PSmad3+/Olig2− expression defines a subpopulation of gfap-GFP+/Sox9+ neural progenitors and radial glia-like cells in mouse dentate gyrus through embryonic and postnatal development

In mouse dentate gyrus, radial glia-like cells (RGLs) persist throughout life and play a critical role in the generation of granule neurons. A large body of evidence has shown that the combinatorial expression of transcription factors (TFs) defines cell types in the developing central nervous system (CNS). As yet, the identification of specific TFs that exclusively define RGLs in the developing mouse dentate gyrus (DG) remains elusive. Here we show that phospho-Smad3 (PSmad3) is expressed in a subpopulation of neural progenitors in the DG. During embryonic stage (E14-15), PSmad3 was predominantly expressed in gfap-GFP-positive (GFP+)/Sox2+ progenitors located at the lower dentate notch (LDN). As the development proceeds (E16-17), the vast majority of PSmad3+ cells were GFP+/Sox2+/Prox1low+/Ki67+ proliferative progenitors that eventually differentiated into granule neurons. During postnatal stage (P1–P6) PSmad3 expression was observed in GFP+ progenitors and astrocytes. Subsequently, at P14–P60, PSmad3 expression was found both in GFP+ RGLs in the subgranular zone (SGZ) and astrocytes in the molecular layer (ML) and hilus. Notably, PSmad3+ SGZ cells did not express proliferation markers such as PCNA and phospho-vimentin, suggesting that they are predominantly quiescent from P14 onwards. Significantly PSmad3+/GFP+ astrocytes, but not SGZ cells, co-expressed Olig2 and S100β. Together, PSmad3+/Olig2− expression serves as an exclusive marker for a specific subpopulation of GFP+ neural progenitors and RGLs in the mouse DG during both embryonic and postnatal period.


Introduction
Using gfap-GFP transgenic mice expressing GFP under the control of mouse gfap promoter, it has previously been shown that gfap-GFP+ (GFP+) progenitors around the dentate notch (DN) contribute to granule neurons in the dentate gyrus (DG) during development (Altman and Bayer, 1990;Seki et al., 2014).The GFP+ progenitors have also been suggested to contribute to other cell types: radial glia-like cells (RGLs) and protoplasmic astrocytes (Brunne et al., 2013).After birth, neurogenesis takes place mainly in the hilus and its border from the granule cell layer (GCL), becoming confined to the subgranular zone (SGZ) by postnatal day 14 (P14; Namba et al., 2005;Seki et al., 2014).Some GFP+ progenitors are converted to adult type RGLs immediately after birth (Matsue et al., 2017).However, the mechanism by which RGLs are specified during DG development is largely unknown.
There is considerable evidence that combinatorial expressions of TFs under the influence of signaling molecules define progenitor cell types in the developing CNS.However, it is currently unknown what TF code distinguishes RGLs from astrocytes, both of which appear mostly during the early postnatal period.Molecular markers for RGLs such as Sox9 are important to maintain RGLs in adulthood.However, they are also expressed in astrocytes, making it difficult to understand the mechanism by which RGLs and astrocytes develop in different ways.
Previous studies have shown that varieties of signals control the development of the dentate gyrus (DG) both in the embryo and the adult.Both BMP7 and Wnt signals regulate the expression of transcription factor Prox1, thereby governing the fate of granule neurons in the DG (Galceran et al., 2000;Choe et al., 2013).Sox2-dependent Shh signaling plays a key role both in the development of RGLs and sustained neurogenesis at the SGZ in adult mice (Li et al., 2013).ALK5dependent TGFβ signaling through pSmad2 maintains late events during adult hippocampal neurogenesis (He et al., 2014).Smad3 is crucial for neuronal survival and adult neurogenesis in the hippocampus (Tapia-González et al., 2013).However, lack of TGFβ-Smad signaling does not affect the development of dentate granule neurons (Choe et al., 2013).These studies suggest that TGFβ-Smad2/3 signaling is essential for the development of adult but not embryonic granule neurons in the hippocampus.However, it remains unclear whether TGFβ-Smad signaling plays a role for other cell types that derived from GFP+ progenitors, namely astrocytes in the developing DG.
Phospho-Smad3 (PSmad3) has been shown to regulate not only progenitor specification and neuronal differentiation in the embryonic spinal cord (García-Campmany and Martí, 2007).As yet its expression pattern in the developing mouse DG has not been well studied.A recent study pointed out that TGFβ-PSmad3 pathway controls the glioblastoma stemness (Ikushima et al., 2009).Given the migratory and proliferative properties of the GFP+ DG progenitors (Seki et al., 2014), they may share some properties.In this study, we therefore examined the expression patterns of PSmad3 in the developing mouse DG.We show that PSmad3 is expressed in both a subpopulation of GFP+/Sox9+ progenitors, RGLs, and astrocytes throughout the embryonic and postnatal period.It is worthy of note that Olig2 expression distinguishes astrocytes from the RGLs.Together, PSmad3+/Olig2− expression defines a subpopulation of GFP+/Sox9+ neural progenitors and RGLs at the SGZ in both embryonic and postnatal mouse DG.

