Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

Domain: neuroscience
Published: 2026-02-09
Editorial outcome: major_revision

Abstract
Adult neurogenesis in the subgranular zone (SGZ) has been implicated in cognitive and affective functions. The role of neuroinflammation and reactive microglia in SGZ neurogenesis is not well understood. TGF-β signaling is critical to maintaining microglia homeostasis in the adult brain. To investigate the role of microglia in SGZ neurogenesis, using microglia-specific inducible knockout (iKO) mice for TGF-β1 ligand or receptor (Alk5 or Tgfbr2), here we show that TGF-β-deficient microglia increase adult neurogenesis in the SGZ, accompanied by altered anxiety-like behavior in KO mice. Single-cell RNA seq (ScRNAseq) analysis shows decreased PTEN signaling, and immunohistochemistry shows increased mTOR activity in DCX+ newly born neuroblasts at the SGZ in iKO mice. Inhibition of mTOR signaling by rapamycin reverses the heightened SGZ neurogenesis in iKO mice. This study reveals the role of microglia in regulating hippocampal adult neurogenesis via the PTEN-mTOR pathway and its potential implications for behavioral and affective functions.

Introduction
The process by which new neurons are generated in the adult brain is known as adult neurogenesis1, which predominantly takes place in two neurogenic regions: the subventricular zone (SVZ) and the subgranular zone (SGZ) in the adult mouse brain. The SVZ adult neurogenesis is crucial for processing olfactory sensory information2,3 and neurorepair after injury4–6, while the SGZ plays an essential role in pattern separation, forgetting, and affective function regulation7–11. Adult neurogenesis is a highly dynamic process that can be regulated by exercise12, an enriched environment13,14, and pathological conditions such as stroke4,6 and epilepsy15. However, enhanced neurogenesis has been shown to be both beneficial5,16,17 or detrimental under pathological conditions15. Similarly, the role of microglia and neuroinflammation in adult neurogenesis has been under debate. Specifically, it has been speculated that neuroinflammation resulting from CNS injury or disease might be detrimental to adult neurogenesis6,18–21. However, CNS injuries (stroke or traumatic brain injury (TBI)) or pathological conditions (epilepsy), which are accompanied by neuroinflammation and reactive microglia, can transiently stimulate neurogenesis15,22,23. Additionally, while the role of microglia in subventricular zone (SVZ) neurogenesis has recently been explored during brain development and adulthood24–27, its role in SGZ neurogenesis under physiological and pathological conditions is less understood28,29. Understanding the role and mechanisms of microglia in regulating adult SGZ neurogenesis and dentate gyrus (DG) sprouting might provide additional therapeutic targets to enhance beneficial or inhibit detrimental neurogenesis.

