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. 2025 Jan 29;21(3):1151–1161. doi: 10.4103/NRR.NRR-D-24-00623

Cell type–dependent role of transforming growth factor-β signaling on postnatal neural stem cell proliferation and migration

Kierra Ware 1, Joshua Peter 1, Lucas McClain 1, Yu Luo 1,2,3,*
PMCID: PMC12296437  PMID: 39885664

graphic file with name NRR-21-1151-g001.jpg

Keywords: adult neurogenesis, doublecortin, hippocampus, migration, neural stem cells, proliferation, transforming growth factor-β

Abstract

Adult neurogenesis continuously produces new neurons critical for cognitive plasticity in adult rodents. While it is known transforming growth factor-β signaling is important in embryonic neurogenesis, its role in postnatal neurogenesis remains unclear. In this study, to define the precise role of transforming growth factor-β signaling in postnatal neurogenesis at distinct stages of the neurogenic cascade both in vitro and in vivo, we developed two novel inducible and cell type-specific mouse models to specifically silence transforming growth factor-β signaling in neural stem cells in (mGFAPcre-ALK5fl/fl-Ai9) or immature neuroblasts in (DCXcreERT2-ALK5fl/fl-Ai9). Our data showed that exogenous transforming growth factor-β treatment led to inhibition of the proliferation of primary neural stem cells while stimulating their migration. These effects were abolished in activin-like kinase 5 (ALK5) knockout primary neural stem cells. Consistent with this, inhibition of transforming growth factor-β signaling with SB-431542 in wild-type neural stem cells stimulated proliferation while inhibited the migration of neural stem cells. Interestingly, deletion of transforming growth factor-β receptor in neural stem cells in vivo inhibited the migration of postnatal born neurons in mGFAPcre-ALK5fl/fl-Ai9 mice, while abolishment of transforming growth factor-β signaling in immature neuroblasts in DCXcreERT2-ALK5fl/fl-Ai9 mice did not affect the migration of these cells in the hippocampus. In summary, our data supports a dual role of transforming growth factor-β signaling in the proliferation and migration of neural stem cells in vitro. Moreover, our data provides novel insights on cell type–specific-dependent requirements of transforming growth factor-β signaling on neural stem cell proliferation and migration in vivo.

Introduction

During CNS development, neural stem cells proliferate, differentiate, and mature into new neurons through a process called neurogenesis (Amanollahi et al., 2023). It is now widely accepted that neurogenesis also occurs in adulthood in many mammalian species at two niches in the brain, the subgranular zone (SGZ) of the dentate gyrus in the hippocampus and the subventricular zone (SVZ) of the lateral ventricles (Amanollahi et al., 2023). The process of the neurogenic cascade starts with quiescent neural stem cells becoming activated to proliferate and divide into transient amplifying progenitor cells and differentiate into neuroblasts which eventually survive, migrate, and mature into functional neurons (Kase et al., 2020). The journey of quiescent neural stem cells to a mature neuron in adult neurogenesis takes approximately 6–8 weeks in the SGZ of the hippocampus (Wang et al., 2021). These adult-born neurons integrate into the neuronal circuitry of the hippocampus and have been implicated in important functions such as spatial and object recognition memory (Portero-Tresserra et al., 2023), pattern separation (Yagi et al., 2023), forgetting (Wang et al., 2020; Scott et al., 2021), and emotion (Li et al., 2022).

Adult neurogenesis is tightly regulated by its microenvironment with a complex interplay of cellular and molecular mechanisms yet to be fully characterized. One mechanism known to play a role in regulating proliferation and differentiation in different cell types is the transforming growth factor-β (TGF-β) pathway (Tan et al., 2021; Wang et al., 2022b). The TGF-β signaling pathway is known to be crucial for neurogenesis during development (Meyers and Kessler, 2017) while its part in postnatal neurogenesis remains elusive. The TGF-β superfamily pathway contains 7 type I receptors, termed activin-like kinase 1-7 (ALK1-ALK7), 5 different type II receptors, and over 30 ligands that bind different combinations of these receptors. Specifically, for the TGF-β subfamily, the binding of a TGF-β ligand brings together two pairs of type 1 (TGFβR1- also known as ALK5) and type 2 receptors (TβRII) to form a heterotetrametric complex and initiate its signaling cascade. In this cascade, R-Suppressor of Mothers Against Decapentaplegic (R-SMAD) proteins (SMAD2 and SMAD3) become phosphorylated and form a co-SMAD complex with Smad4 to translocate into the nucleus and regulate the transcription of target genes (Tzavlaki and Moustakas, 2020). The precise role of TGF-β signaling in adult neurogenesis in vivo is unclear due to the early lethal phenotype in the global TGF-β1 knockout (KO) mice (Brionne et al., 2003). Due to the lack of neural stem cell (NSC)-specific gene KO models, previous studies using pharmacological molecules or non-NSC-specific conditional KO (cKO) mice have led to some conflicting results (Buckwalter et al., 2006; Wachs et al., 2006; He et al., 2014; Kandasamy et al., 2014). Specifically, enhancing TGF-β signaling by chronic infusion leads to inhibition of neurogenesis (Wachs et al., 2006) while ablation of TGF-β signaling in CAMK2A+ mature neurons and immature neuroblasts also leads to compromised neurogenesis in the adult brain (He et al., 2014; Kandasamy et al., 2014). Factors that could contribute to the conflicting results are that pharmacological modulations affect multiple cell types simultaneously, potentially leading to indirect effects on NSCs. Similarly, some of the previously utilized Cre mouse models are not specific to adult NSCs, which results in the deletion of the Alk5 gene (the type I receptor of the TGF-β ligand) in mature neurons. Since neuronal activity is known to indirectly modulate adult neurogenesis (Van der Borght et al., 2005; Luo et al., 2021), this could make the results from previous studies difficult to interpret. Therefore, although these studies support the importance of TGF-β signaling in regulating adult neurogenesis, to understand the precise and causal role of this signaling pathway in distinct stages of neurogenesis, it is critical to develop cell type-specific and inducible gene modulation models to study TGF-β signaling in adult neurogenesis. In this study, we developed novel neonatal-specific or adult inducible cell-specific mouse lines to study TGF-β signaling during different stages of the neurogenic cascade, focusing on proliferation and migration both in vitro and in vivo.

Methods

Animals

All animal protocols were approved by the Institutional Animal Care and Use Committee at University of Cincinnati (protocol # 24-03-06-01, approval date: April 12, 2024) and performed strictly in accordance with the National Institutes of Health guidelines on animal use and care. Mice are housed in the Laboratory Animal Medical Services facility on a 14-hour light/10-hour dark cycle. Food and water were provided ad libitum. We used the DCXCreERT2 transgenic mouse line (Zhang et al., 2010) (MGI: 5438982) to specifically delete Alk5 signaling in immature neurons. We used the iSuRe-Cre transgenic mouse line (PMID: 31118412) to increase recombination specificity and efficiency in the DCXCreERT2 inducible line. C57BL/6J WT mice (RRID: IMSR_JAX:000664), mGFAPCre (RRID: IMSR_JAX:012886), Ai9 R26-CAG-tdTomato (RRID: IMSR _JAX:007909) and ALK5fl/fl (RRID: IMSR_JAX:028701) mice from Jackson Laboratory were used to generate mGFAPcre-ALK5WT/WT-Ai9 control or mGFAPcre-ALK5fl/fl-Ai9 KO mice to assess the contributions of ALK5-mediated TGF-β signaling on adult neurogenesis. Both male and female mice were used for all mouse lines between the ages of 6–8 weeks. All animals were used in the data analysis unless tissue quality was poor due to unsuccessful perfusion or cryoprotection. Please refer to the key resource table (Table 1) for all animal RRID, stock numbers, and suppliers.

