PROJ 2: NEUROANATOMY: POSTNATAL ANATOMIC AND CONNECTIONAL EFFECTS OF DISRUPTION

最近的证据表明，候选阅读障碍易感基因(CDSG)在阅读障碍中起作用 大脑皮层的发育，尤指神经元的迁移和成熟。在项目II中，我们 将调查胚胎引起的神经元迁移障碍的出生后解剖学后果 针对CDSG同源物Dyxld、KIAA0319或 DCDC2在大鼠大脑皮层的表达。根据初步结果，因为目前还不知道 人类是否所有这些基因变异都会导致功能丧失，我们还将调查其影响 CDSG过度表达。由于在目标1中，所有CDSG都与阅读障碍有关联 我们将讨论解剖RNAi和过度表达的表型，这些表型似乎在 基因？即双峰分布的转基因细胞，它们要么迁移不足，要么迁移超过其 预期的层流位置。我们将使用分子和出生日期标记来评估表型 这些错位迁移的神经元，无论层数是否合适。此外，我们还将联合转染Gain和 小麦胚芽凝集素转基因导致功能神经元丧失，这将使精确确定 对照和实验病例中转基因神经元的连通性。我们将比较 大鼠大脑半球内和半球间、皮质-皮质、皮质-丘脑和丘脑-皮质的联系 分别用不同的CDSG shRNA转染，以及实验组和对照组之间。在 期望这项工作能够指导阅读障碍亚型的研究，目标2将专注于系统性 胚胎转染发夹状RNA或过表达的大鼠大脑中的差异 每一个CDSG同源物的质粒。在胚胎完成工作后，我们将在原位使用 杂交和免疫组织化学方法比较基因的时间和空间表达 出生后大鼠的模式。我们还将评估转基因神经元的神经元形态和 他们的过程。目标3考察了解剖组织的广泛变化，这些变化包括 假设直接由shRNA或过表达构建体的局部转染产生，并作为 二次塑性相关效应的结果。我们将使用高效准确的体视学探头来 估计整个新皮质和丘脑的神经元数量、神经元大小和区域体积。一个 对前脑解剖结构的准确描述，这种解剖结构是由基因敲除或过度表达引起的 CDSGs的大鼠同系物，细胞自主和次级效应，以及它们的过程 发展，是遗传和行为之间的一座很好的桥梁，将有助于摆脱更广泛的 了解人类发育性阅读障碍的神经生物学基础。我们将链接结果 从这个项目到项目I中研究的发育和分子机制，直到 行为变化将在项目III中描述。

Recent evidence indicates that candidate dyslexia susceptibility genes (CDSGs) have roles in the development of the cerebral cortex, especially in neuronal migration and maturation. In Project II, we will investigate postnatal anatomic consequences of neuronal migration disorders induced by embryonic transfection with small hairpin RNAs (shRNA) targeted against CDSG homologs Dyxld, Kiaa0319, or Dcdc2 in the rat cerebral cortex. Based on preliminary results, and because it is not yet known in humans whether all of these gene variants result in loss of function, we will also investigate the effects of CDSG overexpression. Since all CDSGs share among them an association with dyslexia, in Aim 1 we will address anatomical RNAi and overexpression phenotypes that appear to be shared among the genes¿namely a bimodal distribution of transfected cells that either undermigrate or migrate past their expected laminar locations. We will use molecular and birthdate markers to assess the phenotypes of these mismigrated neurons, whether or not layer appropriate. In addition, we will co-transfect gain and loss of function neurons with a wheat germ agglutinin transgene that will allow precise determination of the connectivity of transfected neurons in both control and experimental cases. We will compare the intra- and inter-hemispheric, cortico-cortical, cortico-thalamic, and thalamo-cortical connections in rats transfected with different CDSG shRNAs, as well as between experimentals and controls. In the expectation that this work can guide research on dyslexia subtyping, Aim 2 will focus on systematic differences that are seen in the brains of rats embryonically transfected with shRNA or overexpression plasmids for each of the CDSG homologs. Following completed work in embryos, we will use in situ hybridization and immunohistochemistry to compare the genes' temporal and spatial expression patterns in the postnatal rat. We will also assess the neuronal morphology of transfected neurons and their processes. Aim 3 examines widespread changes in anatomic organization, which are hypothesized to arise directly from local transfections of shRNA or overexpression constructs and as a result of secondary plasticity-related effects. We will use efficient and accurate stereologic probes to estimate neuron number, neuron size, and regional volume throughout the neocortex and thalamus. An accurate description of the forebrain anatomy that results from either knockdown or overexpression of rat homologs of CDSGs, both cell autonomous and secondary effects, and the course of their development, serve as a good bridge between genetics and behavior and will help to shed a broader light on the neurobiological substrates underlying developmental dyslexia in humans. We will link results from this project down to developmental and molecular mechanisms studied in Project I and up to behavioral changes to be characterized in Project III.