Animals
Gfap-GFP transgenic mice that express GFP under the control of mouse gfap promoter (Suzuki et al., 2003) were housed under a standard condition (12-h light/dark cycle) in the animal care facility of Tokyo Medical University.All experiments were carried out in accordance with the guideline of the Institutional Animal Care and Use Committees and conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23) revised in 1996.All efforts were made to minimize the number of animals used and their suffering.Embryos and pups of the gfap-GFP transgenic mice above were used.The day on which a vaginal plug was found was designated as embryonic day 0.5 (E0.5), and the day of birth was designated as postnatal day 0.5 (P0.5).

Tissue preparation
Embryos were harvested at E14.5−E18.5, and postnatal mice were anesthetized and perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH7.4,at room temperature.The brains were removed and washed with PBS and immersed in 30% sucrose/0.1 M PB.The forebrains were embedded into OCT compound and stored at -70°C.Cryosections were cut at 25 μm thickness.

Immunohistochemistry
Cryosections were processed for immunohistochemistry as described previously (Ohyama et al., 2005;Seki et al., 2014).Briefly, cryosection of the hippocampus were incubated with primary antibodies overnight at 4°C.For some antibody labeling experiments (Ki67, phospho-vimentin), antigen retrieval with Histo VT One (Nacalai, Japan) was carried out following manufacturer's instructions.After wash with PBS three times, the sections were incubated with secondary antibodies for 45 min at room temperature.After wash with PBS three times, the sections were mounted with Vectashield (Vector lab).Images were taken with a Zeiss confocal microscope LSM700.In some cases, fluorescence images were digitally zoomed at 0.5x to 2x.Stacks of optical sections (1.8 μm in thickness/optical section) were obtained at 0.9 μm increments on the z-axis using x20 objective.The images were corrected for brightness and contrast and composed using Zeiss Image Browser, ZEN software (Zeiss, Thomwood, NY) and Adobe Photoshop CS6 (San Jose, CA).Mice (n = 3-6) were examined for individual experiments, and, for quantification of some experiments, 9 sections at least were analyzed for each using Fiji of image J. Mean ± SE was indicated in the results.

Results
Gfap-GFP+ cells contribute to RGLs at the SGZ and astrocytes in the molecular layer and hilus Using gfap-GFP mice (Seki et al., 2014;Matsue et al., 2017), we first monitored the contributions of gfap-GFP-positive (GFP+) progenitors in the developing mouse dentate gyrus (DG).GFP+ progenitors were found in the ventricular zone (VZ) around the dentate notch (DN) and in the primordium of the DG at E14-E16 (Figures 1A,B).At postnatal day1 (P1), many GFP+ progenitors accumulate in the developing DG (Figure 1C).By P6 hilus, granule cell layer (GCL), and molecular layer (ML) become apparent (Figure 1D).GFP+ cells were observed not only in the hilus of the DG, but also in the ML.At P14-21, in addition to the GFP+ astrocytes in the hilus and ML, GFP+ cells were also found at the SGZ where RGLs reside postnatally (Figures 1E,F).Taken together, our data indicate that GFP+ progenitors contribute to both RGLs at the SGZ and astrocytes in the hilus and ML over time (Supplementary Figure 1).

