Etv5 Is Required for Proper Maturation and Dendritic Development of Adult‐Born Granule Cells in the Dentate Gyrus

ABSTRACT

Adult neurogenesis in the dentate gyrus (DG) of the hippocampus is a dynamic and tightly regulated process that is finely regulated by a diverse array of transcription factors. While the transcription factor Etv5, a member of the Erythroblast Transformation Specific (ETS) family, has been implicated in embryonic development by regulating cell proliferation and differentiation across various tissues, its specific role in adult hippocampal neurogenesis remains unexplored. Here, we show that conditional ablation of Etv5 specifically in adult‐born granule cells (GCs) increases the proportion of Doublecortin (DCX)‐positive immature GCs at the expense of mature neurons, without affecting the proportion of neither radial glia‐like cells (RGCs) nor SOX2⁺ progenitor cells within the neurogenic niche. Furthermore, Etv5 conditional mutant mice exhibit reduced dendritic complexity and defects in spine development, indicating impaired neuronal maturation and suggesting deficits in synaptic integration of adult‐born GC neurons.

The adult brain retains a remarkable ability to generate new neurons in specific regions, such as the dentate gyrus (DG) of the hippocampus. This process, known as adult hippocampal neurogenesis, is essential for functions such as memory and mood regulation, and involves a carefully regulated balance between the proliferation of neural progenitor cells and the proper maturation and integration of newborn neurons. In this study, we investigated the role of the transcription factor Etv5, an ETS family member known for its involvement in developmental processes, in adult‐born neurons of the hippocampus. Using a conditional genetic mouse model, we show that loss of Etv5 disrupts the maturation of newborn neurons without altering the initial stages of neurogenesis. Etv5 deletion results in reduced dendritic complexity and impaired spine development, indicating defects in neuronal maturation. Altogether, these findings reveal a previously unrecognized function of Etv5 in the late stages of adult neurogenesis and highlight its importance for the structural integration of adult‐born neurons into the hippocampal circuitry. SGZ, subgranular zone; GCL, granule cell layer; ML, molecular layer.

Abbreviations

BDNF

brain‐derived neurotrophic factor

CREB

cAMP response element‐binding protein

DCX

doublecortin

DG

dentate gyrus

dpi

days post injection

Er81

ETS‐related 81

ERM

ETS‐related molecule

ETS

erythroblast transformation specific

Etv1

ETS translocation variant 1

Etv4

ETS translocation variant 4

Etv5

ETS translocation variant 5

GCL

granular cell layer

GCs

granular cells

GDNF

glial cell line‐derived neurotrophic factor

GFAP

glial fibrillary acidic protein

GLAST

Glutamate–Aspartate Transporter

IPCs

intermediate progenitor cells

MFBs

mossy fiber boutons

NeuN

neuronal nuclei

NPC

neural stem/progenitor cells

NT‐3

neurotrophin 3

Pea3

polyoma enhancer activator 3

RGCs

radial glia‐like cells

RRID

Research Resource Identifier

SGZ

subgranular zone

SOX2

(sex determining region Y)‐box 2

TAM

tamoxifen

TOM

tdTomato

1. Introduction

The hippocampal dentate gyrus (DG) is one of the regions in the adult mammalian brain where neurogenesis persists throughout life, including humans. Adult hippocampal neurogenesis contributes to the plasticity of the hippocampal circuits and plays a critical role in cognitive functions such as learning, memory, and mood regulation (Goncalves et al. 2016; Toda et al. 2019; van Praag et al. 2002). This complex process requires the precise coordination of neural stem/progenitor cells (NPCs) proliferation, differentiation, and integration of newborn granule cells (GCs) into preexisting neural networks. Cell proliferation in the DG is tightly controlled by different factors, which regulate the balance between quiescent and active neural progenitor cells. Once generated, adult‐born GCs undergo a stereotypical sequence of morphological and functional maturation that resembles neuronal development during the perinatal period. This maturation and integration process spans approximately 8 weeks for proper DG function, suggesting that immature neurons contribute uniquely to hippocampal function before reaching full maturity (Esposito et al. 2005; Kempermann et al. 2015; Kropff et al. 2015; Laplagne et al. 2006).

Functional integration of adult‐born GCs is orchestrated by intrinsic factors, such as transcriptional regulators, as well as extrinsic signals, including locally secreted neurotrophic factors. Neurotrophic factors, like brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT3), and glial cell line‐derived neurotrophic factor (GDNF), play key roles in regulating adult neurogenesis by supporting the survival, maturation, and synaptic integration of new neurons within hippocampal circuits (Bergami et al. 2008; Bonafina et al. 2020; Lu and Chang 2004; Vilar and Mira 2016).

Transcription factors play a pivotal role regulating gene expression, orchestrating diverse cellular processes such as proliferation, differentiation, and survival. Among these, E26 transformation‐specific (ETS) transcription variant 5 (Etv5), also known as ETS‐related molecule (ERM), exerts versatile functions in normal physiological processes including embryonic development, cell metabolism, and neural system development. Additionally, Etv5 is overexpressed in oncogenic processes, where it is involved in proliferation and cancer invasion (Qi et al. 2020; Wei et al. 2023). While much of the research on Etv5 has focused on its role in these oncogenic processes, its involvement in cellular differentiation has begun to attract growing interest, particularly in the context of the nervous system (Arber et al. 2000; de Nooij et al. 2013; Fontanet et al. 2013; Fontanet et al. 2018; Livet et al. 2002; Patel et al. 2003). Thus, one of the most intriguing aspects of Etv5 is its dual involvement in both the proliferative and differentiative stages of cellular development. This makes it particularly relevant to the study of adult hippocampal neurogenesis, a process that involves both proliferation of neural progenitor cells and their subsequent differentiation into functional neurons.

Etv5 belongs to the Pea3 subfamily of ETS transcription factors which comprises three members: Etv1, Etv4, and Etv5, also named Er81, Pea3 and ERM, respectively. The three proteins have been described as being expressed in different neuronal subpopulations in the central and peripheral nervous systems. Generally, Etv4 and Etv5 are associated with similar functions, whereas Etv1 appears to be implicated in distinct physiological roles (Oh et al. 2012). Interestingly, ETS transcription factors have been shown to be induced by neurotrophic factors and to mediate many of their downstream biological effects (Arber et al. 2000; de Nooij et al. 2013; Fontanet et al. 2013; Livet et al. 2002; Patel et al. 2003). In particular, Etv4 and Etv5 are upregulated by BDNF in hippocampal pyramidal neurons, where they play a crucial role in promoting dendritic complexity and spine maturation (Fontanet et al. 2018).

