Quantseq from: A circular RNA expressed from the FAT3 locus regulates neural development

Circular RNAs (circRNAs) are key regulators of cellular processes, are abundant in the nervous system, and have putative regulatory roles during neural differentiation. However, the knowledge about circRNA functions in brain development is limited. Here, using RNA-sequencing, we show that circRNA levels increase substantially over the course of differentiation of human embryonic stem cells into rostral and caudal neural progenitor cells (NPCs), including three of the most abundant circRNAs, ciRS-7, circRMST, and circFAT3. Knockdown of circFAT3 during early neural differentiation resulted in alterations in the expression of genes involved in insulin signaling and resistance. Single-cell transcriptomic analysis of 30 and 90 days differentiated cerebral organoids deficient in circFAT3 showed a loss of telencephalic radial glial cells and mature cortical neurons, respectively. Furthermore, non-telencephalic NPCs in cerebral organoids showed changes in the expression of genes involved in neural differentiation and migration, including FAT4, ERBB4, UNC5C, and DCC. In vivo depletion of circFat3 in mouse prefrontal cortex using in utero electroporation led to alterations in the positioning of the electroporated cells within the neocortex. Overall, these findings suggest a conserved role for circFAT3 in neural development involving the formation of anterior cell types, neuronal differentiation, and migration. Loss-of-function studies of ciRS-7, circRMST, and circFAT3 in H9 human embryonic stem cells (hESCs) differentiation into rostral and caudal neural progenitor cells (rNPCs and cNPCs, respectively) using AGO-dependent short hairpin RNAs (agoshRNAs) targeting the circRNA backsplicing junction. Five days prior to the 11 days of differentiation into rNPCs and cNPCs, H9 hESCs were transduced with lentiviral vectors encoding the agoshRNAs or empty control vectors (A244).