Table 1.

Key resources

Source Identifier
Antibodies
DCX (1:1000, host: rabbit) Cell signaling, Danvers, MA, USA Cat# 4604s
NeuN (1:1000, host: mouse) BioLegend, Sab Diego, CA, USA Cat# 834501
Alexa Fluor 647 AffiniPure Donkey anti-Mouse IgG (H+L) Jackson ImmunoResearch, West Grove, PA, USA Cat# 715-605-151
RRID: AB_2340863
Donkey anti-Rabbit IgG (H+L) Highly Cross-Absorbed Secondary Antibody, Alexa FluorTM Plus 488 Thermo Fisher Scientific, Waltham, MA, USA Cat# A32790
RRID: AB_2762833
p-Smad 2 Cell signaling mAb# 3108
Total Smad 2 Cell signaling mAb# 5339
p-Smad 3 Cell signaling mAb# 9520
Total Smad 3 Cell signaling mAb# 9523
Critical commercial assays and components
BCA assay Thermo Fisher Scientific Cat# 23227
PierceTM ECL Western kit Thermo Fisher Scientific Cat# 32106
4%–20% Mini-PROTEAN TGXTM Precast Protein Gels, 10-well, 50 µL BioRad, Hercules, CA, USA Cat# 4561094
10× Tris /Glycine /SDS Buffer BioRad #1610732
10× Tris/Glycine BioRad #1610734
Stripping buffer Thermo Fisher Scientifc Cat# 46428
20× TBS Tween 20 Thermo Fisher Scientifc Cat # 28360
Nitrocellulose Membrane, Roll, 0.45 µm BioRad Cat# 1620115
Methanol Thermo Fisher Scientific A433P-4
Laemmli Sample Buffer BioRad Cat# 161-0747
RIPA lysis buffer, 10× Sigma, St. Louis, MO, USA Cat# 20-188
Blotting-Grade Blocker BioRad Cat# 170-6404
Chemiluminesence Thermo Fisher Scientifc Cat# 32106
Countess II FL Thermo Fisher Scientific AMQAF1000
Cell counter slides Thermo Fisher Scientific C10228
Trypan blue Thermo Fisher Scientific T10282
CytoFlex S cell sorter Beckman Coulter, Indianapolis, IN, USA C09766
Neural Basal Media Thermo Fisher Scientifc Cat# 21103049
EGF Peprotech (through Thermo Fisher Scientific), Waltham, MA, USA Cat# 315-09
bFGF Peprotech (through Thermo Fisher Scientific) Cat# 450-33
Heparin Sigma Cat# H3393
Pen/Strep Sigma Cat# P4333
B27 Thermo Fisher Scientifc Cat# 17504044
Glutamax Thermo Fisher Scientifc Cat# 35050061
Accutase Thermo Fisher Scientific Cat# NC9839010
Poly-D-lysine Thermo Fisher Scientifc Cat# A3890401
Chemicals, peptides, and recombinant proteins
Tamoxifen Sigma T5648-5G
Sunflower seed oil Sigma S5007
100% ethanol Sigma 493546-1L
2,2,2-Tribromoethanol Sigma T48402-25G
2-Methyl-2-butanol Sigma 240486-100ml
DAPI Sigma D9542
SB-431542 Sigma S4317
TGF-β1 R & D Systems, Minneapolis, MN, USA 240-B-002/CF
Software
ImageJ NIH, Bethesda, MD, USA https://doi.org/10.1038/nmeth.2089
RRID: SCR_003070
Zen 3.4 (Blue edition) Zeiss, Oberkochen, Germany RRID: SCR_013672
Cytoflex Beckman Coulter, Indianapolis, IN, USA RRID: SCR_017217
Other
Confocal Microscope Leica, Wetzlar, Germany Stellaris 8
EVOS M500 Microscope Thermo Fisher Scientifc Cat# AMF5000
Experimental models: Organisms/strains
Mouse: DCXCreER Chinese Academy of Sciences, Shanghai, China MGI: 5438982 (Not commercially available)
Mouse: iSuReCre National Center for Cardiovascular Research, Spain PMID: 31118412 (Not commercially available)
Mouse: mGFAPCre The Jackson Laboratory, Farmington, CT, USA ISMR Cat# 012886
RRID: IMSR_JAX:012886
Mouse: B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J The Jackson Laboratory ISMR Cat# 007909
RRID: ISMR_JAX:007909
Mouse: Tgfbr1tm1.Karl/KulJ The Jackson Laboratory ISMR Cat# 028701
RRID: ISMR_JAX:028701
Oligonucleotides
Primers: ALK5 delta F: CCT GCA GTA AAC TTG GAA TAA GAA G Integrated DNA Technologies, Coralville, IA, USA N/A
Primers: ALK5 delta R: GGA ACT GGG AAA GGA GAT AAC Integrated DNA Technologies N/A
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bFGF: Basic fibroblast growth factor; DAPI: 4′,6-diamidino-2-phenylindole; DCX: doublecortin; EGF: epidermal growth factor; NeuN: neuron-specific nuclear protein; N/A: not applicable.

Tamoxifen treatment

For in vivo analysis, all 6–7-week-old DCXcreERT2-ALK5wt/wt-Ai9, DCXcreERT2-iSuRe-ALK5wt/wt, DCXcreERT2-ALK5fl/fl-Ai9, and DCXcreERT2-iSuRe-ALK5fl/fl male and female mice were given tamoxifen (TAM) dissolved in 90% of sunflower oil and 10% of ethanol via gavage. Mice were given 5 consecutive gavage injections at 180 mg/kg per day and harvested the brain at 5 days or 4 weeks after the last TAM treatment for immunohistochemical analysis.