Here, we show that Etv5 is expressed by adult‐born hippocampal neurons. Given the well‐established role of Etv5 in mediating BDNF signaling in pyramidal neurons (Fontanet et al. 2018), we investigated its potential function in the development of newborn granule cells in the adult DG, a context in which BDNF is also known to regulate critical aspects of neuronal maturation. Considering the known involvement of Etv5 in cellular proliferation and differentiation, it is of great interest to explore its function within the unique context of adult hippocampal neurogenesis.

Understanding how this transcription factor influences the balance between neural progenitors and their differentiation into granule neurons could provide significant insights into both the basic biology of neurogenesis and its potential implications for neurodevelopmental and neurodegenerative disorders.

2. Materials and Methods

2.1. Animals

Etv5 ^f/f and Etv4 ^−/− mice were generously provided by Dr. Olivia Bermingham‐McDonogh (University of Washington, Seattle, USA). The Etv5‐conditional mutant mice were described previously (Zhang et al. 2009), and the Etv4‐mutant mice were described in detail in Livet et al. (2002). The genetically modified driver mouse lines Nestin ^Cre (Tronche et al. 1999) and Glast ^CreERT2 (Mori et al. 2006), as well as the conditional reporter line CAG ^{floxStop‐tdTomato} (Ai14) (B6;129S6‐Gt(ROSA)26Sor ^{tm14(CAG‐tdTomato) Hze/J} ) conditional reporter line (Madisen et al. 2010), were kindly provided by Dr. G. Lanuzza (Fundación Instituto Leloir, IIBBA‐CONICET, Argentina). Etv5 ^f/f mice were crossed with either Nestin ^Cre or with Glast ^CreERT2 :CAG ^{floxStop‐tdTomato} mice. All transgenic strains were genotyped using a PCR‐based strategy, which is available upon request. Tamoxifen (TAM, Sigma‐Aldrich, Cat# T5648) dissolved in corn oil (Sigma‐Aldrich, Cat# C8267) was administered intraperitoneally to 2‐month‐old mice at a dose of 150 μg/g/day for two consecutive days to achieve appreciable Tomato (TOM) expression in adult‐born GCs (Yang et al. 2015). Mouse brains were collected at the indicated time points after TAM administration. All experimental procedures were approved by the Institutional Animal Care and Use Committee (CICUAL) of the Leloir Institute (Protocol number: FIL90) and were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals of the Council for International Organizations for Medical Sciences and the Guide for the Care and Use of Laboratory Animals. Mice of either sex were housed in groups of four to five per cage under standard laboratory conditions, with ad libitum access to food and water. All mice were maintained on a CD1 genetic background. A total of 35–40 mice were used in this study, including three to four mice per experimental group. No randomization was performed to allocate subjects in the study, and no exclusion criteria were used. For histological analyses, mice were subjected to intracardiac perfusion fixation. Animals were deeply anesthetized with ketamine (150 μg/g) and xylazine (15 μg/g), administered intraperitoneally (i.p.), to ensure complete loss of pain perception and reflexes.

2.2. Immunofluorescence

Immunostaining was performed on free‐floating 50 μm‐thick coronal sections obtained from adult mouse brains. Sections were blocked in 10% normal donkey serum (Jackson ImmunoResearch Laboratories) diluted in phosphate buffer (PBS) containing 0.3%–1.5% Triton X‐100. Immunofluorescence staining was performed using the following primary antibodies: anti‐Etv5 (1:1000, Proteintech, Cat# 13011‐1‐AP; RRID: AB_2278092), anti‐Etv4 (1:400, Santa Cruz Biotechnology, Cat# sc‐166629; RRID: AB_2278090), anti‐DCX (1:350, Santa Cruz Biotechnology, Cat# sc‐8066; RRID: AB_2088494), anti‐Ki67 (1:1000, Leica Biosystems, Cat# NCL‐Ki67p; RRID: AB_442102), anti‐GFAP (1:600, DSHB, Cat# N206A/8; RRID: AB_2877343), anti‐SOX2 (1:1000, BD Pharmingen, Cat# 561469; RRID: AB_10694256), anti‐NeuN (1:1000, Millipore, Cat# MAB377; RRID: AB_2298772), anti‐Nestin (1:300, BD Pharmingen, Cat# 556309; RRID: AB_396354) and anti‐Calbindin (1:25, Cat# BCN96.1.1C8; RRID: AB_3073528). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories and used at a dilution of 1:300. The following secondary antibodies were used: Cy2‐donkey anti‐mouse IgG (H + L) Jackson ImmunoResearch Labs Cat# 715‐225‐150; RRID: AB_2340826; Cy2‐donkey anti‐rabbit IgG (H + L) Jackson ImmunoResearch Labs Cat# 711‐225‐152; RRID: AB_2340612; Cy3‐donkey anti‐mouse IgG (H + L) Jackson ImmunoResearch Labs Cat# 715‐165‐150; RRID: AB_2340813; Cy5‐donkey anti‐goat IgG (H + L) Jackson ImmunoResearch Labs Cat# 705‐175‐147; RRID: AB_2340415; Cy5‐donkey anti‐rabbit IgG (H + L) Jackson ImmunoResearch Labs Cat# 711‐175‐152; RRID: AB_2340607; Cy2‐donkey anti‐goat IgG (H + L) Jackson ImmunoResearch Labs Cat# 705‐001‐003; RRID: AB_2340383. The sections were counterstained with the nuclear dye DAPI (4′,6‐diamidino‐2‐phenylindole; 1:10000, Sigma‐Aldrich, Cat# D9542; RRID: AB_2307445).

