Genetic tools to study CNS development and function

 描述（由申请方提供）：谱系分析用于确定胚胎发育中的祖细胞-子代关系。这些分析告诉我们的类型和选择的时间，祖细胞对产生的复杂性，在成年生物体中观察到的细胞命运的理解。在胚胎中枢神经系统中，谱系分析提供了一个基本的基础上建立模型的分数，并提供了一个了解胚胎神经管祖细胞，通过一系列的分子指导的决定，产生巨大的复杂性的细胞类型中观察到的成年中枢神经系统。 所有谱系追踪的方法都依赖于一个基本原则--能够不可磨灭地标记一个祖先或祖先的子集。最近，这已经通过使用位点特异性重组酶来实现。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。在这些细胞中，重组酶以永久的方式激活报告基因，从而允许通过随后的发育阶段跟踪这些细胞及其后代谱系。基于酶的谱系分析已经改变了我们对胚胎CNS发育的理解。 基于内切酶的谱系分析受到一个关键限制。该方法的特异性完全依赖于用于驱动重组酶的启动子/调节元件的特异性。为了提高这种方法的特异性，我们和其他人已经开发了交叉和诱导的方法来标记更具选择性的祖细胞群。这些改进导致胚胎中枢神经系统不同区域的命运图更加精确。尽管有这些改进，但仍有一个警告-基因及其同源调控元件通常在祖细胞中表达，以及在相关或不相关的有丝分裂后神经元群体中表达。重组酶驱动因子在有丝分裂后神经元中的表达混淆了谱系分析，因为重组发生在有丝分裂后细胞中，而不管它是哪种祖细胞。 来源于。因此，在这种情况下，不能准确地确定祖先-后代关系。 在这里，我们建议开发一个新的平台谱系分析，称为祖锚定交叉命运映射（PRISM），它规避了上述限制。在具体目标1中，我们将建立PRISM方法的有效性。在具体目标2中，我们将使用这种方法在原理验证实验中解决腹侧前脑中关键分子Shh的谱系，其中Shh在祖细胞和有丝分裂后神经元中同时表达。 提出的菌株添加到不断增长的谱系分析条件工具箱。由于大多数重组酶驱动程序受到限制，这种方法将广泛适用于研究重要的谱系相关问题。

 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.