Tissue culture

Primary NSCs were isolated from 6-week-old mGFAPcre-ALK5WT/WT-Ai9 or mGFAPcre-ALK5fl/fl-Ai9 by microdissection to extract the SVZ in the lateral ventricles of the adult mouse brain to prepare for primary NSCs cultures. After processing the tissue and culturing the cells in growth media containing epidermal growth factor and basic fibroblast growth factor as described in previous studies (Luo et al., 2020, 2022), we used neurospheres between P3 and P8 to assess the proliferation and migration of NSCs. Primary NSCs cultures were prepared from several individual mGFAPcre-ALK5WT/WT-Ai9 or mGFAPcre-ALK5fl/fl-Ai9 to repeat the in vitro experiments. Each data point for the in vitro experiments is the average of multiple technical replicates from an independent experiment as a single biological replicate. After dissociating the neurospheres, individual cells were plated at a density of 3.2 × 103 cells/well in a 96-well round bottom plate to form individual spheres for proliferation quantification or placed in a 24-well poly-D-lysine coated plate once the diameter of the sphere reached 250–350 µm for in vitro migration analysis. On day 1, neurospheres used for proliferation were treated with neurobasal control media TGF-β1, or SB-431542 (inhibitor of ALK4, ALK5, and ALK7 signaling). On day 4, images of each sphere were taken using Evos M500 microscope (Thermo Fisher Scientific, Waltham, MA, USA) and quantified using ImageJ (https://imagej.nih.gov/ij/). 4–20 replicate wells were quantified for each treatment group. Migrating neurospheres were treated with neurobasal control media, TGF-β1, SB-431542, or TGF-β1 + SB-431542. Each well was imaged every 24, 48, 72, and 96 hours with each treatment group containing 4–6 replicates and quantified the area migrated using ImageJ. Experiments were replicated at least three times showing similar results. The migration index used to quantify migrating neurospheres was defined as dividing the total area of migrated NSCs at 48 or 96 hours by the area of the neurospheres at 2 hours. Table 1 provides supplier information.

Fluorescence-activated cell sorting

Cells were grown in a neurosphere culture as described in the methods and dissociated into individual cells to be plated in 96-well round bottom plates at a density of 3.2 × 103 cells/well. Cells were treated as described in the methods previously (Bedolla et al., 2024a) and collected into 15 mL conical tubes for dissociation using accutase. Dissociated single cell resuspension was subjected to analysis of cell number and cell size using the CytoFlex S cell sorter (Cat# C09766). Additionally, we used the CytoFlex S cell sorter (Cat# C09766) to accurately measure cell size. Each tube was set to sort cells at a rate of 10 µL/min for 5 minutes. Gating was determined by the separation of debris size to cell size to separate out any debris. Table 1 provides supplier information.

Western blotting

Individual NSCs were plated on poly-D-lysine coated 12-well plates and treated with neurobasal control media, TGF-β1 or SB-431542, and homogenized using RIPA lysis buffer 2 hours after plating. To analyze protein levels in migrating neurospheres, 10–12 Individual neurospheres were plated on poly-D-lysine coated 6 well plates and harvested in RIPA lysis buffer. Protein concentration was determined using the Pierce BCA protein assay kit according to the manufacturer’s instructions (Cat# 23227, Thermo Fisher Scientific). Equal amounts of protein were loaded onto 4%–20% SDS-PAGE gradient gels (Cat# 4561094, BioRad) and electrophoretically transferred to nitrocellulose membranes (Cat# 1620115, BioRad). The membranes were blocked in 1% milk in TBST for 1 hour at room temperature. Primary antibodies were incubated overnight at 4°C followed by 1-hour secondary antibody incubation at room temperature. The following primary antibodies were used: p-Smad2 (rabbit, 1:250, mAb# 3108, Cell Signaling, Danvers, MA, USA), total Smad2 (rabbit, 1:1000, mAb# 5339, Cell Signaling). Enhanced chemiluminescence was performed with a PierceTM ECL Western kit (Thermo Fisher Scientific) and the density of the bands was quantified using ImageJ. All experiments were repeated at least three times yielding similar results. Table 1 provides for supplier information.

Immunohistochemistry

Mice were anesthetized with 1x Avertin (containing 2,2,2-tribromoethanol and 2-methyl-2-butanol (Sigma)) via intraperitoneal injections and perfused with ice-cold PBS followed by 4% paraformaldehyde. The brains were left in paraformaldehyde overnight followed by subsequent 20% and 30% sucrose overnight incubations. Following the sucrose incubations, the brains were sectioned using a Leica Cryostat at a thickness of 30 µm. 4% BSA/0.3% Triton X-100 was used to block sections for 1 hour followed by incubation with primary antibodies (16–48 hours) and their respective secondary antibodies conjugated with Alexa fluorescence 488 (rabbit, 1:1000, Thermo Fisher Scientific, RRID: AB_2762833) or 647 (mouse, 1:500, Jackson ImmunoResearch, West Grove, PA, USA, RRID: AB_2340863). In this study, we used the following primary antibodies: doublecortin (DCX; rabbit, 1:1000, Cat# 4604s, Cell Signaling) and neuron-specific nuclear protein (NeuN) (mouse, 1:1000, Biolegend, San Diego, CA, USA, Cat# 834501). A Leica Stellaris 8 confocal microscope was used for confocal imaging. Quantification of the percentage of migrated cells was carried out using Zen Blue 3.4 software (Zeiss) by measuring the distance to the middle of the cell from the bottom of the SGZ layer and dividing that by the distance of the bottom to the top of the SGZ layer. Each cell was then organized into respective layers based on the distance it had migrated. At least three coronal sections containing similar regions of interest were quantified for each mouse and averaged values for each mouse were considered as one data point and converted to percentage for statistical analysis. Table 1 provides supplier information.

Statistical analysis

All studies were analyzed using SigmaPlot 12.0 (Systat Software Inc., San Jose, CA, USA, RRID: SCR_003210). Results are expressed by mean ± SEM of the indicated number of experiments. Statistical analysis was performed using the Student’s t-test, and one- or -two-way analysis of variance (ANOVA), as appropriate, with Tukey’s post hoc tests. A P-value of or less than 0.05 was considered significant.

Results

Primary adult neural stem cells directly respond to transforming growth factor-β1 ligand via the pSMAD2/3 signaling pathway which is dependent on the ALK5 receptor

