Molecular control of brain size

 描述(申请人提供)：小头畸形症(“异常小脑”)是一种神经发育障碍，会导致神经系统症状，如智力残疾、语言障碍和癫痫。已有许多致病基因被报道，其中大部分编码中心体蛋白。中心体蛋白的突变是如何导致小头畸形症的确切原因尚不清楚。之前使用苍蝇和小鼠模型进行的研究表明，中心体蛋白的突变可能会扰乱神经前体细胞的增殖，或者以牺牲神经前体细胞为代价诱导提前分化为神经元。然而，目前可用的小头畸形动物模型具有相当轻微的表型，这使得很难解决哪些分子和细胞机制是人类严重小头畸形的关键。该项目旨在开发和建立具有强健表型的小头畸形症新的动物模型。我们有两个假设：(1)由于许多小头症基因产物共存于中心体，其中一些可能存在生化和遗传上的相互作用。例如，ASPM(异常纺锤体样，与小头畸形相关)和WDR62(WD重复区域62)是人类小头畸形的两个最常见的突变原因，它们相互作用。因此，一种本身没有表型的小头畸形基因的杂合缺失，可能会增强另一种小头畸形基因纯合敲除小鼠的温和表型；(2)与小鼠不同，雪貂有一个增大的大脑，其中包含外径向神经胶质细胞，这是一种在人类皮质中高度丰富的神经胶质细胞。因此，与相同基因的敲除小白鼠相比，小头畸形症基因敲除的雪貂可能表现出较强的表型。在目标1中，我们将检查Aspm-/-；Wdr62+/-小鼠的大脑比任何对照组小鼠(Aspm+/+；Wdr62+/-和Aspm-/-；Wdr62+/+小鼠)，这些小鼠的表型可以忽略不计。我们将描述这两种蛋白质之间的相互作用，以及在Aspm-/-；Wdr62+/-小鼠的发育皮质中母子中心体的不对称遗传。在目标2中，我们将建立和表征我们最近使用一种新的基因组编辑工具TALEN培育的Aspm基因敲除雪貂。初步数据显示，他们患有严重的小头畸形症。利用免疫组织化学和腺病毒绿色荧光蛋白感染后的时间推移成像，我们将检测不同的鼻祖细胞在Aspm基因敲除和Wile类型的雪貂中的丰度和行为。这项拟议的工作将为小头症和皮质畸形提供令人兴奋的新动物模型，在大脑皮层发育和进化领域创建一个创新的实验系统。随着人类遗传学研究中基因清单的不断增加，我们的方法将演示当基因敲除的小鼠没有表现出强大的表型时，如何研究该基因的功能意义。此外，它应该解决目前关于外径向胶质细胞在大脑皮层发育中的作用的争论，并有可能识别正常皮质发育的新机制。

 DESCRIPTION (provided by applicant): Microcephaly ("abnormally small brain") is a neurodevelopmental disorder that causes neurological symptoms, such as intellectual disability, language delay, and epilepsy. A number of causative genes have been reported, the majority of which encode centrosomal proteins. Exactly how mutations in centrosomal proteins cause microcephaly is not well understood. Previous studies using fly and mouse models suggest that mutations in centrosomal proteins may disrupt proliferation of neural progenitor cells (NPCs) or induce premature differentiation into neurons at the expense of NPCs. However, currently available animal models of microcephaly have pretty mild phenotypes, making it hard to address which molecular and cellular mechanisms are critical to severe microcephaly in humans. This project seeks to develop and establish new animal models for microcephaly with robust phenotypes. We have two hypotheses: (1) because many microcephaly gene products colocalize in the centrosome, some of them may interact biochemically and genetically. For example, ASPM (abnormal spindle-like, microcephaly-associated) and WDR62 (WD repeat domain 62), the two most common causes for human microcephaly when mutated, interact with each other. Thus, heterozygous deletion of one microcephaly gene, which has no phenotype at all by itself, may enhance the mild phenotype in homozygous knockout mice of another microcephaly gene; (2) Unlike mice, ferrets have an enlarged brain, which contains outer radial glial cells, a type of NPCs that is highly abundant in the human cortex. Thus, knockout ferrets of a microcephaly gene may show robust phenotypes compared to knockout mice of the same gene. In Aim 1, we will examine Aspm-/-; Wdr62+/- mice that have a significantly smaller brain than any control mice (Aspm+/+; Wdr62+/- and Aspm-/-; Wdr62+/+ mice), which have negligible phenotypes. We will characterize interaction between the two proteins as well as asymmetric inheritance of mother versus daughter centrosomes in the developing cortex of Aspm-/-; Wdr62+/- mice. In Aim 2, we will establish and characterize Aspm-knockout ferrets that we have recently generated using TALEN, a new genome-editing tool. Preliminary data show that they have severe microcephaly. Using immunohistochemistry and adenoviral green fluorescent protein infection followed by time-lapse imaging, we will examine abundance and behaviors of diverse NPCs in Aspm-knockout and wile-type ferrets. The proposed work will provide exciting new animal models for microcephaly and cortical malformation in general, creating an innovative experimental system in the field of cerebral cortical development and evolution. With ever-increasing list of genes from human genetic studies, our approach will demonstrate how to study functional meanings of a gene when knockout mice of the gene do not show robust phenotypes. In addition, it should resolve the current debate over roles of outer radial glial cell during cerebral cortical development, and has the potential to identify novel mechanisms of normal cortical development.