2.3. Confocal Microscopy

Images were acquired using a Zeiss LSM 880 Airyscan confocal microscope (Carl Zeiss, Jena, Germany) with identical acquisition settings for control and experimental samples. Images were acquired with a 63X oil‐immersion objective (NA 1.4), and marker colocalization was assessed in z‐stacks comprising 22 optical sections per soma. For dendritic complexity analysis, images were acquired from 50 μm‐thick sections using a 63X objective. Z‐stacks consisted of 22 optical sections collected at 0.12 μm intervals. Dendritic length and branching were quantified from projections of the central 30 μm portions of three‐dimensional (3D) reconstructions onto a single plane in DG granule cells expressing DCX and/or tdTomato (TOM). For spine analysis, images were acquired using a Zeiss LSM 880 Airyscan microscope (63×) from 50 μm thick sections. Z‐stacks spanning 5–10 μm were collected at 0.1 μm intervals with the Airy unit set to 1. Three‐dimensional (3D) reconstructions of dendritic segments were generated, and dendritic spines were manually counted on dendritic fragments located within the middle third of the molecular layer. Mushroom spines were defined as spines with a prominent head and a long neck. Measurements of dendritic complexity were performed as previously described (Sholl 1953; Irala et al. 2016) using the NeuronJ plugin ImageJ software. Images of TOM‐labeled mossy fiber boutons (MFBs) in the CA3 region were acquired using a Zeiss LSM 880 Airyscan microscope with a 63× objective and a 2× digital zoom. Bouton area and filopodia number were quantified from projections of 3D reconstructions onto a single plane. Mossy fiber boutons larger than 3 μm and connected to the mossy fiber tract at least at one end were included in the analysis. Filopodia were identified as protrusions arising from the large mossy terminals. All image acquisition and morphological analyses were performed blind to experimental group identity.

2.4. PCR Analysis

Animals were euthanized by cervical dislocation, and the DG was dissected under a stereomicroscope. For PCR analysis, total RNA was isolated from the DG of WT mice using RNeasy Mini Kit columns (Qiagen), following the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using the High‐Capacity cDNA Reverse Transcription Kit with MultiScribe reverse transcriptase (Applied Biosystems). PCR analysis was performed using the following primers: mouse Etv5‐Forward: 5′‐AGA TGT CAG AGC CCG TTG TC‐3′; mouse Etv5‐Reverse: 5′‐CAT AGA GCG GGT CGT GGT AT‐3′; mouse Etv4‐Forward: 5′CAG CCC TTT TCC AGG GCA GAA C‐3′; mouse Etv4‐Reverse: 5′‐GCT GGA AGA CGG AGC TGT GCT C‐3′; mouse TATA‐binding protein (Tbp)‐Forward: 5′‐GGG GAG CTG TGA TGT GAA GT‐3′; Tbp‐Reverse: 5′‐CCA GGA AAT TCT GGC TCA‐3′.

2.5. Quantification and Statistical Analysis

All analyses were performed blind to the experimental condition. To ensure unbiased analysis, brain samples were coded with arbitrary numbers by a lab member not involved in the experiments. All quantifications were performed blind to genotype, which was revealed after data analysis was completed. Statistical tests used throughout the paper are described in the figure legends and in the text. Briefly, at least three 50 μm sections from the dorsal hippocampus were collected from each animal. Images were acquired from both hemispheres in the middle region of the suprapyramidal blade of the DG for analysis. Data were analyzed using GraphPad Prism 8.0.1, and are expressed as mean ± SEM. The n for each experiment is indicated in the figure legends, and statistical significance is shown as follows: *p < 0.05; **p < 0.01; ***p < 0.001. No statistical method was used to predetermine sample sizes, which were selected based on those commonly used in the field (Bonafina et al. 2020). Normality of residuals pooled across genotypes was assessed using the Kolmogorov–Smirnov test following Rochon et al. (2012) (Table S1). Two‐tailed unpaired Student‘s test was performed to assess statistical significance between two independent groups. For comparisons involving two independent variables, two‐way ANOVA followed by Sidak‘s multiple comparisons test was performed. No statistical test for outlier identification was applied, and all data points obtained were included in the analysis without exclusion.

3. Results

3.1. Etv5 Ablation Increases the Number of Immature Granule Cells in the Adult Dentate Gyrus

Based on previous findings showing Etv5 expression in different hippocampal regions, including the DG (Fontanet et al. 2018), one of the neurogenic niches in the adult brain, we investigated whether this transcription factor plays a role in adult hippocampal neurogenesis. To reveal whether newly generated cells express Etv5, we performed immunostaining on adult mouse hippocampal sections using antibodies against Doublecortin (DCX), a marker of immature neurons.

Etv5 expression was detected in all immature DCX⁺ neurons, suggesting that this factor may be involved in hippocampal adult neurogenesis (Figure 1A). We also analyzed the expression of Etv4, another member of the Pea3 family of transcription factors, which has been shown to be expressed in the DG (Fontanet et al. 2018). Our findings indicate that Etv4 is expressed in mature GC neurons of the DG, but not in newly generated DCX⁺ neurons (Figure S1).

FIGURE 1.

Deletion of Etv5 in neuronal precursors increases the proportion of immature DCX⁺ neurons in the dentate gyrus (DG). (A) Expression of Etv5 in the adult DG. Representative images showing Etv5 localization in coronal sections of the dentate gyrus from 2‐month‐old mice. Left: immunostaining for Etv5 (green) and DCX (doublecortin, magenta). Right: higher‐magnification images of the region outlined in the left panel. Insets show enlarged views of the boxed areas, showing Etv5 expression in the nuclei of DCX‐expressing neurons. Arrowheads indicate the Etv5⁺/DCX⁺ immature neurons. Scale bars: 100 μm (left panel) and 20 μm (right panels). (B) Representative images showing DCX⁺ immature neurons (red) in coronal sections of the DG from 2‐month‐old control (Nestin ^Cre :Etv5 ^+/+) and Etv5‐deficient (Nestin ^Cre :Etv5 ^f/f ) mice. Nuclei were counterstained with DAPI (blue). Higher‐magnification images of the outlined regions are shown. Scale bars: 200 and 50 μm respectively. (C) Quantification of the proportion of DCX⁺ neurons (DCX⁺/DAPI) in control and Etv5‐deficient mice. Bars represent mean ± SEM (n = 3 mice/genotype), *p < 0.05, two‐tailed Student's t‐test: t(4) = 3.387; p = 0.0276. (D) Representative images showing proliferating Ki67⁺ progenitor cells in coronal sections of DG from 2‐month‐old control and Etv5‐deficient mice. Nuclei were counterstained with DAPI (blue). Higher‐magnification images of the outlined regions containing Ki67⁺ nuclei are shown. Scale bars: 200 and 50 μm, respectively. (E) Quantification of the proportion of Ki67⁺ cells (Ki67⁺/DAPI) in control and Etv5‐deficient mice. Bars represent mean ± SEM (n = 3 mice/genotype), ns, not significant, two‐tailed Student's t‐test: t(4) = 0.5771; p = 0.594.

