TARGET-DEPENDENT INTERACTIONS IN EARLY CNS DEVELOPMENT

(调查摘要)：这项建议旨在研究 用一种独特的转基因小鼠作为小鼠的橄榄小脑环路 一个模范系统。这一系列动物携带的转基因包括 白喉毒素基因的毒性α亚基在 浦肯野细胞特异性基因L7的调控。因为. 其时间监管的特殊性，L7监管区域 驱动标记基因以一种最初的异质性方式表达 一系列的矢状带，最先出现在中线和 随后横向扩散。成年后，所有的浦肯野细胞都是 阳性。当使用L7调节序列来驱动a- 白喉毒素基因(ADT)，它们产生一个自主的浦肯野细胞， 早发型细胞死亡开始于中线并扩散到 半个半球。作为这种细胞死亡的明显后果， 突触前下橄榄和颗粒细胞逆行 退化。最后，几乎没有颗粒细胞，只有极少数 下层橄榄神经元仍然存在。 为了推行这一模式系统，申请者提出了五个具体目标： 为了研究浦肯野细胞丢失对颗粒细胞行为的影响， 包括颗粒细胞死亡的性质和时间，以及 增殖行为。PI将尝试将 外颗粒细胞层(EGL)，浦肯野内有越来越大的间隙 单元层。将评估EGL细胞的成熟状态。 两个分子标记Tag-1的原位杂交 (成熟/迁移颗粒细胞)和GABA受体。扩散 将通过BrdU抑制EGL细胞的运输来衡量 或许，对浦肯野与人类之间关系的定量分析 细胞和分裂颗粒细胞。颗粒细胞死亡将被检测到 在一定程度上是通过隧道技术。 为了研究Bergmann神经胶质细胞作为媒介的潜在作用 蒲肯野细胞丢失与颗粒细胞改变之间的关系。初步 研究表明，Bergmann胶质细胞变厚并扭曲为 早在P8和P24，就不再有任何GFAP阳性的Bergmann 突变体中可见胶质细胞。L7ADT小鼠将被GFAP和 一种暂时性的，Bergmann神经胶质特异性细胞标志物，BLBP。使用这些 试剂，神经胶质细胞的状态将与 EGL来寻找相关性。列出了潜在的体内范例， 包括用Arac分离神经胶质细胞。 检查下层细胞丢失的时间和空间模式 橄榄在突变体中具有特殊的“异质效应” 关于橄榄的结构，可能与特定的 已经退化的浦肯野细胞“。TUNEL标记将 也可以在这个系统中使用。 构建L7：ADT小鼠5‘端长度较短的附加系。 在LacZ融合基因中使用这种启动子的经验表明 导致浦肯野细胞死亡的时间应该早于 目前的动物品系。 来创造和分析L7基因敲除。基于的本地化 L7蛋白和信使核糖核酸，申请人假设这些动物 应该有浦肯野细胞树突状发育缺陷。至 方便对空鼠的分析，PI汇编了一份清单 包括霍夫曼的理查德·斯迈恩博士在内的几位合作者- 拉罗什(谁将创造基因敲除老鼠)，卡尔·席林(谁将创造) 对来自L7基因敲除大脑的细胞进行“各种测试”)。 大卫·林登(他将测试 脑片中的L7-/-细胞。

(Investigator's Abstract): The proposal is aimed at the study of the olivocerebellar circuit of the mouse using a unique transgenic mouse as a model system. The transgene carried by this line of animals consists of the toxic alpha subunit of the diphtheria toxin gene under the regulatory control of the Purkinje cell specific gene, L7. Because of the peculiarities of its temporal regulation, the L7 regulatory regions drive marker gene expression in an initially heterogeneous fashion a series of sagittal bands that are first apparent at the midline and subsequently spread laterally. By adulthood, all Purkinje cells are positive. When the L7 regulatory sequences are used to drive the a- diphtheria toxin gene (ADT), they produce a Purkinje cell autonomous, early onset cell death that begins at the midline and spreads to the hemispheres. As an apparent consequence of this cell death, the presynaptic inferior olive and granule cells suffer a retrograde degeneration. In the end, virtually no granule cells and very few inferior olive neurons remain. To pursue this model system, the applicant proposes five specific aims: To study the effects of the Purkinje cell loss on granule cell behavior, including the nature and timing of granule cell death and changes in proliferative behavior. The PI will attempt to correlate gaps in the external granule cell layer (EGL) with the growing gaps in the Purkinje cell layer. The state of maturation of the EGL cells will be assessed by in situ hybridization of two molecular markers, TAG-1 (maturing/migrating granule cells) and the GABA receptor. Proliferation will be measured by BrdU incorportation by the EGL cells with qualitative and, perhaps, quantitative analysis of the relationship between Purkinje cells and dividing granule cells. Granule cell deaths will be detected in part via the TUNEL technique. To study the potential role of the Bergmann glial cell as an intermediary between the Purkinje cell loss and the granule cell changes. Preliminary studies show that the Bergmann glia become thickened and distorted as early as P8 and by P24, there are no longer any GFAP positive Bergmann glia visible in the mutant. L7ADT mice will be stained with GFAP and a transient, Bergmann glial-specific cell marker, BLBP. Using these reagents, the state of the glia will be compared with the state of the EGL to look for correlations. Potential in vivo paradigms are listed, including disrtuption of the glial cells with araC. To examine the timing and spatial pattern of cell loss in the inferior olive in the mutants with particular regard to "heterogeneous effects on olive structures that could be correlated with specific sets of Purkinje cells that have undergone degeneration". TUNEL labeling will be used in this system as well. To construct additional lines of L7:ADT mice with shorter 5' regions. Experience with such promoters in lacZ fusion genes suggests that the resulting Purkinje cell death should be at earlier times than in the current line of animals. To create and analyze an L7 gene knockout. Based on the localization of the L7 protein and mRNA, the applicant hypothesizes that these animals should have defects in Purkinje cell dendritic development. To facilitate the analysis of the null mice, the PI has assembled a list of several collaborators, including Drs. Richard Smeyne, of Hoffman- LaRoche (who will create the knockout mice), Karl Schilling (who will perform "various tests" on cells from the L7 knockout brain), and Dr. David Linden (who will test the electrophysiological properites of the L7-/- cells in brain slices.