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），是几乎所有皮层回路的一部分。正常的皮质功能严重依赖于这些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细协调。项目1研究了细胞周期成分在哺乳动物脑的模式和功能中的作用，发现两个g1期活性细胞周期蛋白cD1和cD2在MGE中不同的祖细胞亚群中表达。cD2的消融导致皮质PV+神经元的丢失，但不会导致SST+中间神经元的丢失。我们假设cD1的功能促进放射状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可能促进中间祖细胞的对称分裂，主要产生PV+中间神经元

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.