To assess whether Etv5 could play a role in this process, we evaluated the number of DCX⁺ cells in adult Etv5‐deficient mice. To this end, conditional Etv5 mutant mice were generated by crossing Nestin ^Cre mice (Tronche et al. 1999), which express Cre recombinase in neuronal progenitors, with mice carrying floxed Etv5 alleles (Etv5 ^f/f ) (Zhang et al. 2009). This strategy resulted in Nestin ^Cre :Etv5 ^f/f mice which represent a model in which Etv5 is deleted in the whole neural lineage due to recombination in neural stem progenitor cells during development (Fontanet et al. 2018).

We then compared the number of DCX⁺ neurons in the adult DG of Nestin ^Cre :Etv5 ^f/f mice and control littermates (Nestin ^Cre :Etv5 ^+/+ ). While Etv5‐deficient mice exhibited a significant increase in the number of DCX⁺ cells (Figure 1B,C), no differences were observed in the number of mature NeuN⁺ neurons or GFAP⁺ (Glial Fibrillary Acidic Protein) astrocytes (Figure S2). Similar analysis was done on Etv4‐deficient mice (Livet et al. 2002) and consistent with the undetectable expression of Etv4 in newly generated DCX⁺ neurons, no change in their number was observed in Etv4 ^−/−mutant mice (Figure S3).

In order to analyze whether the increased number of immature neurons observed in the Nestin ^Cre :Etv5 ^f/f could be due to altered proliferation of neuronal progenitors, we analyzed the subgranular zone (SGZ) for dividing cells. We used Ki67, a marker of actively cycling cells, to identify proliferating progenitors. No differences in the proportion of Ki67⁺ cells were detected between control and Etv5‐deficient animals, indicating that the increase in DCX⁺ cells is not due to enhanced proliferation of adult hippocampal progenitors (Figure 1D,E).

3.2. Etv5 Is Mainly Expressed in Immature and Mature Adult‐Born Granule Cells in the Dentate Gyrus

To analyze the expression of Etv5 specifically in adult‐born GCs, we utilized a tamoxifen (TAM)‐inducible Cre line (Glast ^CreERT2 ), in which CreERT2 is expressed under the control of the astrocyte‐specific glutamate transporter (GLAST) (Mori et al. 2006). GLAST is known to be expressed in neural stem cells in the adult brain and in GFAP⁺ astrocytes (Bonaguidi et al. 2012; NA et al. 2013). This line was mated with the Rosa26‐tdTomato (CAG ^{floxStoptdTom} ) reporter transgenic mice (Madisen et al. 2010), allowing conditional expression of the fluorescent reporter tdTomato (TOM) in cells undergoing recombination.

Following TAM administration, TOM signal enabled us to trace the development of newborn GCs in the DG. These labeled cells were analyzed over time based on their location, morphology, and expression of cell‐type‐specific markers (Figure S4).

To characterize Etv5 expression during the transition from stem/progenitor cells to mature granule neurons in the adult DG, we performed immunostaining on brain sections from two‐month‐old Glast ^CreER2Tom mice, 21 days post TAM injection (dpi). To examine whether radial glia‐like cells (RGCs) express Etv5, we performed co‐immunostaining for GFAP, a marker expressed in the adult brain by both astrocytes and RGCs. RGCs were identified in the DG based on their characteristic morphology with the cell bodies located in the subgranular zone (SGZ) and a single radial process extending through the granule cell layer (GCL) with branching processes reaching the outer GCL and molecular layer (ML), and some basal processes projecting into the hilus (Gebara et al. 2016; Kriegstein and Alvarez‐Buylla 2009). We did not observe nuclear Etv5 expression in GFAP⁺ RGCs. Intriguingly, approximately 40% of GFAP⁺ processes showed Etv5 staining (Figure 2A–C).

FIGURE 2.

Characterization of Etv5 expression in adult‐born granule cells of the dentate gyrus (DG). (A) Schematic representation of the adult hippocampal neurogenic lineage, including radial glia‐like cells (RGC), intermediate progenitor cells (IPCs), newly generated immature and mature granule cells. The expression patterns of markers used to characterize the different stages of neuronal maturation are indicated. ML, molecular layer; GCL, granular cell layer; SGZ, subgranular zone. (B) Analysis of Etv5 expression in adult‐born cells at different stages of maturation in the dentate gyrus (DG). Tamoxifen (TAM) was administered intraperitoneally (i.p.) to 2‐month‐old Glast ^CreERT2Tom mice, and animals were analyzed at 21 days post‐injection (dpi) by immunofluorescence and confocal microscopy. Representative images show Etv5 expression (gray) together with stage‐markers (green) in coronal sections of the DG. Nuclei were counterstained with DAPI (blue), and adult‐born cells expressing tdTomato (TOM) are shown in red. Scale bar: 15 μm. White arrows indicate TOM⁺ cells positive or negative for the indicated marker (green). Red arrows indicate the same TOM⁺ cells and highlight the presence or absence of nuclear Etv5 expression (gray). (C) Quantification of the proportion of the adult‐born cells (TOM⁺) expressing nuclear Etv5 at different stages of maturation (Etv5⁺GFAP⁺/GFAP⁺, Etv5⁺SOX2⁺/SOX2⁺, Etv5⁺DCX⁺/DCX⁺, Etv5⁺NeuN⁺/NeuN⁺). Data are presented as mean ± SEM.

To assess Etv5 expression in neurogenic progenitor populations, we analyzed SOX2⁺ cells, which label both radial glia‐like and early intermediate progenitor cells (IPCs). Notably, Etv5 immunoreactivity was very low or undetectable in the vast majority of SOX2⁺ cells, with only a small minority of nuclei showing detectable Etv5 expression, indicating that similar to our findings in GFAP⁺ RGCs, Etv5 is expressed in only a minor subset of SOX2⁺ progenitor populations. We then assessed Etv5 expression in postmitotic adult‐born neurons. Etv5 was prominently detected in the nuclei of TOM⁺ neurons that expressed either DCX (immature GCs) or NeuN (mature GCs). In addition, Etv5 was clearly localized in the nuclei of preexisting hippocampal granule neurons (Figure 2).

Together, these results indicate that Etv5 is expressed in only a subset of RGCs and neural progenitors but becomes upregulated as neurons exit the cell cycle and begin the maturation process. This observation also supports a role for Etv5 in the postmitotic stages of adult hippocampal neurogenesis.

