In utero electroporation uncovers a distinct population of early dentate gyrus progenitors

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Shinohara, Tokiharu Takahashi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6508491/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Nov, 2025 Read the published version in Cellular and Molecular Neurobiology → Version 1 posted 13 You are reading this latest preprint version Abstract 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 striking differences between two distinct populations: 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, 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 direct comparison identifies a functionally distinct subset of early progenitors that rapidly differentiate, revealing previously unrecognized temporal and functional heterogeneity in dentate development. This study demonstrates how stage-specific in utero electroporation uncovers diverse progenitor populations potentially underrepresented by existing genetic approaches, providing new insights into the cellular diversity that shapes hippocampal structure and function. Clinical trial number: not applicable dentate gyrus in utero hippocampal electroporation dentate progenitor cells granule cells dentate migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The hippocampus plays fundamental roles in learning, memory formation, and emotional processing as a key component of the limbic system. Its complex structure, comprising the hippocampal pyramidal cells layer of the cornu ammonius (CA1, CA2, and CA3 regions) and the granule cells layer (GCL) of the dentate gyrus (DG), develops through intricate cellular migrations and differentiations. Among these regions, the subgranular zone of the DG is particularly notable as a unique neurogenic niche where neurogenesis continues throughout life [ 1 – 4 ]. During development, undifferentiated cells migrate from the dentate notch (DN) through the dentate migratory stream (DMS) to establish the GCL [ 5 , 6 ], although the precise behaviors and properties of these migrating progenitors remain to be fully elucidated. The developmental process of the DG follows a remarkably unique and complex trajectory, involving cell migration along pathways that differ significantly from those of pyramidal cells in the neocortex and CA1 [ 7 ]. Previous research [ 8 – 11 ] has shown that neural progenitors that later differentiate into granule cells (GCs) in the DG exhibit a unique behavioral pattern, migrating between three distinct proliferative matrices: the primary dentate proliferative matrix (PDM) in the ventricular zone, the secondary dentate proliferative matrix (SDM) in the limbus, and the tertiary dentate proliferative matrix (TDM) in the hilus, thereby changing their proliferative zones (as illustrated in Fig. 1 A). The spatial organization of these matrices follows a specific pattern: the PDM resides in the neuroepithelium superior to the fimbria, adjacent to the DN. Following initial proliferation in the PDM, progenitor cells migrate above the cortical hem to the fimbrio-dentate junction (FDJ) along the pial membrane. Subsequently, these cells proceed along the pial membrane toward the prospective DG via the DMS. During this process, neural progenitor cells undergo proliferation and establish the SDM in the region beneath the hippocampal fissure extending to the pial membrane at the DG limbus [ 5 ]. These SDM-derived neural progenitors generate GCs in the outer shell of the GCL, while their TDM counterparts, which form in the hilus, give rise to GCs in the inner core of the GCL. Our current understanding of these complex migratory patterns mainly comes from studies using transgenic reporter mice, particularly those expressing fluorescent proteins under specific promoters. For example, mice with the Gfap promoter-driven reporter have shown that neural stem/progenitor cells emerge at E14.5 and contribute significantly to DG formation [ 11 ]. Although genetic approaches, particularly Cre-lox-based lineage tracing with tamoxifen-inducible control [ 10 , 12 , 13 ], have provided valuable insights, they are inherently limited by their dependence on pre-selected promoters of known genetic markers. This reliance on specific molecular markers potentially overlooks progenitor populations that lack expression of these canonical genes, particularly during earlier developmental stages when regulatory networks may be distinct. Complementary approaches that can label and track cells independently of specific gene expression would help eliminate potential biases and provide a more comprehensive understanding of DG development. Direct labeling approaches, particularly in utero electroporation, have served as a pioneering method in developmental neurobiology since their introduction in the early 2000s. This technique first revealed crucial insights into neocortical development, including characterization of the inside-out migration pattern and other fundamental developmental processes [ 14 – 21 ]. A particular strength of this technique lies in its ability to enable precise spatiotemporal targeting along the ventricular surface at defined developmental stages while providing unbiased labeling of progenitors regardless of their molecular characteristics. Moreover, the sparse labeling pattern achieved through electroporation allows detailed observation of individual cell morphology and migration trajectories at single-cell resolution, providing an excellent complement to genetic and retroviral approaches. While in utero electroporation has proven instrumental in studying hippocampal pyramidal neuron development [ 7 , 22 – 24 ], its application to DG development remains relatively unexplored despite its early demonstration [ 22 , 25 , 26 ]. Given the complex nature of DG development and the distinct advantages of each methodology, the integration of diverse experimental approaches – genetic, viral, and electroporation-based – will be crucial for building a comprehensive understanding of DG development. In this study, we set out to perform systematic analysis of DG development using in utero electroporation, examining multiple embryonic stages from E11.5 onward. While technical limitations restricted reproducible labeling to E12.5, this approach enabled immediate visualization of an early progenitor population through targeted cellular labeling during a critical period of DG development. By establishing a novel dual-labeling system that combines direct cell labeling with transgenic Gfap -GFP reporter mice, we were able to directly compare, within the same animal, the behavior of electroporated cells with the well-characterized Gfap -expressing progenitor population. This comparative approach revealed a previously unrecognized population that diverges from the Gfap -expressing dentate progenitors and may represent an earlier wave of dentate development not adequately captured by current genetic models. These labeled cells displayed unique characteristics: exclusively populating the outer GCL without reaching the TDM and showing greater propensity for differentiation into GCs compared to Gfap -expressing progenitors that maintain an undifferentiated state. Our findings reveal heterogeneity among dentate progenitors, with early-labeled progenitors potentially establishing the structural scaffold of this critical hippocampal structure. Materials and Methods Animals C57BL/6J mice and Gfap- GFP transgenic mice were used for in utero electroporation experiments. C57BL/6J mice were obtained from Charles River Laboratories Japan (Yokohama, Japan). The Gfap- GFP mice on a C57BL/6NCr background express green fluorescent protein (GFP) under the control of the glial fibrillary acidic protein (Gfap) promoter and have been described previously [27]. All mice were maintained under standard conditions with a 12-hour light/dark cycle in the animal care facilities of Tokyo Medical University. All animal procedures were approved by the Institutional Animal Care and Use Committees of Tokyo Medical University and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996). Efforts were made to minimize the number of animals used and their suffering. For timed mating, the day of vaginal plug detection was defined as embryonic day 0.5 (E0.5). In utero electroporation In utero electroporation of mouse embryos was performed as previously described [28], with modifications. The pCAG -mRFP construct (a gift from Dr. Masanori Uchikawa at Osaka University) was used to visualize neuronal progenitors and neurons by red fluorescent protein (RFP) expression, though signal intensity may decrease through successive cell divisions. Briefly, timed pregnant mice at E11.5 (n=8), E12.5 (n=91), E14.5 (n=140), and E15.5 (n=14) were anesthetized and the uterine horns were exposed by laparotomy. The plasmid solution containing the pCAG -mRFP construct was injected into the lateral ventricle of embryos (7.29 ± 1.95 embryos/dam; range 0-10 embryos) using a glass micropipette. Square electrical pulses (30 V, 50 ms duration, four pulses) were then applied to the embryos using a tweezer-type electrode (CUY650P3, BEX, Japan) connected to an electroporator (CUY21 Vivo-SQ, BEX). The electrode was positioned to target the dentate neuroepithelium. These electrical pulse parameters were optimized to achieve efficient transfection while minimizing tissue damage, based on preliminary experiments. After electroporation, the uterine horns were repositioned and the abdominal cavity was closed. Electroporated embryos were harvested and fixed at various developmental stages including E13.5, E14.5, E15.5, E17.5 and E18.5 for further analysis. Immunofluorescence analysis of cell differentiation markers Cell differentiation states were analyzed by immunofluorescence staining as previously described [28] with minor modifications. Antibodies were selected based on their specificity and previous validation in similar developmental studies of the DG. At embryonic stages E13.5, E14.5, E15.5, E17.5 and E18.5, mouse embryos were transcardially perfused with 4% paraformaldehyde (PFA) in phosphate-buffered saline (pH 7.4). The developing DG primordium was dissected and further fixed in 4% PFA at 4°C overnight. DG tissues were cryosectioned perpendicular to the septotemporal axis of the hippocampus using a cryostat (CM1850, Leica Biosystems, Wetzlar, Germany) to preserve the processes of dentate neural progenitor cells. The sections were then immersed in a blocking solution containing 2% bovine serum albumin in Tris-buffered saline with 0.1% Triton X-100 (TBST, Sigma, St. Louis, MO) for 1 hour at room temperature (RT). Sections were then incubated with primary antibodies overnight at 4 ̊C, washed with TBST and then incubated with the appropriate fluorescence-conjugated secondary antibodies for 1 hour at RT. The primary and secondary antibodies used are listed in Table S1. Confocal imaging was performed using a confocal microscope (LSM700, Carl Zeiss) and the acquired images were processed using the Zeiss LSM Image Browser and ZEN software to adjust brightness and contrast and to generate composite images. Results Early migration and differentiation of E12.5 dentate progenitors To study the development of dentate neural progenitors (DNPs), we performed in utero electroporation targeting the DN region using the pCAG -mRFP construct, which drives ubiquitous expression regardless of cell fate in targeted cells (Fig. 1B). Following previously established protocol [22], we systematically examined developmental stages from E11.5 onward, the earliest stage at which the DN hippocampus region can be targeted by this method. Electroporation at E11.5 resulted in poor embryonic survival; in contrast, E12.5 consistently yielded viable embryos with reproducible DNP labeling. While electroporation from E14.5 successfully labeled cells in different hippocampal regions, it showed dramatically reduced efficiency in specifically targeting DNPs, with only occasional sporadic success (Supplementary Fig. 1). This stage-dependent efficiency suggests a critical developmental window for DNP labeling by this method. Therefore, we focused our detailed analysis on DNPs labeled at E12.5, where we achieved reproducible results that enabled us to track their migratory behavior and fate. For molecular marker analysis, we used Sox2 to identify undifferentiated neural stem/progenitor cells and Tbr2 to detect committed neural precursors to specify the differentiation status of the labeled cells. We focused our analysis on RFP-positive cells maintaining detectable expression levels throughout development, noting that some labeled cells became undetectable through proliferation or showed different behaviors. The in utero electroporation at E12.5 combined with these molecular markers allowed us to identify a distinct subpopulation with strong differentiation tendency. One day post-labeling (E12.5->E13.5), RFP+ cells were distributed in the ventricular zone (VZ) and displayed undifferentiated characteristics, as evidenced by Sox2 expression (Figs. 1C; D1-4, white arrows). These cells had a bipolar morphology. Some RFP+ cells, which had migrated towards the pial surface, did not express Sox2 but instead expressed Tbr2, a marker of intermediate neuronal precursors (Figs. 1D1-4, green arrowheads). These slightly differentiated neural precursors exhibited a multipolar morphology. Two days post-labeling (E12.5->E14.5), the RFP+ cells on the ventricular surface (VS) side remained Sox2+ and undifferentiated (Figs. 1E; F1-4, white arrows). In contrast, RFP+ cells on the pial side expressed Tbr2, indicating their progression towards a neuronal