Microglia are the innate immune cells in the brain and act as a first line of defense when the brain is under duress30. The transition from a homeostatic state to a reactive phenotype in microglia is a key hallmark of their sensitivity to the surrounding microenvironment31–34. Their unparalleled capacity to sense and respond to changes in the brain’s microenvironment via pattern recognition receptors (PRRs), and specialized receptors such as purinergic receptors (P2RY) or Triggering Receptor Expressed On Myeloid Cells 2 (TREM2) make microglia master regulators of various processes, ranging from immune responses to synaptic pruning. Recently, microglia have been implicated in playing a critical role in regulating adult SVZ neurogenesis in mice24–27. Specifically, microglia ablation studies have been used to assess their functions in adult neurogenesis in the SVZ. Microglia ablation globally26 or in the SVZ locally24 did not reduce the proliferation and early differentiation of SVZ neural stem cells (NSCs). Rather, microglia might be critical for the migration of newly born neuroblasts and the pruning of dendritic spines of newly born neurons, as microglia-ablated mice show reduced migration of newly born neurons to the olfactory bulb24 (OB) and reduced synaptic dendritic spines in the newly born neurons in OB25. Overall, the precise roles and mechanisms of microglia in regulating SGZ adult neurogenesis under both homeostatic and reactive states remain largely unknown. However, there is key evidence that supports a direct regulatory role of microglia in adult hippocampal neurogenesis. For example, one study reported that microglia phagocytose apoptotic neuroblasts in the SGZ29, which was later shown to be critical in maintaining adult SGZ neurogenesis28. Additionally, under pathological conditions, microglia ablation abolished epilepsy-induced SGZ neurogenesis35, suggesting that in epilepsy, reactive microglia might contribute to the increased SGZ neurogenesis, possibly through the function of P2RY1235. In summary, these findings underscore a potentially critical role of microglia in regulating adult neurogenesis under physiological and pathological conditions. However, due to the complex cellular environment in vivo, especially after injury, the precise contribution of microglia to neurogenesis and the signaling pathways that mediate these interactions remain challenging to decipher. One common factor in conditions where microglia might be critical for neurogenesis is a reactive microglial profile (e.g., after injury, stroke, or pathological conditions in epilepsy) or a developmentally regulated microglial profile during embryonic development. This prompts the question of whether a reactive or developmentally regulated microglia profile could causally and directly enhance adult neurogenesis. Recent studies by us36 and others37,38 showed that altering microglia-derived TGF-β signaling, either through ligand or receptor knockout (KO) in microglia, leads to loss of microglia homeostasis. This shift of microglial homeostatic status results in transcriptomic profiles resembling those of aged, disease-related, and injury-related microglia36. Using these microglia-specific mouse models targeting TGF-β signaling, we aim to determine whether manipulating the TGF-β signaling pathway in microglia can directly affect adult neurogenesis in the SGZ. Our results show that, indeed, abolishing TGF-β signaling in microglia via the loss of the TGF-β1 ligand or via loss of function of the ALK5 (type 1) or TβRII (type 2) receptors all lead to an altered microglia profile that is sufficient to transiently stimulate SGZ adult neurogenesis. This finding establishes a direct role of microglial profile in regulating SGZ neurogenesis, independent of disease models like stroke, TBI, or epilepsy, providing a clean model to study microglia-NSC crosstalk. In our study, using this as a microglia-NSC crosstalk model, we also investigate the functional outcome of this heightened SGZ neurogenesis in anxiety-related behavior tests (elevated plus or elevated zero maze), which have been linked to SGZ adult neurogenesis8,10. Additionally, using scRNAseq analysis and functional validation by double KO mouse models and pharmacological inhibition, we investigate several candidate molecular pathways that could potentially mediate this cell-cell crosstalk, identified by scRNAseq and cell-cell interaction analysis. In summary, in contrast to the prevailing hypothesis that inflammatory microglia are, in general, detrimental to SGZ adult neurogenesis, our results show that microglia made reactive by silencing of TGF-β signaling can stimulate adult SGZ neurogenesis. Our data also show that microglia-derived Insulin-like Growth Factor 1 (IGF-1) or Tumor Necrosis Factor-Alpha (TNF-α) are not required for this stimulatory effect; instead, it is mediated by the upregulation of epilepsy-related genes and alterations in the PTEN-mTOR pathways in the iKO mice.

Results
The absence of Alk5-mediated TGF-β signaling in microglia results in the downregulation of microglia homeostatic genes and microgliosis, but not astrogliosis, in the adult mouse brain

