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

使用 RNA-seq 识别神经元迁移中的 Hox 转录靶标

• 批准号: 8015905
• 负责人: Erik A Lundquist
• 金额: $ 29.06万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8015905
• 关键词: Address; Affect; Animal Model; Animals; Anterior; Axon; Bilateral; Birth Place; Brain; Brain region; Caenorhabditis elegans; Candidate Disease Gene; Cells; Cues; Development; Drosophila genus; Emerging Technologies; Environment; Event; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Genome; Goals; Growth Cones; Immigration; Lead; Left; Location; Mediating; Mental Retardation; Molecular; Nematoda; Nervous system structure; Neuraxis; Neurons; Pathway interactions; Pattern; Population; Process; Proteins; RNA; RNA Interference; RNA Sequences; Right-On; Sampling; Schizophrenia; Signal Transduction; Specific qualifier value; Staging; Sum; Techniques; Testing; Transcript; Transcriptional Regulation; Whole Organism; autism spectrum disorder; brain shape; cell growth; cell motility; cell type; developmental disease; efficacy testing; extracellular; gain of function; gain of function mutation; insight; instrument; interest; loss of function; migration; mutant; nervous system development; neuroblast; programs; promoter; public health relevance; response; transcription factor

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.

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。In C.在秀丽隐杆线虫中，双侧对称的Q成神经细胞产生依赖于Wnt信号传导向前（在右侧）和向后（在左侧）迁移的后代。虽然导致转录变化的典型Wnt途径已被理解，但不太清楚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定。在这个建议中，我们试图了解Wnt诱导的转录变化的机制，在指导成神经细胞迁移在C。优雅的在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介导了这种对Wnt的后向引导，并可能调节其他基因，这些基因指定了后向与前向迁移。我们将利用这个相对简单的指导决定，以确定基因的MAB-5控制后与前迁移。我们将在Illumina Genome AnalyzerII仪器上使用新兴的转录测序技术（RNA-seq）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来鉴定这些候选基因中的哪一个控制Q后代迁移。这将代表一个独特的进步和洞察分化的转录调控，通过识别下游分化因子转录因子调节。这些研究还将测试新兴的RNA-seq技术通过突变体分析识别转录因子的转录靶点的有效性，这是一个通常用微阵列解决的问题。虽然有技术可用于从模型生物体中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有限或回收的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检测从整个生物体分离的RNA群体中基因表达变化的灵敏度。这一提议的重要性在于，它将在引导细胞迁移中鉴定MAB-5 Hox靶标，并且它将测试RNA-seq新兴技术在鉴定模型生物体中转录靶标中的功效和灵敏度，其中细胞特异性RNA库通常无法获得。 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发育障碍和智力迟钝，如无脑畸形，也可能导致精神分裂症和自闭症谱系障碍。本提案的目的是利用模式生物线虫C. elegans了解神经元迁移的基本分子机制，这可能会提供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RNA-seq新兴技术在识别基因表达差异和转录因子靶点方面的功效。