The role of maternal and paternal genomes in neuronal networks

In early imprinting research, Androgenetic and parthenogenetic mouse chimera models demonstrated that imprinted genes are essential in early neural lineage commitment and play a crucial role during brain development. Indeed, proper timing of brain development and the organization of brain regions require a fine dosage of the imprinted gene expression. Moreover, recent work has demonstrated the key role of imprinted genes in the 24-hour-timed brain functions, such as circadian rhythms. However, the biological role of many imprinted genes in the developing and adult brain remain elusive. In this framework, we investigated the specific contribution of the maternal or paternal genome in cortical development and the maintenance of the circadian clock, with a particular focus on the transition from individual cells to a comprehensive neuronal network. To this aim, we took advantage of androgenetic (AG) (double paternal genome) and parthenogenetic (PG) (double maternal genome) mESC lines, generating two experimental models with increasing complexity, the 2D neuron cultures, as well as a newly developed 3D cortical organoid (cOrgs). We profiled the imprinting status of all cell lines at the stem state and during the corticogenesis. We evidenced strict maintenance of the parent-of-origin expression of IGs in the androgenetic line and the biparental-like expression of most of the IGs in the PGs, as previously reported. We found that the cells carrying double maternal genomes were characterized by strong downregulation of Notch signaling at the stem state. Moreover, the maternal or paternal genome did not influence the ability to generate mature and functional neurons. However, we highlighted a delay in the differentiation timing at the neurogenesis stage of the PG line. On the other hand, the characterization of the 3D model revealed an increased size of the AG- and PG- cOrgs compared to the wild type. The assessment of spontaneous activity of 2D-derived neurons revealed, for the first time, an influence of the maternal or paternal genome on the activity within the neuronal network. AG-derived neurons presented a highly synchronized individual bursting activity and more extensive connection topology. In contrast, PG- derived neurons were connected in smaller, less synchronized networks but still characterized by bursting behavior. Moreover, we identified highly specialized neurons, known as network leaders, driving the network activity in both AG and PG cultures. We demonstrated opposite influences of the carried genome on the AG and PG leader's firing 2 behavior. Finally, we found that the maternal and paternal genomes differentially influenced clock genes' rhythmicity and transcriptional level of core and complementary clock loops. We found a complete loss of Ror-a, Rev-a, and Nfil3 rhythmicity in AG 2D and 3D models and the loss of Bmal1 rhythmicity in PG models. Moreover, we found that the alteration in a gene's transcriptional level is unrelated to its rhythmicity and vice versa. Overall, this study highlighted the difference between the maternal and paternal genomes in contributing to neuronal differentiation and organoid assembly. The identified 3D differentiation protocol will be instrumental in the imprinting research and will overcome the lethality found in living organisms like the mice chimeras.