Defining the role of Tox3 in congenital cerebellar hypoplasia and ataxia

项目摘要/摘要 小脑是一种微妙的层状结构，调节平衡、自主运动协调和 通过与前脑的连接来调节语言和认知。坐在前脑的正下方， 据估计，小脑含有大约80%的人脑神经元。相应地，小脑 发育不全、病理或功能障碍与一系列疾病或障碍有关，包括共济失调， 自闭症和智力缺陷。大多数小脑神经元是一种称为小脑颗粒细胞(CGCs)的亚型， 它们是从一种名为菱形唇(RL)的结构胚胎中产生的--一种位于 脑室带(VZ)和第四脑室顶板之间的界面。主导机制 对RL生发龛和后续一代颗粒细胞的规范仍不完全 明白了。具体地说，脑室区域潜在的表观遗传和转录变化 前体细胞选择成为RL在很大程度上是未知的。我们已经生成了一个新的条件 Tox3的基因敲除(CKO)小鼠，Tox3是TOX转录因子家族中的一个成员 与T细胞表观遗传学的调节有关。在RL规范之前丢失TOX3导致几乎完成 CGCs发育不全和100%穿透性共济失调。我们假设TOX3介导一种表观遗传开关 从菱形唇部生成小脑颗粒细胞前体所必需的及其相关 脑室带--脑室带缺失会导致发育性共济失调。利用我们成熟的生物信息学 管道和单细胞方法，结合老鼠和人类模型系统，我们将 建议研究Tox3调节小脑组织发生的机制。 我们建议分两部分进行这项工作。具体目标1的重点是询问表观遗传学 用多模式单细胞RNA和ATAC测序研究Tox3CKO的转录后果 结合单细胞切割和标签确定调节VZ/RL前体和CGC的遗传网络 创世纪。特定目标2的主要目标是确定TOX3在小鼠小脑谱系中的作用 可诱导的Cre驱动程序和体细胞突变。

PROJECT SUMMARY / ABSTRACT The cerebellum is an exquisitely laminated structure regulating the balance, voluntary motor coordination and modulating language and cognition through connections with the forebrain. Sitting just under the forebrain, cerebellum is estimated to contain roughly 80% of the neurons in the human brain. Accordingly, cerebellar dysgenesis, pathology, or dysfunction is associated with a host of diseases or disorders, including ataxia, autism, and intellectual deficit. Most cerebellar neurons are a subtype called cerebellar granule cells (CGCs), which are generated embryonically from a structure termed the rhombic lip (RL)—a germinal zone residing at the interface between the ventricular zone (VZ) and roof plate of the fourth ventricle. The mechanisms leading to the specification of the RL germinal niche and subsequent generation of granule cells remain incompletely understood. Specifically, the epigenetic and transcriptomic changes underlying how the ventricular zone precursor cells choose the to become RL are largely unknown. We have generated a novel conditional knockout (cKO) mouse for Tox3, a member of the TOX family of transcription factors previously associated with the regulation of epigenetics in T Cells. Loss of TOX3 prior to RL specification leads to almost complete agenesis of CGCs and 100% penetrant ataxia. We hypothesize that TOX3 mediates an epigenetic switch necessary for the generation of cerebellar granule cell precursors from the rhombic lip and associated ventricular zone—the loss of which results in developmental ataxia. Using our mature bioinformatics pipelines and single-cell approaches in combination with both mouse and human models systems, we will propose to investigate the mechanisms by which Tox3 regulates cerebellar histogenesis. We propose to carry out this work in two parts. The focus of Specific Aim 1 is to interrogate the epigenetic and transcriptomic consequences of cKO of Tox3 using multimodal single-cell RNA- and ATAC-sequencing combined with single-cell Cut&Tag to determine genetic networks regulating VZ/RL precursors and CGC genesis. The main goal of Specific Aim 2 is to define the role of TOX3 on murine cerebellar lineages using inducible Cre drivers and somatic mutagenesis.