progenitor state (Figs. 1E; G1-4, green arrowheads). This pattern persisted three days post-labeling (E12.5->E15.5). RFP+ cells on the VS side retained a bipolar morphology and continued to express Sox2 (Figs. 1H; I1-4, white arrows), maintaining their undifferentiated state. At the same time, RFP+ cells that had migrated towards the pial surface continued to express Tbr2, further confirming their commitment to a neuronal fate (Figs. 1H; J1-4, green arrowheads). These results demonstrate that some E12.5-labeled neural progenitor cells differentiated into neuronal progenitors as they migrated towards the pial surface, while others remained in an undifferentiated state on the VS side. Maturation of E12.5-labeled progenitors into granule cells To determine whether E12.5-labeled DNPs can progress to terminal differentiation and acquire granule cell identity, we examined the expression of mature granule cell markers at later developmental stages. We performed immunostaining at E15.5 (E12.5->E15.5) for Prox1, a homeodomain transcription factor essential for GC identity and a marker of mature GCs. As shown in Fig. 2A1, RFP+ cells were observed extending from the VS to the emerging DG primordium. The RFP+ cells in the marginal zone were Tbr2-positive (Figs. 2A2-5, green arrowheads) and also expressed Prox1, indicating a more advanced differentiation state. In contrast, RFP+ cells in the hilus were Prox1-positive but Tbr2-negative (Figs. 2A2-5, green arrows), indicating their further differentiation into mature GCs. Five days post-labeling (E12.5->E17.5), RFP+ cells along the hippocampal fissure (HF) and pial surface remained Tbr2-positive (Figs. 2B1; B2-5, white arrowheads). RFP+ cells along the medial side of the HF expressed Prox1 (Figs. 2B1; B2-5, green arrows), suggesting their progression towards mature GC identity. These Prox1+/RFP+ cells were positioned more medially than the Tbr2+/RFP+ cells. This spatial organization shows that E12.5-labeled cells differentiate in a distinctive pattern, with cells migrating from the outer regions toward more medial positions as they mature. Six days post-labeling (E12.5->E18.5), RFP+ cells in the hilus, rather than just below the HF, were Prox1-positive but Tbr2-negative (Figs. 2C1; C2-5 green arrows). This Prox1+/Tbr2- GC phenotype persisted at both 5 and 6 days post-labeling. Notably, these Prox1+/RFP+ GCs also expressed the mature neuronal marker NeuN (Figs. 2D1; D2-5, white arrows). The co-expression of Prox1 and NeuN confirms the acquisition of mature granule cell identity, representing the terminal stage in the differentiation process of E12.5-labeled progenitors. These results demonstrate that DNPs labeled at E12.5 are capable of differentiating into fully mature GCs within a relatively short developmental window, highlighting the rapid differentiation potential of this early progenitor population. Comparative analysis of E12.5-labeled and Gfap-GFP+ progenitor populations DNPs have been characterized using various approaches, including classical methods and transgenic reporter lines [8-11, 12, 13]. These different studies have consistently revealed similar migratory behaviors and differentiation trajectories of DG progenitors. To evaluate how our electroporation-based labeling method at E12.5 relates to these established approaches and to potentially identify progenitor populations that may have been unrecognized by existing genetic labeling methods, we compared our labeled cells with Gfap -GFP+ progenitors that emerge at E14.5, representing one of the well-characterized genetic marking systems. We introduced the pCAG -mRFP construct into the DG primordium of Gfap -GFP mice at E12.5 and analyzed the labeled cells at different time points. Two days post-labeling (E12.5->E14️.5), RFP+ cells displayed an undifferentiated Sox2-positive phenotype within the VZ (Figs. 3A1-5, B1-5) and Tbr2-positive neuronal precursor phenotype on the pial side (Figs. 3A1-5; C1-5, white arrowheads), consistent with our earlier observations in wild-type mice. At E14.5, Gfap -GFP+ cells observed were distributed throughout the VZ up to the pial membrane (Figs. 3A2; 3B1-5, orange arrows; C1-5, orange arrowheads). This suggests that while E12.5-labeled progenitors were already migrating and differentiating by E14.5, Gfap -GFP+ cells emerging at this stage still populated the entire DG primordium. Notably, we observed minimal overlap between the two populations, as Gfap -GFP+ cells were barely labeled by the RFP reporter after in utero electroporation at E12.5 (Figs. 3A1-2, A5, B1-2 orange arrows; C1-2 orange arrowheads). The Gfap -GFP+ cells located on the VS side at E14️.5 were Sox2-positive but Tbr2-negative, indicating that they remained undifferentiated (Figs. 3B1-5 orange arrowheads). Consistent with the observation in Fig. 1, the E12.5-labeled RFP+ cells that had migrated to the pial membrane side displayed a Tbr2-positive, Sox2-negative neuronal progenitor phenotype (Figs. 3C1-5, white arrowheads). Interestingly, a similar distribution and marker profile was also observed for Gfap -GFP+ cells at this E14.5 time point, with Gfap -GFP+ cells located on the pial membrane side also exhibiting Tbr2 positivity and Sox2 negativity (Figs. 3C1-5, orange arrowheads). These findings suggest that both populations initially follow similar migratory routes, though their differentiation kinetics later diverge. Three days post-labeling (E12.5->E15.5), both RFP+ and Gfap -GFP+ cells were distributed within the developing DG region (Figs. 3D1-5) [11]. Only a few undifferentiated RFP+ cells, characterized by Sox2 positivity and Tbr2 negativity, were found within the hilus (Figs. 3E1-5, white arrow). In contrast, undifferentiated Gfap -GFP+ cells with a Sox2+/Tbr2- profile were considerably more abundant in the hilus (Figs. 3E1-5, orange arrows). At the same developmental stage, Tbr2-positive, Sox2-negative RFP+ cells were arranged in a V-shaped pattern along the pial membrane and prospective HF (Figs. 3E1-5, white arrowheads), indicating a more differentiated neuronal progenitor state. Notably, the Gfap -GFP+ cells showed a similar distribution, with Sox2-/Tbr2+ population lining up along the pial membrane and HF (Figs. 3E1-5, orange arrowheads). To confirm whether these progenitor populations differentiated into mature GCs, immunostaining was performed with the Prox1 antibody (Fig. 3F). The differentiation profiles of the two populations showed clear differences at this stage. RFP+ cells on the pial side exhibited weak Prox1 expression while retaining Tbr2 positivity (Figs. 3F1; F2-5, white arrows), suggesting an intermediate state of differentiation. In contrast, RFP+ cells along the HF strongly expressed Prox1 (Figs. 3F2-5, white arrowheads), indicating progression towards a mature GC identity. Gfap -GFP+ cells near the FDJ expressed Tbr2 but were negative for Prox1 (Figs. 3F1; F2-5, orange arrows), indicating an intermediate progenitor state. However, only a minute subset of Gfap -GFP+ cells on the pial side showed Tbr2 expression along with weak Prox1 immunoreactivity (Figs. 3F2-5, orange arrowheads), suggesting that they had progressed further towards a mature GC identity. These comparative analyses demonstrate distinct developmental trajectories for the two progenitor populations. While both E12.5-labeled and Gfap -GFP+ cells can generate GCs, they differ significantly in their differentiation kinetics. Specifically, E12.5-labeled cells exhibited more advanced GC differentiation, as evidenced by extremely robust Prox1 expression along the HF. In contrast, Gfap -GFP+ cells appeared to be slightly delayed in their differentiation trajectory, with a significant proportion retaining an intermediate progenitor phenotype near the FDJ. Late-stage developmental divergence of E12.5-labeled and Gfap-GFP+ populations To determine whether the differentiation differences observed at E15.5 persist and potentially become more pronounced at later developmental stages, we analyzed both cell populations at E17.5 (5 days post-labeling) and E18.5 (6 days post-labeling). At E17.5, both populations showed similar positioning of Tbr2-positive neuronal progenitors just below the HF and along the pial membrane (Figs. 4A; B1-5, white arrowheads indicate the progenitors derived from E12.5-labeled cells and orange arrowheads from Gfap -GFP+ cells, respectively). Undifferentiated Sox2-positive cells were found within the hilus in both populations, with a notable prevalence among Gfap -GFP+ cells (Figs. 4A; B1-5, white and orange arrows). Furthermore, Prox1 immunostaining (Figs. 4C-J) revealed distinct spatial patterns of GC differentiation between the two populations. E12.5-derived Prox1-positive RFP+ cells were observed within the hilus (Figs. 4C; D1-5, white arrows), while Prox1+/ Gfap -GFP+ cells were predominantly located on the pial membrane side (Figs. 4D1-5, orange arrows). By E18.5, the spatial segregation between the two populations became more pronounced. E12.5-derived mature GCs were preferentially localized within the molecular layer, particularly on the HF side (Figs. 4F1-5, white arrows). On the pial membrane side, these RFP+ GCs were not only found in the densely packed GCL, but also extended into the hilus region (Figs. 4E, G1-5, H, I1-5, white arrows). A subset of undifferentiated Sox2-positive cells derived from E12.5 labeling were still present within the hilus (Figs. 4G1-5, white arrowheads). In contrast to this distribution pattern, GCs derived from Gfap -GFP+ population were mainly distributed along the hilar side or within the hilus of the densely packed GCL (dashed line) (Figs. 4F1-5, G1-5, I1-5, J1-5, orange arrows). Notably, undifferentiated cells persisted not only in the hilus but also within the densely packed GC region (Figs. 4F1-5, G1-5, orange arrowheads). These observations highlight key differences in the migratory patterns and differentiation states of the two progenitor populations. E12.5-labeled cells showed more advanced differentiation and integration into the molecular layer, while a substantial subset of Gfap -GFP+ cells remained undifferentiated within the GCL itself. Both E12.5-labeled RFP+ cell-derived and Gfap -GFP-derived Tbr2+ neuronal progenitors were present within the molecular layer at this stage (Figs. 4I1-5, white and orange arrowheads, respectively). To quantitatively assess the differentiation status of both populations, we analyzed the percentage of RFP+ and Gfap -GFP+ cells expressing Sox2 (undifferentiated) and Prox1 (GC) markers within the DG primordium. This analysis revealed striking differences: 75.5% ± 3.5% (n=3) of E12.5-derived RFP+ cells had differentiated into Prox1+ GCs, whereas only 24.5% ± 4.0% of Gfap -GFP+ cells showed GC identity (Fig. 4K). Conversely, 64.8% ± 9.3% of Gfap -GFP+ cells maintained an undifferentiated Sox2+ state (Fig. 4K). These quantitative data provide further evidence that E12.5-labeled progenitors and Gfap -GFP+ cells exhibit divergent behaviors during later stages of DG development. While the majority of E12.5-derived cells had progressed to a mature GC fate, a substantial proportion of the Gfap -GFP+ population maintained an undifferentiated progenitor state, retaining their stem-like properties even as the GCL was being established. The distinct behaviors of E12.5-labeled cells and Gfap -GFP+ cells - with the former predominantly generating mature GCs and the latter maintaining an undifferentiated state - establish the existence of two separate progenitor populations with different developmental programs in the developing DG. Discussion Stage-specific labeling by in utero electroporation enables direct visualization of early dentate progenitors While transgenic reporter mice have been instrumental in revealing the molecular identity and lineage relationships of dentate progenitors [10-13, 30], direct visualization and tracking of early progenitors from their point of origin has remained challenging. In utero electroporation, which enables precise spatiotemporal targeting of ventricular progenitors and visualization of individual cell migration at single-cell resolution, has been successfully applied to the studies of hippocampal pyramidal neurons [7, 22-24]. However, its systematic application to early DG development has not been reported. In this study, we set out to perform comprehensive stage-dependent analysis using in utero electroporation from E11.5 onward. While targeting efficiency varied across stages, E12.5 emerged as a critical time point allowing reproducible labeling of early dentate progenitors. Targeting efficiency declined substantially from E14.5 onward, proving particularly challenging despite previous reports [22]. Similarly, electroporation at E11.5 resulted in poor embryonic survival, preventing reliable analysis. This stage-dependent technical limitation may explain the paucity of electroporation studies targeting the developing DG in the current literature, and likely reflects the increasing anatomical complexity around the developing DN region. The unique morphological transitions of the primordial DN present significant challenges to current electroporation approaches. Our findings highlight the need to develop new, specialized electroporation strategies that are specifically optimized for the distinct anatomical features of the developing DN. Despite these technical challenges, successful labeling at E12.5 proved particularly informative, as the sparse labeling and single-cell resolution provided by electroporation allowed us to follow the dynamic behavior of early dentate progenitors in detail. As visualized in Fig. 5, this approach revealed distinct patterns of progenitor migration