Proliferation, Specification & Brain Function

描述（由申请人提供）：本项目研究发育中的内侧神经节隆起（MGE）中增殖和中间神经元命运决定的相互作用，并探讨改变中间神经元亚群的功能后果。两大类神经元（谷氨酸能兴奋性神经元和gaba能抑制性神经元）几乎构成了大脑皮层的所有神经回路。它们的形态、生化成分、电生理特性和突触连接可以区分不同的中间神经元亚型。尽管这些gaba能细胞对大脑功能很重要，但令人惊讶的是，人们对它们的产生是如何在细胞或分子水平上受到调节的知之甚少。项目1 （Ross PI）研究细胞周期成分在哺乳动物脑的模式和功能中的作用。他们发现两种gl期活性细胞周期蛋白cDI和cD2在MGE中不同的祖细胞亚群中表达，其中cD2的基因消融会导致皮质PV+神经元的损失，但不会导致SST+中间神经元的损失。实验探讨了cDI主要促进放射状胶质细胞（RGCs）不对称分裂的假设，其中一些RGCs产生SST+中间神经元。相反，cD2可能促进主要产生PV+中间神经元的中间祖细胞（IPCs）的对称分裂，项目2 （Anderson PI）研究了Notch、Wnt和Shh信号系统在调节MGE产生的神经元数量和亚型中的相互作用。他们发现Notch信号的调节增强了背侧MGE （dMGE）中cD2的表达，并假设这将以牺牲SST+中间神经元为代价增加PV+输出。试点数据暗示Shh和Wnt信号之间的相互作用调节Notch活性影响中间神经元的产生，这些关系将被探索，项目3 （Shi PI）使用最先进的时间推移视频显微镜和免疫组织化学技术在子宫内室内注射逆转录病毒荧光蛋白来检查细胞内在和外部机制调节MGE的分裂。这些研究与项目1和项目2中的细胞周期和信号传导研究紧密结合。Core B （Moore Dir.）将在该项目中生成的小鼠模型中确定涉及不同中间神经元亚型和解剖区域的选择性中间神经元缺陷的功能意义。中间神经元亚群损失对行为、脑结构和生理的影响是寻求的。人们普遍认识到，Notch、Shh和Wnt等关键信号通路和D-cyclins等细胞周期调节因子广泛相互作用，调节神经元的产生和命运。然而，相互作用的复杂性、腹侧前脑来源神经元命运的多样性以及该区域基因操作的挑战构成了对MGE进行全面研究的主要障碍。该计划通过4个Pis的共同努力，使用尖端的方法来阐明发育信号如何调节这些至关重要的神经元的命运和输出，从而解决了这种复杂性。

DESCRIPTION (provided by applicant): This Program examines the interaction of proliferation and interneuron fate determination in the developing medial ganglionic eminence (MGE), and probes functional consequences of altering interneuron subpopulations. Two broad classes of neurons}}}glutamatergic excitatory and GABAergic inhibitory}}} comprise virtually every neural circuit in cerebral cortex. Their morphology, biochemical constituents, electrophysiological properties and synaptic connections can distinguish a remarkable variety of interneuron subtypes. Despite the importance of these GABAergic cells to brain function, surprisingly little is known about how their production is regulated on a cellular or molecular level. Project 1 (Ross PI) studies the roles of cell cycle constituents in the patterning and function of mammalian brain. They found that two Gl-phase active cyclins, cDI and cD2, are expressed in different progenitor subsets in the MGE where genetic ablation of cD2 produces a loss of cortical PV+ but not SST+ interneurons. Experiments probe the hypothesis that cDI functions primarily to promote asymmetric divisions of radial glial cells (RGCs), some of which generate SST+ interneurons. In contrast, cD2 may promote the symmetric divisions of intermediate progenitor cells (IPCs) that will primarily generate PV+ interneurons Project 2 (Anderson PI) investigates the interacting roles of Notch, Wnt and Shh signaling systems to regulate the number and subtypes of neurons generated from the MGE. They have found that modulation of Notch signaling enhances cD2 expression in dorsal MGE (dMGE) and hypothesize that this will increase PV+ output at the expense of SST+ interneurons. Pilot data implicate interactions between Shh and Wnt signaling regulate Notch activity to impact interneuron production and these relationships will be explored Project 3 (Shi PI) uses in utero intraventricular injection of retroviral fluorescent proteins with state-of- the-art time-lapse videomicroscopy and immunohistochemistry to examine cell intrinsic and extrinsic mechanisms regulating divisions in the MGE. These istudies are heavily integrated with cell cycle and signaling investigations in Projects 1 and 2. Core B (Moore Dir.) will determine the functional significance of selective interneuron deficits that involve different interneuron subtypes and anatomical regions in mouse models generated within the Program. Consequences of interneuron subset loss on behavior, brain structure and physiology are sought. It is widely appreciated that key signaling pathways like Notch, Shh, and Wnt and cell cycle regulators like D-cyclins extensively interact to regulate neuronal generation and fate. However the complexity of the interactions, diversity of ventral forebrain-derived neuronal fates and challenges for gene manipulation in this region pose major impediments to comprehensive study in the MGE. This Program tackles this complexity through the combined efforts of 4 Pis using cutting edge approaches to the elucidation of how developmental signals regulate fate and output of these critically important neurons. PUBLIC HEALTH RELEVANCE: Interneuron deficits have been implicated in the pathobiology of major neurological and psychiatric illnesses, including epilepsy, anxiety disorders, autism and schizophrenia. While a great deal has been learned over the last 20 years about proliferation of excitatory, glutamatergic precursors in cortex, a number of challenges have slowed the pace of discovery for studies of the ventral niches that generate interneurons. This Program strives to address this knowledge gap and our work over the past 4 years positions us well to succeed. PROJECT 1 Principal Investigator: M. Elizabeth Ross Title: Cell Cycle Regulation in Interneuron Genesis & Cortical Construction Description (provided by applicant): 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. Public Health Relevance: The mechanisms linking cell division to neural specification, particularly in subcortical brain, are appreciated at only a rudimentary level. That VZ and SVZ cells use different cell cycle components and that disturbing this balance can alter the interneuron composition in the cerebral cortex adds to the rich complexity of ways neurogenesis is regulated in the developing brain. This Program brings together 4 laboratories with the advanced capabilities that place us in an unprecedented position to understand the mechanisms regulating genesis of these neuron subtypes that are targets of many neuropsychiatric diseases.