3.3. Etv5 Is Required for Proper Maturation of Adult‐Born Granule Cells

To investigate the role of Etv5 in the maturation of newly generated neurons in the adult hippocampus, we generated a conditional‐inducible mouse model for the deletion of Etv5 in the newborn cells by crossing Glast ^CreERT2Tom line with Etv5 ^f/f mice to generate Glast ^CreERT2Tom :Etv5 ^f/f animals (Figure S5A). This strategy enabled the conditional deletion of Etv5 in newly born granule neurons with lineage tracing, allowing us to track their development through the expression of specific markers, morphology, and synaptic maturation. As controls, we used Glast ^CreERT2Tom :Etv5 ^+/+ mice. Etv5 ablation in Glast ^CreERT2Tom :Etv5 ^f/f mice was confirmed by anti‐Etv5 immunostaining in the DG (Figure S5B).

To determine whether Etv5 ablation affects the total number of newly generated GCs, the number of TOM⁺ cells was quantified in two‐month‐old Glast ^CreERT2Tom :Etv5 ^f/f and control (Glast ^CreERT2Tom :Etv5 ^+/+ ) mice injected with TAM at 21 and 28 dpi. No significant differences were observed in the total number of TOM⁺ cells between the groups, suggesting that Etv5 is not essential for the generation/survival of adult‐born GCs (Figure 3A).

FIGURE 3.

Etv5 is required for the correct maturation of adult‐born granule cells (GCs). (A) Bar graphs show the number of cells expressing the reporter marker tdTomato (TOM)/area in control (Glast ^CreERT2Tom :Etv5 ^+/+), indicated as Etv5 ^+/+ and Etv5‐deficient (Glast ^CreERT2Tom :Etv5 ^flox/flox ) mice, indicated as Etv5 ^f/f , at 21 and 28 dpi (days post‐injection). Data are presented as mean ± SEM (n = 3–4 mice per genotype). ns, not significant, two tailed Student's t‐test. 21 dpi graph: t(6) = 0.619, p = 0.558.28 dpi graph: t(4) = 0.963, p = 0.390. (B, C) Representative confocal images at 21 dpi showing TOM⁺ cells labeled with GFAP in control (Etv5 ^+/+ ) and Etv5‐deficient mice (Etv5 ^f/f ) (B). Blue corresponds to DAPI‐stained nuclei. Arrowheads indicate TOM⁺GFAP⁺. Scale bar: 20 μm. The bar graph shows the proportion of total TOM⁺ cells expressing GFAP in Etv5 ^+/+ and Etv5 ^f/f mice (C). Data are presented as mean ± SEM (n = 3 mice per genotype); ns indicates no significant difference by Student's t‐test: t(4) = 0.683, p = 0.167. (D‐E) Representative confocal images at 21 dpi showing TOM⁺ cells labeled with SOX2 in control (Etv5 ^+/+ ) and Etv5‐deficient mice (Etv5 ^f/f ) (D). Blue corresponds to DAPI‐stained nuclei. Arrowheads indicate TOM⁺SOX2⁺. The bar graph shows the proportion of total TOM⁺ cells expressing SOX2 in Etv5 ^+/+ and Etv5 ^f/f mice (E). Data are presented as mean ± SEM (n = 3 mice per genotype); ns indicates no significant difference by Student's t‐test: t(4) = 0.623, p = 0.566. (F and H) Representative confocal images of GCs expressing the reporter marker TOM stained with DCX (cyan) at 21 (F) and 28 (H) dpi in control (Etv5 ^+/+ ) and Etv5 ^f/f mice. Higher magnification images taken from the section outlined containing DCX⁺ immature neurons, are also shown. Arrowheads indicate TOM⁺DCX⁺ cells. Scale bars: 50 and 20 μm respectively. (G and I) Bar graphs show the proportion of newly generated immature neurons (TOM⁺DCX⁺/TOM⁺). Data are presented as mean ± SEM (n = 3–4 mice per genotype). *p < 0.05, by Student's t‐test: t(6) = 3.233, p = 0.017 (G); t(4) = 2.967, p = 0.041 (I). (J) Representative confocal images at 28 dpi showing TOM‐expressing cells labeled with NeuN (green) in Etv5 ^+/+ and Etv5 ^f/f animals. (J´) Higher magnification images taken from the section outlined in J. The images show staining with NeuN (green) and DCX (cyan). White arrows indicate TOM⁺NeuN⁺ neurons with mature dendritic processes outside the granule cell layer (*). Yellow arrows indicate immature DCX⁺NeuN⁻ cells. Scale bars: 40 and 20 μm. (K) Bar graphs show the proportion of newly generated mature neurons, defined as TOM⁺DCX⁻ cells displaying mature dendritic morphology/TOM⁺ at 21 and 28 dpi. The mature neuronal identity of these cells was confirmed by positive NeuN immunoreactivity, as shown in J. Data are presented as mean ± SEM (n = 3–4 mice per genotype). *p < 0.05, **p < 0.01 by Student's t‐test: t(6) = 5.421, p = 0.0016 (21 dpi); t(4) = 2.932, p = 0.0427 (28 dpi).

Consistent with the absence of Etv5 expression in the nuclei of both quiescent and proliferating neuronal progenitors, we found no differences in either the density of RGCs labeled with GFAP and TOM or the proportion of SOX2⁺/TOM⁺ cells between Glast ^CreERT2Tom :Etv5 ^f/f and control mice at 21 dpi (Figure 3B–E). Additionally, we found no differences in the proportion of another RGC marker, Nestin, between control and Etv5‐deficient mice (Figure S6). This time point was chosen because it represents a window during which cells at various stages of maturation can be observed within the neurogenic lineage.

Given our previous observation indicating that neuronal Etv5 deletion in Nestin ^Cre :Etv5 ^f/f mice leads to an increased number of DCX⁺ immature neurons, we further examined DCX⁺ cells in animals with Etv5 deletion restricted to adult‐born neurons. We assessed the number of DCX⁺TOM⁺ neurons at 21 and 28 dpi, and observed a significant increase in the proportion of newly generated immature neurons at both developmental stages (Figure 3F–I). Interestingly, we also found a marked reduction in the number of newly generated mature neurons in Glast ^CreERT2Tom :Etv5 ^f/f mice, defined as TOM⁺DCX⁻ cells exhibiting mature dendritic morphology, as evidenced by well‐elaborated dendritic arbors and positive immunolabeling for the neuronal marker NeuN (Figure 3J,K). These findings suggest a defect in the developmental transition from DCX‐expressing immature neurons to mature neuronal populations.

Overall, our results suggest that while Etv5 is not required for the survival or early specification of adult‐born GCs, its absence delays the transition of immature GCs (DCX⁺) into mature neurons, thereby leading to an accumulation of immature DCX⁺ neurons.

