Combinatorial function of Foxp1/2/4 in Purkinje cell diversification and cerebellar development

总结 小脑发育异常，特别是浦肯野细胞的病理和功能障碍， 与多种神经发育疾病有关，包括共济失调、自闭症谱系 精神分裂症和语言障碍。作为最早出生的小脑细胞群之一， 浦肯野细胞被认为在小脑的发育、功能和发病机制中起作用。 有证据表明存在具有不同分子特征的浦肯野细胞亚型。但 浦肯野细胞多样化的分子机制仍然知之甚少。 因此，我们缺乏一个条目来评估单个浦肯野细胞亚型的作用。通过单细胞 通过RNA和染色质可及性分析，我们发现了至少九种分子上不同的亚型， 小鼠小脑发育中的浦肯野细胞。这些浦肯野细胞亚型有助于不同的 发育中的小脑中的隔室。值得注意的是，浦肯野细胞亚型显示出 Foxp 1、Foxp 2和Foxp 4的特征性组合表达，它们属于 叉头盒转录因子家族。人类FOXP 1或FOXP 2的突变与语言有关 自闭症谱系障碍和智力残疾，表明这些蛋白质协调了 与认知疾病相关的神经回路的发展。体外研究表明，FoxP 蛋白质形成具有可变转录靶点和活性物质的二聚体或低聚体，具体取决于 结合伴侣我们假设Foxp 1/2/4形成组合的“FoxP码”，以指定不同的 浦肯野细胞亚型，这反过来又控制小脑的形态发生。AIM 1将联合收割机 常规表达分析、空间转录组学和体积成像，以确定 PC亚型的发展与小脑的形态发生有关。目标2将 从小鼠小脑中单独和组合地删除Foxp 1/2/4。我们将使用组织学， 单细胞RNA-seq和行为研究，以评估单个和 复合Foxp 1/2/4突变对小脑发育和行为功能的影响。Aim 3将使用多- 用组学方法研究组合FoxP基因调控细胞凋亡的分子机制， 用于浦肯野细胞分化的转录程序。在这个项目完成后，我们预计将有 确定了Foxp 1/2/4在小脑发育中的个体和组合作用。本研究将 不仅对小脑发育的基础知识， 对大量未探索的小脑相关疾病的分子基础的理解。

SUMMARY Abnormalities in cerebellar development, especially pathology and dysfunction of Purkinje cells, have been implicated in a wide variety of neurodevelopmental diseases, including ataxia, autism spectrum disorder, schizophrenia, and language impairment. Being one of the earliest-born cerebellar cell groups, Purkinje cells are believed instrumental in the development, function, and pathogenesis of the cerebellum. Evidence suggests the existence of Purkinje cell subtypes with distinct molecular features. However, the molecular mechanisms underlying the diversification of Purkinje cells remain poorly understood. Consequently, we lack an entry to assess the role of individual Purkinje cell subtypes. Through single-cell RNA and chromatin accessibility analyses, we uncovered at least nine molecularly distinct subtypes of Purkinje cells in the developing mouse cerebellum. These Purkinje cell subtypes contribute to different compartments in the developing cerebellum. Remarkably, the Purkinje cell subtypes display a characteristic combinatorial expression of Foxp1, Foxp2, and Foxp4, which belong to a subgroup of the forkhead-box transcription factor family. Mutations of human FOXP1 or FOXP2 are linked to speech disorders, autism spectrum disorder, and intellectual disability, indicating that these proteins coordinate the development of the neural circuits related to cognitive diseases. In vitro evidence shows that FoxP proteins form dimers or oligomers with variable transcriptional targets and actives depending on the binding partner. We hypothesize that Foxp1/2/4 form combinatorial “FoxP codes” to specify distinct Purkinje cell subtypes, which in turn control the morphogenesis of the cerebellum. Aim 1 will combine conventional expression analysis, spatial transcriptomics, and volume imaging to determine the development of PC subtypes in relation to the morphogenesis of the cerebellum. Aim 2 will delete Foxp1/2/4, individually and in combinations, from the mouse cerebellum. We will use histology, single-cell RNA-seq, and behavioral studies to evaluate the impacts of single and compound Foxp1/2/4 mutations on cerebellar development and behavioral function. Aim 3 will use a multi- omic approach to study the molecular mechanism by which combinatorial FoxP genes regulate the transcription program for Purkinje cell differentiation. At the completion of this project, we expect to have identified the individual and combinatorial roles of Foxp1/2/4 in cerebellar development. This study will have a significant positive impact, not only on the basic knowledge of cerebellar development but also on the understanding of the molecular basis of the vast number of unexplored cerebellum-related diseases.