We previously showed that the deletion of microglia-derived Tgfb1 ligand results in the loss of microglial homeostatic morphology and transcriptomic profile36. We also showed that this loss of homeostasis in microglia is due to direct loss of TGF-β signaling in microglia and not that of astrocytes, as deletion of the type 1 TGF-β receptor Alk5 in microglia leads to a similar phenotype in microglia, while Alk5 deletion in astrocytes does not36. To investigate how the loss of TGF-β signaling in microglia and its subsequent induction of reactive profiles might affect adult neurogenesis, we first utilize the microglia-specific receptor knockout mice in this study to avoid potential caveats of the direct effect of loss of microglia-derived TGF-β ligand on adult neurogenesis. Microglia-specific Alk5 inducible knockout mice were generated by crossing the Cx3cr1CreER line with the Alk5fl/fl line to ablate Alk5-mediated signaling in myeloid cells (Microglia, boarder-associated macrophage-BAMs, and peripheral monocytes/macrophages) during adulthood upon tamoxifen (TAM) administration (Fig. 1A). A R26-YFP (yellow fluorescent protein) reporter allele is also bred in both Cx3cr1CreER-Alk5WT/WT and Cx3cr1CreER-Alk5fl/fl mice to label recombined microglia and facilitate microglia sorting. Young adult Cx3cr1CreER-Alk5WT/WT or Cx3cr1CreER-Alk5fl/fl mice were harvested 3 weeks post TAM treatment (Fig. 1A), a timepoint allowing the majority of peripheral monocytes/macrophages to replenish from CX3CR1-negative progenitors, to minimize the effect of gene knockout on monocytes. Our previous study shows that this TAM regimen in the Cx3cr1CreER-Alk5fl/fl iKO mice efficiently deletes floxed Alk5 alleles in brain macrophages36 but shows no differences in Alk5 mRNA levels in blood or splenic monocytes (Supplementary Fig. 1). Compared to Cx3cr1CreER-Alk5WT/WT control mice (Fig. 1B), microglia displayed a reduction in homeostatic markers such as P2ry12 and Tmem119 in Cx3cr1CreER-Alk5fl/fl mice (Fig. 1C). Consistently, from sorted brain microglia and BAMs (YFP+), we demonstrate a significant decrease in Alk5, Tmem119, and P2ry12 mRNA levels (Fig. 1D) in Cx3cr1CreER-Alk5fl/fl mice in comparison to control microglia from Cx3cr1CreER-Alk5WT/WT (Fig. 1E–G) mice. Moreover, the pro-inflammatory cytokine Tnf was upregulated in microglia sorted from Cx3cr1CreER-Alk5fl/fl mice (Fig. 1H). Tnf expression levels are not altered in the blood or splenic monocytes (Supplementary Fig. 1), supporting the CNS-specific modulation of this pathway in our mouse model. Taken together, the downregulation of homeostatic markers and the upregulation of pro-inflammatory cytokines indicate a reactive phenotype of microglia. Additionally, Igf1, an essential growth factor to embryonic and adult neurogenesis, was also significantly upregulated in Cx3cr1CreER-Alk5fl/fl (Fig. 1I), consistent with our previous results from the MG-Tgfb1 iKO microglia, suggesting a potential effect in the regulation of adult neurogenesis of these young adult mice, given previous stipulations on IGF-1’s role in neurogenesis39–41. Consistent with our report in MG-Tgfb1 iKO mice, total microglia (IBA1+) cells in cortex, striatum, and hippocampus increase in MG-Alk5 iKO mice compared to control mice (Fig. 1L, N, O) while total astrocytes (S100β+) number remains the same in MG-Alk5 iKO mice (Fig. 1M). Corroborating an increased number of IBA1+ microglia in the iKO mice, there was a significant increase in the number of Ki67+ cells (Fig. 1K, N). Additionally, the majority of Ki67+ cells colocalized with IBA1+ cells (Fig. 1K, O) in Cx3cr1CreER-Alk5fl/fl (Fig. 1K), and total IBA1+/Ki67+ cells also increased in the MG-Alk5 iKO mice. This is consistent with previous studies suggesting impaired TGF-β signaling in microglia leads to microgliosis36,42 (Fig. 1L, N, O) but not astrogliosis.

