Cell Cycle Regulation in Interneuron Genesis & Cortical Construction

抑制性皮层中间神经元，大多数起源于内侧神经节隆起（MGE），几乎是每个皮层回路的一部分。正常的皮质功能关键取决于产生这些GABA能细胞的数量和亚型与兴奋性投射神经元的适当比例。这需要祖细胞亚型增殖与分化的精确协调。项目1研究了细胞周期组分在哺乳动物脑的模式和功能中的作用，并表明两种G1期活性细胞周期蛋白，cD 1和cD 2，在MGE的不同祖细胞亚群中表达。cD 2的消融导致皮质PV+但不是SST+中间神经元的损失。我们假设，CD 1的功能，以促进放射状胶质细胞，其中一些产生SST+中间神经元的不对称分裂。相比之下，cD 2可以促进主要产生PV+中间神经元的中间祖细胞的对称分裂 目标1。将使用子宫内这些细胞周期蛋白的急性过表达和敲低，以及细胞位置、形态学分析和与增殖和有丝分裂后亚群标记物的共标记，检查cD 2与cD 1在MGE分裂中的不同作用。通过与项目3的合作，WT、cD 1- / -和cD 2-/- MGE中的时间推移成像将研究cD 2或cD 1的丢失如何影响对称分裂与不对称分裂。我们将利用荧光标记方法比较cD 2-/-与cD 1-/- MGE的细胞周期时相持续时间。将检验cD 2表达有利于对称分裂而cD 1促进不对称分裂的假设。 目标2.将使用两种不同的方法研究cD 2 + MGE祖细胞的转录组，以从转基因小鼠中的cD 2 + MGE细胞中捕获RNA;翻译核糖体亲和纯化（TRAP）或荧光激活细胞分选（FACS），然后进行微阵列。数据将定义cD 2在MGE中运行的分子背景。这些阵列的解释将极大地促进了项目2研究的见解，这些研究已经确定了Notch信号传导和背侧MGE中cD 2表达调控之间的联系，而Wnt和Shh信号传导对增殖有影响，可能在Notch的上游。因此，在本发明中， 潜在有意义的表达模式将更容易识别。 目标3.诱导型cD 2-CreER[T2]将用于绘制cD 2+祖细胞的命运结果，而Nkx2.1-Cre：cD 2fl/fl和Dlx 1/2-Cre：cD 2fl/fl模型中的条件失活将探测cD 2对中间神经元特化的贡献。我们假设PV+中间神经元主要来自SVZ中的cD 2+祖细胞，而SST+中间神经元主要来自VZ中的神经源性分裂。项目2的专业知识将是必不可少的，因为我们建立MGE衍生的CD 2+祖细胞的命运图。 将在神经行为分析核心中测试MGE内选择性cD 2丢失对中间神经元分布和功能的影响，以探测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.