Defining the role of Tox3 in congenital cerebellar hypoplasia and ataxia

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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.