To begin characterizing the role of TGF-β signaling on adult neurogenesis, we first examined whether TGF-β signaling components were expressed in NSCs. Widely used primary NSCs neurosphere culture (Luo et al., 2022; Wang et al., 2022a) was established from 6-8 week-old wild-type (WT) mice and total RNA collected from these NSCs to examine the expression levels of key components of the TGF-β signaling via RNA-seq (Figure 1A and B). Our data shows that NSCs express both type 1 (Alk5) and type 2 (Tgfβr2) receptors required for ligand binding and signaling. Additionally, they also express Smad2, Smad3, and Smad4, indispensable proteins to the downstream signaling of TGF-β (Figure 1B). Furthermore, these cells also show expression of integrins (Itga5, Itgb8) and Lrrc32, a critical milieu molecule, which are necessary for the activation of latent TGF-β1 (Qin et al., 2018; Duan et al., 2022). Evidence of these critical TGF-β signaling components supports that NSCs are capable of direct signaling in response to TGF-β ligands. Additionally, we generated NSC-specific Alk5 cKO cells utilizing the Cre-loxP system to effectively ablate ALK5-mediated signaling. mGFAPCre driver line (mouse glial fibrillary acidic protein promoter in which Cre recombinase is expressed at early postnatal days in astrocytes and NSCs (Garcia et al., 2010; Wang et al., 2022a) is crossed with the Alk5fl/fl mice in which two loxP sites flank the exon 3 of the Alk5 gene (Figure 1C). However, the in vitro neurosphere culture system supports the survival and proliferation of NSCs but not astrocytes. To ensure that exon 3 was successfully deleted, essential for ALK5 activation, primers were designed to flank the loxP sites which enable us to detect the floxed allele or recombined allele in genomic DNA via PCR (Figure 1C). Primary NSC cultures are established from control (mGFAPcre-ALK5wt/wt) or mGFAPcre-ALK5fl/fl mice at the age of 6–8 weeks. Genomic DNA was extracted and subjected to PCR. The floxed allele is detected in control NSCs (~2 kb) and in mGFAPcre-ALK5fl/fl NSCs, only a recombined allele (< 0.5 kb) is detected (Figure 1D), indicating successful DNA recombination in NSCs. Functional ALK5-mediated TGF-β signaling was then analyzed in control and Alk5 cKO cells by treating them with either exogenous TGF-β1 ligand, SB-431542 (a small molecule inhibitor of TGF-β type 1 receptors ALK4, 5, and 7) (Inman et al., 2002), or a combination of ligand and SB-431542 (Figure 1E). Exogenous TGF-β1 ligand treatment induces active TGF-β signaling (measured by phosphorylation of SMAD2 protein) in WT neurospheres while it had no effect on cKO neurospheres, demonstrating ALK5-mediated TGF-β signaling in WT NSCs that is abolished in the Alk5 cKO cells (Figure 1F and G). Treatment of SB-431542 alone inhibited the low basal signaling observed in WT NSCs and reduced TGF-β1-induced signaling by at least 88% in the TGF-β1+SB-431542 combination group (Figure 1F and G). Taken together, we demonstrate genetic and pharmacological stimulation or inhibition of ALK5-mediated TGF-β signaling specifically in NSCs derived from young adult mice.

Figure 1.

Figure 1

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Primary adult neural stem cells directly respond to TGF-β1 ligand via the ALK5 receptor-dependent pSMAD2/3 signaling pathway.

(A) Adult neurosphere cultures are established from the SVZ in the adult mouse brain. (B) Bulk RNA-seq of wildtype (WT) neurospheres for TGF-β signaling components in fragments per kilobase of transcript per million mapped reads (FPKM). (C) Model for targeting Alk5 gene deletion in the neural stem cells (NSC) (red arrows denote loxP sites) and (D) validation of Alk5 knockout (KO) in mGFAPcre-ALK5fl/fl-Ai9 mice cKO derived primary cells. Blue arrows indicate the location of polymerase chain reaction (PCR) primers. (E) Experimental design depicting plating and treatment of exogenous TGF-β1 and SB-431542 for western blotting. (F) Representative image for chemiluminescence probing of p-SMAD2 and total SMAD2 with quantification in G. Data are expressed as the Mean ± SE. For panel B, neurospheres were collected from WT culture and three samples were subjected to RNA-seq and each sample was plotted as an individual data point. For panel G, each data point represents the average from an independent biological replicate (with three technical replicates for each biological replicate). Two-way analysis of variance with Tukey’s post hoc test was used for statistical analysis of western blot quantification (n = 3), *P < 0.05, **P < 0.01. cKO: Conditional KO; KO: knockout; SB: SB-431542 (inhibitor of ALK4, ALK5, and ALK7 signaling); TGF-β: transforming growth factor-β; WT: wild-type.

Transforming growth factor-β1 inhibits the proliferation of primary adult neural stem cells via ALK5-mediated transforming growth factor-β signaling

We next examined the role of TGF-β signaling on the proliferation of NSCs in vitro. Utilizing WT primary NSCs, we disassociated neurospheres into single cells and plated them into 96-well round bottom plates (3000 cells per well), allowing them to aggregate in a 3D neurosphere (Figure 2A) and proliferate in the presence of epidermal growth factor and bFGF. Cells were treated with exogenous TGF-β1 ligand (20 ng/mL) or SB-431542 (10 µM) for 4 days and quantified the diameter of each sphere to calculate the average volume of the single sphere. Interestingly, TGF-β1 ligand inhibited 3D neurosphere growth while SB-431542 stimulated growth (Figure 2D and G) suggesting an anti-proliferative role of TGF-β signaling in NSCs during their proliferation. However, changes in the volume of the sphere could be attributed to either changes of cell size or changes in cell numbers in each sphere. To investigate whether the increase in neurosphere volume is due to increased cell size, neurospheres were dissociated into single-cell suspension and subjected to fluorescence-activated cell sorting after 4 days of treatment to determine the average cell size by the forward scatter of each group (Figure 2B). Our data show that there was no difference in cell size among the groups (Figure 2E). Consistent with this, neurospheres were dissociated from each treatment group and the number of individual cells was counted (Figure 2C), data show that the TGF-β1 treated group had decreased total cell numbers in comparison to control (P < 0.05) while the SB treated group had increased total cell numbers compared with the TGF-β1 treated group (P < 0.0001; Figure 2F). While the anti-proliferative effects of TGF-β signaling pathways on a variety of cell types have been reported before (Wu and Li, 2022), our data supports a direct inhibitory role of TGF-β signaling on primary NSCs proliferation in vitro in a 3D culturing system.

Figure 2.

Figure 2

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Treatment with exogenous TGF-β1 results in a decrease of neurosphere size due to the inhibition of proliferation.

(A) Experimental design of individual neurosphere aggregation and growth in a 96-well round bottom plate for flow cytometry (B), single cell counting (C), and quantification for 3D sphere size (D). Quantification for forward scatter (E) total cell number (F) and sphere volume (G). Each data point for the forward scatter graph is the average of 8–20 neurospheres per condition (n = 4 biological replicate). Each data point for the number of cells graph is the average of each condition of an equal amount of neurospheres per group (n = 8–9). (G) Representative results of one experiment for sphere volume where each data point represents a single neurosphere (experiments were repeated four times with similar results). Data are expressed as the Mean ± SEM. One-way analysis of variance with Tukey’s post hoc test was used as statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns: Not significant; SB: SB-431542 (inhibitor of ALK4, ALK5, and ALK7 signaling); TGF-β1: transforming growth factor-β1.