and differentiation that enhance our understanding of early DG development. Developmental trajectory of E12.5-labeled dentate progenitors Our in utero electroporation studies provided detailed visualization of early dentate progenitor cells at E12.5, enabling us to specifically label and track progenitors at their point of DN origin along the ventricular surface. As demonstrated in Fig. 5A, the migratory behavior of these E12.5-labeled cells followed a characteristic inward migration pattern (red arrows): First, these progenitors migrated from the ventricular surface towards the FDJ at the pial membrane. They then distributed just below the pial membrane and the hippocampal fissure (HF), forming a secondary proliferative matrix at the prospective margins of the DG. Finally, these early-born E12.5-labeled cells differentiated into mature GCs, establishing the initial framework of the GCL. Critically, as shown in Fig. 5A, unlike Gfap -GFP+ cells that eventually populate the TDM in the hilus, E12.5-labeled cells were never observed in the TDM, providing compelling evidence for the fundamentally distinct developmental properties of this early progenitor population. This process followed a distinctive outside-in pattern, with cells progressively populating the GCL from the molecular layer side inwards towards the hilus (Fig. 5A, red region). Interestingly, this inward migration pattern during early development parallels the outside-in layering of the DG that has been previously reported in the adult brain, where comparisons of granule cell distribution after embryonic versus postnatal stages demonstrated that earlier-born cells predominantly occupy outer positions in the granule cell layer, while later-born cells populate inner regions adjacent to the hilus [30]. Our direct visualization of E12.5-labeled progenitors provides cellular-level evidence for the developmental origins of this layering principle. While previous studies using Nestin and Gfap reporter mice have suggested that subpial progenitors contribute to GC generation during migration to the hilus [10,11], our work provides the first direct visualization of this process at single-cell resolution, revealing a continuous series of cell movements originating from the ventricular surface at E12.5. The combination of precise spatiotemporal targeting and sparse labeling enabled by electroporation has allowed us to track individual cell behavior during this critical developmental period, providing new insights into the cellular dynamics of early dentate development. Temporal heterogeneity and functional divergence of progenitor pools in dentate gyrus development Among the cells labeled at E12.5, we identified a distinct subpopulation that maintained detectable RFP expression and showed unique properties. While other labeled cells exhibited different behaviors or became undetectable due to signal dilution through proliferation, this particular subpopulation demonstrated temporal heterogeneity from the later emerging Gfap -expressing progenitors, as illustrated in Fig. 5B. The striking contrast between these populations is one of our most significant findings – the E12.5-labeled cells that we characterized in detail showed a robust propensity for neuronal differentiation, exhibiting rapid migration and giving rise to the initial GCL of the DG (red dots in the Fig. 5B right panel). In contrast, a substantial proportion of the later-born Gfap -GFP+ cells maintained an undifferentiated state (dark green dots in the Fig. 5B middle panel), suggesting their potential role in expanding the progenitor pool and contributing to the subsequent increase in GCL thickness through delayed differentiation. While our electroporation approach enabled tracking of a specific subpopulation showing accelerated differentiation, the full heterogeneity of E12.5 progenitors may not be captured due to signal dilution through proliferation and the presence of other labeled cells showing different behaviors. Nevertheless, the identification of this differentiation-prone population that lacks Gfap expression represents a significant finding in understanding dentate gyrus development. The semi-quantitative analysis depicted in Fig. 5B clearly shows that E12.5-labeled RFP+ cells are predominantly found as differentiated Prox1+ granule cells (red dots in right panel), while disproportionately fewer maintain a Sox2+ progenitor state (magenta dots in middle panel). This stark cellular distribution pattern highlights a functional divergence between early and late progenitors, suggesting a carefully orchestrated developmental programme that balances immediate neurogenesis with the maintenance of a progenitor reservoir for sustained growth. These divergent behaviors align with the two proposed migration routes previously described for dentate progenitors. In the first route, the PDM in the ventricular zone near the DN continues along the pial membrane to form the SDM just below the HF, which then differentiates into GCs. Subsequently, a subset of progenitors reaches the future hilus to form the TDM [5,6,11,12]. This ‘outside-in’ pattern, where earlier migrating progenitors seed the molecular (outer) side of the GCL and later arrivals populate the inner hilus region, is directly confirmed by our observations in Fig. 5A, which clearly demonstrates that the E12.5 progenitors exclusively contribute to the outer GCL (red) without reaching the TDM, whereas the later Gfap -GFP+ population (green) establishes the TDM and has a more pronounced inside component [10,31-33]. Additionally, as indicated in Fig. 5A, some Gfap -GFP+ cells are known to take a more direct route toward the hilus, contributing to TDM formation, a second migration pathway that is not observed in E12.5-labeled cells. Interestingly, our results are also consistent with previous reports indicating that the suprapyramidal blade (SPB) of the DG forms before the infrapyramidal blade (IPB), with the SPB outer shell being established between E13.5 and P0.5, whereas the IPB outer shell emerges after E16.5 [8-10,34]. Our observations suggest that E12.5 progenitors in the DN contribute to the initial formation of the SPB outer shell, as evidenced by their early migration pattern (Fig. 5B). Understanding the diversity and properties of dentate progenitors has relied heavily on genetic lineage tracing approaches, with Nestin-Cre systems revealing progenitor emergence at E14.5 [10, 12] and Hopx-CreERT2 demonstrating labeling as early as E10.5 [13]. While these approaches have been invaluable for identifying specific NSC-derived lineages, they are inherently limited in their ability to capture the precise timing of emergence and initial migratory behavior of dentate progenitors. Our application of in utero electroporation at E12.5 has revealed a previously uncharacterized population of early dentate progenitors. These cells exhibit distinct properties from known populations, notably showing accelerated differentiation kinetics without maintaining neural stem cell characteristics, as clearly evidenced by the predominance of RFP+Prox1+ cells over RFP+Sox2+ cells in Fig. 5b. While current technical limitations restricted our analysis to E12.5, these findings demonstrate the value of combining direct labeling with genetic approaches to uncover the full complexity of dentate development. Future technical refinements will likely enable broader developmental analysis, potentially revealing additional progenitor diversity during DG formation. Declarations Funding This work was supported by grants from the Japan Society for the Promotion of Science KAKENHI (grant numbers 16K18983 and 20K07233, both to HMS) and the Centre for Diversity at Tokyo Medical University (TMUCD-202202, TMUCD-202301, TMUCD-202409 to HMS). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions HMS designed and performed all experiments and prepared all the Figs. HMS wrote the first draft, and TT extensively revised and refined the manuscript. All authors reviewed the manuscript. Data Availability All data included in this study are available upon request by contact with the corresponding author. Ethics approval All animal procedures were approved by the Institutional Animal Care and Use Committees of Tokyo Medical University and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996). Efforts were made to minimize the number of animals used and their suffering. Acknowledgements We are very grateful to Dr Seiji Shiota (Hoshi University) for generating the Gfap- GFP + transgenic mice expressing GFP under the control of mouse Gfap promoter. We thank Professor Emeritus Tatsunori Seki (Tokyo Medical University) for his generous support. We also thank Keiko Toda, Mio Hayashida, Chikako Miyazaki and Mieko Utsugi (Tokyo Medical University) for technical assistance. This work was supported by grants from the Japan Society for the Promotion of Science KAKENHI (grant numbers 16K18983 and 20K07233, both to HMS) and the Centre for Diversity at Tokyo Medical University (TMUCD-202202, TMUCD-202301, TMUCD-202409 to HMS). References Altman J (1963) Autoradiographic investigation of cell proliferation in the brains of rats and cats. Anat Rec 145: 573-591. 10.1002/ar.1091450409. Seki T, Arai Y (1993) Highly polysialylated neural cell adhesion molecule (NCAM-H) is expressed by newly generated granule cells in the dentate gyrus of the adult rat. J Neurosci 13: 2351-2358. 10.1523/jneurosci.13-06-02351. Alvarez-Buylla A, Lim DA (2004) For the long run: maintaining germinal niches in the adult brain. 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Biol Open 2: 739-749. 10.1242/bio.20135231. Caramello A, Galichet C, Rizzoti K, Lovell-Badge R (2021) Dentate gyrus development requires a cortical hem-derived astrocytic scaffold. Elife 10: 10.7554/eLife.63904. Mathews EA, Morgenstern NA, Piatti VC, Zhao C, Jessberger S, Schinder AF, Gage FH (2010) A distinctive layering pattern of mouse dentate granule cells is generated by developmental and Adult Neurogenesis. J Comp Neurol 518: 4479–4490. 10.1002/cne.22489. Rakic P, Nowakowski RS (1981) The time of origin of neurons in the hippocampal region of the rhesus monkey. J Comp Neurol 196: 99-128. 10.1002/cne.901960109. Altman J, Bayer SA (1990) Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. J Comp Neurol 301: 365-381.10.1002/cne.903010304. Noguchi H, Castillo JG, Nakashima K, Pleasure SJ (2019) Suppressor of fused controls perinatal expansion and quiescence of future dentate adult neural stem cells. Elife 8: 10.7554/eLife.42918. Nelson BR, Hodge RD, Daza RA, Tripathi PP, Arnold SJ, Millen KJ, Hevner RF (2020) Intermediate progenitors support migration of neural stem cells into dentate gyrus outer neurogenic niches. Elife 9: 10.7554/eLife.53777. Additional Declarations No competing interests reported. Supplementary Files Fig.S1ShinoharaTakahashi250220.tif Supplementary Fig. 1 Limited labeling efficiency of dentate progenitors by in utero electroporation after E14.5 (A) Immunofluorescence staining of hippocampal sections at E17.5 following in utero electroporation at E14.5, showing minimal RFP-positive cells (red) in the developing dentate gyrus (DG). Sections were immunostained for Sox2 (green) and Prox1 (blue) to identify the dentate neural progenitors (DNPs) and granule cells (GCs), respectively. (A2, 3) Higher magnification images of the boxed regions in A1, demonstrating the presence of labeled pyramidal cells in the ventricular surface (A2) and very slightly labeled cells in developing DG (A3). (B, C) Representative showing the scarcity of RFP-positive cells (red) in the developing DG at E17.5 following in utero electroporation at E15.5. Sections were counterstained with DAPI (blue). (B2, B3) Higher magnification images of the boxed regions in B1, demonstrating the presence of labeled pyramidal cells in the ventricular surface (B2) and the absence of labeled cells in developing DG (B3). (C2, C3) Higher magnification images of the boxed regions in C1 showing the ventricular surface (C2) and developing DG (C3) further illustrating the inefficient labeling of dentate progenitors at this developmental stage. Scale bars: C, E, H, 100 μm. TableS1.2503172.docx Cite Share Download PDF Status: Published Journal Publication published 06 Nov, 2025 Read the published version in Cellular and Molecular Neurobiology → Version 1 posted Editorial decision: Revision requested 27 May, 2025 Reviews received at journal 26 May, 2025 Reviews received at journal 26 May, 2025 Reviews received at journal 22 May, 2025 Reviews received at journal 20 May, 2025 Reviewers agreed at journal 15 May, 2025 Reviewers agreed at journal 14 May, 2025 Reviewers agreed at journal 12 May, 2025 Reviewers agreed at journal 12 May, 2025 Reviewers invited by journal 12 May, 2025 Editor assigned by journal 27 Apr, 2025 Submission checks completed at journal 25 Apr, 2025 First submitted to journal 22 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6508491","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":462565423,"identity":"ebaf21a9-f07e-4345-abed-afc7ba8d8d32","order_by":0,"name":"Hiroshi M. Shinohara","email":"","orcid":"","institution":"Tokyo Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"M.","lastName":"Shinohara","suffix":""},{"id":462565424,"identity":"50653513-8328-4ee6-a0ef-9553811d2bd0","order_by":1,"name":"Tokiharu Takahashi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYFACNobDIIqfgYEZKpIAEcYFeGBaJNtI0QJWanAMVQtuYM/elni4oOZe4ub7zYeNeSoYovnZExg//GDgy8NpC8+xA4dnHCtO3HaMLTmZ5wxD7syeB8ySPQxsxTi1SKQ3HOZhSwBq4TE+zNv2P3fDjQQGaaBfEhvwavmXkLi5DayFIXf/jQTm3/i1pB0AqkxI3MDGY5wM0rJBIoENvy1njiUc5u1LMJ5xLC3ZcA7QLzPOPGyz7DHA7Rf29jbjzzzfEmT7mw8flnhTwZDb3558+MaPimM4QwwFMPGAKcYGUDQlEKWF8QeCXUOcllEwCkbBKBgJAAB17lVVAeVcCQAAAABJRU5ErkJggg==","orcid":"","institution":"Tokyo