PSmad3 expression in GFP+ RGLs in early developing mouse DG
PSmad3 has been shown to control the proliferative and migratory behavior of glioblastoma (Ikushima et al., 2009).GFP+/Sox2+ neural progenitors also possess both proliferative and migratory properties (Seki et al., 2014).This led us to examine whether PSmad3 is expressed in the GFP+/Sox2+ neural progenitors in developing mouse DG.At E14, PSmad3 expression was found in the ventricular zone (VZ) of the lower part of the DN and in a migrating stream of GFP+/Sox2+ progenitors toward the DG primordium (n = 3; Figures 2A1−B8).

Development of PSmad3+ RGLs
Gfap-GFP+ (GFP+) DG progenitors express Sox2 and migrate away from the VZ of the DN, and they are found in the SGZ, ML, and hilus in the postnatal DG (Figure 1).These data suggest that both the GFP+ RGLs in the SGZ and the astrocytes in the ML and hilus are derived from the DN.Consistent with this, PSmad3 expression was found in both GFP+/Prox1 + low progenitors, GFP+ RGLs, and GFP+/Olig2+ astrocytes.Given the Prox1 low + expression, the PSmad3+/ GFP+ progenitors seem to possess a property of neurogenic cells that give rise to Prox1 high + dentate granule neurons.Interestingly, PSmad3+/ GFP+/Prox1 low + progenitors were also observed in early postnatal DG (data not shown).However, the developmental origin of PSmad3+ RGLs and astrocytes remains elusive.Given that PSmad3+ cells are observed mostly in the lower DN and fimbriadentate junction at E14-17, the PSmad3+ RGLs are likely to originate from the lower DN.Consistent with this, PSmad3 is expressed in a subpopulation of GFP+/Sox9+ cells that are likely to originate from a broader region around the DN (Figure 6).While our data imply that PSmad3+/ GFP+/Prox1 low + neurogenic progenitors may originate from the lower DN, a genetic lineage tracing analysis will be necessary to further clarify this issue.
It has previously been shown that expansion of RGLs is prominent during the first week after birth (Matsue et al., 2017).In support of this notion, the expansion of PSmad3+/GFP+ RGLs becomes evident during the perinatal period (Figure 5; Supplementary Figure 3).The majority of the PSmad3+ RGLs co-express Sox2, Sox9, pVim, and PCNA (Figures 3, 5, 6; Supplementary Figure 3), supporting their proliferative capacity in the first postnatal week.
The TGFβ-pSmad2/3-Sox2 pathway has been implicated in regulating the stemness of glioma-initiating cells (Ikushima et al., 2009).Recent studies have also shown that PSmad3 can bind to Sox9 and activate Sox9-dependent transcription (Furumatsu et al., 2009), and that Sox9 and Slug co-operate to maintain cancer stem cells (Guo et al., 2012).While Slug is not expressed in the developing brain   Marin and Nieto, 2004), Snail1, a cousin of Slug controls the number of neural progenitors at the SGZ of the hippocampus (Zander et al., 2014).Similarly, Zeb1 at the downstream of PSmad3 maintains RGLs in the DG (Gupta et al., 2021).PSmad3 may contribute to control the proliferative property of RGLs in the DG through the regulation of the EMT-TFs Sox9, Snail1, and Zeb1.Sufu acts as a positive regulator of Shh signal, thereby RGLs expansion in the DG (Noguchi et al., 2019).TGFβ2 induces Gli1 expression in a PSmad3-dependent manner after ischemia/ reperfusion injury (Peng et al., 2019).Future analysis of the crosstalk between the TGFb-PSmad3 pathway and Shh signaling will also shed light on the mechanism controlling the expansion of postnatal RGLs in the DG.
Tapia-Gonzalez et al. showed that Smad3 is expressed in early intermediate progenitors but not in Sox2+ RGLs in adult mouse DG.Smad3 mRNA was observed in the granule cell layer (GCL) in addition to the SGZ.Our data show that PSmad3 is expressed in GFP+/Sox2+/Sox9+ progenitors and RGLs.These data appear to be somewhat contradictory each other.Tapia-Gonzalez et al. analyzed the DG of 3-4 month old female mice.In our data, PSmad3 expression was observed in the SGZ, hilus, and ML in male mice at P60 (Figure 12).It is also worth noting that PSmad3 was very weakly expressed in the GCL of the mouse DG at P60 but not at P30 (Figures 7,12).Intriguingly, at 6 months old of age, PSmad3 was strongly expressed in the GCL in addition to the SGZ, hilus, and ML (data not shown).It seems that the expression pattern of PSmad3 is somehow slightly different between adolescent and adult mouse DG.
Our data clearly show that PSmad3 is expressed in subpopulation of GFP+ cells.This also explain why in Tapia-Gonzalez et al., PSmad3 expression was not observed in Sox2+ and GFAP+ RGLs at the SGZ of 3-4 month old mouse DG (Tapia-González et al., 2013).GFP+ expression can label many more GFAP-expressing cells, compared to immunolabeling with GFAP antibody.Given our data that some GFP+ cells at the SGZ express Tbr2 (unpublished data), and that Tbr2+ intermediate progenitors at the SGZ express Ascl1 (Tapia-González et al., 2013), this may explain why we were able to detect PSmad3+/GFP+ RGLs at the SGZ, whereas PSmad3 expression was detected in Ascl1+ progenitors (Tapia-González et al., 2013).
Taken together, PSmad3 is expressed in a subpopulation of GFP+/ Sox2+/Sox9+ dentate progenitors in the embryo, and the RGLs and astrocytes in the postnatally developing mouse DG.Although this also seems to be the case in the adult, further careful analysis of PSmad3 expression in combination with various markers of distinct cell types

