LINC00461 contributes to neural progenitor organisation and regulates developmental transcription progression in human brain models

Long non-coding RNAs (lncRNAs) are increasingly recognised as important regulators of human brain development, contributing to the control of gene expression, chromatin organisation and developmental transitions. Their strong tissue specificity and dynamic expression during neural differentiation make them particularly relevant for the study of human neurodevelopment, yet the function of most brain-enriched lncRNAs remains poorly understood. Human pluripotent stem cell-derived neural models, including neural progenitor cultures and brain organoids, provide a powerful platform to investigate these transcripts in a human developmental context, allowing both transcriptional regulation and three-dimensional tissue organisation to be explored. Within this framework, this thesis focused on LINC00461 (also known as MIR9-2HG), a chromatin-associated lncRNA whose expression is enriched in the human central nervous system. LINC00461 was selected as a candidate regulator of neural development because of its induction during in vitro neural differentiation, its preferential expression in brain tissues and in developing cortical populations, and its genomic location within the 5q14.3 region, which has been implicated in neurodevelopmental and psychiatric disorders. Its proximity to MEF2C, together with previous evidence from the homologous murine NDIME–Mef2c axis, further supported the hypothesis that LINC00461 may participate in regulatory networks relevant to human neural lineage progression. To assess the functional contribution of LINC00461, two independent human embryonic stem cell lines carrying a premature transcriptional termination signal within the locus were generated and analysed during neural progenitor and brain organoid differentiation. LINC00461 loss was associated with broad alterations in neural developmental programmes and impaired organisation of neural progenitors, with the phenotype becoming more evident in three-dimensional brain organoids. LINC00461 disruption was also consistently associated with increased expression of the neighbouring transcription factor MEF2C. However, this increase occurred in 1 a transcriptional context characterised by reduced neural developmental programmes, suggesting that MEF2C abundance and functional output may become uncoupled following LINC00461 loss. Overall, this study identifies LINC00461 as a functionally relevant component of human neural development and shows that its loss affects both developmental transcriptional programmes and neural tissue organisation. The results further suggest a regulatory connection between the human LINC00461 locus and MEF2C, providing a basis for future mechanistic studies of this non-coding regulatory axis.