Medical University","correspondingAuthor":true,"prefix":"","firstName":"Tokiharu","middleName":"","lastName":"Takahashi","suffix":""}],"badges":[],"createdAt":"2025-04-23 03:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6508491/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6508491/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10571-025-01616-3","type":"published","date":"2025-11-06T15:57:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89449421,"identity":"7c5874a6-81b0-4991-a8ab-b9a6d0515a91","added_by":"auto","created_at":"2025-08-20 05:59:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":560665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eE12.5-labeled neural progenitors migrate towards the pial surface during differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic representation of the developing hippocampus, which consists of the pyramidal cell layer (PCA) of the CA regions and granule cell layer (GCL) of the dentate gyrus (DG). The DG has three distinct proliferative matrices: the primary dentate proliferative matrix (PDM) in the ventricular zone near the dentate notch (DN), the secondary dentate proliferative matrix (SDM) in the limbus, and the tertiary dentate proliferative matrix (TDM) in the hilus. The GCL is divided into the outer region (outer shell) and the inner region (inner core), and further into suprapyramidal blade (SPB) and infrapyramidal blade (IPB). Progenitor cells originating from the PDM lining the ventricular surface migrate across the fimbria towards the fimbriodentate junction (FDJ), giving rise to the SDM along the hippocampal fissure HF and pia. Additional TDM are then generated within the hilus, and the GCL is finally established from these proliferative matrices. The solid red line shows the GCL and the dotted red line shows the boundary between the outer shell and the inner core. (\u003cstrong\u003eB\u003c/strong\u003e) Schematic of the \u003cem\u003epCAG\u003c/em\u003e-mRFP plasmid used to label cells transfected at E12.5 in the mouse brain. (\u003cstrong\u003eC\u003c/strong\u003e-\u003cstrong\u003eJ\u003c/strong\u003e) Immunofluorescence staining for RFP (magenta), the neural stem marker Sox2 (blue) and neuronal progenitor marker Tbr2 (yellow) in perpendicular sections to the septo-temporal axis of the DG at 1 day (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e), 2 days (\u003cstrong\u003eE\u003c/strong\u003e-\u003cstrong\u003eG\u003c/strong\u003e) and 3days (\u003cstrong\u003eH\u003c/strong\u003e-\u003cstrong\u003eJ\u003c/strong\u003e) after \u003cem\u003ein utero\u003c/em\u003eelectroporation at E12.5. (\u003cstrong\u003eD1\u003c/strong\u003e-\u003cstrong\u003e4\u003c/strong\u003e) Boxed area in \u003cstrong\u003eC\u003c/strong\u003e at higher magnification. (\u003cstrong\u003eF1\u003c/strong\u003e-\u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003eG1\u003c/strong\u003e-\u003cstrong\u003e4\u003c/strong\u003e) Boxed regions near the ventricular surface (\u003cstrong\u003eF1\u003c/strong\u003e-\u003cstrong\u003e4\u003c/strong\u003e) and pial surface (\u003cstrong\u003eG1\u003c/strong\u003e-\u003cstrong\u003e4\u003c/strong\u003e) in \u003cstrong\u003ee\u003c/strong\u003e. (\u003cstrong\u003eI1\u003c/strong\u003e-\u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003eJ1\u003c/strong\u003e-\u003cstrong\u003e4\u003c/strong\u003e) Boxed regions near the ventricular surface (\u003cstrong\u003eI1\u003c/strong\u003e-\u003cstrong\u003e4\u003c/strong\u003e) and pial surface (\u003cstrong\u003eJ1\u003c/strong\u003e-\u003cstrong\u003e4\u003c/strong\u003e) in \u003cstrong\u003eH\u003c/strong\u003e. Arrows indicate RFP+Sox2+ neural stem cells; arrowheads indicate RFP+Tbr2+ neuronal progenitors. VS, ventricular surface; Pia, pial surface. Scale bars: \u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e, 50 μm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6508491/v1/d42c1b8f68d0af38a93b0cf5.png"},{"id":89449420,"identity":"75e7247a-3b2a-48d5-aa9a-b22d4710fca0","added_by":"auto","created_at":"2025-08-20 05:59:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":832258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eE12.5-labeled neural progenitor cells migrate to the DG primordium and differentiate into granule cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eC\u003c/strong\u003e) Distribution of RFP+ cells electroporated at E12.5 and then collected at E15.5 (\u003cstrong\u003eA\u003c/strong\u003e), E17.5 (\u003cstrong\u003eB\u003c/strong\u003e) and E18.5 (\u003cstrong\u003eC\u003c/strong\u003e). (\u003cstrong\u003eA2\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e, \u003cstrong\u003eB2\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e, \u003cstrong\u003eC2\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) Higher magnification views of \u003cstrong\u003eA1\u003c/strong\u003e, \u003cstrong\u003eB1\u003c/strong\u003e and \u003cstrong\u003eC1\u003c/strong\u003e, respectively. RFP (magenta, \u003cstrong\u003eA2\u003c/strong\u003e, \u003cstrong\u003eB2\u003c/strong\u003e, \u003cstrong\u003eC2\u003c/strong\u003e), Tbr2 (yellow, \u003cstrong\u003eA3\u003c/strong\u003e, \u003cstrong\u003eB3\u003c/strong\u003e, \u003cstrong\u003eC3\u003c/strong\u003e), Prox1 (blue, \u003cstrong\u003eA4\u003c/strong\u003e, \u003cstrong\u003eB4\u003c/strong\u003e, \u003cstrong\u003eC4\u003c/strong\u003e), immunostaining in the dentate gyrus (DG); merge shown in A5, B5, C5. Green arrows indicate RFP+Prox1+ granule cells. Green arrowheads (\u003cstrong\u003eA2\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) and white arrowheads (\u003cstrong\u003eB2\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) indicate RFP+Tbr2+ neuronal progenitor cells at E15.5 and E17.5, respectively. (\u003cstrong\u003eD\u003c/strong\u003e) Immunofluorescent staining for RFP (magenta), Prox1 (blue) and the mature neuron marker NeuN (yellow) at E18.5. (\u003cstrong\u003eD2\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) Higher magnification views of \u003cstrong\u003eD1\u003c/strong\u003e. (\u003cstrong\u003eD2)\u003c/strong\u003e RFP signal only. (\u003cstrong\u003eD3\u003c/strong\u003e) Prox1 signal only. (\u003cstrong\u003eD4\u003c/strong\u003e) NeuN signal (yellow) only. (\u003cstrong\u003eD5\u003c/strong\u003e) Merged RFP, Prox1 and NeuN channels. White arrows indicate RFP+Prox1+NeuN+ mature granule cells. VS, ventricular surface; HF, hippocampal fissure. Scale bars: \u003cstrong\u003eA\u003c/strong\u003e, 50 μm; \u003cstrong\u003eB\u003c/strong\u003e-\u003cstrong\u003eD\u003c/strong\u003e, 100 μm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6508491/v1/8e18d2af2b926140e29c44a9.png"},{"id":89449766,"identity":"708e4eb4-dfbe-40f4-96d4-898cd93c172e","added_by":"auto","created_at":"2025-08-20 06:07:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":725950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGfap\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-GFP+ cells migrate to the pial surface and show a different distribution than E12.5-labeled cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eE\u003c/strong\u003e) Immunofluorescence for RFP (magenta), GFP (green), Sox2 (blue) and Tbr2 (yellow) in perpendicular sections to the septotemporal axis of the dentate gyrus (DG) at one day (\u003cstrong\u003eA\u003c/strong\u003e), two days (\u003cstrong\u003eA, B\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e) and three days (\u003cstrong\u003eD\u003c/strong\u003e and \u003cstrong\u003eE\u003c/strong\u003e) after E12.5 \u003cem\u003ein utero\u003c/em\u003eelectroporation. (\u003cstrong\u003eA1\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) Overview at low magnification. (\u003cstrong\u003eA1\u003c/strong\u003e) RFP only. (\u003cstrong\u003eA2\u003c/strong\u003e) GFP only. (\u003cstrong\u003eA3\u003c/strong\u003e) Sox2 only. (\u003cstrong\u003eA4\u003c/strong\u003e) Tbr2 only. (\u003cstrong\u003eA5\u003c/strong\u003e) RFP, GFP, Sox2 and Tbr2 channels combined. (\u003cstrong\u003eB1\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e and \u003cstrong\u003eC1\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) boxed regions on ventricular side (\u003cstrong\u003eB1\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) and pial side (\u003cstrong\u003eC1\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) in A1-A5, respectively. (\u003cstrong\u003eB1\u003c/strong\u003e, \u003cstrong\u003eC1\u003c/strong\u003e) RFP only. (\u003cstrong\u003eB2\u003c/strong\u003e, \u003cstrong\u003eC2\u003c/strong\u003e) GFP only. (\u003cstrong\u003eB3\u003c/strong\u003e, \u003cstrong\u003eC3\u003c/strong\u003e) Sox2 only. (\u003cstrong\u003eB4\u003c/strong\u003e, \u003cstrong\u003eC4\u003c/strong\u003e) Tbr2 only. (\u003cstrong\u003eB5\u003c/strong\u003e, \u003cstrong\u003eC5\u003c/strong\u003e) RFP, GFP, Sox2 and Tbr2 channels combined. (\u003cstrong\u003eD1\u003c/strong\u003e-\u003cstrong\u003eD5\u003c/strong\u003e) Overview at low magnification. (\u003cstrong\u003eE1\u003c/strong\u003e-\u003cstrong\u003eE5\u003c/strong\u003e) Higher magnification of the boxed region in D1-D5. (\u003cstrong\u003eD1\u003c/strong\u003e, \u003cstrong\u003eE1\u003c/strong\u003e) RFP only. (\u003cstrong\u003eD2\u003c/strong\u003e, \u003cstrong\u003eE2\u003c/strong\u003e) GFP only. (\u003cstrong\u003eD3\u003c/strong\u003e, \u003cstrong\u003eE3\u003c/strong\u003e) Sox2 only. (\u003cstrong\u003eD4\u003c/strong\u003e, \u003cstrong\u003eE4\u003c/strong\u003e) Tbr2 only. (\u003cstrong\u003eD5\u003c/strong\u003e, \u003cstrong\u003eE5\u003c/strong\u003e) RFP, GFP, Sox2 and Tbr2 channels combined. Orange arrows indicate GFP+Sox2+ cells, white arrowheads indicate RFP+Tbr2+ cells, orange arrowheads indicate GFP+Tbr2+ cells and white arrows indicate RFP+Sox2+ cells. (\u003cstrong\u003eF\u003c/strong\u003e) Immunostaining for RFP (magenta), GFP (green), Tbr2 (yellow), Prox1 (blue) in perpendicular sections to the septotemporal axis of the DG at E15.5 after E12.5 \u003cem\u003ein utero\u003c/em\u003eelectroporation. (\u003cstrong\u003eF1\u003c/strong\u003e) Overview at low magnification (\u003cstrong\u003eF2\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) Higher magnification of the boxed region in \u003cstrong\u003eF1\u003c/strong\u003e. (\u003cstrong\u003eF2\u003c/strong\u003e) RFP and Prox1. (\u003cstrong\u003eF3\u003c/strong\u003e) GFP only. (\u003cstrong\u003eF4\u003c/strong\u003e) Tbr2 only. (\u003cstrong\u003eF5\u003c/strong\u003e) Prox1 only. White arrows indicate RFP+Tbr2+ cells and white arrowheads indicate RFP+Prox1+ cells, orange arrows indicate GFP+Tbr2+ cells and orange arrowheads indicate GFP+Prox1+ cells. VS, ventricular surface; Pia, pial side. Scale bars: 50 μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6508491/v1/41e770f30a9ffdf73a2c3ede.png"},{"id":89449425,"identity":"2eb3e212-e3b5-428c-9983-5a607eb6d632","added_by":"auto","created_at":"2025-08-20 05:59:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":553982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn the DG primordium, E12.5-labeled cells show different distribution and characteristics than \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGfap\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-GFP+ cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eB\u003c/strong\u003e) Immunofluorescence for RFP, (magenta) GFP (green), Sox2 (blue) and Tbr2 (yellow) in perpendicular sections to the septotemporal axis of the dentate gyrus (DG) of \u003cem\u003eGfap\u003c/em\u003e-GFP mouse at E17.5 (\u003cstrong\u003eA\u003c/strong\u003e) after E12.5 \u003cem\u003ein utero\u003c/em\u003e electroporation. (\u003cstrong\u003eB1\u003c/strong\u003e-\u003cstrong\u003eB5\u003c/strong\u003e) The boxed region of \u003cstrong\u003eA\u003c/strong\u003e. (\u003cstrong\u003eB1\u003c/strong\u003e) RFP only. (\u003cstrong\u003eB2\u003c/strong\u003e) GFP only. (\u003cstrong\u003eB3\u003c/strong\u003e) Sox2 only. (\u003cstrong\u003eB4\u003c/strong\u003e) Tbr2 only. (\u003cstrong\u003eB5\u003c/strong\u003e) RFP, GFP, Sox2 and Tbr2 channels combined. White arrows and arrowheads indicate RFP+Sox2+ and RFP+Tbr2+ cells, respectively. Orange arrows and arrowheads indicate GFP+ Sox2+ and GFP+Tbr2+ cells, respectively. (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e-\u003cstrong\u003eJ\u003c/strong\u003e) Immunostaining for RFP, (magenta) GFP (green), Tbr2 (yellow), Prox1 (blue) at E17.5 (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e) at E18.5 (\u003cstrong\u003eH\u003c/strong\u003e-\u003cstrong\u003eJ\u003c/strong\u003e). White arrowheads and arrows indicate RFP+Tbr2+ and RFP+Prox1+ cells, respectively. Orange arrowheads and arrows indicate GFP+ Tbr2+ and GFP+Prox1+ cells, respectively. (\u003cstrong\u003eD1\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) Higher magnification of the boxed region of C. (\u003cstrong\u003eI1\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e, \u003cstrong\u003eJ1\u003c/strong\u003e-\u003cstrong\u003e5\u003c/strong\u003e) Higher magnification of ventricular (\u003cstrong\u003eI1-5\u003c/strong\u003e) and pial (\u003cstrong\u003eJ1-5\u003c/strong\u003e) boxed region in H. (\u003cstrong\u003eD1\u003c/strong\u003e, \u003cstrong\u003eI1\u003c/strong\u003e, \u003cstrong\u003eJ1\u003c/strong\u003e) RFP only. (\u003cstrong\u003eD2\u003c/strong\u003e, \u003cstrong\u003eI2\u003c/strong\u003e, \u003cstrong\u003eJ2\u003c/strong\u003e) GFP only. (\u003cstrong\u003eD3\u003c/strong\u003e, \u003cstrong\u003eI3\u003c/strong\u003e, \u003cstrong\u003eJ3\u003c/strong\u003e) Tbr2 only. (\u003cstrong\u003eD4\u003c/strong\u003e, \u003cstrong\u003eI4\u003c/strong\u003e, \u003cstrong\u003eJ4\u003c/strong\u003e) Prox1 only. (\u003cstrong\u003eD5\u003c/strong\u003e, \u003cstrong\u003eI5\u003c/strong\u003e, \u003cstrong\u003eJ5\u003c/strong\u003e) RFP, GFP, Tbr2 and Prox1 channels combined. (\u003cstrong\u003eE\u003c/strong\u003e-\u003cstrong\u003eG\u003c/strong\u003e) Immunostaining for RFP, (magenta) GFP (green), Sox2 (blue) and Prox1 (yellow) at E18.5. (\u003cstrong\u003eE1\u003c/strong\u003e) Overview at low magnification. (\u003cstrong\u003eF1\u003c/strong\u003e- \u003cstrong\u003eF5\u003c/strong\u003e, \u003cstrong\u003eG1\u003c/strong\u003e-\u003cstrong\u003eG5\u003c/strong\u003e) Ventricular (\u003cstrong\u003eF1\u003c/strong\u003e-\u003cstrong\u003eF5\u003c/strong\u003e) and pial (\u003cstrong\u003eG1\u003c/strong\u003e-\u003cstrong\u003eG5\u003c/strong\u003e) boxed regions in \u003cstrong\u003eE\u003c/strong\u003e. White arrows and arrowheads indicate RFP+Prox1+ and RFP+Sox2+ cells. Orange arrows and arrowheads indicate GFP+Prox1+ and GFP+ Sox2+ cells. VS, ventricular surface, Pia, pial side and HF, hippocampal fissure. Scale bars: 100 μm.