The increase in DCX⁺ neurons observed in TAM‐inducible Glast ^CreERT2Tom :Etv5 ^f/f mice, in which Etv5 is specifically deleted in newly born neurons of the adult DG, together with the previously reported increase in DCX⁺ neurons observed in Nestin ^Cre :Etv5 ^f/f mice (Figure 1B), indicates that the effect of Etv5 on these immature cells is cell‐autonomous and does not rely on its deletion in pre‐existing cells within the DG niche.

3.4. Etv5 Deletion Disrupts the Morphological Development of Newborn Granule Cells in Mature Neural Circuits

The integration of newborn GCs into the DG circuitry follows a well‐characterized developmental trajectory, progressing through distinct morphological and functional stages (Esposito et al. 2005; Piatti et al. 2011). Given that Etv5 has been involved in regulating dendritic maturation in hippocampal pyramidal neurons (Fontanet et al. 2018), we examined whether its ablation impacts dendritic development in adult‐born GCs.

To investigate whether Etv5 deletion affects the morphological maturation of adult‐born hippocampal neurons, we analyzed dendritic arborization in DCX⁺TOM⁺ neurons from Etv5‐deficient (Glast ^CreERT2Tom : Etv5 ^f/f ) and control mice at 21 and 28 dpi. Although total dendritic length remained unchanged at both time points, a reduction in dendritic complexity was observed in Etv5‐deficient GCs at 28 dpi, as evidenced by Sholl analysis (Sholl 1953) (Figure 4A–G). These findings support a role for Etv5 in modulating the morphological maturation of newly generated granule cells as they progress through distinct stages of postmitotic development.

FIGURE 4.

Etv5 is required for proper dendritic development in adult‐born GCs. (A) Diagram representing the input and output connections of newly generated GCs in the hippocampus. DG, dentate gyrus; GCs, granule cells; MFBs, mossy fiber boutons. (B and E) Representative drawings of DCX⁺TOM⁺ adult‐born GCs derived from Control (Etv5 ^+/+ ) and Etv5‐deficient (Etv5 ^f/f ) mice at 21 (B) and 28 (E) dpi (days post‐injection). Scale bar: 10 μm. (C and F) Graphs showing the quantification of dendritic length in control (Etv5 ^+/+ ) and Etv5 mutant (Etv5 ^f/f ) littermates at 21 (C) and 28 (F) dpi. The results are shown as mean ± SEM. About 8–15 neurons per mouse were analyzed from 3 to 4 mice per genotype; ns indicates no significant difference, by Student's t‐test: t(60) =1.489, p = 0.141 (21 dpi); t(57) = 1.331; p = 0.188 (28 dpi). (D and G) Sholl analysis of the dendritic arbors of DCX⁺TOM⁺ cells in control (Etv5 ^+/+ ) and Etv5‐deficient mice (Etv5 ^f/f) at 21 (D) and 28 (G) dpi. Results are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 were determined by Two‐way ANOVA, followed by Sidak's multiple comparison test: 28 dpi; t(38) = 3.622, 4.850, 7.062, 7.062, 6.380, 4.531 and 3.349. p = 0.0169, 0.0004, < 0.0001, < 0.0001, < 0.0001, 0.0011, and 0.0361. (H) Representative low‐magnification confocal image of calbindin‐immunostained (green) coronal sections of the hippocampal CA3 region of control animals at 28 dpi. Dotted lines demarcate the Stratum Pyramidale (SP) and Stratum Lucidum (SL). Scale bar: 50 μm. A higher magnification image of the boxed CA3 region is shown on the right. Arrows indicate Mossy Fiber Boutons (MFBs, red) within the SL. Nuclei are stained with DAPI (blue, *). Scale bar: 30 μm. (I) Representative confocal images of MFB (TOM⁺) in CA3 of control and Etv5‐deficient mice at 28 dpi. Arrowheads indicate MFBs (red) and asterisks (*) mark the nuclei of CA3 cells stained with DAPI (blue). Scale bar: 10 μm. (J) The bar graphs show the quantification of the MFB area at 21 and 28 dpi. Three animals from each genotype were analyzed. Results are presented as mean ± SEM and analyzed by two‐tailed Student's t‐test: t(4) =1.462, p = 0.217 (21 dpi); t(4) = 1.533, p = 0.2001 (28 dpi). ns indicates no significant difference.

Next, we studied the output connections from newly generated DG neurons to the CA3 region. Granule cells establish glutamatergic excitatory synapses onto CA3 pyramidal neurons through large mossy fiber boutons (MFBs), whereas thin filopodial extensions from these MFBs contact nearby GABAergic interneurons, mediating feedforward inhibition (Acsady et al. 1998; Lawrence and McBain 2003; Restivo et al. 2015; Toni et al. 2008). To analyze the output connectivity of newly generated GCs, we used confocal imaging to quantify the size of MFBs and the associated filopodial extensions in the stratum lucidum (SL), where the axons originating from the DG granule cells (Calbindin⁺) form synaptic contacts with the apical dendrites of CA3 pyramidal neurons (Figure 4H). Our analysis revealed no significant differences in the number of filopodia between control and Etv5‐deficient mice at either of the two developmental time points examined (data not shown). Although not statistically significant, we observed a trend toward reduced MFB area (Figure 4I,J) that might reflect an immature status of these connections.

3.5. Etv5 Is Required for the Maturation of Excitatory Synaptic Contacts in Newborn Granule Cells

Dendritic spines are the principal postsynaptic sites through which granule neurons receive glutamatergic inputs, and the timing of their formation and maturation may reflect a critical aspect of their functional integration. In neurons generated in the adult hippocampus, dendritic spines are clearly present at 21 dpi. On the other hand, it has also been reported that BDNF signaling, which regulates the expression of Etv5 in hippocampal pyramidal neurons, is essential for the proper development of dendritic spines in newly generated neurons in the adult DG (Bergami et al. 2008). To investigate the development of dendritic spines in newborn neurons, we focus on spine density and shape. Dendritic spines exhibit substantial morphological diversity and are commonly categorized into four types based on their morphology: mushroom, thin, stubby, and filopodia (Nimchinsky et al. 2002; Sala 2002). Filopodia and stubby spines are often associated with early stages of neuronal development, whereas mushroom spines are more abundant in mature neurons (Nimchinsky et al. 2002). We analyzed spine density and morphology in TOM⁺ newborn GCs from Glast ^CreERT2Tom :Etv5 ^f/f and control mice at 21 and 28 dpi. Our findings showed no significant difference in total spine density between control and Etv5‐deficient mice. While the total spine density did not differ significantly between groups, we observed a marked reduction in the density of mushroom‐shaped spines in Etv5‐deficient neurons. This reduction in the density of more mature spines was accompanied by a significant increase in the number of stubby‐type spines in Etv5‐deficient GCs (Figure 5A–F). As expected, spine density increased over the course of maturation from 21 to 28 dpi.

