In Utero Electroporation Uncovers an Early-Differentiating Subset of Dentate Gyrus Progenitors

The dentate gyrus of the hippocampus develops through complex cellular migrations and differentiations, which have been primarily characterized using genetic lineage tracing approaches. Through systematic application of in utero electroporation across developmental stages, we found that labeling was most effective at embryonic day 12.5 (E12.5), as earlier stages resulted in embryonic lethality while later stages showed markedly reduced efficiency. To directly compare these cells with genetically-defined progenitor populations, we established a novel dual-visualization system, combining electroporation with transgenic reporter mice (Gfap-GFP). This approach revealed marked differences in developmental trajectories: Gfap-GFP+ cells maintain undifferentiated neural stem/progenitor characteristics with persistent Sox2 expression, while E12.5-labeled cells predominantly differentiate into Prox1-positive granule cells by E18.5. These early-labeled cells display characteristic migration patterns, with 60.9% differentiating into Prox1-positive granule cells compared to only 22.8% of Gfap-GFP+ cells (P < 0.001), exclusively following an outside-in trajectory to establish the initial framework of the granule cell layer, without reaching the tertiary dentate matrix. In contrast, Gfap-GFP+ cells populate the tertiary dentate matrix and serve as a sustained progenitor reservoir. Molecular marker analysis reveals sequential expression of Sox2, Tbr2, and Prox1, demonstrating progressive differentiation during migration. Our findings identify an early-differentiating subset of dentate progenitors with accelerated neurogenic progression, revealing previously unrecognized temporal and functional heterogeneity in dentate development. This study demonstrates how stage-specific in utero electroporation can complement genetic approaches by uncovering progenitor subsets with rapid differentiation kinetics, providing new insights into the cellular diversity that shapes hippocampal structure and function.