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eK\u003c/strong\u003e) Quantification of Sox2+ and Prox1+ cells among RFP+ (E12.5 electroporated) and \u003cem\u003eGfap\u003c/em\u003e-GFP+ (E14.5) cells. The red area represents the Prox1+ mature granule cell population. The magenta area represents the Sox2+ stem cell population. The blue area represents the Prox1+ mature granule cell population. The green area represents the Sox2+ stem cell population. Compared to the E14.5 \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells, the E12.5 electroporated cells showed a higher proportion of differentiated neuronal progenitors.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6508491/v1/9c580fc5cfea26e144b7908d.png"},{"id":89449426,"identity":"88704444-b842-4e95-a720-33137803ba1c","added_by":"auto","created_at":"2025-08-20 05:59:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":174836,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistinct distribution patterns and differentiation fates of E12.5-labeled and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGfap\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-GFP+ progenitors in dentate gyrus formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) E12.5-labeled cells (colored by red) are observed in the primary dentate matrix (PDM) near the dentate notch (DN) and migrate over the fimbria to reach the fimbriodentate junction (FDJ). They then establish the secondary dentate matrix (SDM) extending along the pia and hippocampal fissure (HF). These progenitors migrate inward (red arrows) toward the hilus and differentiate into granule cells, forming the outer shell of the granule cell layer (GCL, red). Similarly, \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells (green) begin their migration from the PDM region two days later, following comparable migratory routes (green arrows), but differentiate into granule cells that populate the inner core of the GCL (blue). A key distinction is seen in undifferentiated Sox2+ and \u003cem\u003eGfap\u003c/em\u003e-GFP+ progenitors (dark green dots), which display a broader distribution pattern, populating not only the migratory stream along the pia and HF, but also establishing residence within the hilus and GCL. These hilus-resident progenitors are thought to constitute the tertiary dentate matrix (TDM). Some \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells are known to take a direct route toward the hilus (also indicated by green arrows), contributing to TDM formation. In contrast, E12.5-labeled cells are not observed in the TDM, reflecting their pronounced tendency toward rapid differentiation.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Immunofluorescence analysis and schematic representation of E12.5-labeled and \u003cem\u003eGfap\u003c/em\u003e-GFP+ cell populations in the developing dentate gyrus (DG) at E18.5. The left panel shows original immunostaining data with RFP (E12.5-labeled cells) in red, GFP (\u003cem\u003eGfap\u003c/em\u003e-GFP+ cells) in green, Sox2 in magenta, and Prox1 in blue. The middle and right panels display summary diagrams of the boxed DG region in the left panel, with corresponding colored dots representing these cell populations. The middle panel (neural stem/progenitors) demonstrates that E12.5-labeled RFP+Sox2+ neural progenitors (magenta) are markedly scarce and show restricted localization primarily within the presumptive granule cell layer (GCL,dotted line). In striking contrast, \u003cem\u003eGfap\u003c/em\u003e-GFP+Sox2+ progenitors (dark green) are substantially more abundant and exhibit a widespread distribution throughout multiple zones including the hilus, presumptive GCL, and molecular layers, highlighting their stem cell-like undifferentiated state. The right panel (granule cells) shows that despite these different progenitor distribution patterns, both RFP+Prox1+ cells (red, numerically dominant) and \u003cem\u003eGfap\u003c/em\u003e-GFP+Prox1+ cells (blue) differentiated granule cells predominantly localize within the boundaries of the presumptive GCL. A small population of double-labeled RFP+\u003cem\u003eGfap\u003c/em\u003e-GFP+Prox1+ cells (orange) is also present. Collectively, these patterns emphasize that E12.5-labeled cells predominantly undergo differentiation, while \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells largely maintain an undifferentiated stem cell-like state.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6508491/v1/5859dcf6ca4e568626686f92.png"},{"id":95564019,"identity":"4c1462fc-0b51-42dd-965c-0afb4c9fa2dd","added_by":"auto","created_at":"2025-11-10 16:06:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3804879,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6508491/v1/a3b5fac4-6a3b-42ba-8c98-40b553b957b9.pdf"},{"id":89449457,"identity":"a98a6314-c989-4864-b6b7-9e296436abed","added_by":"auto","created_at":"2025-08-20 05:59:53","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":35030616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1 Limited labeling efficiency of dentate progenitors by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein utero\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e electroporation after E14.5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Immunofluorescence staining of hippocampal sections at E17.5 following \u003cem\u003ein utero\u003c/em\u003eelectroporation at E14.5, showing minimal RFP-positive cells (red) in the developing dentate gyrus (DG). Sections were immunostained for Sox2 (green) and Prox1 (blue) to identify the dentate neural progenitors (DNPs) and granule cells (GCs), respectively. (\u003cstrong\u003eA2\u003c/strong\u003e,\u003cstrong\u003e 3\u003c/strong\u003e) Higher magnification images of the boxed regions in \u003cstrong\u003eA1\u003c/strong\u003e, demonstrating the presence of labeled pyramidal cells in the ventricular surface (\u003cstrong\u003eA2\u003c/strong\u003e) and very slightly labeled cells in developing DG (\u003cstrong\u003eA3\u003c/strong\u003e). (\u003cstrong\u003eB, C\u003c/strong\u003e) Representative showing the scarcity of RFP-positive cells (red) in the developing DG at E17.5 following \u003cem\u003ein utero\u003c/em\u003e electroporation at E15.5. Sections were counterstained with DAPI (blue). (\u003cstrong\u003eB2, B3\u003c/strong\u003e) Higher magnification images of the boxed regions in \u003cstrong\u003eB1\u003c/strong\u003e, demonstrating the presence of labeled pyramidal cells in the ventricular surface (\u003cstrong\u003eB2\u003c/strong\u003e) and the absence of labeled cells in developing DG (\u003cstrong\u003eB3\u003c/strong\u003e). (\u003cstrong\u003eC2, C3\u003c/strong\u003e) Higher magnification images of the boxed regions in \u003cstrong\u003eC1\u003c/strong\u003e showing the ventricular surface (\u003cstrong\u003eC2\u003c/strong\u003e) and developing DG (\u003cstrong\u003eC3\u003c/strong\u003e) further illustrating the inefficient labeling of dentate progenitors at this developmental stage. Scale bars: \u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e, 100 μm.\u003c/p\u003e","description":"","filename":"Fig.S1ShinoharaTakahashi250220.tif","url":"https://assets-eu.researchsquare.com/files/rs-6508491/v1/b0678cbe4385de3be0a59332.tif"},{"id":89449422,"identity":"e50b7694-2d67-4595-a150-999221a5cec4","added_by":"auto","created_at":"2025-08-20 05:59:51","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17306,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.2503172.docx","url":"https://assets-eu.researchsquare.com/files/rs-6508491/v1/c98371577569f4d1ab4642a5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"In utero electroporation uncovers a distinct population of early dentate gyrus progenitors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe hippocampus plays fundamental roles in learning, memory formation, and emotional processing as a key component of the limbic system. Its complex structure, comprising the hippocampal pyramidal cells layer of the cornu ammonius (CA1, CA2, and CA3 regions) and the granule cells layer (GCL) of the dentate gyrus (DG), develops through intricate cellular migrations and differentiations. Among these regions, the subgranular zone of the DG is particularly notable as a unique neurogenic niche where neurogenesis continues throughout life [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. During development, undifferentiated cells migrate from the dentate notch (DN) through the dentate migratory stream (DMS) to establish the GCL [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], although the precise behaviors and properties of these migrating progenitors remain to be fully elucidated.\u003c/p\u003e \u003cp\u003eThe developmental process of the DG follows a remarkably unique and complex trajectory, involving cell migration along pathways that differ significantly from those of pyramidal cells in the neocortex and CA1 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previous research [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] has shown that neural progenitors that later differentiate into granule cells (GCs) in the DG exhibit a unique behavioral pattern, migrating between three distinct proliferative matrices: the primary dentate proliferative matrix (PDM) in the ventricular zone, the secondary dentate proliferative matrix (SDM) in the limbus, and the tertiary dentate proliferative matrix (TDM) in the hilus, thereby changing their proliferative zones (as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The spatial organization of these matrices follows a specific pattern: the PDM resides in the neuroepithelium superior to the fimbria, adjacent to the DN. Following initial proliferation in the PDM, progenitor cells migrate above the cortical hem to the fimbrio-dentate junction (FDJ) along the pial membrane. Subsequently, these cells proceed along the pial membrane toward the prospective DG via the DMS. During this process, neural progenitor cells undergo proliferation and establish the SDM in the region beneath the hippocampal fissure extending to the pial membrane at the DG limbus [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These SDM-derived neural progenitors generate GCs in the outer shell of the GCL, while their TDM counterparts, which form in the hilus, give rise to GCs in the inner core of the GCL.\u003c/p\u003e \u003cp\u003eOur current understanding of these complex migratory patterns mainly comes from studies using transgenic reporter mice, particularly those expressing fluorescent proteins under specific promoters. For example, mice with the \u003cem\u003eGfap\u003c/em\u003e promoter-driven reporter have shown that neural stem/progenitor cells emerge at E14.5 and contribute significantly to DG formation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although genetic approaches, particularly Cre-lox-based lineage tracing with tamoxifen-inducible control [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], have provided valuable insights, they are inherently limited by their dependence on pre-selected promoters of known genetic markers. This reliance on specific molecular markers potentially overlooks progenitor populations that lack expression of these canonical genes, particularly during earlier developmental stages when regulatory networks may be distinct. Complementary approaches that can label and track cells independently of specific gene expression would help eliminate potential biases and provide a more comprehensive understanding of DG development.\u003c/p\u003e \u003cp\u003eDirect labeling approaches, particularly \u003cem\u003ein utero\u003c/em\u003e electroporation, have served as a pioneering method in developmental neurobiology since their introduction in the early 2000s. This technique first revealed crucial insights into neocortical development, including characterization of the inside-out migration pattern and other fundamental developmental processes [\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19 CR20\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. A particular strength of this technique lies in its ability to enable precise spatiotemporal targeting along the ventricular surface at defined developmental stages while providing unbiased labeling of progenitors regardless of their molecular characteristics. Moreover, the sparse labeling pattern achieved through electroporation allows detailed observation of individual cell morphology and migration trajectories at single-cell resolution, providing an excellent complement to genetic and retroviral approaches.