Cell-Type-Specific Splicing of Transcription Regulators and Ptbp1 by Rbfox1/2/3 in the Developing Neocortex

This study investigates how master splicing regulators interact and the extent to which transcription regulators are spliced differently during brain development. Cell-type-specific RNA-Seq analyses of the developing neocortex uncover variable expression of the Rbfox1/2/3 genes and enriched splicing events in transcription regulators, altering protein isoforms or inducing nonsense-mediated mRNA decay. Transient expression of Rbfox proteins in radial glial progenitors induces neuronal splicing events preferentially in transcription regulators such as Meis2 and Tead1. Surprisingly, Rbfox proteins promote the inclusion of a mammal-specific alternative exon and a previously undescribed poison exon in Ptbp1. Simultaneous ablation of Rbfox1/2/3 in the neocortex downregulates neuronal isoforms and disrupts radial neuronal migration. Furthermore, the progenitor isoform of Meis2 promotes Tgfb3 transcription, while the Meis2 neuron isoform promotes neuronal differentiation. These observations indicate that transcription regulators are differentially spliced between cell types in the developing neocortex. This study includes RNA-seq data from four sets of experiments: [1] RNA-Seq of enriched mouse cortical neural progenitor and neuron populations isolated from embryonic 14.5 day (E14.5) dorsal forebrains, using Tg(Eomes/Tbr2:EGFP) and Tg(Tubb3:EGFP) BAC transgenic lines: Tbr2_EGFP_negative (2 replicates, enriched for radial glial cells or RGCs); Tbr2_EGFP_positive (2 replicates, enriched for intermediate progenitor cells, or IPCs); Tubb3_EGFP_positive( 2 replicates, enriched for neurons). [2] Single-cell Long-Read RNA-sequencing (PacBio): Wild-type E14.5 dorsal cortex. [3] Rbfox3 overexpression and RNA-Seq in the developing brain: In utero electroporation on E13.5 and RNA-Seq of FACS-sorted EGFP-positive cells on E14.5: Control pCAG-IRES-EGFP empty vector (2 replicates); Rbfox3 overexpression, pCAG-Rbfox3-IRES-EGFP (2 replicates). [4] Meis2 isoform overexpression and RNA-Seq in the Neuro2a cell line: Control empty vector (4 replicates); Meis2 neuron-isoform overexpression (4 replicates); Meis2 RGC-isoform overexpression (4 replicates)