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

最近的证据表明，候选的阅读障碍易感基因（CDSGs）在阅读障碍的发生中起作用。 大脑皮层的发育，特别是神经元的迁移和成熟。在项目II中，我们 将研究胚胎发育引起的神经元迁移障碍的出生后解剖后果， 用靶向CDSG同源物Dyxld、Kiaa 0319或 dcdc 2在大鼠大脑皮层的表达。根据初步结果，由于目前尚不清楚 人类是否所有这些基因变异导致功能丧失，我们还将调查其影响 CDSG过度表达。由于所有的CDSG都与阅读障碍有关，在目标1中， 我们将讨论解剖学RNAi和过表达表型，这些表型似乎是在 基因即转染细胞的双峰分布，这些细胞要么迁移不足，要么迁移超过它们的基因。 预期层流位置。我们将使用分子和出生日期标记来评估表型 这些错误迁移的神经元，是否层适当。此外，我们将共转染增益， 用麦胚凝集素转基因治疗功能丧失的神经元， 在对照和实验情况下转染的神经元的连通性。我们将比较 大鼠半球内和半球间、皮质-皮质、皮质-丘脑和丘脑-皮质连接 转染不同的CDSG shRNA，以及实验和对照之间。在 希望这项工作能指导阅读障碍亚型的研究，目标2将集中在系统的 在胚胎转染shRNA或过表达的大鼠大脑中观察到的差异 每个CDSG同源物的质粒。在胚胎中完成工作后，我们将在原位使用 杂交和免疫组化比较基因的时空表达 出生后老鼠的行为模式。我们还将评估转染的神经元的神经元形态， 他们的进程。目的3检查解剖组织的广泛变化， 假设直接来自shRNA或过表达构建体的局部转染， 与塑性相关的二次效应的结果。我们将使用高效准确的体视学探头， 估计整个新皮层和丘脑的神经元数量、神经元大小和区域体积。一个 准确描述前脑解剖结构，结果无论是敲低或过度表达 大鼠同源的CDSGs，细胞自主和继发性的影响，以及他们的过程 发展，作为遗传学和行为之间的良好桥梁，将有助于摆脱更广泛的 阐明人类发展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， 在项目III中描述的行为变化。

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.