Functional characterization of long non-coding RNAs involved in the epigenetic regulation of human neural development

The aim of this thesis is to provide a comprehensive investigation into the role of long non-coding RNAs (lncRNAs) in human brain development and neuronal specifi cation, using as experimental models both human embryonic stem cell (hESC)-derived cortical organoids (hCOs) and canonical in vitro hESCs-derived neural progenitor cells (NPCs) and terminal diff erentiated cortical neurons (TDCNs). We employed a computational workfl ow to identify a repertoire of the most promising lncRNA candidates. The initial analysis prioritized spatio-temporal expression patterns and sequence characteristics, as the primary criteria for the selection. lncRNA genes were required to be upregulated during the later stages of neural diff erentiation compared to pluripotency, enriched in the chromatin fraction, and transcribed as poly-exonic transcripts. This approach identifi ed a set of 63 lncRNAs, with the lncRNA MIAT emerging as the top candidate. CRISPR/Cas9-mediated knockout (KO) of lncRNA MIAT revealed its critical role in orchestrating proper cortical development and cellular subtype specifi cation. In the absence of lncRNA MIAT, we observed defective neuronal networking, including axon guidance and synaptic organization, accompanied by altered extracellular matrix remodelling events and induced expression of caudal and ventral signatures in mutant cells, suggesting an early cell mispatterning during the development. Coherently with this observation, MIAT KO organoids exhibit reduced brain expansion, specifi cally in the outer sub-ventricular zone (oSVZ), where an impact on human-enriched populations of radial glial cells resembles a gaining of “ape-like” traits and archaic gene regulatory network. These fi ndings propose the lncRNA MIAT as a key regulator of human-specifi c events occurring in cell patterning, forebrain regionalization, lineage identity acquisition and cortical expansion during the fi rst trimester of the human brain development