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

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.