Molecular control of brain size

 描述（由申请人提供）：小头畸形（“异常小的大脑”）是一种神经发育障碍，可引起神经系统症状，如智力残疾、语言延迟和癫痫。许多致病基因已被报道，其中大多数编码中心体蛋白。中心体蛋白质的突变究竟是如何导致小头畸形的还没有很好的理解。先前使用苍蝇和小鼠模型的研究表明，中心体蛋白的突变可能会破坏神经祖细胞（NPC）的增殖或诱导过早分化为神经元，以牺牲NPC为代价。然而，目前可用的小头畸形动物模型具有相当温和的表型，使得难以解决哪些分子和细胞机制对人类严重小头畸形至关重要。该项目旨在开发和建立具有稳健表型的新的小头畸形动物模型。我们有两个假设：（1）由于许多小头症基因产物共定位于中心体，其中一些可能在生化和遗传上相互作用。例如，ASPM（异常纺锤体样，小头畸形相关）和WDR 62（WD重复结构域62），这两种突变时人类小头畸形的最常见原因，相互作用。因此，一个小头畸形基因的杂合缺失，其本身根本没有表型，可能会增强另一个小头畸形基因的纯合敲除小鼠的轻度表型;（2）与小鼠不同，雪貂有一个扩大的大脑，其中含有外放射状胶质细胞，一种在人类皮层中高度丰富的NPC。因此，与相同基因的敲除小鼠相比，小头畸形基因敲除的雪貂可能显示出稳健的表型。在目标1中，我们将检查Aspm-/-; Wdr 62 +/-小鼠，其具有比任何对照小鼠（Aspm+/+; Wdr 62 +/-和Aspm-/-; Wdr 62 +/+小鼠）显著更小的脑，其具有可忽略的表型。我们将表征两种蛋白质之间的相互作用以及Aspm-/-; Wdr 62 +/-小鼠发育中皮质中母亲与女儿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TALEN（一种新的基因组编辑工具）生成的Aspm敲除雪貂。初步数据显示他们患有严重的小头畸形。使用免疫组织化学和腺病毒绿色荧光蛋白感染，然后通过延时成像，我们将研究Aspm基因敲除和野生型雪貂中不同NPC的丰度和行为。拟议的工作将为小头畸形和皮质畸形提供令人兴奋的新动物模型，在大脑皮质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的不断增加，我们的方法将展示如何在基因敲除小鼠没有表现出强大的表型时研究基因的功能意义。此外，它应该解决目前关于外放射状胶质细胞在大脑皮质发育过程中的作用的争论，并有可能确定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.