\u003c/p\u003e \u003cp\u003eWhile \u003cem\u003ein utero\u003c/em\u003e electroporation has proven instrumental in studying hippocampal pyramidal neuron development [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], its application to DG development remains relatively unexplored despite its early demonstration [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Given the complex nature of DG development and the distinct advantages of each methodology, the integration of diverse experimental approaches \u0026ndash; genetic, viral, and electroporation-based \u0026ndash; will be crucial for building a comprehensive understanding of DG development.\u003c/p\u003e \u003cp\u003eIn this study, we set out to perform systematic analysis of DG development using \u003cem\u003ein utero\u003c/em\u003e electroporation, examining multiple embryonic stages from E11.5 onward. While technical limitations restricted reproducible labeling to E12.5, this approach enabled immediate visualization of an early progenitor population through targeted cellular labeling during a critical period of DG development. By establishing a novel dual-labeling system that combines direct cell labeling with transgenic \u003cem\u003eGfap\u003c/em\u003e-GFP reporter mice, we were able to directly compare, within the same animal, the behavior of electroporated cells with the well-characterized \u003cem\u003eGfap\u003c/em\u003e-expressing progenitor population. This comparative approach revealed a previously unrecognized population that diverges from the \u003cem\u003eGfap\u003c/em\u003e-expressing dentate progenitors and may represent an earlier wave of dentate development not adequately captured by current genetic models. These labeled cells displayed unique characteristics: exclusively populating the outer GCL without reaching the TDM and showing greater propensity for differentiation into GCs compared to \u003cem\u003eGfap\u003c/em\u003e-expressing progenitors that maintain an undifferentiated state. Our findings reveal heterogeneity among dentate progenitors, with early-labeled progenitors potentially establishing the structural scaffold of this critical hippocampal structure.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimals\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6J mice and \u003cem\u003eGfap-\u003c/em\u003eGFP transgenic mice were used for \u003cem\u003ein utero\u003c/em\u003e electroporation experiments. C57BL/6J mice were obtained from Charles River Laboratories Japan (Yokohama, Japan). The \u003cem\u003eGfap-\u003c/em\u003eGFP mice on a C57BL/6NCr background express green fluorescent protein (GFP) under the control of the glial fibrillary acidic protein (Gfap) promoter and have been described previously [27]. All mice were maintained under standard conditions with a 12-hour light/dark cycle in the animal care facilities of Tokyo Medical University. All animal procedures were approved by the Institutional Animal Care and Use Committees of Tokyo Medical University and were performed in accordance with the National Institutes of Health \u003cem\u003eGuide for the Care and Use\u003c/em\u003e of Laboratory Animals (NIH Publication No. 80-23, revised 1996). Efforts were made to minimize the number of animals used and their suffering. For timed mating, the day of vaginal plug detection was defined as embryonic day 0.5 (E0.5). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn utero electroporation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn utero\u003c/em\u003e electroporation of mouse embryos was performed as previously described [28], with modifications. The \u003cem\u003epCAG\u003c/em\u003e-mRFP construct (a gift from Dr. Masanori Uchikawa at Osaka University) was used to visualize neuronal progenitors and neurons by red fluorescent protein (RFP) expression, though signal intensity may decrease through successive cell divisions. Briefly, timed pregnant mice at E11.5 (n=8), E12.5 (n=91), E14.5 (n=140), and E15.5 (n=14) were anesthetized and the uterine horns were exposed by laparotomy. The plasmid solution containing the \u003cem\u003epCAG\u003c/em\u003e-mRFP construct was injected into the lateral ventricle of embryos (7.29 \u0026plusmn; 1.95 embryos/dam; range 0-10 embryos) using a glass micropipette. Square electrical pulses (30 V, 50 ms duration, four pulses) were then applied to the embryos using a tweezer-type electrode (CUY650P3, BEX, Japan) connected to an electroporator (CUY21 Vivo-SQ, BEX). The electrode was positioned to target the dentate neuroepithelium. These electrical pulse parameters were optimized to achieve efficient transfection while minimizing tissue damage, based on preliminary experiments. After electroporation, the uterine horns were repositioned and the abdominal cavity was closed. Electroporated embryos were harvested and fixed at various developmental stages including E13.5, E14.5, E15.5, E17.5 and E18.5 for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunofluorescence analysis of cell differentiation markers\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell differentiation states were analyzed by immunofluorescence staining as previously described [28] with minor modifications. Antibodies were selected based on their specificity and previous validation in similar developmental studies of the DG. At embryonic stages E13.5, E14.5, E15.5, E17.5 and E18.5, mouse embryos were transcardially perfused with 4% paraformaldehyde (PFA) in phosphate-buffered saline (pH 7.4). The developing DG primordium was dissected and further fixed in 4% PFA at 4\u0026deg;C overnight. DG tissues were cryosectioned perpendicular to the septotemporal axis of the hippocampus using a cryostat (CM1850, Leica Biosystems, Wetzlar, Germany)\u0026nbsp;to preserve the processes of dentate neural progenitor cells. The sections were then immersed in a blocking solution containing 2% bovine serum albumin in Tris-buffered saline with 0.1% Triton X-100 (TBST, Sigma, St. Louis, MO) for 1 hour at room temperature (RT).\u003c/p\u003e\n\u003cp\u003eSections were then incubated with primary antibodies overnight at 4 ̊C, washed with TBST and then incubated with the appropriate fluorescence-conjugated secondary antibodies for 1 hour at RT. The primary and secondary antibodies used are listed in Table S1. Confocal imaging was performed using a confocal microscope (LSM700, Carl Zeiss) and the acquired images were processed using the Zeiss LSM Image Browser and ZEN software to adjust brightness and contrast and to generate composite images.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEarly migration and differentiation of E12.5 dentate progenitors\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the development of dentate neural progenitors (DNPs), we performed \u003cem\u003ein utero\u003c/em\u003e electroporation targeting the DN region using the \u003cem\u003epCAG\u003c/em\u003e-mRFP construct, which drives ubiquitous expression regardless of cell fate in targeted cells (Fig. 1B). Following previously established protocol [22], we systematically examined developmental stages from E11.5 onward, the earliest stage at which the DN hippocampus region can be targeted by this method. Electroporation\u0026nbsp;at E11.5 resulted in poor embryonic survival; in contrast, E12.5 consistently yielded viable embryos with reproducible DNP labeling. While\u0026nbsp;electroporation\u0026nbsp;from E14.5 successfully labeled cells in different hippocampal regions, it showed dramatically reduced efficiency in specifically targeting DNPs, with only occasional sporadic success (Supplementary Fig. 1).\u0026nbsp;This stage-dependent efficiency suggests a critical developmental window for DNP labeling by this method.\u0026nbsp;Therefore, we focused our detailed analysis on DNPs labeled at E12.5, where we achieved reproducible results that enabled us to track their migratory behavior and fate. For molecular marker analysis, we used Sox2 to identify undifferentiated neural stem/progenitor cells and Tbr2 to detect committed neural precursors to specify the differentiation status of the labeled cells.\u0026nbsp;We focused our analysis on RFP-positive cells maintaining detectable expression levels throughout development, noting that some labeled cells became undetectable through proliferation or showed different behaviors. The \u003cem\u003ein utero\u003c/em\u003e electroporation at E12.5 combined with these molecular markers\u0026nbsp;allowed us to identify a distinct subpopulation with strong differentiation tendency.\u003c/p\u003e\n\u003cp\u003eOne day post-labeling (E12.5-\u0026gt;E13.5), RFP+ cells were distributed in the ventricular zone (VZ) and displayed undifferentiated characteristics, as evidenced by Sox2 expression (Figs. 1C; D1-4, white arrows). These cells had a bipolar morphology. Some RFP+ cells, which had migrated towards the pial surface, did not express Sox2 but instead expressed Tbr2, a marker of intermediate neuronal precursors (Figs. 1D1-4, green arrowheads). These slightly differentiated neural precursors exhibited a multipolar morphology.\u003c/p\u003e\n\u003cp\u003eTwo days post-labeling (E12.5-\u0026gt;E14.5), the RFP+ cells on the ventricular surface (VS) side remained Sox2+ and undifferentiated (Figs. 1E; F1-4, white arrows). In contrast, RFP+ cells on the pial side expressed Tbr2,\u0026nbsp;indicating their progression towards a neuronal progenitor state (Figs. 1E; G1-4, green arrowheads).\u003c/p\u003e\n\u003cp\u003eThis pattern persisted three days post-labeling (E12.5-\u0026gt;E15.5). RFP+ cells on the VS side retained a bipolar morphology and continued to express Sox2 (Figs. 1H; I1-4, white arrows), maintaining their undifferentiated state. At the same time, RFP+ cells that had migrated towards the pial surface continued to express Tbr2, further confirming their commitment to a neuronal fate (Figs. 1H; J1-4, green arrowheads).\u003c/p\u003e\n\u003cp\u003eThese results demonstrate that some E12.5-labeled neural progenitor cells differentiated into neuronal progenitors as they migrated towards the pial surface, while others remained in an undifferentiated state on the VS side.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMaturation of E12.5-labeled progenitors into granule cells\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether E12.5-labeled DNPs can progress to terminal differentiation and acquire granule cell identity, we examined the expression of mature granule cell markers at later developmental stages. We performed immunostaining at E15.5 (E12.5-\u0026gt;E15.5) for Prox1, a homeodomain transcription factor essential for GC identity and a marker of mature GCs. As shown in Fig. 2A1, RFP+ cells were observed extending from the VS to the emerging DG primordium. The RFP+ cells in the marginal zone were Tbr2-positive (Figs. 2A2-5, green arrowheads) and also expressed Prox1, indicating a more advanced differentiation state. In contrast, RFP+ cells in the hilus were Prox1-positive but Tbr2-negative (Figs. 2A2-5, green arrows), indicating their further differentiation into mature GCs.\u003c/p\u003e\n\u003cp\u003eFive days post-labeling (E12.5-\u0026gt;E17.5), RFP+ cells along the hippocampal fissure (HF) and pial surface remained Tbr2-positive (Figs. 2B1; B2-5, white arrowheads). RFP+ cells along the medial side of the HF expressed Prox1 (Figs. 2B1; B2-5, green arrows), suggesting their progression towards mature GC identity. These Prox1+/RFP+ cells were positioned more medially than the Tbr2+/RFP+ cells.\u0026nbsp;This spatial organization shows that E12.5-labeled cells differentiate in a distinctive pattern, with cells migrating from the outer regions toward more medial positions as they mature.\u003c/p\u003e\n\u003cp\u003eSix days post-labeling (E12.5-\u0026gt;E18.5), RFP+ cells in the hilus, rather than just below the HF, were Prox1-positive but Tbr2-negative (Figs. 2C1; C2-5 green arrows). This Prox1+/Tbr2- GC phenotype persisted at both 5 and 6 days post-labeling. Notably, these Prox1+/RFP+ GCs also expressed the mature neuronal marker NeuN (Figs. 2D1; D2-5, white arrows). The co-expression of Prox1 and NeuN confirms the acquisition of mature granule cell identity, representing the terminal stage in the differentiation process of E12.5-labeled progenitors. These results demonstrate that DNPs labeled at E12.5 are capable of differentiating into fully mature GCs within a relatively short developmental window, highlighting the rapid differentiation potential of this early progenitor population.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eComparative analysis of E12.5-labeled and Gfap-GFP+ progenitor populations\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNPs have been characterized using various approaches, including classical methods and transgenic reporter lines [8-11, 12, 13]. These different studies have consistently revealed similar migratory behaviors and differentiation trajectories of DG progenitors. To evaluate how our electroporation-based labeling method at E12.5 relates to these established approaches and to potentially identify progenitor populations that may have been unrecognized by existing genetic labeling methods, we compared our labeled cells with \u003cem\u003eGfap\u003c/em\u003e-GFP+ progenitors that emerge at E14.5, representing one of the well-characterized genetic marking systems.