Engrailed genes and cerebellum morphology, spatial gene expression and circuitry

描述(申请人提供)：越来越多的证据表明，大脑的许多区域，包括小脑，都与自闭症谱系障碍(ASD)有关。小脑不仅在熟练的运动表现中起着关键作用，而且各种研究都表明它与包括语言在内的非运动活动有关。一系列等位基因的小鼠渐进型(EN)突变体显示出小脑的形态缺陷和传入通路的地形图中断。因此，更好地了解EN1/2如何调节小脑发育应该有助于洞察具有大体形态变化的人类小脑畸形综合征，以及具有更细微的形态异常但可能具有显著脑回路功能障碍的ASD等疾病。ASD和EN基因之间另一个可能的联系是人类EN2的RFLP，它与自闭症有关。此外，EN2突变小鼠在社会互动方面也存在缺陷。需要一种新的全局方法来研究小脑在形态、模式基因表达和功能特定的传入通路水平上是如何组织的，因为叶和矢状面旁基因表达的模式反映了每条传入通路的地形。此外，叶化的区域差异反映了功能的专门化，因为哺乳动物的小脑沿着内侧轴被分离成主要容纳运动回路的中央蚯蚓，以及主要容纳脑小脑回路的周围半球。EN基因将被用作遗传切入点，以确定矢状面旁基因表达和叶状结构是如何在遗传和细胞水平上形成模式的，以便深入了解小脑回路是如何铺设的。此外，还将确定介导这些过程的下游效应器，因此这可能是候选的小脑疾病基因。由于基本的叶状结构和矢状面旁的基因表达在哺乳动物中是保守的，我们在小鼠身上的发现应该为评估人类小脑的发育和疾病提供了基础。其具体目的是：1)研究EN1/2如何调节蚯蚓和大脑半球不同的叶化模式，以及如何调控矢状面旁基因表达和苔藓纤维地貌：i)通过分析时间和细胞类型特异的EN1/2条件敲除突变体，确定负责叶化、基因表达和传入地形图的关键阶段和细胞类型。Ii)通过检测EN1/2条件功能丧失和获得突变体中的浦肯野细胞和颗粒细胞的扩张和迁移、空间基因表达和传入发育来确定受EN1/2调控的细胞过程。2)确定和测试候选基因，包括EN1/2下游在构图两个坐标系和/或建立传入拓扑图中具有作用的Eph/ephins：i)鉴定在en2阳性或en2阴性细胞中差异表达的受EN1/2调控的基因。ii)改变候选基因和Eph/eparins在发育中的浦肯野细胞或颗粒细胞中的表达。

DESCRIPTION (provided by applicant): There is mounting evidence that many regions of the brain, including the cerebellum, are involved in autism spectrum disorder (ASD). The cerebellum not only plays a critical role in skilled motor performance, but a variety of studies have implicated it in non-motor activities, including language. An allelic series of mouse Engrailed (En) mutants display both morphological defects in the cerebellum and topographic disruptions of afferent pathways. A better understanding of how En1/2 regulate cerebellum development should therefore provide insight into human cerebellum malformation syndromes with gross morphological changes, as well as diseases such ASD with more subtle morphological abnormalities but that could have significant cerebrocerebellar circuit dysfunction. An additional possible link between ASD and the En genes is RFLPs of human EN2 that are associated with autism. Also, En2 mutant mice have deficits in social interactions. A new global approach to studying the cerebellum is needed to relate how it is organized at the level of morphology, patterned gene expression and functionally specific afferent pathways, because the pattern of the folia and parasagittal gene expression reflects the topography of each afferent pathway. Furthermore, regional differences in foliation reflect functional specializations, as the mammalian cerebellum is segregated along the medial-lateral axis into a central vermis housing mainly motor circuits and surrounding hemispheres housing mainly cerebrocerebellar circuits. The En genes will be used as a genetic entry point to determine how parasagittal gene expression and foliation are patterned at the genetic and cellular levels in order to gain insight into how cerebellum circuitry is laid down. In addition, downstream effectors will be identified that mediate these processes and thus that could be candidate cerebellum disease genes. Since the basic foliation pattern and parasagittal gene expression is conserved across mammals, our findings in mice should provide a foundation for assessing human cerebellum development and disease. The Specific aims are: 1) Study how En1/2 regulate the distinct foliation patterns in the vermis and hemispheres, and also pattern parasagittal gene expression and mossy fiber topography by: i) Identifying the critical stages and cell types responsible for patterning foliation, gene expression and afferent topography by analyzing temporal and cell type specific En1/2 conditional knock-out mutants. ii) Identifying cellular processes regulated by En1/2 by examining Purkinje cell and granule cell expansion and migration, spatial gene expression and afferent development in En1/2 conditional loss- and gain-of-function mutants. 2) Identify and test candidate genes including Eph/Ephrins that have roles downstream of En1/2 in patterning the two coordinate systems and/or establishing afferent topography by: i) Identifying genes differentially expressed in En2 positive or En2 negative cells that are regulated by En1/2. ii) Altering expression of candidate genes and Eph/Ephrins in developing Purkinje cells or granule cells.