During evolution, the mammalian cerebral cortex has undergone a considerable increase in size and complexity. The emergence of the cortical structure begins during embryonic development when neural stem cells initially undergo proliferative division to expand their pool and then switch to neurogenic division, generating differentiating progenitors that will give rise to neurons. Although the intrinsic molecular mechanisms instructing the switch from proliferative to neurogenic division have been well-studied, most work to date has focused on gene expression. However, as a consequence of transcriptional and post-transcriptional regulation, different transcripts can arise from a single gene. In particular, the process of alternative splicing occurs at a high frequency in the nervous system and can lead to changes in protein output regardless of gene expression. In the past years, the role of post- transcriptional mechanisms in neuronal maturation and function have been extensively investigated, mostly focusing on the function of specific isoforms or RNA binding proteins. Yet, the role of alternative splicing in generating transcript and protein diversity during neurogenic commitment is still unknown. Therefore, I used a combination of different RNA sequencing technologies and bioinformatic tools to reveal the transcript and protein diversity of proliferating progenitors, differentiating progenitors, and neurons isolated from double reporter mouse line. I identified widespread isoform diversity and many novel transcripts amongst expressed genes in the developing cortex. To date, this analysis represents the most comprehensive characterization of full-length transcript diversity at different stages of the neurogenic lineage in the developing mouse cortex. The resulting transcriptome annotation was used to quantify changes in exon inclusion across cells of the neurogenic lineage and identified alternative splicing events potentially involved in neurogenic commitment. These alternative splicing events were enriched in the coding sequence of isoforms, indicating that they might be relevant for protein diversity generation in the developing cortex. During neurogenesis, alternative splicing events were less enriched in regions that could disrupt or strongly affect protein structure and function, such as transmembrane regions, active sites, and domains. Interestingly, my results indicated that alternative splicing enables increased functional diversity by modulating protein-protein interaction sites and signaling properties of proteins. Still, further studies are required to delineate the causal relationship between these alternative splicing choices and cell-fate commitment. Applying a similar approach to other mammalian species, including humans, has the potential to uncover species-specific innovations and conserved features that underlie evolutionary cortex expansion. Moreover, understanding the function of isoforms during neural development could provide important insights into the molecular mechanisms involved in the onset of neurodevelopmental disorders. Therefore, the higher cell-specificity offered by the present dataset, compared to similar studies, allowed not only a better understanding of transcript and protein diversity generated by alternative splicing in the nervous system and highlighted potential functional consequences, but also shed light on the advantages of applying such strategy to address different biological questions.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:90287 |
| Date | 03 June 2024 |
| Creators | Cardoso de Toledo, Beatriz |
| Contributors | Müller-Planitz, Felix, Becker, Catherina G., Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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