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.