Cx3cr1CreER-Alk5 iKO mice show a transient increase in hippocampal neurogenesis in young adult mice

CNS injuries (such as stroke or TBI) have been reported to induce microglia reactivity and increase adult neurogenesis in the SGZ43,44. To investigate whether there is a direct causal relationship between microglial TGF-β signaling and levels of SGZ adult neurogenesis, we evaluated the impact of Alk5 deletion in microglia on adult neurogenesis in the hippocampus (SGZ). Cx3cr1CreER-Alk5WT/WT or Cx3cr1CreER-Alk5fl/fl mice were harvested at 3-, 6- and 12-weeks post TAM to follow the full maturation cycle of the adult neurogenic cascade (Fig. 2A). BrdU was administered at one-week post TAM injections (Fig. 2A) for 8 days to label dividing cells and subsequently track a cohort of adult-born neurons from proliferation to maturation. MG-Alk5 iKO mice showed a significant increase (>66%) in the number of doublecortin (DCX+) cells as compared to control mice (Fig. 2B, E). Consistently, the number of BrdU+ cells increased by greater than 36% in MG-Alk5 iKO mice at 3 weeks post-TAM (Fig. 2F). Interestingly, by 6- and 12-weeks post-TAM, we no longer observed any significant changes in the amount of DCX+ cells in the SGZ (Fig. 2E), suggesting the increase in a cohort of newly born immature neurons in the SGZ is not sustained. However, BrdU+/NeuN+ cells remained increased at 6 and 12 weeks after TAM (Fig. 2F), supporting that the increased population of immature neuroblasts (DCX+) cells at 3 weeks indeed led to an increased number of adult-born mature neurons at the 6-week and 12-week time points. We also note that there is a significant decrease in total DCX+ cell numbers at 12 weeks post-TAM compared to 3 weeks, consistent with the previously reported age-related decline in SGZ neurogenesis45,46. It has been previously reported that only ~30% of newly born cells (identified by BrdU pulse labeling) at SGZ survive to mature neurons47 and this is recapitulated in our studies as we observe a steady decline of BrdU+ double positive cells over the 3–12-week time course in control mice which show ~30% survival rate in total BrdU+ cells (Fig. 2B–F). In contrast, MG-Alk5 iKO mice displayed an increased survival rate of ~50% in BrdU+ cells during the 3–12-week timeframe (Fig. 2F), with both WT and MG-Alk5 iKO mice showing the expected switch of BrdU+ cells from more immature neurons (BrdU+/DCX+ colocalization) at 3 weeks (Fig. 2G) to more mature neurons (BrdU+/NeuN+ colocalization) at 6 and 12 weeks post TAM (Fig. 2H), indicating maturation of neuroblasts to neurons over time. Similarly, the ventral hippocampus exhibits an increase in DCX+ cells and dendritic arborizations at 3 weeks post TAM but recovers to the control level at 6- and 12-weeks post TAM (Supplementary Fig. 2). Furthermore, we observed increased DCX+ dendritic arborization in the hippocampus of the Cx3cr1CreER-Alk5fl/fl mice (Fig. 2I), consistent with increased DCX+ immature neuron numbers. Taken together, our results show that microglia reactivity caused by loss of microglial TGF-β signaling stimulates adult hippocampal neurogenesis transiently and results in a net gain of a cohort of mature neurons by 6-12 weeks post-TAM. The increase in DCX+ immature neurons observed at 3 weeks in the MG-Alk5 iKO mice returned to a comparable level to control mice at 6- and 12-weeks post Alk5 gene deletion in microglia. To examine whether the return of DCX+ cells to the control levels in later time points is due to the recovery of microglia phenotype in the Cx3cr1CreER-Alk5fl/fl iKO mice, we analyzed the phenotype of microglia from 3 to 12 weeks post TAM treatment and found that, indeed, there is a recovery of morphology, homeostatic marker P2RY12 expression which correlates with recovered nuclear phosphorylated Mothers against decapentaplegic homolog 3 (pSMAD3) levels in IBA1+ cells (Supplementary Fig. 3) at 12 weeks post TAM, supporting that the neurogenesis phenotype is directly linked to the microglia phenotype in the Cx3cr1CreER-Alk5fl/fl iKO mice.

