TARGET-DEPENDENT INTERACTIONS IN EARLY CNS DEVELOPMENT

（研究者摘要）：该提案旨在研究 使用独特的转基因小鼠作为 一个模型系统这一系动物携带的转基因包括 白喉毒素基因的毒性α亚单位的 浦肯野细胞特异性基因L7的调控。因为 其时间调节的特殊性，L7调节区域 以最初的异质方式驱动标记基因表达， 一系列矢状带，首先在中线出现， 随后横向扩散。到了成年期，所有的浦肯野细胞 肯定的：第当L7调节序列用于驱动a- 白喉毒素基因（ADT），它们产生一个浦肯野细胞自主， 早发型细胞死亡，开始于中线，并蔓延到 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遭受逆行 退化最后，几乎没有颗粒细胞， 下橄榄神经元保留。 为了推行这一示范制度，申请人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 浦肯野氏束外颗粒细胞层（EGL），间隙增大 细胞层将评估EGL细胞的成熟状态 通过两种分子标记物TAG-1的原位杂交， （成熟/迁移颗粒细胞）和GABA受体。增殖 将通过EGL细胞的BrdU掺入进行测量， 或许还可以定量分析浦肯野氏病 细胞和分裂的颗粒细胞。将检测到颗粒细胞死亡 部分通过TUNEL技术。 研究Bergmann胶质细胞作为中介细胞的潜在作用 浦肯野细胞丢失和颗粒细胞改变之间的联系。初步 研究表明，伯格曼神经胶质变得增厚和扭曲， 早在P8和P24，不再有任何GFAP阳性Bergmann 突变体中可见胶质细胞。L7 ADT小鼠将用GFAP染色， 一种短暂的Bergmann神经胶质特异性细胞标记物BLBP。使用这些 试剂，神经胶质的状态将与神经胶质的状态进行比较。 EGL寻找相关性。列出了潜在的体内范例， 包括用araC破坏神经胶质细胞。 检查下丘脑细胞丢失的时间和空间模式， 特别是关于“异质效应”， 橄榄结构上，可能与特定的 浦肯野细胞已经经历了变性”。TUNEL标记将 在这个系统中也可以使用。 构建具有较短5'区域的L7：ADT小鼠的额外系。 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早于 目前的动物。 制造并分析L7基因敲除。基于本地化的 L7蛋白和mRNA，申请人假设这些动物 浦肯野细胞树突发育有缺陷。到 为了便于对无效小鼠进行分析，PI已收集了一份清单， 包括霍夫曼的理查德·斯梅恩博士在内的几位合作者的研究成果 LaRoche（谁将创造敲除小鼠），Karl Schilling（谁将 对来自L7基因敲除大脑的细胞进行“各种测试”。 大卫林登（谁将测试的电生理特性， 脑切片中的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.