Engrailed genes and cerebellum morphology, spatial gene expression and circuitry

描述（由申请人提供）：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，而且各种研究表明它在非运动活动中也起作用，包括语言。等位基因系列的小鼠Engrailed （En）突变体显示小脑形态缺陷和传入通路的地形破坏。因此，更好地了解En1/2如何调节小脑发育，将有助于了解具有明显形态变化的人类小脑畸形综合征，以及具有更细微形态异常但可能具有显著脑小脑回路功能障碍的疾病，如ASD。ASD和En基因之间的另一个可能的联系是与自闭症相关的人类EN2的RFLPs。此外，En2突变小鼠在社交互动方面存在缺陷。由于叶状叶和旁矢状叶基因表达的模式反映了每个传入通路的地形，因此需要一种新的全局方法来研究小脑在形态学、基因表达模式和功能特异性传入通路水平上的组织方式。此外，叶状结构的区域差异反映了功能的特化，因为哺乳动物小脑沿着内侧-外侧轴被分离成主要容纳运动回路的中央蚓部和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，以确定副矢状胶质基因的表达和叶面形成是如何在遗传和细胞水平上形成的，从而深入了解小脑回路是如何形成的。此外，下游效应物将被确定介导这些过程，因此可能是候选的小脑疾病基因。由于基本叶状结构模式和副矢状突基因表达在哺乳动物中是保守的，我们在小鼠中的发现应该为评估人类小脑的发育和疾病提供基础。具体目标是：1)研究En1/2如何调节蚓部和半球不同的叶状结构模式，并通过分析时间和细胞类型特异性的En1/2条件敲除突变体，确定负责叶状结构、基因表达和传入地形的关键阶段和细胞类型。ii)通过检测En1/2条件丧失和功能获得突变体中浦肯野细胞和颗粒细胞的扩增和迁移、空间基因表达和传入发育，确定En1/2调控的细胞过程。2)鉴定和测试候选基因，包括Eph/Ephrins，它们在En1/2下游参与两个坐标系的模式化和/或建立传入地形：1)鉴定受En1/2调控的En2阳性和En2阴性细胞中差异表达的基因。ii)在发育中的浦肯野细胞或颗粒细胞中改变候选基因和Eph/ ephrin的表达。公共卫生相关性：小脑整合四肢和身体运动的位置、速度和力量等信息，所有这些都是进行平稳、协调运动所必需的。它还与语言等认知功能有关。更好地理解Engrailed （En）基因的突变是如何导致一系列小脑形态缺陷和神经回路中断的，应该为解释人类疾病提供新的原理，这些疾病包括小脑体积缩小，以及自闭症谱系障碍（ASD）等疾病，其中存在更细微的结构改变，但仍可能导致神经回路的严重功能障碍。

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. PUBLIC HEALTH RELEVANCE: The cerebellum integrates information such as the position, speed and force at which the limbs and body are moving, all of which are necessary to carry out smooth, coordinated movements. It also is implicated in cognitive functions like language. A better understanding of how mutations in the Engrailed (En) genes result in a range of morphological cerebellum defects as well as disruptions of neural circuits should provide new principals to aid in the interpretation of human diseases which involve reduction in the size of the cerebellum, as well as diseases such as autism spectrum disorder (ASD) where there are more subtle structural alterations but could nevertheless have profound dysfunction of neural circuits.