Differentiation stage-specific expression of transcriptional regulators for epithelial mesenchymal transition in dentate granule progenitors

During the development of the mouse dentate gyrus (DG), granule neuronal progenitors (GNPs) arise from glial fibrillary acidic protein (GFAP)-expressing neural stem cells in the dentate notch. However, the transcriptional regulators that control their stepwise differentiation remain poorly defined. Since neurogenesis involves epithelial-to-mesenchymal transition (EMT)-like processes, we investigated the spatio-temporal expression profiles of the EMT transcription factors Zeb1, Scratch2 (Scrt2) and Nkx6-2 in relation to known GNP markers. Our results show that Zeb1 and Scrt2 exhibit sequential, but partially overlapping expression across embryonic and postnatal stages of GNP differentiation. Zeb1 is highly enriched in gfap-GFP+/Sox2+ neural stem/progenitor pools and subsets of Tbr2+/Prox1+/NeuroD+ intermediate GNPs, whereas Scrt2 predominates in Tbr2+/Prox1+/NeuroD+ GNPs. Strikingly, the neuronal EMT regulator Nkx6-2 shows selective expression in postnatal Tbr2+/Prox1+ GNPs, but it is excluded from embryonic counterparts. This temporally coordinated yet distinct expression of Zeb1, Scrt2 and Nkx6-2 reveals discrete transcriptional programs orchestrating GNP differentiation and neurogenic progression at embryonic versus postnatal stages of DG neurogenesis.


Introduction
In the developing central nervous system, the expression of transcription factors (TFs) not only defines progenitor cell types but also orchestrates their differentiation.A previous study showed that gfap-GFP+ cells arising around the dentate notch (DN) first express Sox2 and give rise to Tbr2+ intermediate progenitors, which then contribute to the formation of the Prox1+ granule neuronal cell layer (Seki et al., 2014).Some gfap-GFP+ cells maintain their stemness, at later stages, and contribute to neural stem/progenitor cells in the subgranular zone (SGZ) of the postnatal DG (Matsue et al., 2017).Prox1 not only regulates a granule cell fate over a pyramidal neuronal fate in the hippocampus, but also neuronal differentiation and maintenance of post-mitotic states (Iwano et al., 2012).To better understand the mechanism for the progressive differentiation of dentate granule neurons, it is important to identify in more detail the temporally regulated expression of TFs in the granule neuronal lineage.
Emerging evidence suggests that different epithelial mesenchymal transition (EMT)-TFs play distinct roles in controlling stemness and the onset of neuronal differentiation (Singh and Solecki, 2015).We have recently shown that pSmad3, a key mediator of EMT is expressed in DG stem/progenitors and RGL cells in the developing DG (Ohyama et al., 2023).Zeb1, downstream of pSmad3, contributes to maintaining the stemness of cancer-initiating cells (CICs) by inhibiting cellular senescence (Ansieau et al., 2008;Chaffer et al., 2013).Zeb1 also controls the stemness of GNPs in the cerebellum through the downregulation of cell polarity genes (Singh et al., 2016).Conversely, inhibition of Zeb1 leads to upregulation of cell polarity genes, resulting in cerebellar granule neuronal differentiation (Singh et al., 2016).Another EMT-TF, Snail, regulates the number of neural progenitors throughout life (Zander et al., 2014).Scrt2, a member of the Snail family TFs, inhibits cell cycle re-entry through the regulation of miR25, de-represses the expression of the cyclin-dependent kinase inhibitor p57, thereby triggering the onset of neuronal differentiation in the developing spinal cord (Rodríguez-Aznar et al., 2013).Scrt2 also controls the onset of neuronal migration in the mouse cerebral cortex (Itoh et al., 2013).In addition, Nkx6-2 has been identified as a neuronal EMT-TF (Liang et al., 2016).These studies suggest that different EMT-TFs play time-dependent roles during neuronal differentiation.
Here, we examined the expression patterns of the EMT-TF Zeb1, Scrt2, and Nkx6-2 in the developing DG, and compared them with the temporal expression profiles of previously identified TFs that are expressed in the dentate GNPs.