Transforming growth factor-β1 enhances the migration of primary adult neural stem cells via ALK5-mediated transforming growth factor-β signaling

Migration of NSCs and their progenies is an important yet understudied process in adult neurogenesis, especially the role of TGF-β signaling in postnatal NSCs. Therefore, we next investigated the role of TGF-β signaling in the migration of WT and Alk5 cKO NSCs. Using a previously established in vitro migration assay (Luo et al., 2022), single neurosphere was grown in individual 96 well-round bottom plates (starting from the same number of dissociated NSCs) and harvested when their size was between 250–300 µm diameter and plated on Poly-D-Lysine coated 24-well plates to be treated with either exogenous TGF-β1, SB-431542, or TGF-β1 + SB-431542 (Figure 3A). We observed enhanced migration of primary WT NSCs at 48 hours in the presence of exogenous TGF-β1 and decreased migration in the presence of SB-431542 as defined by the migration index (total area of migrated cells at x hour/ total area at 2 hours; x is defined as the elapsed number of hours). Interestingly, the effects of TGF-β1 were abolished by co-incubation of SB-431542 (Figure 3B and C). At 48 hours, there is no change in the migration of primary cKO NSCs treated with exogenous TGF-β1 (Figure 3C) compared with vehicle-treated cKO cells and a significant decrease in migration for each treatment group compared with their respective WT counterparts (TGF-β1, SB, and TGF-β1 + SB treated P < 0.001; Figure 3C). Moreover, it takes primary cKO NSCs 96 hours to reach a migration index similar to that of WT cells at 48 hours (Figure 3D) indicating a decreased basal migration. Treatment of TGF-β1 had no effects on Alk5 cKO cells confirming ALK5-dependent effects of TGF-β1 signaling on the migration of NSCs. Interestingly, SB-431542 still showed an inhibitory effect on the migration of Alk5 cKO cells, suggesting that ALK4 or ALK7 receptors might also have an important role in the migration of these cells (Figure 3D). In summary, our data suggest that TGF-β signaling in primary WT NSCs inhibits the proliferation of cells while simultaneously enhancing the migration of primary WT NSCs. Inhibition of TGF-β signaling via genetic ablation of Alk5 or chemical inhibitor had opposite effects compared with TGF-β ligand treatment on these phenotypes. Overall, our findings support an anti-proliferative but pro-migration role of TGF-β signaling in vitro, supporting our hypothesis of a dual role for TGF-β signaling in adult neurogenesis.

Figure 3.

Figure 3

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Primary NSCs treated with exogenous TGF-β1 enhance their migration while deletion of Alk5 or SB-431542 treatment inhibits migration.

(A) Experimental design used to examine the migration of neural stem cells (NSCs) in vitro. (B) Representative images of neurospheres plated on a coated 24-well plate over the course of 96 hours to observe the migration of NSCs (n = 2–5). Note that wildtype (WT) spheres migrate beyond the imaging field and therefore are only quantified up to 48 hours. (C, D) Quantified by their migration index. Graphs are representative results from one of three replicate experiments (n = 2–5). Two-way analysis of variance with Tukey’s post hoc pairwise analysis was used when comparing WT and knockout (KO) cells within different treatment groups (C). One-way analysis of variance was used when comparing treatment groups within KO alone (D). Mean ± SEM. **P < 0.01, ***P < 0.001. KO: Knockout; ns: not significant; SB: SB-431542 (inhibitor of ALK4, ALK5, and ALK7 signaling); TGF-β: transforming growth factor-β.

The role of transforming growth factor-β signaling on the post-natal hippocampal neurogenesis in vivo

The TGF-β signaling pathway is a dynamic system that needs to be studied in a precise time and context-dependent manner due to its multi-functional roles in many different cell types, especially with the correct cellular context in vivo. To this end, we developed two novel inducible and cell type-specific mouse models to specifically silence TGF-β signaling in NSCs (mGFAPcre-ALK5fl/fl-Ai9) or immature neuroblasts (DCXcreERT2-ALK5fl/fl-Ai9) to investigate how the lack of TGF-β signaling affects NSCs or adult-born immature neuroblasts migration in vivo. The Cre-inducible tandem dimer tomato (tdTomato) fluorescent protein (Ai9 allele) enables us to genetically label NSCs in vivo and fate map their survival and morphology at different stages of the neurogenic cascade. In mGFAPcre-ALK5fl/fl-Ai9 mice (Figure 4A), Cre recombinase becomes active around early postnatal stages (Ganat et al., 2006; Chow et al., 2008) in NSCs (and astrocytes) and has previously been used by us and many other groups to study postnatal neurogenesis (Garcia et al., 2010; Wang et al., 2022a). Given the previous study showing the potential role of ALK5 in hippocampal adult neurogenesis (He et al., 2014), we focused on the hippocampal niche in our study. In the mGFAPcre-ALK5fl/fl-Ai9 mice cKO mouse line, at 8 weeks of age (a time point that allows cKO aNSCs to mature into new neurons from the onset of Cre activity), we did not observe any differences in the total number of doublecortin (DCX)+ immature neuroblasts suggesting that loss of TGF-β signaling in NSCs does not affect the net generation of immature neuroblasts (Figure 4B and C). There is a significant decrease in the migration of neurons throughout the granule cell layer (Figure 4D and F) without changes in the total number of tdTomato+ cells (Figure 4D and E), indicating that the increase in cells at the inner layer (SGZ) and decrease in cells at the outer layer is not due to change of total tdTomato+ cells but due to the aberrant migration of KO cells. This data supports the role of TGF-β signaling in the migration of newly born neurons in this early post-natal stage (from P0–P60 of age). The mGFAPcre driver line deletes floxed genes around postnatal day 0 (P0) (Garcia et al., 2010) in mice and targets NSCs and leads to Alk5 gene deletion throughout the whole neurogenic cascade (including RGLs-radial glial cells, IPCs-intermediate progenitor cells, immature neurons, and mature neurons). Therefore, to further investigate the role of TGF-β signaling specifically in adult newborn immature neuroblasts (DCX+) in the hippocampus, we generated inducible DCXcreERT2-ALK5fl/fl-Ai9 iKO mice to specifically target TGF-β signaling in immature neuroblasts (Figure 5A). To validate the specificity of this Cre driver line (Zhang et al., 2010), 6-week-old DCXCreERT2-Ai9 mice were treated with 180 mg/kg of TAM once a day for 5 days and harvested the mice at 5- or 30 days post-TAM (Figure 5B) to examine the recombination of the Ai9 reporter allele in a cohort of DCX+ immature neurons longitudinally. Our data show that at 5 days post-TAM, most tdTomato+ cells were DCX+ (Figure 5C–G) while at 30 days most tdTomato+ cells were NeuN+ (Figure 5C–G), indicating accurate labeling of immature neurons and successful tracking of these cells through maturation into NeuN+ neurons. While we did not observe a change in the total amount of tdTomato+ cells, there was a significant difference in total DCX+ cells between post-TAM-5 day and post-TAM-30 day brains (Figure 5E). This difference in total DCX+ cells in DG could be attributed to the differences in the age of the mice at the time of harvest, suggesting that neurogenesis is more active in the 8-week-old brain compared with the 11-week-old brain. Nevertheless, after the specificity and efficiency of the DCXCreERT2 line were validated, we next investigated how the lack of TGF-β signaling in immature newly born neuroblasts affects the migration of these cells. To this end, we generated DCXCreERT2-ALK5fl/fl-Ai9 iKO and control (DCXCreERT2 -ALK5wt/wt- Ai9) mice (Figure 6A). Control and iKO mice were treated between 5–6 weeks of age with the same 5XTAM dosage, and the hippocampus was harvested and analyzed for the number and location of tdTomato+ cells 30 days later. Interestingly, there are no significant differences in the migration of adult-born neurons in the DCXcreERT2-ALK5fl/fl-Ai9 iKO mice when compared with WT mice (Figure 6B–H) despite previous studies reporting TGF-β signaling to be important for later stages of adult neurogenesis (He et al., 2014; Gradari et al., 2021). In line with our data, we observed no changes in total tdTomato+ or DCX+ cells (Figure 6D and E) between control and DCXcreERT2-ALK5fl/fl-Ai9 iKO mice. Additionally, there were no significant differences in tdTomato+/DCX+ double positive cells or tdTomato+/NeuN+ double positive cells when comparing DCXcreERT2-ALK5fl/fl-Ai9 iKO to control mice (Figure 6F and G). Taken together, our data suggests that TGF-β signaling is not essential to the migration of adult-born neurons when ablated during the later stages of the neurogenic cascade in young adult mice.

