Cell Cycle Regulation in Interneuron Genesis & Cortical Construction

抑制性皮质中间神经元，大多数起源于内侧神经节隆起(MGE)，几乎是每个皮质环路的一部分。正常的皮质功能关键依赖于产生数量和亚型与兴奋性投射神经元比例适当的GABA能细胞。这需要祖细胞亚型增殖与分化的精细协调。项目1研究了细胞周期成分在哺乳动物大脑模式和功能中的作用，并表明两个G1期活性周期蛋白CD1和CD2在MGE中的不同祖细胞亚群中表达。消融CD2可导致皮质PV+中间神经元的丢失，而不是SST+中间神经元的丢失。我们假设CD1功能促进放射状神经胶质细胞的不对称分裂，其中一些细胞产生SST+中间神经元。相反，CD2可能促进中间前体细胞的对称分裂，这些中间前体细胞将主要产生PV+中间神经元 目的1.CD2和CD1在MGE分裂中的不同作用将通过子宫内这些细胞周期蛋白的急性过度表达和敲除，结合细胞位置、形态和标记增殖和有丝分裂后亚群的标记来检验。通过与Project 3的合作，WT、c D I-/-和CD2-/-MGE中的时间流逝成像将检查CD2或CD1的丢失如何影响对称与非对称划分。我们将利用一种荧光标记方法来比较CD2-/-和CD1-/-MGE的细胞周期时长。CD2表达有利于对称性，而CD1促进不对称分裂的假设将得到检验。 目的2.用两种不同的方法从转基因小鼠的CD2+MGE细胞中捕获RNA，即翻译核糖体亲和纯化(TRAP)或荧光激活细胞分选(FACS)，然后用微阵列技术研究CD2+MGE前体细胞的转录组。数据将定义CD2在MGE中运行的分子环境。来自项目2研究的见解将极大地促进对这些阵列的解释，这些研究已经确定了Notch信号与背侧MGE中CD2表达的调节之间的联系，而Wnt和Shh信号对增殖有影响，可能在Notch的上游。因此， 潜在的有意义的表达模式将更容易识别。 目的3.可诱导的CD2-Creer[T2]将被用于定位CD2+前体细胞的命运，而在Nkx2.1-Cre：cD2fl/fl和Dlx1/2-Cre：cD2fl/fl模型中的条件失活将探索CD2对神经元间质指定的贡献。我们推测，PV+中间神经元主要来源于SVZ的CD2+前体细胞，而SST+中间神经元主要来源于VZ的神经源性分裂。在我们建立MGE来源的CD2+祖细胞的命运图谱时，项目2的专业知识将是至关重要的。在MGE内选择性地丢失CD2对神经元间分布和功能的结果将在神经行为分析核心中进行测试，以探索由于这些中间神经元亚群丢失而导致的认知变化。

Inhibitory cortical interneurons, most originating in the medial ganglionic eminence (MGE), are part of virtually every cortical circuit. Normal cortical function critically depends on generating these GABAergic cells in numbers and subtypes in proper proportion to excitatory projection neurons. This requires exquisite coordination of progenitor subtype proliferation with differentiation. Project 1 studies the roles of cell cycle constituents in the patterning and function of mammalian brain and showed that two G1-phase active cyclins, cD1 and cD2, are expressed in distinct progenitor subsets in the MGE. Ablation of cD2 results in loss of cortical PV+ but not SST+ interneurons. We hypothesize that cD1 functions to promote asymmetric divisions of radial glial cells, some of which generate SST+ interneurons. In contrast, cD2 may promote the symmetric divisions of intermediate progenitor cells that will primarily generate PV+ interneurons Aim 1. The distinct roles of cD2 vs. cD1 in MGE divisions will be examined using acute overexpression and knockdown of these cyclins in utero, together with analyses of cell position, morphology, and colabeling with markers of proliferative and post-mitotic subpopulations. Via collaboration with Project 3, timelapse imaging in WT, c D I - / - and cD2-/- MGE will examine how loss of cD2 or cD1 affects symmetric vs. asymmetric divisions. We will take advantage of a fluorescence tagging method to compare cell cycle phase duration in cD2-/- vs. cD1-/- MGE. The hypothesis that cD2 expression favors symmetric while cD1 promotes asymmetric divisions will be tested. Aim 2. The transcriptome of cD2+ MGE progenitors will be investigated using two different approaches to capture RNA from cD2+ MGE cells in transgenic mice; Translating Ribosome Affinity Purification (TRAP) or fluorescence activated cell sorting (FACS) followed by microarray. Data will define the molecular context in which cD2 is operating in the MGE. Interpretation of these arrays will be greatly facilitated by insights from Project 2 studies that have identified a connection between Notch signaling and regulation of cD2 expression in the dorsal MGE, while Wnt and Shh signaling have effects on proliferation, likely upstream of Notch. Thus, potentially meaningful expression patterns will be more readily recognizable. Aim 3. Inducible cD2-CreER[T2] will be used to map the fate outcomes of cD2+ progenitors while conditional inactivation in Nkx2.1-Cre:cD2fl/fl and Dlx1/2-Cre:cD2fl/fl models will probe contributions of cD2 to interneuron specification. We hypothesize that PV+ interneurons arise primarily from cD2+ progenitors in the SVZ while SST+ interneurons derive primarily from neurogenic divisions in the VZ. Project 2 expertise will be essential as we establish fate maps of MGE-derived cD2+ progenitors. Outcomes of cD2 loss selectively within the MGE on interneuron distribution and function will be tested in the Neurobehavioral Analysis Core, to probe cognitive changes due to loss of these interneuron subsets.