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

描述（由申请人提供）：神经元迁移是塑造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 信号传导向前（右侧）和向后（左侧）迁移的后代。虽然导致转录变化的经典 Wnt 途径已被了解，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。在本提案中，我们试图了解 Wnt 诱导的转录变化指导秀丽隐杆线虫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部。 Hox 转录因子 MAB-5 介导这种后向引导以响应 Wnt，并可能调节其他指定后向与前向迁移的基因。我们将利用这个相对简单的指导决策来识别受 MAB-5 调节的基因，这些基因控制后向与前向迁移。我们将在 Illumina Genome AnalyzerII 仪器上使用新兴的转录测序 (RNA-seq) 技术分析野生型和 mab-5 功能丧失和功能获得突变体的转录组，以识别响应 MAB-5 活性而上调或下调的基因。然后我们将使用高通量 RNAi 来识别这些候选基因中哪些控制 Q 后代迁移。通过识别转录子因子调节的下游分化因子，这将代表着对分化转录调节的独特进步和洞察。这些研究还将测试新兴的 RNA-seq 技术通过突变分析识别转录因子转录靶点的功效，这通常是通过微阵列解决的问题。虽然现有技术可从模型生物体的特定细胞群中分离 RNA 库，但由于细胞特异性启动子可用性的限制或回收的 RNA 量较低，这些技术可能不适用于所有细胞类型。在本提案中，我们将测试 RNA-seq 的敏感性，以检测从整个生物体中分离的 RNA 群体中基因表达的变化。该提案的意义重大，因为它将鉴定引导细胞迁移中的 MAB-5 Hox 靶点，并且将测试新兴的 RNA-seq 技术在通常无法获得细胞特异性 RNA 池的模型生物体中鉴定转录靶点的有效性和敏感性。 公共卫生相关性：神经元迁移是大脑和中枢神经系统发育的关键过程。这一过程的缺陷可能导致智力低下的发育障碍，例如无脑畸形，也可能导致精神分裂症和自闭症谱系障碍。该提案的目标是利用模型生物线虫线虫来了解神经元迁移的基本分子机制，这可能有助于深入了解与中枢神经系统发育障碍相关的潜在分子机制。该提案还将测试新兴的 RNA-seq 技术识别基因表达差异和转录因子目标的功效。

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.