Animals
Gfap-GFP mice on a C57BL6/NCrl background (Suzuki et al., 2003) were housed under standard conditions (12 h light/dark cycle) at the animal care facility of Tokyo Medical University.All experiments were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committees and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals , revised in 1996.Every effort has been made to minimize the number of animals used and their suffering.Embryos and pups of gfap-GFP transgenic and C57BL6 wild-type mice were used.The day on which a vaginal plug was found was referred to as embryonic day 0.5 (E0.5) and the day of birth was referred to as postnatal day 0.5 (P0.5).

Immunohistochemistry
Cryosections were processed for immunohistochemistry as previously described (Ohyama et al., 2023).Briefly, embryos and pups of gfap-GFP transgenic mice and sibling controls were fixed in 4% PFA at 4°C overnight.After fixation, samples were immersed in 30% sucrose in 0.1 M PB at 4°C overnight and then embedded in OCT compound.Cryosections of the hippocampus in the coronal plane were incubated with primary antibodies at 4°C overnight.After three washes with PBS, sections were incubated with secondary antibodies for 45 min at room temperature.After three washes with PBS, the sections were coverslipped with Vectashield (Vector Laboratories, H-1200, CA).Images were captured using a Zeiss LSM700 confocal microscope with Zeiss Image Browser, ZEN software (Zeiss, Thomwood, NY).Images were corrected for brightness and contrast and composited using Adobe Photoshop CS6 (San Jose, CA).Mice (n = 3-5) were examined and, for quantification of some experiments, at least 6 sections were analyzed for each using Fiji of image J. Mean ± SE is given in the results.

Quantification using Fiji
To quantify marker expression, we used Fiji-ImageJ software.First, we opened the TIFF file containing the red (e.g., Zeb1) and green (e.g., Sox2) fluorescence using Fiji.We set the threshold color to white to distinguish the area of interest and adjusted the brightness as needed to improve contrast and reduce the background noise.Using the Hue sliders, we isolated the specific signal, such as Zeb1+Sox2+ cells and used the Analyse>Measure function to count these cells.We repeated this process to count Sox2+ cells.The resulting cell counts were then transferred to an Excel spreadsheet where we calculated the percentage of, for example, Zeb1+Sox2+ cells relative to Zeb1+ cells.This approach was taken by two individuals through blinding and ensured consistent and accurate quantification of marker expression across samples.
Previous studies have shown that Tbr2+ progenitors differentiate not only into dentate granule neurons, but also into reelin+ Cajal-Retzius (CR) neurons at the hippocampal fissure (Hodge et al., 2013).This raised the question of whether Scrt2 is also expressed in a Tbr2+ CR neuronal lineage at the hippocampal fissure.However, Scrt2 and p73 expression did not overlap, while some p73+/Tbr2+ cells were found at the hippocampal fissure (Figures 7A1-A8,B1-B8,C1-C8; Supplementary Figure S4, data not shown).In addition, Scrt2 was not expressed in reelin+ Cajal-Retzius (CR) neurons (data not shown).Our data also showed that reelin+ CR neurons at the hippocampal fissure did not express gfap-GFP (Figures 7D1-D8).Thus, neither gfap-GFP+ cells nor Scrt2+ progenitors contribute to CR neurons.Consistent with this, both p73 and Lhx1/5, markers of CR neuronal progenitors were not expressed in gfap-GFP+ progenitors (Figures 7D1-D8, data not shown).These data  indicate that Scrt2+/Tbr2+ cells are GNPs but not CR neuronal progenitors.
Scrt2 is also expressed in some Zeb1+/ Sox2+ GNPs The vast majority of gfap-GFP+ cells express the neural stem/ progenitor cell marker Sox2 both in the VZ and in the parenchyma of the developing DG (Figure 1).While some of these differentiate into Prox1+ dentate granule neurons (Figure 1), many Sox2+/gfap-GFP+ RGL progenitors were retained (Figure 1).Given our data that Scrt2 is expressed in some gfap-GFP+ cells (Supplementary Figure S3), this raised the possibility that Scrt2 is co-expressed with Zeb1 in Sox2+ progenitors.Indeed, co-expression of Scrt2 and Zeb1 was found in Sox2 low + at E18 and P6 (Figures 8A1-A7,B1-B7).