Loss of microglial type 2 TGF-β receptor or TGF-β1 ligand in microglia recapitulates the phenotype of increased immature neurons at the adult hippocampal niche

Both type 1 and type 2 receptors are required for the formation of the heterotetramer receptor complex that mediates TGF-β signaling48. While the type 1 receptor, ALK5, can mediate and is required for TGF-β signaling, it is well documented that other TGF-β superfamily ligands can also signal through this receptor, including ligands from the activin subclass49. In contrast, the type 2 receptor, TβRII, exclusively mediates signaling through the TGF-β ligands50. Therefore, to further investigate whether the loss of TGF-β signaling or loss of activin signaling via the ALK5 receptor is the cause of the increase in adult neurogenesis we observed in adult mice, we also generated the Cx3cr1CreER-Tgfbr2fl/fl iKO mouse line (Supplementary Fig. 4A) to not only maximize recombination efficiency but also mitigate the deletion of TGF-β signaling down to just one subclass containing the TGF-β1, 2, and 3 ligands. Following the same experimental timelines, Cx3cr1CreER-Tgfbr2WT/WT and Cx3cr1CreER-Tgfbr2fl/fl mice were treated with TAM and harvested at 3 weeks post-TAM (Supplementary Fig. 4A). Our results are consistent with the MG-Alk5 iKO mice (Figs. 1, 2) in that homeostatic markers Tmem119 and P2ry12 for microglia are downregulated (Supplementary Fig. 4B, C, quantification in D–E) and microglia morphology resembles an activated state (Supplementary Fig. 4B, C). We also confirm that deletion of TβRII in CX3CR1+ cells specifically affects downstream signaling in IBA1+ cells and not IBA1- cells indicated by the nuclear localization of pSMAD3 (Supplementary Fig. 4B, C, F–G). Furthermore, loss of type 2 receptor (TβRII) in CX3CR1+ myeloid cells recapitulates the same findings of increased DCX+ cells and dendritic arborization in these Cx3cr1CreER-Tgfbr2fl/fl iKO mice (Supplementary Fig. 4H–K). In summary, these data suggest that it is not loss of activin, but specifically TGF-β signaling in microglia that mediates the increase in adult SGZ neurogenesis. Next, we also generated Cx3cr1CreER-Tgfb1fl/fl ligand iKO mice which also recapitulate the same phenotype of increased SGZ adult neurogenesis compared to control mice at 3 weeks post TAM treatment (Supplementary Fig. 5). Additionally, in contrast to the recovery of microglia phenotype in the Cx3cr1CreER-Alk5fl/fl receptor iKO mice, our results show that Cx3cr1CreER-Tgfb1fl/fl ligand iKO mice maintain the microglia reactive phenotype and loss of pSMAD3 signal in microglia up to 12 weeks post TAM treatment (Supplementary Fig. 6). Consistent with the sustained microglia reactive phenotype, DCX+ cells remain increased at 12 weeks after TAM treatment in the Cx3cr1CreER-Tgfb1fl/fl ligand iKO mice (Supplementary Fig. 5). Taken together, our data strongly support that microglia reactivity in the iKO directly contributes to the observed increase in the DCX+ cells in the adult mouse hippocampus.

P2ry12CreER driver line results in deletion of Alk5 in both microglia and choroid plexus macrophages, which lead to similar increases in immature neurons in the hippocampus as compared to the Cx3cr1 driver line