Figure 4.

Figure 4

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Abolishing ALK5-mediated TGF-β signaling in neonatal mice in the mGFAPcre-ALK5fl/fl-Ai9 cKO mice alters the migration of postnatal-born neurons.

(A) The mouse model used to examine the migration of postnatal-born neurons at 8 weeks post ablation of Alk5. (B) Representative images of total DCX+ immature neuroblasts at the subgranular zone in the dentate gyrus of WT or cKO mice. (C) Quantification of total DCX+ cells. (D) Representative images of the migration of postnatal-born neurons (tdTomato+ from P0–P60) in the hippocampus. Green dashed lines denote the top and bottom boundaries of the granule cell layer while the white dashed lines mark the inner, middle, and outer layers used to quantify the migration of cells. Quantification of total tdTomato+ cells (E) and (F) migration distance (percentage in each layer). Each data point is the average of 3–6 brain sections from each mouse (n = 5–6). Data are expressed as the Mean ± SE. Student’s t-test (C and E) or two-way analysis of variance with Tukey’s post hoc test (F) was used for statistical analysis. **P < 0.01, cKO: Conditional KO; DAPI: 4′,6-diamidino-2-phenylindole; DCX: doublecortin; IHC: immunohistochemistry; KO: knockout; ns: not significant; SB: SB-431542 (inhibitor of ALK4, ALK5, and ALK7 signaling); TGF-β: transforming growth factor-β; WT: wild-type.

Figure 5.

Figure 5

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DCXcreERT2 driver line effectively recombines floxed alleles in immature neurons in the dentate gyrus of the hippocampus.

(A) The mouse model used to characterize the labeling efficiency and recombination of the DCXcreERT2 driver line at (B) different time points after TAM treatment. (C) Representative images showing immunohistochemistry staining (for tdTomato, DCX, NeuN, DAPI) with white arrows denoting tdTomato+/white arrows denoting tdTomato+/DCX+ cells and yellow arrows denoting the tdTomato+/NeuN+ cells in the hippocampus. Quantification for (D) total tdTomato+ cells, (E) total DCX+ cells, (F) total tdTomato+/DCX+ double positive cells and (G) total tdTomato+/NeuN+ double positive cells. Each data point is the average of 3–6 brain sections per mouse (n = 5–6). Student’s t-test was used for statistical analysis. Data are expressed as the Mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001. DAPI: 4′,6-Diamidino-2-phenylindole; DCX: doublecortin; IHC: immunohistochemistry; NeuN: neuron-specific nuclear protein; ns: not significant; TAM: tamoxifen.

Figure 6.

Figure 6

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Late-stage ablation of TGF-β signaling in a DCXcreERT2-ALK5fl/fl-Ai9 iKO mice does not affect the migration of adult-born neurons in the dentate gyrus of the hippocampus.

(A) The mouse model used to examine the migration of adult-born neurons in the DCXcreERT2-ALK5fl/fl-Ai9 mouse line at (B) 30 days post TAM. (C) Representative images showing immunohistochemistry staining (for tdTomato, DCX, NeuN, DAPI) with purple dashed lines denoting the top and bottom boundaries of the granule cell layer (GCL) while the white dashed lines mark the inner, middle, and outer layers for the migration of cells in the hippocampus. Yellow arrows mark the tdTomato+/NeuN+ cells. Quantification for (D) total tdTomato+ cells, (E) total DCX+ cells, (F) total tdTomato+/DCX+ double positive cells, (G) total tdTomato+/NeuN+ double positive cells, and (H) migration distance (percentage in each layer). Each data point is the average of 3–6 brain sections per mouse (n = 8–9). Data are expressed as the Mean ± SE. Student’s t-test (D–G) or two-way analysis of variance with Tukey’s post hoc test (H) for statistical analysis. DAPI: 4′,6-Diamidino-2-phenylindole; DCX: doublecortin; KO: knockout; NeuN: neuron-specific nuclear protein; ns: not significant; TAM: tamoxifen; TGF-β: transforming growth factor-β; WT: wild-type.

Substantial efforts have been made in recent years to enhance the accuracy and efficiency of Cre-mediated recombination and tracking of KO cells by fluorescent proteins. This is especially important to ensure that reporter-positive cells are truly tracking KO cells when antibodies are not available. Recent reports by others and us show that recombination of a single floxed reporter allele does not always demonstrate successful recombination of both alleles of the targeted genes, depending on the size of the floxed region (Faust et al., 2023; Bedolla et al., 2024b). To further enhance the rigor of our research and to increase the accuracy of the tracking of KO cells, we turned to a recently developed new tool, iSuRe mice developed by the Molecular Genetics of Angiogenesis from the Benedito lab (Fernández-Chacón et al., 2019). This mouse model contains an inducible dual reporter-cre that, upon recombination, produces a constitutively active CRE protein, with high recombination efficiency. Additionally, this novel iSuRe-Cre construct contains a Cre-dependent membranous tomato reporter that is expressed along with the constitutively active Cre, via P2A, enabling more accurate genetic labeling and tracking of the target cell population (Figure 7A). Recent work characterizing this model to ensure proper recombination efficiency and specificity was done when crossing the DCXcreERT2 driver line to generate the DCXcreERT2-iSuRe mouse line (Bedolla et al., 2024b). We therefore further generated the DCXcreERT2-iSuRe-ALK5fl/fl iKO mouse line and subjected these mice to the same treatment paradigm as the DCXcreERT2-ALK5fl/fl-Ai9 iKO mouse line and harvested mice 30 days post-TAM (Figure 7B). Similarly, our results show no differences in the migration of adult-born neurons in the DCXcreERT2-iSuRe-ALK5fl/fl iKO mice (Figure 7C and H). We also report no significant differences between total tdTomato+ or DCX+ cells as well as no change in tdTomato+/DCX+ and tdTomato+/NeuN+ cells (Figure 7C–G), suggesting that TGF-β signaling in DCX+ immature neurons are not critical for their survival or maturation. Taken together, our data supports that loss of TGF-β signaling in adult-born immature neuroblasts during later stages of adult neurogenesis is not required for the survival or proper migration of these newly born neurons, highlighting the need for precise cell-specific models to study TGF-β signaling in highly dynamic and complex systems.

Figure 7.

Figure 7

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Late-stage ablation of TGF-β signaling in the DCXcreERT2-iSuRe-Alk5fl/fl iKO mice does not affect the migration of adult-born neurons in the dentate gyrus of the hippocampus.

