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。在秀丽隐杆线虫中，双侧对称Q神经细胞产生后代，这些后代迁移（右侧）和后方（左侧）取决于Wnt信号传导。尽管了解导致转录变化的规范WNT途径，但尚不清楚WNT引起的这些转录变化如何导致特定的细胞和生长锥引导决策。在此提案中，我们试图理解Wnt诱导的转录变化的机制，在秀丽隐杆线虫中神经细胞迁移的指导中。在没有Wnt信号的情况下，Q神经细胞后代迁移到前部。 HOX转录因子MAB-5介导了对Wnt的后验指导，并可能调节指定后验和前迁移的其他基因。我们将利用这一相对简单的指导决定来识别由MAB-5调节的基因，该基因控制后迁移与前迁移。我们将使用在Illumina Genome Analyserii仪器上使用新兴转录测序技术（RNA-SEQ）来分析野生型和MAB-5功能丧失以及功能突变体的增益的转录组，以识别对MAB-5活性响应响应的基因。然后，我们将使用高吞吐量RNAi来识别这些候选基因控制Q后代迁移的哪个。这将代表通过识别由转录子因子调节的下游分化因子来对分化的转录调节的独特进步和见解。这些研究还将测试RNA-Seq的新兴技术通过突变分析鉴定转录因子的转录靶标，这是通常用微阵列解决的问题。尽管可以将RNA池与模型生物的特定细胞群体分离出来，但由于限制了细胞特异性启动子的可用性或恢复低量的RNA，它们可能并不可行。在此提案中，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。该提案很重要，因为它将在引导细胞迁移中识别MAB-5 HOX靶标，并且它将测试RNA-Seq新兴技术在模型生物体中转录靶标识别细胞特异性RNA库中经常无法获得转录靶标的功效和敏感性。 公共卫生相关性：神经元迁移是大脑和中枢神经系统发展的关键过程。此过程中的缺陷会导致患有智力低下的发育障碍，例如Lissencephalies，也可能导致精神分裂症和自闭症谱系障碍。该建议的目的是使用模型生物线虫虫秀丽隐杆线虫了解神经元迁移的基本分子机制，这可能会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的新兴技术以鉴定基因表达差异和转录因子靶标。