Single cell isoform expression across mouse brain regions and development Much of the mouse brain's biology is defined by the action of long RNA and protein isoforms in distinct cell types and brain regions. RNA-isoforms are altered on individual variable sites [transcription start site (TSS)- choice, alternative exon inclusion, RNA-modifications and polyA-site choice] during development. Currently, a simpler view of gene expression summarizing each gene's activity in a single number (“how much a gene talks”) and the status of single variable sites is being explored widely – also in development. Yet, which multiple individual sites are regulated individually and which as complex units, the complete isoforms (“the complete phrases a gene says”) and their cell-type specific expression are poorly understood in mouse brain development. As a result, there is a critical gap in our understanding of the brain. Without this knowledge, pinpointing the molecular causes of neurodevelopmental disorders will remain difficult. Our overall objective is to distinguish isoforms that are broadly expressed across multiple cell types from those that are specific to cell populations to enable an understanding of these isoforms' action. The central hypothesis is that many brain-region and development specific isoforms are generated by specific cell populations. Aim 1 will reveal the cell-type specific isoforms in all cell types across five mouse brain regions in order to understand variability in isoform usage between brain regions and cell types. This will distinguish for each brain-region specific isoform between multiple models that can cause its specific expression pattern. Aim 1 will also develop new methods to monitor isoform expression in 1,000,000 cells. Aim 2 will define full-length mouse RNA molecules across all hippocampal and cerebellar cell types during postnatal development, as well as full-length isoforms specifically expressed at specific time points. We will distinguish between distinct models that explain such time specific isoforms for each specifically expressed isoform. We provide a new innovative view of isoform biology across brain regions of young adult mice and from early to late postnatal development. This adds the dimension of isoform abundance to single-cell approaches in the brain and reveals which of three distinct mechanisms causes isoform expression differences observed in bulk samples. Our work also provides clues as to which isoform, cell type and time point are of importance in neurodevelopmental diseases.

The function of brain regions is in parts defined by the specific cell types that they comprise and by the specific molecular repertoires these cell types generate. However, the extent to which brain-region and development specific RNA isoforms are also highly cell-type specific is not fully understood. Here, we untangle for each isoform specific to brain-regions or developmental time points, to which extent it is specific of one or more cell types.