(A) The mouse model used to examine the migration of adult-born neurons in the DCXcreERT2-iSuRe-Alk5fl/fl mouse line at (B) 30 days post-TAM. (C) Representative images showing immunohistochemistry staining (for tdTomato, DCX, NeuN, DAPI) with purple dashed lines denoting the top and bottom boundaries of the granule cell layer while the white dashed lines mark the inner, middle, and outer layers for the migration of cells in the hippocampus. Quantification for (D) total DCX+ cells, (E) total tdTomato+ cells, (F) total tdTomato+/DCX+ double positive cells, (G) total tdTomato+/NeuN+ double positive cells, and (H) migration distance (percentage in each layer). Yellow arrows mark the tdTomato+/NeuN+ cells. Each data point is the average of 3–6 brain sections per mouse (n = 4). Student’s t-test (D–G) or two-way analysis of variance with Tukey’s post hoc test (H) for statistical analysis. Data are expressed as the Mean ± SE. DAPI: 4′,6-Diamidino-2-phenylindole; DCX: doublecortin; NeuN: neuron-specific nuclear protein; ns: not significant; TAM: tamoxifen; TGF-β: transforming growth factor-β.

Discussion

With over seven type I receptors, five different type II receptors, and more than 30 ligands that bind different combinations of these receptors, numerous combinations can be made in the TGF-β signaling pathway, contributing to its complex and dynamic signaling paradigms. With this pathway’s many different receptor and ligand combinations, it can regulate many different genes in different cell types, making it a difficult pathway to properly characterize in any given cell type. Currently, a major focus of the field is to improve the precise modulation of TGF-β signaling while limiting confounding variables often seen in models used to study its effects on the neurogenic cascade, particularly proliferation and migration of NSCs and adult-born neurons. While it is known that TGF-β signaling is a key player in neurogenesis during development, its role in adult neurogenesis in vivo remains controversial. Global KO of TGF-β produces a lethal phenotype (Shull et al., 1992; Larsson et al., 2001; Massagué and Sheppard, 2023), proposing a formidable problem when studying the role of TGF-β signaling in adult neurogenesis. Pharmacological studies have shown contradictory results of TGF-β signaling in adult neurogenesis (Buckwalter et al., 2006; Wachs et al., 2006; He et al., 2014; Kandasamy et al., 2014). Some studies show that inhibiting crucial components of TGF-β signaling enhanced the proliferation of adult NSCs (Wachs et al., 2006) while decreasing the maturation and survival of adult-born neurons (Pineda et al., 2013; He et al., 2014). Conversely, other studies have reported that chronic delivery of TGF-β in vivo inhibits neurogenesis (Buckwalter et al., 2006). While these studies support a potential role of TGF-β signaling in adult neurogenesis, the discrepancy between different studies could be partially due to the lack of cell type-specific effects of pharmacological manipulation. Other CNS cells can indirectly modulate NSCs and the lack of NSC-specific or neurogenic stage-specific- animal models for TGF-β signaling makes it difficult to precisely elucidate the role of TGF-β signaling in adult neurogenesis (Al-Onaizi et al., 2020; Araki et al., 2021). In this study, temporal and cell-type specific control of the TGF-β signaling pathway enabled us to more rigorously interrogate the precise regulation of ALK5-mediated TGF-β signaling on postnatal neurogenesis. To investigate the potential dual role hypothesis in a cell-specific and neurogenic stage-specific manner, we utilized mGFAPcre-ALK5fl/fl-Ai9 cKO or DCXcreERT2-ALK5fl/fl-Ai9 iKO mice to examine the effect of TGF-β signaling on proliferation and migration both in vitro and migration in vivo.

Utilizing the mouse primary neurosphere 3D culturing model, our data show that TGF-β1 ligand inhibits and SB-431542 stimulates the proliferation of NSCs in vitro. It has been consistently reported that TGF-β signaling is a negative modulator of proliferation in multiple cell types (Zhang et al., 2017). Embryonic stem cells (ES) are a great tool to utilize in the study of TGF-β signaling due to their importance not only during development but also their ability to self-renew indefinitely. It has been reported that treatment with SB-431542 decreases the proliferation of ES cells in vitro (Ogawa et al., 2007; Watabe and Miyazono, 2009). Consistent with this, it has been reported that in primary rat NSCs, TGF-β signaling also modulated NSC quiescence/proliferation as well as differentiation and survival (Wachs et al., 2006; Kandasamy et al., 2014). In agreement with this previous work, our data obtained using the mouse primary NSCs supports TGF-β1 signaling as a negative regulator of proliferation for NSCs in vitro. Mechanistically, it is proposed that TGF- β signaling regulates the proliferation of NSCs via cell cycle regulation, specifically by cell cycle arrest during the G1 to S phase (Mukherjee et al., 2010; Zhang et al., 2017). TGF-β is known to regulate cell proliferation through the suppression of transcription factors like c-Myc and the Id proteins (Id1, Id2, Id3) (Pietenpol et al., 1990 p.1; Kang et al., 2003; Zhang et al., 2017). C-Myc inhibits the expression of CDK inhibitors like p21 where the subsequent repression of c-Myc allows for increased expression of p21 (Gartel et al., 2001). CDKs are required for the transition of the G1 phase into the S phase at the restriction point and TGF-β signaling induces the expression of CDK inhibitors which further suppresses growth-promoting signaling (Zhang et al., 2017; Decker et al., 2021). Interestingly, we did not observe a change in total tdTomato+ cells in the mGFAPcre-ALK5fl/fl-Ai9 cKO line in vivo which indicates no net change of cells surviving to maturity following deletion of the TGF-β type 1 receptor (Alk5) in mGFAP+ cells from P0 neonatal stages. One potential limitation of the mGFAPcre driver is the deletion of Alk5 in GFAP+ astrocytes which could introduce confounding variables in this model. However, the in vitro NSC neurosphere culture preferentially enriches NSCs over astrocytes, especially at passages larger than P3, allowing us to evaluate the direct role of TGF-β signaling specifically in NSCs. Nevertheless, this mGFAPcre-ALK5fl/fl-Ai9 cKO model allows us to investigate the combined effects of loss of ALK5 function during the whole process of the neurogenic cascade.