\u003c/p\u003e\n\u003cp\u003eWe introduced the \u003cem\u003epCAG\u003c/em\u003e-mRFP construct into the DG primordium of \u003cem\u003eGfap\u003c/em\u003e-GFP mice at E12.5\u0026nbsp;and analyzed the labeled cells at different time points. Two days post-labeling (E12.5-\u0026gt;E14️.5), RFP+ cells displayed an undifferentiated Sox2-positive phenotype within the VZ (Figs. 3A1-5, B1-5) and Tbr2-positive neuronal precursor phenotype on the pial side (Figs. 3A1-5; C1-5, white arrowheads), consistent with our earlier observations\u0026nbsp;in wild-type mice. At E14.5, \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells observed were distributed throughout the VZ up to the pial membrane (Figs. 3A2; 3B1-5, orange arrows; C1-5, orange arrowheads). This suggests that while E12.5-labeled progenitors were already migrating and differentiating by E14.5, \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells emerging at this stage still populated the entire DG primordium.\u003c/p\u003e\n\u003cp\u003eNotably, we observed minimal overlap between the two populations, as \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells were barely labeled by the RFP reporter after \u003cem\u003ein utero\u003c/em\u003e electroporation at E12.5 (Figs. 3A1-2, A5, B1-2 orange arrows; C1-2 orange arrowheads). The \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells located on the VS side at E14️.5 were Sox2-positive but Tbr2-negative, indicating that they remained undifferentiated (Figs. 3B1-5 orange arrowheads).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsistent with the observation in Fig. 1, the E12.5-labeled RFP+ cells that had migrated to the pial membrane side displayed a Tbr2-positive, Sox2-negative neuronal progenitor phenotype (Figs. 3C1-5, white arrowheads). Interestingly, a similar distribution and marker profile was also observed for \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells at this E14.5 time point, with \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells located on the pial membrane side also exhibiting Tbr2 positivity and Sox2 negativity (Figs. 3C1-5, orange arrowheads). These findings suggest that both populations initially follow similar migratory routes, though their differentiation kinetics later diverge.\u003c/p\u003e\n\u003cp\u003eThree days post-labeling (E12.5-\u0026gt;E15.5), both RFP+ and \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells were distributed within the developing DG region (Figs. 3D1-5) [11]. Only a few undifferentiated RFP+ cells, characterized by Sox2 positivity and Tbr2 negativity, were found within the hilus (Figs. 3E1-5, white arrow). In contrast, undifferentiated \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells with a Sox2+/Tbr2- profile were considerably more abundant in the hilus (Figs. 3E1-5, orange arrows).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt the same developmental stage, Tbr2-positive, Sox2-negative RFP+ cells were arranged in a V-shaped pattern along the pial membrane and prospective HF (Figs. 3E1-5, white arrowheads), indicating a more differentiated neuronal progenitor state. Notably, the \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells showed a similar distribution, with Sox2-/Tbr2+ population lining up along the pial membrane and HF (Figs. 3E1-5, orange arrowheads).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo confirm whether these progenitor populations differentiated into mature GCs, immunostaining was performed with the Prox1 antibody (Fig. 3F). The differentiation profiles of the two populations showed clear differences at this stage. RFP+ cells on the pial side exhibited weak Prox1 expression while retaining Tbr2 positivity (Figs. 3F1; F2-5, white arrows), suggesting an intermediate state of differentiation. In contrast, RFP+ cells along the HF strongly expressed Prox1 (Figs. 3F2-5, white arrowheads),\u0026nbsp;indicating progression towards a mature GC identity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGfap\u003c/em\u003e-GFP+ cells near the FDJ expressed Tbr2 but were negative for Prox1 (Figs. 3F1; F2-5, orange arrows), indicating an intermediate progenitor state. However, only a minute subset of\u0026nbsp;\u003cem\u003eGfap\u003c/em\u003e-GFP+ cells on the pial side showed Tbr2 expression along with weak Prox1 immunoreactivity (Figs. 3F2-5, orange arrowheads), suggesting that they had progressed further towards a mature GC identity.\u003c/p\u003e\n\u003cp\u003eThese comparative analyses demonstrate distinct developmental trajectories for the two progenitor populations. While both E12.5-labeled and \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells can generate GCs, they differ significantly in their differentiation kinetics. Specifically, E12.5-labeled cells exhibited more advanced GC differentiation, as evidenced by extremely robust Prox1 expression along the HF. In contrast, \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells appeared to be slightly delayed in their differentiation trajectory, with a significant proportion retaining an intermediate progenitor phenotype near the FDJ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLate-stage developmental divergence of E12.5-labeled and Gfap-GFP+ populations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether the differentiation differences observed at E15.5 persist and potentially become more pronounced at later developmental stages, we analyzed both cell populations at E17.5 (5 days post-labeling) and E18.5 (6 days post-labeling).\u003c/p\u003e\n\u003cp\u003eAt E17.5, both populations showed similar positioning of Tbr2-positive neuronal progenitors just below the HF and along the pial membrane (Figs. 4A; B1-5, white arrowheads indicate the progenitors derived from E12.5-labeled cells and orange arrowheads from \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells, respectively). Undifferentiated Sox2-positive cells were found within the hilus in both populations, with a notable prevalence among \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells (Figs. 4A; B1-5, white and orange arrows). Furthermore, Prox1 immunostaining (Figs. 4C-J) revealed distinct spatial patterns of GC differentiation between the two populations. E12.5-derived Prox1-positive RFP+ cells were observed within the hilus (Figs. 4C; D1-5, white arrows), while Prox1+/\u003cem\u003eGfap\u003c/em\u003e-GFP+ cells were predominantly located on the pial membrane side (Figs. 4D1-5, orange arrows).\u003c/p\u003e\n\u003cp\u003eBy E18.5, the spatial segregation between the two populations became more pronounced. E12.5-derived mature GCs were preferentially localized within the molecular layer, particularly on the HF side (Figs. 4F1-5, white arrows). On the pial membrane side, these RFP+ GCs were not only found in the densely packed GCL, but also extended into the hilus region (Figs. 4E, G1-5, H, I1-5, white arrows). A subset of undifferentiated Sox2-positive cells derived from E12.5 labeling were still present within the hilus (Figs. 4G1-5, white arrowheads).\u003c/p\u003e\n\u003cp\u003eIn contrast to this distribution pattern, GCs derived from \u003cem\u003eGfap\u003c/em\u003e-GFP+ population were mainly distributed along the hilar side or within the hilus of the densely packed GCL (dashed line) (Figs. 4F1-5, G1-5, I1-5, J1-5, orange arrows). Notably, undifferentiated cells persisted not only in the hilus but also within the densely packed GC region (Figs. 4F1-5, G1-5, orange arrowheads).\u003c/p\u003e\n\u003cp\u003eThese observations highlight key differences in the migratory patterns and differentiation states of the two progenitor populations.\u0026nbsp;E12.5-labeled cells showed more advanced differentiation and integration into the molecular layer, while a substantial subset of \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells remained undifferentiated within the GCL itself.\u003c/p\u003e\n\u003cp\u003eBoth E12.5-labeled RFP+ cell-derived and \u003cem\u003eGfap\u003c/em\u003e-GFP-derived Tbr2+ neuronal progenitors were present within the molecular layer at this stage (Figs. 4I1-5, white and orange arrowheads, respectively). To quantitatively assess the differentiation status of both populations, we analyzed the percentage of RFP+ and \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells expressing Sox2 (undifferentiated) and Prox1 (GC) markers within the DG primordium. This analysis revealed striking differences: 75.5%\u0026nbsp;\u0026plusmn;\u0026nbsp;3.5% (n=3) of E12.5-derived RFP+ cells had differentiated into Prox1+ GCs, whereas only 24.5%\u0026nbsp;\u0026plusmn;\u0026nbsp;4.0% of \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells showed GC identity (Fig. 4K). Conversely, 64.8%\u0026nbsp;\u0026plusmn;\u0026nbsp;9.3% of \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells maintained an undifferentiated Sox2+ state (Fig. 4K).\u003c/p\u003e\n\u003cp\u003eThese quantitative data provide further evidence that E12.5-labeled progenitors and \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells exhibit divergent behaviors during later stages of DG development. While the majority of E12.5-derived cells had progressed to a mature GC fate, a substantial proportion of the \u003cem\u003eGfap\u003c/em\u003e-GFP+ population maintained an undifferentiated progenitor state, retaining their stem-like properties even as the GCL was being established. The distinct behaviors of E12.5-labeled cells and \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells - with the former predominantly generating mature GCs and the latter maintaining an undifferentiated state - establish the existence of two separate progenitor populations with different developmental programs in the developing DG.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStage-specific labeling by in utero electroporation\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e\u003cem\u003eenables direct visualization of early dentate progenitors\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile transgenic reporter mice have been instrumental in revealing the molecular identity and lineage relationships of dentate progenitors [10-13, 30],\u0026nbsp;direct visualization and tracking of early progenitors from their point of origin has remained challenging. \u003cem\u003eIn utero\u0026nbsp;\u003c/em\u003eelectroporation, which enables precise spatiotemporal targeting of ventricular progenitors and visualization of individual cell migration at single-cell resolution, has been successfully applied to the studies of hippocampal pyramidal neurons [7, 22-24]. However, its systematic application to early DG development has not been reported.\u003c/p\u003e\n\u003cp\u003eIn this study, we set out to perform comprehensive stage-dependent analysis using \u003cem\u003ein utero\u003c/em\u003e electroporation from E11.5 onward. While targeting efficiency varied across stages, E12.5 emerged as a critical time point allowing reproducible labeling of early dentate progenitors. Targeting efficiency declined substantially from E14.5 onward, proving particularly challenging despite previous reports [22]. Similarly, electroporation at E11.5 resulted in poor embryonic survival, preventing reliable analysis. This stage-dependent technical limitation may explain the paucity of electroporation studies targeting the developing DG in the current literature, and likely reflects the increasing anatomical complexity around the developing DN region. The unique morphological transitions of the primordial DN present significant challenges to current electroporation approaches. Our findings highlight the need to develop new, specialized electroporation strategies that are specifically optimized for the distinct anatomical features of the developing DN.\u003c/p\u003e\n\u003cp\u003eDespite these technical challenges, successful labeling at E12.5 proved particularly informative, as the sparse labeling and single-cell resolution provided by electroporation allowed us to follow the dynamic behavior of early dentate progenitors in detail. As visualized in Fig. 5, this approach revealed distinct patterns of progenitor migration and differentiation that enhance our understanding of early DG development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDevelopmental trajectory of E12.5-labeled dentate progenitors\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur \u003cem\u003ein utero\u003c/em\u003e electroporation studies provided detailed visualization of early dentate progenitor cells at E12.5, enabling us to specifically label and track progenitors at their point of DN origin along the ventricular surface. As demonstrated in Fig. 5A, the migratory behavior of these E12.5-labeled cells followed a characteristic inward migration pattern (red arrows): First, these progenitors migrated from the ventricular surface towards the FDJ at the pial membrane. They then distributed just below the pial membrane and the hippocampal fissure (HF), forming a secondary proliferative matrix at the prospective margins of the DG. Finally, these early-born E12.5-labeled cells differentiated into mature GCs, establishing the initial framework of the GCL. Critically, as shown in Fig. 5A, unlike \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells that eventually populate the TDM in the hilus, E12.5-labeled cells were never observed in the TDM, providing compelling evidence for the fundamentally distinct developmental properties of this early progenitor population.\u003c/p\u003e\n\u003cp\u003eThis process followed a distinctive outside-in pattern, with cells progressively populating the GCL from the molecular layer side inwards towards the hilus (Fig. 5A, red region). Interestingly, this inward migration pattern during early development parallels the outside-in layering of the DG that has been previously reported in the adult brain, where comparisons of granule cell distribution after embryonic versus postnatal stages demonstrated that earlier-born cells predominantly occupy outer positions in the granule cell layer, while later-born cells populate inner regions adjacent to the hilus [30].