Genetic tools to study CNS development and function

研究中枢神经系统发育和功能的遗传工具

• 批准号: 9036059
• 负责人: Rajeshwar B Awatramani
• 金额: $ 23.18万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9036059
• 关键词: Adult; Anterior; Behavior; Birth; Cell Lineage; Cells; Complex; Confusion; Development; Embryo; Embryonic Development; Event; Generations; Genes; Genetic; Genetic Recombination; Goals; Label; Maps; Mediating; Methods; Mitotic; Movement; Mus; Neural tube; Neuraxis; Neurons; Neurosciences; Organism; Population; Prosencephalon; Regulatory Element; Reporter; Resources; Series; Site; Specificity; Staging; Stem cells; Time; Transgenes; Transgenic Organisms; base; cell fate specification; cell type; cost effective; design; efficacy testing; improved; migration; model building; nerve stem cell; nervous system development; progenitor; promoter; public health relevance; recombinase; research study; tool

 DESCRIPTION (provided by applicant): Lineage analysis serves to define progenitor-progeny relationships in embryonic development. These analyses inform our understanding of the types and timing of choices that progenitors make towards generating the complexity of cell fates observed in the adult organism. In the embryonic CNS, lineage analysis has provided a fundamental substrate on which scores of models are built upon, and have provided an understanding of how the embryonic neural tube progenitors, through a series of molecularly guided decisions, generate the enormous complexity of cell types observed in the adult CNS. All methods of lineage tracing rely on a single fundamental principle - that of being able to indelibly label a progenitor, or subset of progenitors. Most recently, this has been accomplished through the use of site- specific recombinases. Site-specific recombinases can be driven by gene-specific promoters in discrete regions of the developing neural tube. In these cells, the recombinase activates a reporter in a permanent manner, thereby permitting tracking of those cells, and their descendent lineages, through subsequent developmental stages. Recombinase based lineage analysis has transformed our understanding of embryonic CNS development. Recombinase based lineage analysis is subject to one critical limitation. The specificity of the method is wholly reliant on the specificity of the promoter/regulatory elements used to drive the recombinase. Towards improving the specificity of this method, we and others have developed intersectional and inducible methods to label more selective groups of progenitors. These improvements have led to even more refined fate maps in various regions of the embryonic CNS. Despite these improvements, one caveat remains - genes and their cognate regulatory elements are often expressed in progenitors, as well as in related or unrelated postmitotic neuron populations. Expression of recombinase drivers in postmitotic neurons obfuscates lineage analysis, as recombination occurs in the postmitotic cell regardless of which progenitor it was derived from. Thus, in such cases, progenitor-progeny relationships cannot be accurately ascertained. Here we propose to develop a new platform for lineage analysis, termed Progenitor anchored intersectional fate mapping (PRISM) which circumvents the aforementioned limitations. In Specific Aim 1, we will establish the validity of the PRISM approach. In Specific Aim 2, we will use this approach in proof-of- principle experiments to resolve the lineage of the key molecule Shh in the ventral forebrain wherein Shh is expressed contemporaneously in progenitor cells as well as in postmitotic neurons. The proposed strains add to the growing conditional toolbox for lineage analysis. Since most recombinase drivers are subject to limitations, this method will be broadly applicable for studying important lineage related questions.

 描述（由申请人提供）：谱系分析用于定义胚胎发育中的祖代关系。这些分析使我们了解祖细胞为产生在成体生物体中观察到的细胞命运的复杂性而做出的选择的类型和时间。胚胎中枢神经系统，谱系分析提供了建立数十个模型的基本基础，并提供了对胚胎神经管祖细胞如何通过一系列分子指导决策产生巨大复杂性的理解成体中枢神经系统中观察到的细胞类型的所有谱系追踪方法都依赖于一个基本原则——能够不可磨灭地标记祖细胞或祖细胞子集，最近，这已经通过使用位点特异性来实现。位点特异性重组酶可以由发育中的神经管的离散区域中的基因特异性启动子驱动，重组酶以永久的方式激活报告基因，从而允许追踪这些细胞。基于重组酶的谱系分析改变了我们对胚胎中枢神经系统发育的理解，该方法的特异性完全依赖于启动子/调节元件的特异性。为了提高这种方法的特异性，我们和其他人开发了交叉和诱导方法来标记更多选择性的祖细胞群，这些改进导致了更加精细的命运。尽管有这些改进，但仍然需要注意的是，基因及其同源调控元件通常在祖细胞以及相关或不相关的有丝分裂后神经元群体中表达，这会混淆谱系分析。 ，因为无论是哪个祖细胞，重组都发生在有丝分裂后细胞中 因此，在这种情况下，祖先-后代关系无法准确确定。在这里，我们建议开发一个新的谱系分析平台，称为祖锚定交叉命运图谱（PRISM），它可以规避上述限制。 ，我们将确定 PRISM 方法的有效性，在具体目标 2 中，我们将在原理验证实验中使用该方法来解析腹侧关键分子 Shh 的谱系。前脑证明，Shh 在祖细胞和有丝分裂后神经元中同时表达，由于大多数重组酶驱动程序受到限制，因此该方法将广泛适用于研究重要的谱系相关。问题 。