FIGURE 5.

Etv5 is required for proper dendritic spine development in adult‐born granule cells (GCs). (A and D) Representative confocal images of dendritic segments from adult GCs derived from control and Etv5‐deficient mice at 21 (A) and 28 (D) dpi (days post injection). Arrowheads indicate mushroom spines. Scale bar: 5 μm. (B and E) Bar graphs show the quantification of total spine along 100 μm of dendrite length in neurons from control (Etv5 ^+/+) and Etv5‐deficient (Etv5 ^f/f ) mice at 21 (B) and 28 (E) dpi. Data represent mean ± SEM (n = 3 animals/genotype). Statistical analysis was performed using a two‐tailed Student's t‐test: t(4) = 1.113, p = 0.328 (21 dpi); t(4) = 1.027, p = 0.362 (28 dpi). ns: indicates no significant difference. (C and F) Quantification of the four main types of dendritic spines (mushroom, thin, stubby, and filopodium) along 100 μm of dendritic length in neurons from control (Etv5 ^+/+) and Etv5‐deficient (Etv5 ^f/f ) mice. Data represent the mean ± SEM (n = 3 animals/genotype). *p < 0.05; **p < 0.01; ns indicates no significant difference. Statistical analysis was performed using a two‐tailed Student's t‐test: 21 dpi: t(4) = 2.992, p = 0.040 (mushroom); t(4) = 1.021, p = 0.365 (thin); t(4) = 5.125, p = 0.006 (stubby); t(4) = 1.732, p = 0.158 (filopodium). 28 dpi: t(4) = 4.858, p = 0.008 (mushroom); t(4) = 0.541, p = 0.617 (thin); t(4) = 3.787, p = 0.019 (stubby); t(4) = 2.162, p = 0.096 (filopodium).

Altogether, these results suggest that Etv5 plays a critical role in the proper maturation of dendritic spines in adult‐born GCs, suggesting that it could be involved in the integration of newborn GCs in the preexisting hippocampal circuits.

4. Discussion

In the present study, we investigated the impact of Etv5 deletion on adult hippocampal neurogenesis, a process implicated in memory acquisition, particularly in tasks that require pattern separation, the ability to distinguish between similar experiences or stimuli by generating distinct memory representations. Here, we demonstrate that the expression of Etv5 is essential for the proper maturation of adult‐born hippocampal neurons. Using Nestin ^Cre :Etv5 ^f/f mice, in which Etv5 is deleted in all cells derived from neural precursors, we observed an increased number of immature neurons (DCX⁺) in the DG of the adult hippocampus. However, the number of proliferating cells was not affected, ruling out enhanced proliferation as the cause of the increase in immature neurons (Figure 1). Analysis of Etv5 expression in newly generated neurons revealed its presence in the nucleus of developing neuronal cohorts in the DG, with expression observed mainly in the nuclei of both immature granule neurons (DCX⁺) and mature neurons (Figure 2). In contrast to its known association with proliferative processes, this transcription factor was not detected in SOX2⁺ neural progenitors. Intriguingly, Etv5 immunoreactivity was detected in the radial processes of a subset of GFAP⁺ RGCs, an observation that will require further investigation.

Targeted deletion of Etv5 specifically in adult‐born hippocampal neurons using Glast ^CreERT2Tom :Etv5 ^f/f resulted in delayed maturation, evidenced by an accumulation of DCX⁺ neurons, indicating that this effect is cell‐autonomous and not due to changes in the neurogenic niche (Figure 3).

Morphological analysis further revealed defects in neuronal differentiation. Reduced dendritic complexity, as well as impairments in synaptic spine development, were observed in Etv5‐deficient GCs (Figures 4 and 5). Although overall spine density was not significantly altered, we observed a significant reduction in the density of mature spines and an increase in immature spines in mutant mice (Figure 5).

4.1. Etv5 in Nervous System Development

Etv5 is a transcription factor highly expressed in the developing brain, whose expression is tightly regulated under physiological conditions through transcriptional and post‐translational mechanisms. Its dysregulation has been associated with the initiation and progression of various cancers (Wei et al. 2023). Depending on cellular context, Etv5 can promote either proliferation or differentiation (Qi et al. 2020; Wei et al. 2023), and together with Etv4, has been shown to regulate both processes in embryonic stem cells (Akagi et al. 2015).

The observation of Etv5 in the radial processes of a proportion of RGCs raises interesting possibilities beyond its established nuclear role. This could reflect a cytoplasmic function, as described for other transcription factors such as p27Kip1, which promotes actin dynamics and process outgrowth through interactions with RhoA and Stathmin (Besson et al. 2004; Serres et al. 2012), or the ETS family member ESE‐1, which transforms mammary epithelial cells through a non‐transcriptional cytoplasmic mechanism (Prescott et al. 2011). Alternatively, cytoplasmic localization may represent a nucleocytoplasmic shuttling as a means of regulating transcriptional activity. Distinguishing between these possibilities will require further investigation.

As a member of the Pea3 subfamily, Etv5 acts alongside Etv1 and Etv4 in nervous system development, with overlapping and independent expression patterns that suggest both functional cooperation and unique roles (Kandemir and Kurnaz 2025). The three subfamily members are target genes and mediators of different neurotrophic factors. Thus, Etv1 is induced by neurotrophin‐3 (NT3) in dorsal root ganglion (DRG) proprioceptive neurons (de Nooij et al. 2013), Etv4 by GDNF in spinal motor neurons, and both Etv4 and Etv5 by BDNF and NGF through the MEK/MAPK pathway in hippocampal pyramidal and DRG sensory neurons, respectively (Fontanet et al. 2013, 2018; Haase et al. 2002). Loss‐of‐function studies of Etv4 or Etv5 in hippocampal pyramidal neurons and in sensory neurons reveal defects in neuritic growth, indicating that these factors exert non‐redundant roles that cannot be fully compensated by other Pea3 members (Fontanet et al. 2013, 2018). Nevertheless, we acknowledge that compensatory effects cannot be entirely excluded in other neuronal systems.