\u0026nbsp;Our direct visualization of E12.5-labeled progenitors provides cellular-level evidence for the developmental origins of this layering principle.\u003c/p\u003e\n\u003cp\u003eWhile previous studies using \u003cem\u003eNestin\u003c/em\u003e and \u003cem\u003eGfap\u003c/em\u003e reporter mice have suggested that subpial progenitors contribute to GC generation during migration to the hilus [10,11],\u0026nbsp;our work provides the first direct visualization of this process at single-cell resolution, revealing a continuous series of cell movements originating from the ventricular surface at E12.5. The combination of precise spatiotemporal targeting and sparse labeling enabled by electroporation has allowed us to track individual cell behavior during this critical developmental period, providing new insights into the cellular dynamics of early dentate development.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTemporal heterogeneity and functional divergence of progenitor pools in dentate gyrus development\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the cells labeled at E12.5, we identified a distinct subpopulation that maintained detectable RFP expression and showed unique properties. While other labeled cells exhibited different behaviors or became undetectable due to signal dilution through proliferation, this particular subpopulation demonstrated temporal heterogeneity from the later emerging \u003cem\u003eGfap\u003c/em\u003e-expressing progenitors, as illustrated in Fig. 5B. The striking contrast between these populations is one of our most significant findings \u0026ndash; the E12.5-labeled cells that we characterized in detail showed a robust propensity for neuronal differentiation, exhibiting rapid migration and giving\u0026nbsp;rise to the initial GCL of the DG (red dots in the Fig. 5B right panel). In contrast, a substantial proportion of the later-born \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells maintained an undifferentiated state (dark green dots in the Fig. 5B middle panel), suggesting their potential role in expanding the progenitor pool and contributing to the subsequent increase in GCL thickness through delayed differentiation.\u003c/p\u003e\n\u003cp\u003eWhile our electroporation approach enabled tracking of a specific subpopulation showing accelerated differentiation, the full heterogeneity of E12.5 progenitors may not be captured due to signal dilution through proliferation\u0026nbsp;and the presence of other labeled cells showing different behaviors. Nevertheless, the identification of this differentiation-prone population that lacks\u003cem\u003e\u0026nbsp;Gfap\u003c/em\u003e expression represents a significant finding in understanding dentate gyrus development.\u0026nbsp;The semi-quantitative analysis depicted in Fig. 5B clearly shows that E12.5-labeled RFP+ cells are predominantly found as differentiated Prox1+ granule cells (red dots in right panel), while disproportionately fewer maintain a Sox2+ progenitor state (magenta dots in middle panel). This stark cellular distribution pattern highlights a functional divergence between early and late progenitors, suggesting a carefully orchestrated developmental programme that balances immediate neurogenesis with the maintenance of a progenitor reservoir for sustained growth.\u003c/p\u003e\n\u003cp\u003eThese divergent behaviors align with the two proposed migration routes previously described for dentate progenitors. In the first route, the PDM in the ventricular zone near the DN continues along the pial membrane to form the SDM just below the HF, which then differentiates into GCs. Subsequently, a subset of progenitors reaches the future hilus to form the TDM [5,6,11,12]. This \u0026lsquo;outside-in\u0026rsquo; pattern, where earlier migrating progenitors seed the molecular (outer) side of the GCL and later arrivals populate the inner hilus region, is directly confirmed by our observations in Fig. 5A, which clearly demonstrates that the E12.5 progenitors exclusively contribute to the outer GCL (red) without reaching the TDM, whereas the later \u003cem\u003eGfap\u003c/em\u003e-GFP+ population (green) establishes the TDM and has a more pronounced inside component [10,31-33]. Additionally, as indicated in Fig. 5A, some \u003cem\u003eGfap\u003c/em\u003e-GFP+ cells are known to take a more direct route toward the hilus, contributing to TDM formation, a second migration pathway that is not observed in E12.5-labeled cells.\u003c/p\u003e\n\u003cp\u003eInterestingly, our results are also consistent with previous reports indicating that the suprapyramidal blade (SPB) of the DG forms before the infrapyramidal blade (IPB), with the SPB outer shell being established between E13.5 and P0.5, whereas the IPB outer shell emerges after E16.5 [8-10,34].\u0026nbsp;Our observations suggest that E12.5 progenitors in the DN contribute to the initial formation of the SPB outer shell, as evidenced by their early migration pattern (Fig. 5B).\u003c/p\u003e\n\u003cp\u003eUnderstanding the diversity and properties of dentate progenitors has relied heavily on genetic lineage tracing approaches, with Nestin-Cre systems revealing progenitor emergence at E14.5 [10, 12] and Hopx-CreERT2 demonstrating labeling as early as E10.5 [13]. While these approaches have been invaluable for identifying specific NSC-derived lineages, they are inherently limited in their ability to capture the precise timing of emergence and initial migratory behavior of dentate progenitors.\u003c/p\u003e\n\u003cp\u003eOur application of \u003cem\u003ein utero\u003c/em\u003e electroporation at E12.5 has revealed a previously uncharacterized population of early dentate progenitors. These cells exhibit distinct properties from known populations, notably showing accelerated differentiation kinetics without maintaining neural stem cell characteristics, as clearly evidenced by the predominance of RFP+Prox1+ cells over RFP+Sox2+ cells in Fig. 5b. While current technical limitations restricted our analysis to E12.5, these findings demonstrate the value of combining direct labeling with genetic approaches to uncover the full complexity of dentate development. Future technical refinements will likely enable broader developmental analysis, potentially revealing additional progenitor diversity during DG formation.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Japan Society for the Promotion of Science KAKENHI (grant numbers 16K18983 and 20K07233, both to HMS) and the Centre for Diversity at Tokyo Medical University (TMUCD-202202, TMUCD-202301, TMUCD-202409 to HMS).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHMS designed and performed all experiments and prepared all the Figs. HMS wrote the first draft, and TT extensively revised and refined the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data included in this study are available upon request by contact with the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the Institutional Animal Care and Use Committees of Tokyo Medical University and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996). Efforts were made to minimize the number of animals used and their suffering.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are very grateful to Dr Seiji Shiota (Hoshi University) for generating the\u003cem\u003e\u0026nbsp;Gfap-\u003c/em\u003eGFP\u003cem\u003e+\u003c/em\u003e transgenic mice expressing GFP under the control of mouse \u003cem\u003eGfap\u003c/em\u003e promoter. We thank Professor Emeritus Tatsunori Seki (Tokyo Medical University) for his generous support. We also thank Keiko Toda, Mio Hayashida, Chikako Miyazaki and Mieko Utsugi (Tokyo Medical University) for technical assistance. This work was supported by grants from the Japan Society for the Promotion of Science KAKENHI (grant numbers 16K18983 and 20K07233, both to HMS) and the Centre for Diversity at Tokyo Medical University (TMUCD-202202, TMUCD-202301, TMUCD-202409 to HMS).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAltman J (1963) Autoradiographic investigation of cell proliferation in the brains of rats and cats. Anat Rec 145: 573-591. 10.1002/ar.1091450409.\u003c/li\u003e\n\u003cli\u003eSeki T, Arai Y (1993) Highly polysialylated neural cell adhesion molecule (NCAM-H) is expressed by newly generated granule cells in the dentate gyrus of the adult rat. J Neurosci 13: 2351-2358. 10.1523/jneurosci.13-06-02351.\u003c/li\u003e\n\u003cli\u003eAlvarez-Buylla A, Lim DA (2004) For the long run: maintaining germinal niches in the adult brain. 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J Comp Neurol 196: 99-128. 10.1002/cne.901960109.\u003c/li\u003e\n\u003cli\u003eAltman J, Bayer SA (1990) Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. J Comp Neurol 301: 365-381.10.1002/cne.903010304.\u003c/li\u003e\n\u003cli\u003eNoguchi H, Castillo JG, Nakashima K, Pleasure SJ (2019) Suppressor of fused controls perinatal expansion and quiescence of future dentate adult neural stem cells. Elife 8: 10.7554/eLife.42918.\u003c/li\u003e\n\u003cli\u003eNelson BR, Hodge RD, Daza RA, Tripathi PP, Arnold SJ, Millen KJ, Hevner RF (2020) Intermediate progenitors support migration of neural stem cells into dentate gyrus outer neurogenic niches. Elife 9: 10.7554/eLife.53777.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cemn","sideBox":"Learn more about [Cellular and Molecular Neurobiology](https://www.springer.com/journal/10571)","snPcode":"10571","submissionUrl":"https://submission.nature.com/new-submission/10571/3","title":"Cellular and Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"dentate gyrus, in utero hippocampal electroporation, dentate progenitor cells, granule cells, dentate migration","lastPublishedDoi":"10.21203/rs.3.rs-6508491/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6508491/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe 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 \u003cem\u003ein utero\u003c/em\u003e 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 (\u003cem\u003eGfap\u003c/em\u003e-GFP). This approach revealed striking differences between two distinct populations: \u003cem\u003eGfap\u003c/em\u003e-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, exclusively following an outside-in trajectory to establish the initial framework of the granule cell layer, without reaching the tertiary dentate matrix. In contrast, \u003cem\u003eGfap\u003c/em\u003e-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 direct comparison identifies a functionally distinct subset of early progenitors that rapidly differentiate, revealing previously unrecognized temporal and functional heterogeneity in dentate development. This study demonstrates how stage-specific \u003cem\u003ein utero\u003c/em\u003eelectroporation uncovers diverse progenitor populations potentially underrepresented by existing genetic approaches, providing new insights into the cellular diversity that shapes hippocampal structure and function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number: \u003c/strong\u003enot applicable\u003c/p\u003e","manuscriptTitle":"In utero electroporation uncovers a distinct population of early dentate gyrus progenitors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 05:59:47","doi":"10.21203/rs.3.rs-6508491/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-27T14:00:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-27T01:53:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-26T17:25:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-22T15:21:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-20T11:32:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"243743559513620754385201462009039924881","date":"2025-05-15T06:28:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304030228822978798934539144345297605938","date":"2025-05-14T14:08:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81727870969590102088636925133694174319","date":"2025-05-12T15:21:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"149551581654724599847080767949707096073","date":"2025-05-12T14:14:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-12T13:54:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-27T20:16:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-25T05:49:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Neurobiology","date":"2025-04-23T03:27:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cemn","sideBox":"Learn more about [Cellular and Molecular Neurobiology](https://www.springer.com/journal/10571)","snPcode":"10571","submissionUrl":"https://submission.nature.com/new-submission/10571/3","title":"Cellular and Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"33e109c4-36f1-42ad-984e-62a13b75572e","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T16:00:38+00:00","versionOfRecord":{"articleIdentity":"rs-6508491","link":"https://doi.org/10.1007/s10571-025-01616-3","journal":{"identity":"cellular-and-molecular-neurobiology","isVorOnly":false,"title":"Cellular and Molecular Neurobiology"},"publishedOn":"2025-11-06 15:57:25","publishedOnDateReadable":"November 6th, 2025"},"versionCreatedAt":"2025-08-20 05:59:47","video":"","vorDoi":"10.1007/s10571-025-01616-3","vorDoiUrl":"https://doi.org/10.1007/s10571-025-01616-3","workflowStages":[]},"version":"v1","identity":"rs-6508491","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6508491","identity":"rs-6508491","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}