Discussion
Our results reveal distinct spatio-temporal expression profiles of the EMT-TFs factors Zeb1, Scrt2, and Nkx6-2 during GNP differentiation in the developing mouse DG.We show that Zeb1 and Scrt2 exhibit sequential but partially overlapping expression in embryonic and postnatal GNP populations, whereas Nkx6-2 is selectively expressed in postnatal GNPs.These results highlight temporally coordinated transcriptional programs that control GNP developmental trajectories.

Distinct roles for Zeb1 and Scrt2 in regulating GNP stemness and differentiation
Zeb1 and Sox2 are interdependently involved in the regulation of stemness.Zeb1 inhibits stemness-repressing miRNAs such as miR-200 that target Sox2, thereby maintaining Sox2 expression and stemness (Burk et al., 2008;Singh et al., 2017).In contrast, Zeb1 knockdown decreases the expression level of Sox2 (Pérez et al., 2021).It was also shown that Sox2 can directly bind to the promoter region of Zeb1, indicating a feedback loop (Singh et al., 2017).These data suggest that Zeb1 and Sox2 expressions are mutually dependent.
Regarding Scrt2, its expression in Tbr2+ intermediate progenitors is consistent with its known role in promoting neuronal differentiation.Scrt2 has been shown to regulate the cell cycle exit of neural progenitors by inhibiting cyclin D1 expression and promoting p27 expression (Rodríguez-Aznar and Nieto, 2011).This function may facilitate the transition of Tbr2+ intermediate progenitors to NeuroD1+ neuroblasts in the DG.The postnatal-specific expression of Nkx6-2 in Tbr2+/ Prox1+ cells suggests a potential role in regulating the transition from intermediate progenitors to immature granule neurons.While direct interactions between Nkx6-2 and these differentiation markers have not been extensively studied in the DG, Nkx6-2 has been shown to promote neural migration in other contexts (Toch et al., 2020).Its expression pattern in our study suggests that it may play a similar role in granule neuron development.
The enrichment of Zeb1 in GFAP+/Sox2+ stem/progenitor populations is also consistent with its reported role in inhibiting cellular senescence and maintaining neural progenitor proliferation (Liu et al., 2008;Singh et al., 2016).Zeb1 may repress pro-apoptotic genes such as p73 to maintain the undifferentiated state of dentate GNP-producing progenitors (Bui et al., 2009).In addition, by downregulating cell polarity genes, Zeb1 prevents premature epithelial-mesenchymal transition (EMT) and preserves the stem/ progenitor properties of GNP-generating cells (Singh et al., 2016).

Distinct transcriptional regulation of embryonic versus postnatal GNPs
A striking difference between embryonic and postnatal stages is the selective expression of the neuronal EMT regulator Nkx6-2 in postnatal Tbr2+/Prox1+ GNPs (Figures 9B1-D8).This temporal specificity suggests divergent transcriptional programs governing the dynamics of GNP differentiation in embryonic versus postnatal neurogenic niches.While embryonic GNPs rely on Zeb1 and Scrt2, postnatal GNPs may use Nkx6-2 to facilitate neuronal differentiation through EMT-associated mechanisms.
Recent research has highlighted a transition from radial glia/neural stem cells to radial glial-like cells (RGLs) during postnatal development.For example, Matsue et al. (2017) demonstrated that gfap-GFP+ radial glia transition to gfap-GFP+/BLBP+ RGLs during the postnatal period.Consistent with this, recent single cell RNA-seq data from Hochgerner et al. (2018) revealed a shift in the molecular identity of radial glia after postnatal day 5, which persists into adult stages.In contrast, Hopxexpressing dentate progenitors, which give rise to adult RGLs, retain a consistent molecular signature throughout development (Berg et al., 2019).These data imply that RGLs are a heterogenous cell population.Our recent findings support this notion by showing that phospho-Smad3 is expressed in a subpopulation of RGLs throughout development and in adulthood.
In summary, our data have revealed that the temporally coordinated expression of EMT-TFs Zeb1 and Scrt2 in GNPs is maintained throughout development.In contrast, Nkx6-2 expression is restricted to postnatal GNPs, highlighting a previously unrecognized difference between embryonic and postnatal GNPs during DG development.
expressed in migrating DG progenitors.At E14-P6 Scrt2 expression was found in the cells migrating away from the VZ, and they are aligned in the subpial and hilar regions, corresponding to the prospective GCL and ML, respectively (Figures 6A1-A8,B1-B8,C1-C8; Supplementary Figures S2A1