The Role of DNMT3a in Establishment and Maintenance of Regional SVZ Heterogeneity

While a great deal of literature focuses on how cell fates are established during embryogenesis, how these identities are maintained throughout adulthood is poorly understood. The subventricular zone (SVZ) of the mammalian brain contains a heterogeneous population of adult neural stem cells (NSCs) along its dorsoventral axis that give rise to distinct subtypes of olfactory bulb interneurons. Previous work has shown that this regional heterogeneity is largely cell autonomous. DNA methylation establishes stable and heritable changes in gene expression and is therefore a mechanism by which cellular identity could be “encoded”. Furthermore, mice lacking DNMT3a, a de novo DNA methylase, show a significant decrease in calbindin-positive neurons which originate exclusively from the ventral SVZ. We hypothesize that the ventral identity of adult SVZ NSCs requires DNMT3a-mediated de novo DNA methylation during embryogenesis. To test this, we cultured ventrally-derived SVZ NSCs dissected from postnatal DNMT3a-null and WT mice. After allowing the cultures to differentiate, we performed immunocytochemistry, staining for Tuj1, a neuronal-marker, and calbindin. DNMT3-null cells had a 58% reduction in calbindin-positive neurons, confirming previous results and validating our in vitro system. Rescue of DNMT3a with a DNMT3a-expressing lentivirus in this assay did not re-establish the ventral identity of DNMT3a-null cultures. Although preliminary, this data supports the notion that de novo DNA methylation is required during embryogenesis to encode the ventral identity of NSCs.