Recombineering based analysis of Hox function in kidney development

基于重组工程的肾脏发育中 Hox 功能分析

• 批准号: 9261520
• 负责人: S. Steven Potter
• 金额: $ 33.93万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-08 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9261520
• 关键词: Binding Sites; Biological; Biological Process; Body part; Cell Proliferation; Cells; ChIP-seq; Complement; DNA; DNA Binding; Data; Databases; Development; Diagnosis; Disease; Dissection; Drosophila genus; Enhancers; Failure; Frameshift Mutation; Future; Gene Expression; Gene Expression Profile; Gene Mutation; Gene Targeting; Genes; Genetic; Genetic study; Head; Histology; Homeobox Genes; Hydronephrosis; In Situ Hybridization; Kidney; Lead; Leg; Link; Lower urinary tract; Mammals; Mediating; Mesenchyme; Metanephric Diverticulum; Microfluidics; Modeling; Molecular; Mus; Mutant Strains Mice; Mutate; Mutation; Natural regeneration; Nephrons; Ontology; Organism; Pathway interactions; Pattern; Phenotype; Process; Proteins; Regulation; Research; Resources; Role; Stem cells; Testing; Vesicle; Work; base; design; gene function; imaginal disc; improved; induced pluripotent stem cell; insight; laser capture microdissection; mutant; nephrogenesis; novel; paralogous gene; predictive modeling; public health relevance; transcription factor; transcriptome sequencing

DESCRIPTION (provided by applicant): The Hox genes are among the key genetic regulators of development. Mutations of these genes in simple organisms can cause dramatic transformations of body parts, with legs for example protruding from the head in place of antennae. In mammals, however, mutations generally give considerably milder effects. This is likely the result of the much greater number of Hox genes in mammals and their extensive overlapping functions. In this proposal we describe a novel recombineering strategy that allows us to simultaneously introduce frameshift mutations into sets of contiguous Hox genes, while leaving interspersed shared enhancers intact. We propose to complete the creation of a rich resource of Hox mutant mice that will allow the dissection of both flanking and paralogous Hox gene functional relationships. While 31 Hox genes show robust expression during kidney development, developmental functions have only been identified for six. We propose to unveil the roles of the other Hox genes by creating mice with stepwise reductions in Hox flanking and paralogous function. Preliminary studies of mice already made with mutations in HoxD, 3, 4, 8 and HoxA9, 10, 11, HoxC9, 10, 11 and HoxD9, 10, 11, have identified interesting phenotypes, including dramatic reductions in nephron number, glomerulomegaly, hydronephrosis, and failure to make renal vesicles, discovering the importance of additional Hox genes in kidney development. We also propose an extensive analysis of resulting kidney phenotypes, leveraging our GUDMAP consortium expertise and database. The moleular mechanisms of Hox function will be defined through characterization of overlapping sets of downstream targets and Gene Ontology analysis defining their molecular functions and biological processes. The study of disturbed kidney gene expression patterns in Hox mutants will extend to the single cell level, using novel microfluidics/RNA-Seq approaches. In addition Chip-Seq will be used to define the overlapping sets of Hox DNA binding sites and to distinguish direct targets. The results will improve our understanding of the genetic circuitry of kidney development and will thereby augment future DNA diagnosis and iPS mediated regeneration and/or replacement of diseased or malformed kidneys.

描述（由申请人提供）：Hox 基因是发育的关键遗传调节因子之一。简单生物体中这些基因的突变可能会导致身体部位发生巨大的转变，例如，腿从头部伸出来代替触角。然而，在哺乳动物中，突变通常会产生相当温和的影响。这可能是哺乳动物体内 Hox 基因数量多且功能广泛重叠的结果。在本提案中，我们描述了一种新颖的重组工程策略，该策略允许我们同时将移码突变引入连续的 Hox 基因组中，同时保持散布的共享增强子完好无损。我们建议完成丰富的 Hox 突变小鼠资源的创建，这将允许解剖侧翼和旁系同源的 Hox 基因功能关系。 虽然 31 个 Hox 基因在肾脏发育过程中表现出强劲的表达，但仅确定了其中 6 个基因的发育功能。我们建议通过培育逐步减少 Hox 侧翼和旁系同源功能的小鼠来揭示其他 Hox 基因的作用。对已携带 HoxD、3、4、8 和 HoxA9、10、11、HoxC9、10、11 和 HoxD9、10、11 突变的小鼠进行的初步研究发现了有趣的表型，包括肾单位数量急剧减少、肾小球肿大、肾积水和无法生成肾囊泡，发现了额外的 Hox 基因在肾脏中的重要性发展。我们还建议利用我们的 GUDMAP 联盟专业知识和数据库对所得肾脏表型进行广泛分析。 Hox 功能的分子机制将通过下游靶点重叠组的表征和定义其分子功能和生物过程的基因本体分析来定义。使用新型微流体/RNA-Seq 方法，对 Hox 突变体中受干扰的肾脏基因表达模式的研究将扩展到单细胞水平。此外，Chip-Seq 将用于定义重叠的 Hox DNA 结合位点组并区分直接靶标。这些结果将提高我们对肾脏发育遗传回路的理解，从而增强未来的 DNA 诊断和 iPS 介导的患病或畸形肾脏的再生和/或替代。

项目成果

Key pathways regulated by HoxA9,10,11/HoxD9,10,11 during limb development.

肢体发育过程中由Hoxa9,10,11/Hoxd9,10,11调节的关键途径。

• DOI: 10.1186/s12861-015-0078-5
• 发表时间: 2015-07-19
• 期刊: BMC developmental biology
• 作者: Raines AM;Magella B;Adam M;Potter SS
• 通讯作者: Potter SS