Regarding Etv5, BDNF‐driven expression regulates dendritic arbor complexity and synaptic spine development in hippocampal pyramidal neurons, and Etv5‐deficient animals exhibit significant cognitive and social behavioral deficits (Fontanet et al. 2018). Interestingly, Etv5 also mediates BDNF‐induced neurite outgrowth and neurotransmitter gene expression (Liu et al. 2016). In line with its role in neuronal differentiation, our group has described that NGF induces Etv5 expression to upregulate matrix metalloproteinases required for proper neuronal growth in sensory neurons (Fontanet et al. 2013). Together, these findings establish Etv5 as an essential transducer of neurotrophin signaling for neuronal morphology, synaptic connectivity, and higher brain function.

4.2. Etv5 as a Key Regulator of Adult Hippocampal Neurogenesis

The incorporation and proper integration of adult‐born neurons into hippocampal circuits highlights the brain's remarkable ability for both structural and functional plasticity. Notably, impaired neurogenesis is linked to pathological conditions like depression, Alzheimer's disease, or cognitive decline (Toda et al. 2019; Toda and Gage 2018). This multi‐stage process, encompassing neural stem cell proliferation and differentiation into mature granule neurons, is tightly regulated by both intrinsic transcription factors and extrinsic signals, including trophic factors like BDNF, IGF1, and GDNF, which support the proper integration of the newborn neurons into the preexisting circuit (Bonafina et al. 2020; Toda and Gage 2018).

Given that Etv5 is expressed in newborn hippocampal neurons and has been implicated in both proliferation and differentiation, we hypothesized that it may play a critical role in modulating adult hippocampal neurogenesis. Our results show that Etv5 regulates the maturation of adult‐born neurons without affecting cell survival. Consistent with its role in hippocampal pyramidal neurons, Etv5 deficiency in newborn DG neurons produces similar morphological defects in dendritic complexity and spine development. The phenotypic resemblance between Etv5‐deficient mice and animals lacking TrkB in newborn DG neurons further supports Etv5 as a downstream mediator of BDNF/TrkB signaling in these cells (Bergami et al. 2008).

Capicua (CIC), a direct transcriptional repressor of Pea3 transcription factors including Etv5, plays an important role in hippocampal development. Consistent with our previous findings, loss and gain of function experiments have shown that CIC inhibits dendritic morphogenesis and spine formation in pyramidal hippocampal neurons through regulation of Etv4 and Etv5 (Li et al. 2021). Within the adult neurogenic lineage, CIC and Etv5 display complementary and stage‐specific expression patterns: CIC in proliferating progenitors and mature neurons, Etv5 in immature DCX⁺ neurons and both co‐expressed in mature granule cells of the DG, suggesting convergence on the same regulatory pathway at later stages of maturation. Notably, conditional Cic deletion in glutamatergic precursors produces accumulation of DCX⁺ immature neurons and impaired dendritic arborization (Hourigan et al. 2021), a phenotype strikingly similar to that observed upon Etv5 ablation. Although CIC represses Etv5, its loss does not imply equivalent mechanisms. As CIC controls multiple targets beyond Etv5, causing broad transcriptional deregulation, its deletion affects progenitor stages where Etv5 is not yet expressed, likely through Etv5‐independent mechanisms. Furthermore, the Emx1‐Cre model drives recombination from embryonic stages and deletes CIC across the entire hippocampal niche, altering the adult neurogenic microenvironment. This is fundamentally different from our adult‐specific conditional ablation, which targets Etv5 exclusively in adult‐born granule cells, leaving the surrounding niche intact. Together, these differences likely explain the divergence in findings, and highlight how distinct genetic models can provide complementary insights into the regulation of adult‐born neuron maturation.

It is well established that adult‐born granule cells in the DG require approximately 6–8 weeks to achieve full morphological and functional maturation (Toni et al. 2008; Zhao et al. 2006). The phenotypes we describe, increased DCX⁺ neurons, reduced dendritic complexity and spine deficits, reflect Etv5 deletion during this period. However, whether these phenotypes reflect a true maturation arrest or merely a transient delay, with neurons eventually completing their development, remains an open question. Distinguishing between these possibilities will require future studies extending the analysis to the 6–8 weeks timepoint, by which adult‐born granule cells normally achieve full morphological and functional maturity.

Adult hippocampal neurogenesis plays a crucial role in learning and memory, is implicated in anxiety and depression, supports cognitive flexibility, and is associated with neurodegenerative diseases, making it a field of profound scientific and clinical relevance. Advancing our understanding of the neurogenic process is essential for uncovering both its functional significance and the biological consequences of its dysregulation.

Author Contributions

Antonella S. Ríos: formal analysis, investigation, methodology, writing – review and editing, visualization. Solana F. López: formal analysis, investigation, methodology, writing – review and editing. Fernando Federicci: investigation, methodology, formal analysis, writing – review and editing. Paula A. Fontanet: formal analysis, investigation, methodology, writing – review and editing. Gustavo Paratcha: formal analysis, funding acquisition, methodology, resources, supervision, writing – original draft, writing – review and editing, visualization, project administration. Fernanda Ledda: conceptualization, formal analysis, funding acquisition, methodology, project administration, resources, supervision, writing – original draft, writing – review and editing, visualization.

Funding

This work was supported by grants from the Argentine National Agency for the promotion of Science and Technology‐ANPCyT; PICT 2019‐1472, PICT 2019‐4597, PICT 2020‐1524, PICT 2021‐00627, and Fundación Instituto Leloir. F.L. and G.P. are supported by an Independent Career Position from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). A.S.R., S.F.L., F.F., and P.A.F were supported by ANPCyT and CONICET fellowships.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Expression of Etv4 in the adult DG. Representative images showing localization of Etv4 in coronal sections of dentate gyrus (DG) from 2‐month old mice.

Figure S2: Deletion of Etv5 in neural precursors does not affect the proportion of mature neurons or astrocytes in the dentate gyrus (DG).

Figure S3: Deletion of Etv4 does not alter the proportion of DCX+ immature neurons in the dentate gyrus (DG).

Figure S4: Description of the model used to analyze newborn neurons in the adult hippocampus.

Figure S5: Description and characterization of the conditional Etv5 inactivation in adult‐born dentate gyrus (DG) cells.

Figure S6: Analysis of nestin expression by immunostaining in control and Etv5‐deficient mice during adult hippocampal neurogenesis.

Table S1: Assessment of data normality.

Data Availability Statement

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.