它将测试新兴技术RNA-seq在识别模式生物中转录靶点的有效性和敏感性，因为通常无法获得细胞特异性RNA池。

DESCRIPTION (provided by applicant): Neuronal migration is a key developmental event that shapes the brain and central nervous system. In C. elegans, the bilaterally symmetric Q neuroblasts give rise to descendants that migrate anteriorly (on the right) and posteriorly (on the left) dependent upon Wnt signaling. While canonical Wnt pathways resulting in transcriptional changes are understood, it is less clear how these transcriptional changes induced by Wnt result in specific cell and growth cone guidance decisions. In this proposal we seek to understand the mechanisms of Wnt-induced transcriptional changes in the guidance of neuroblast migration in C. elegans. In the absence of the Wnt signal, the Q neuroblast descendants migrate to the anterior. The Hox transcription factor MAB-5 mediates this posterior guidance in response to Wnt, and might regulate other genes that specify posterior versus anterior migration. We will take advantage of this relatively simple guidance decision to identify genes regulated by MAB-5 that control posterior versus anterior migration. We will analyze the transcriptomes of wild-type and mab-5 loss of function and gain of function mutants using the emerging technology of transcript sequencing (RNA-seq) on the Illumina Genome AnalyzerII instrument to identify genes that are upregulated or downregulated in response to MAB-5 activity. We will then use high throughput RNAi to identify which of these candidate genes control Q descendant migration. This would represent a unique advance and insight into the transcriptional regulation of differentiation by identifying downstream differentiation factors regulated by transcripton factors. These studies will also test the efficacy the emerging technology of RNA-seq to identify transcriptional targets of transcription factors via mutant analysis, a question normally addressed with microarrays. While techniques are available to isolate RNA pools from specific cell populations in model organisms, they might not be feasible for all cells types due to limitations on the availability of cell-specific promoters or the low amount of RNA recovered. In this proposal we will test the sensitivity of RNA-seq to detect changes in gene expression in RNA populations isolated from whole organisms. This proposal is significant in that it will identify MAB-5 Hox targets in guided cell migration, and in that it will test the efficacy and sensitivity of the emerging technology of RNA-seq in the identification of transcriptional targets in model organisms where cell-specific RNA pools often cannot be obtained. PUBLIC HEALTH RELEVANCE: Neuronal migration is a key process in brain and central nervous system development. Deficits in this process can lead to developmental disorders with mental retardation, such as lissencephalies, and also might contribute to schizophrenia and autism spectrum disorders. The goal of this proposal is to use the model organism nematode worm C. elegans to understand the basic molecular mechanisms of neuronal migration, which might provide insight into the underlying molecular mechanisms associated with developmental disorders of the central nervous system. The proposal also will test the efficacy the emerging technology of RNA-seq to identify gene expression differences and transcription factor targets.