Rosenfeld-5R01NS034934-14. Mus musculus

Using targeted gene deletion, we have firmly established that the Class IV POU domain transcription factor Brn-3.2 controls a developmental program regulating axon pathfinding in the mouse visual system. We have isolated and identified downstream gene targets of Brn-3.2, using Representational Difference Analysis (RDA), on cDNA populations derived from wildtype and Brn-3.2-/- retina at the appropriate embryonic stage. One of these candidate genes, the Homeodomain Interacting Protein Kinase 2 (HIPK2), is postulated to be a transcriptional coregulator, based on its in vitro interactions with repressor homeodomain proteins of the NK class, as well as other components of repressor complexes. HIPK2 has also been shown to be involved in post-translational modification of two major transcriptional regulators, p53 and CtBP. Expression of a dominant-negative form of HIPK2 in sensory neurons affects the innervation patterns of their target tissues, suggesting an axon pathfinding defect. The aim of this project is to identify targets for the Homeodomain Interacting Protein Kinase 2 (HIPK2), that we isolated as a downstream gene target of the class IV POU domain transcription factor Brn-3.2, and to investigate their function in the Brn-3.2 dependent pathway regulating axon pathfinding. We hypothesize that the genes regulated by HIPK2 will play a critical role in axon pathfinding and that the results of this study will provide novel insights into the molecular mechanisms of axon pathfinding, and possibly neural plasticity and regeneration, and therefore, be of great interest to the field of neurobiology in general. In order to uncover potential biological role(s) of HIPK2 in neural development, and particularly axon pathfinding, we generated transgenic mouse models for in vivo studies. Our preliminary observations in transgenic mice, indicate that a form of the molecule expected to act as a dominant-negative and designed to be expressed in sensory neurons, affects the innervation patterns of their target tissues, suggesting an axon pathfinding defect. We plan to take advantage of this model to identify genes regulated by HIPK2, and which are likely to be involved in axon pathfinding. To this end, we will compare gene expression profiles in sensory neurons isolated wild-type and transgenic mice. In our experimental paradigm, the trigeminal ganglion represents the ideal sensory structure in which to perform such an analysis: 1) High levels of transgene can be expressed in the trigeminal ganglion, as assessed by the expression of LacZ from the bicistronic construct which contains IRES-LacZ downstream of a dominant negative form of HIPK2. 2) The trigeminal ganglion can be dissected at embryonic day 13.5, when the phenotype is apparent, with ease and free of contamination from surrounding tissues. 3) The amounts of RNA that can be isolated from a single ganglion are in the range of 200-300 ng, which should be sufficient for microarray analysis following linear amplification of RNA. One series will be carried out with transgenic embryos and a control series will be carried out with wildtype littermates. Animals will be prepared and sacrificed by a standard protocol. Tissue will be rapidly dissected from E13.5 trigeminal ganglion, frozen in liquid nitrogen, and stored at -80C until RNA is extracted. RNA will be prepared using RNeasy Micro Kit. We will be providing 4 tissue samples for each of the wildtype and transgenic animals to mitigate any expression differences resulting from mouse to mouse variation. Keywords: other Overall design: as above

通过靶向基因敲除（targeted gene deletion）技术，我们已确凿证实：IV类POU结构域转录因子Brn-3.2可调控小鼠视觉系统中的轴突导向发育程序。我们借助代表性差异分析（Representational Difference Analysis, RDA），针对合适胚胎阶段的野生型与Brn-3.2基因敲除（Brn-3.2-/-）视网膜来源的cDNA群体，分离并鉴定了Brn-3.2的下游基因靶标。其中一个候选基因为同源结构域相互作用蛋白激酶2（Homeodomain Interacting Protein Kinase 2, HIPK2）：基于其与NK类阻遏型同源结构域蛋白及阻遏复合物其他组分的体外相互作用，该基因被推测为转录共调控因子。已有研究表明，HIPK2还参与两种主要转录调节因子p53与CtBP的翻译后修饰。在感觉神经元中表达HIPK2的显性负性突变体，会改变其靶组织的神经支配模式，提示存在轴突导向缺陷。本项目旨在鉴定我们作为IV类POU结构域转录因子Brn-3.2下游靶基因分离得到的同源结构域相互作用蛋白激酶2（HIPK2）的靶基因，并探究这些基因在Brn-3.2依赖的轴突导向调控通路中的功能。我们推测，受HIPK2调控的基因将在轴突导向过程中发挥关键作用；本研究结果将为轴突导向乃至神经可塑性与再生的分子机制提供全新见解，因此对整体神经生物学领域具有重要价值。为揭示HIPK2在神经发育尤其是轴突导向中的潜在生物学功能，我们构建了用于体内研究的转基因小鼠模型。初步的转基因小鼠观察结果显示，该分子的预期显性负性突变体（设计为在感觉神经元中表达）可改变其靶组织的神经支配模式，提示存在轴突导向缺陷。我们计划利用该模型鉴定受HIPK2调控且可能参与轴突导向的基因。为此，我们将对比分离自野生型与转基因小鼠感觉神经元的基因表达谱。在本实验范式中，三叉神经节是开展此类分析的理想感觉结构，原因如下：1） 可在三叉神经节中高效表达转基因，这一点通过包含HIPK2显性负性突变体下游连接IRES-LacZ的双顺反子构建体的LacZ表达得以验证；2） 可在胚胎发育第13.5天（此时表型已显现）轻松分离三叉神经节，且不受周围组织污染；3） 单个神经节可分离得到200~300 ng RNA，经RNA线性扩增后足以满足微阵列分析需求。本研究将设置两组样本：转基因胚胎组与野生型同窝对照组。实验动物将按照标准流程准备并处死。快速摘取E13.5胎鼠的三叉神经节组织，置于液氮中速冻，于-80℃保存直至RNA提取。RNA提取将使用RNeasy Micro Kit。为减小小鼠个体差异导致的表达差异，我们将为野生型与转基因组各提供4份组织样本。关键词：其他 整体实验设计：如上所述