Development of PSmad3+/Olig2+ astrocytes
While PSmad3+ progenitors were found in the DN at E14-E16, very few Olig2+ cells were found in both the DN and DG at E16 (Figures 1, 2, 9).Olig2+ progenitors appear at the upper DN at E18 (Figures 9B1-B6).At P1-6 PSmad3+/GFP+ progenitors were found around both upper and lower DN, and many of them co-expressed Olig2 (data not shown).These data suggest that the Olig2+ astrocytes seem to originate from the VZ around the DN from E18 onwards.
Recent studies have shown that Olig2+ astrocytes are present in both embryonic and adult CNS (Ohayon et al., 2018;Tatsumi et al., 2018;Wang et al., 2021).While it has previously been shown that the majority of Olig2+ astrocytes are GFAP− in the adult CNS (Tatsumi et al., 2018;Wang et al., 2021), we were able to Olig2+/GFP+ astrocytes by using gfap-GFP mice (Figure 9).Nonetheless, there may be Olig2+/GFP-astrocytes in the DG.More careful analysis of Olig2+ astrocytes will be necessary to clarify the heterogeneity of Olig2+ astrocytes.In terms of the function of Olig2+ astrocytes, while Olig2+ OLPs respond to brain injury such as hypoxia and increase in number, the role of Olig2+ astrocytes remains unclear (Allan et al., 2021).It will be interesting to see whether Olig2+ astrocytes act as reactive astrocytes after brain injury.
In conclusion, we have provided evidence that PSmad3 is expressed in a subpopulation of GFP+/Sox9+ neural progenitors, RGLs, and astrocytes in the mouse DG during embryonic and postnatal development, and that Olig2 expression is allocated with astrocytes but not RGLs.While the roles of PSmad3 and Olig2 in the development of RGLs and astrocytes remain elusive, combinatorial expression of these TFs will be useful to identify RGLs and Olig2+ astrocytes to investigate their development and function in the mouse DG.

FIGURE 1
FIGURE 1 Gfap-GFP+ (GFP+) progenitors contribute to RGLs and astrocytes in developing mouse DG (A,B) GFP+ cells are found at the DN and forming DG at E14 and E16.(C,D) Many GFP+ cells are observed in the developing DG at P1 and P6.(E,F) GFP+ cells are located at SGZ and ML of the DG at P14-21.DN, dentate notch; DG, dentate gyrus; GCL, granule cell layer; ML, molecular layer; SGZ, subgranular zone.Scale bars: all 200 μm.