Cortical GABAergic interneurons comprise >20 types of inhibitory cells with distinct morphological and physiological properties. Correct development of this group of neurons is essential for normal neural and cognitive functions, and mistakes in this process have been linked to neurodevelopmental and psychiatric diseases.
Mounting evidence suggests that alternative splicing may facilitate the emergence of distinct neuronal types. However, current analyses of cell type-specific alternative splicing patterns often involve dissociation of brain samples and isolation of individual cells or their compartments. Since these multistep protocols may distort the transcriptomes and proteomes, we will address biological functions of alternative splicing in developing interneurons using a newly established in-situ labelling technology.
Firstly, we will adapt our recently developed hybridization-proximity labelling approach to biotinylate RNAs and proteins in cells expressing commonly used recombinant lineage markers (e.g., EGFP or tdTomato). Secondly, we will use this in situ labelling approach to analyse developmental changes in interneuron type-specific transcriptomes and proteomes by RNA-sequencing and mass-spectrometry. Thirdly, we will address biological functions of the most promising alternative splicing targets identified by this screen by CRISPR/Cas approaches.
Work on the first objective will be carried out during the rotation and finished in the first half of year 2. The second objective will involve performing time-resolved labelling experiments and building a bioinformatics pipeline for processing and integration of the multimodal RNA-sequencing and mass-spectrometry data (years 2-3). Years 3-4 will be spent on the CRISPR/Cas experiments (objective 3) and writing the thesis.
The expertise of the Makeyev group in biochemistry and bioinformatics will ensure successful completion of the first two objectives. The Marin lab is a world leader in interneuron development studies, providing an ideal environment for the third objective. Overall, the project should uncover fundamental mechanisms driving interneuron differentiation and provide cutting-edge tools for further work in this exciting field.