While the few studies on migration in vitro and in vivo have provided some insight into the complexity of TGF-β signaling (primarily in human glioma cells or developing rat cortex) (Platten et al., 2000; Siegenthaler and Miller, 2004; Meyers and Kessler, 2017), the role of this pathway in the importance of late-stage adult neurogenesis remains largely unclear. In this study, we show that exogenous TGF-β1 on neurospheres enhances the migration of NSCs while SB-431542, a small molecule inhibitor, is able to negate this phenotype. Our data suggests that TGF-β signaling is an important regulator of migration for NSCs in vitro. Interestingly, even in the Alk5 cKO cells, SB-431542 was able to further inhibit the basal migration of NSCs compared with vehicle-treated cKO cells. As SB-431542 is known to be an inhibitor of not only ALK5, but ALK4 and ALK7 as well, it is important to consider other TGF-β ligands and receptors that may play a role in regulating the migration of NSCs. ALK4 and ALK7 are both type 1 receptors that can be activated by a multitude of ligands in the activin family subclass like ACTA, ACTB, GDF8, and GDF11 (Goebel et al., 2019, 2022). GDF11 is known to be expressed in the brain during both development and adulthood (Nakashima et al., 1999; Ozek et al., 2018; Mayweather et al., 2021) and while its precise function in adult neurogenesis remains controversial, it has been implicated in the regulation of adult neurogenesis (Ozek et al., 2018; Mayweather et al., 2021). The fact that treatment with SB-431542, even in Alk5 cKO cells, could further inhibit the migration of NSCs suggests a potential mechanism that multiple TGF-β superfamily ligands and receptors could have synergistic or combinatory effects on NSCs migration. Whether activins and GDF11 directly affect the migration of NSCs and whether loss of ALK5 leads to upregulation of ALK4 or ALK7 via a compensatory mechanism warrants further investigation in future studies.

While the neurospheres are grown in a 3D environment, in vitro conditions do not fully recapitulate the proper microenvironments of the neurogenic niches in the adult brain. The effect of TGF-β signaling on the migration of NSCs and adult-born neurons in vivo is an understudied area that has proven difficult to study. One study using a Camk2acre driven TGF-β receptor KO has shown a deficit in late-stage neurogenesis with the caveat that Alk5 is also deleted in the majority of the forebrain mature neurons perinatally in the Camk2aCre-Alk5 cKO mice. Since it is well known that other CNS cells can indirectly modulate NSCs, global or non-NSC-specific KO of TGF-β signaling could produce confounding effects (He et al., 2014). Furthermore, this same study demonstrated that constitutively active ALK5-mediated TGF-β signaling was able to promote the survival of newborn neurons while other studies have shown chronic increase of TGF-β1 reduced the total number of immature neurons in the hippocampus (Buckwalter et al., 2006). Moreover, a recent study showed that overexpression of SMAD2 decreased the proliferation and survival of immature neurons (Gradari et al., 2021). These conflicting studies make it difficult to interpret how TGF-β signaling modulates adult neurogenesis. Since the role of TGF-β signaling in migration remains elusive we sought to explore its effect on migration in a more neurogenic stage-specific manner in vivo. We utilized both the mGFAPcre-ALK5fl/fl-Ai9 cKO or DCXcreERT2-ALK5fl/fl-Ai9 iKO mice to study the effect of ablated TGF-β signaling during both early and late stages of adult neurogenesis respectively. In the mGFAPcre-ALK5fl/fl-Ai9 cKO mouse model we observed impaired migration in cKO mice compared with WT. Conversely, we observed no change in the migration of adult-born neurons in the DCXcreERT2-ALK5fl/fl-Ai9 iKO. The changes observed in migration for the mGFAPcre-ALK5fl/fl-Ai9 cKO but not DCXcreERT2-ALK5fl/fl-Ai9 iKO could be due to the different time points when Alk5 is deleted (neonatal stages vs young adult). This difference could also be attributed to the Alk5 gene deletion in distinct stages of cells in the neurogenic cascade (mGFAPcre targets early NSC lineage while DCXcreERT2 is specific for already differentiated immature neurons). An additional potential contributing factor is the differences in Cre recombinase efficiency in the constitutive mGFAPcre model compared with TAM-regulated Cre-ER protein in the DCXcreERT2 model. However, to rule out this possibility, we crossed the new iSuRe-Cre mouse line with the DCXcreERT2-ALK5fl/fl-Ai9 to generate DCXcreERT2-iSuRe-ALK5fl/fl iKO mice. This enables us to enhance recombination and specificity in DCX+ cells specifically to further confirm whether TGF-β signaling truly is expendable in the late stages of adult neurogenesis. Indeed, we observed no changes in migration for DCXcreERT2-iSuRe-ALK5fl/fl iKO mice, indicating that the role of TGF-β signaling on the migration of adult-born neurons in vivo is not critical to their migration in vivo.

TGF-β signaling is a highly dynamic pathway that requires precision tools to accurately study its role in adult neurogenesis in a context-dependent manner. While it is clear that TGF-β signaling has dual effects on the proliferation and migration in NSC cultures derived from adult mice, the effects were only partially validated in neonatal mice but not in adult immature neurons in vivo. This further highlights the complexity of the TGF-β signaling pathway and warrants future studies to identify molecular targets in different cellular contexts and developmental stages in vivo. Our study provides novel insight into the role of TGF-β signaling in the proliferation and migration of NSCs and adult-born neurons as well as cell-specific and neurogenic-stage-specific tools to more accurately interrogate the molecular and cellular mechanisms at play.

Limitations

Even though we were able to further elucidate the precise role of TGF-β signaling in the proliferation and migration of NSCs in vitro and provide cell-specific tools for future studies, we acknowledge the limitation of our studies. One potential limitation of the mGFAPcre driver is the deletion of Alk5 in GFAP+ astrocytes which could introduce confounding variables in this model. In the DCXcreERT2-ALK5fl/fl-Ai9 iKO mice and DCXcreERT2-iSuRe-ALK5fl/fl iKO mouse models, we only analyzed the survival and migration of tdTomato+/NeuN+ cells at 30 days post-TAM. It is possible that TGF-β signaling is required for the long-term (more than 30 days) survival of the newly born neurons. Additionally, our study did not elucidate the molecular targets of TGF-β signaling regulation in the proliferation or migration of cells either in vitro or in vivo which could be investigated in future studies.

Conclusion

The goal of this study was to elucidate the role of TGF-β signaling in adult neurogenesis at distinct steps during the neurogenic cascade (i.e. proliferation and migration). We also showcase the novel use of mouse lines to study this complex signaling pathway in a time- and context-dependent manner. In summary, we show a dual role of TGF-β signaling directly on aNSC proliferation and migration. Additionally, we provided new insight into the differential roles of TGF-β signaling on NSC proliferation and migration at the early postnatal and young adult stage in vivo.

Acknowledgments:

We thank Chet Closson (Microscopy core manager affiliated with University of Cincinnati) and the University of Cincinnati live imaging core (supported by NIH S10OD030402) for technical support. We would like to thank Dr. Rui Benedito (collaborator affiliated with the National Center for Cardiovascular Research, Spain) for sharing the iSuRe-Cre mouse line and Dr. Zhi-qi Xiong (collaborator affiliated with the Chinese Academy of Sciences) for sharing the DCXCreERT2 mouse line. Graphic abstract was created with BioRender.com (https://BioRender.com/r31n325).

Funding Statement

Funding: This study was supported by NIH grants, Nos. R01NS125074, R01AG083164, R01NS107365, and R21NS127177 (to YL), 1F31NS129204-01A1 (to KW) and Albert Ryan Fellowship (to KW).

Footnotes

Conflicts of interest: The authors declare no competing interests.

C-Editor: Zhao M; S-Editor: Li CH; L-Editors: Li CH, Song LP; T-Editor: Jia Y

Data availability statement:

No additional data are available.

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