We and others have shown that the myeloid-specific driver Cx3cr1CreER mouse line targets peripheral monocytes and macrophages in addition to microglia51,52, which could potentially confound the interpretation of data regarding microglia-specific mechanisms. While the 3-week timepoint allows for the turnover of circulating monocytes and macrophages via non-recombined CX3CR1-negative bone marrow progenitors, the rapid increase in immature neurons we see at 3 weeks could potentially be attributed to indirect influences from either CNS border-associated macrophages (BAMs) or the transient gene deletion in a cohort of KO monocytes or peripheral tissue-resident macrophages. It has also been reported that the knock-in/knockout Cx3cr1CreER mouse lines, where CreER is knocked into the Cx3cr1 locus, disrupt the endogenous Cx3cr1 gene expression53,54. The reduced gene expression of Cx3cr1 in microglia is reportedly able to modulate adult neurogenesis on its own55–57 albeit we used the Cx3cr1CreER-Alk5WT/WT mice as a control to account for this potential caveat. Nevertheless, to circumvent these caveats and to further increase the rigor of our research, we generated the P2ry12CreER/CreER driver line with the ALK5fl/fl line51 (Fig. 3A). This Cre driver line used a P2A sequence to enable CreER expression under the control of endogenous P2ry12 promoter without affecting the expression of endogenous P2ry12 gene and our previous study showed that homozygous CreER in both alleles increased the efficiency of gene deletion in the Alk5 floxed alleles51 and can achieve close to 80% reduction in Alk5 gene expression in sorted brain microglia51. P2ry12CreER/CreER-Alk5fl/fl mice were treated with TAM following the same paradigm and harvested at 3 weeks post-TAM (Fig. 3A), a timepoint where we observed the robust increase in the DCX+ cells in SGZ of the Cx3cr1CreER-Alk5fl/fl mice. Even though there is lower recombination efficiency in P2ry12CreER/CreER-Alk5fl/fl compared to the Cx3cr1CreER-Alk5fl/fl51, we still observe a significant decrease in immunoreactivity of homeostatic markers Tmem119 and P2ry12 in Alk5 iKO microglia when compared to control (Fig. 3B, C, F–G). Additionally, we show that Cx3cr1CreER -Alk5 iKO mice show loss of pSMAD3 in parenchymal microglia, perivascular macrophages, and choroid plexus macrophages (Supplementary Fig. 7) while P2ry12CreER/CreER-Alk5fl/fl mice show reduced pSMAD3 in parenchymal microglia and choroid plexus macrophages but preserve pSMAD3 in perivascular macrophages (Supplementary Fig. 7). These results are consistent with previous studies showing Cre activity in both microglia and choroid plexus in this mouse line51,58. Importantly, the number of DCX+ cells at the SGZ consistently shows an increase in this microglia-specific P2ry12CreER/CreER-Alk5fl/fl mouse model (Fig. 3D, E, H), supporting that reactive microglia and possibly choroid plexus macrophages but not perivascular macrophages or other myeloid cells are responsible for the observed stimulation of adult neurogenesis. Furthermore, we observed increased DCX+ dendritic arborization in the hippocampus of the P2ry12CreER/CreER-Alk5fl/fl mice (Fig. 3D, E, I), consistent with increased DCX+ immature neuron numbers. Taken together, this suggests that Alk5 deletion in microglia and/or choroid plexus macrophages and the resulting loss of TGF-β signaling in this subset of myeloid cells is sufficient to drive an increase in adult neurogenesis and may, in turn, impact synaptic plasticity and functional outcomes.

Evidence of enhanced survival but not proliferation in the Cx3cr1CreER-Alk5fl/fl mouse line suggests necessity of microglia to the maturation and survival of adult-born neurons

The increase in immature neurons we observe in various MG-TGF-β signaling-abolished mouse models raises the question of whether alteration in microglia profile modulates adult neurogenesis via enhanced proliferation of NSCs/intermediate progenitor cells (IPCs) or enhanced survival of newly born immature neurons. To test this, we utilized the Cx3cr1CreER-Alk5fl/fl (Fig. 4A, E) mouse line and either post-labeled (Fig. 4A) or pre-labeled (Fig. 4E) a cohort of newly born cells with BrdU with respect to TAM treatment (induction of gene knockout). The total number of BrdU+ cells at the SGZ line was significantly increased for both pre- and post-labeling paradigms after harvesting mice 3-weeks post TAM (Fig. 4B–H). It is important to note that we are able to analyze these cells by colocalization with either DCX or NeuN but not with IBA1 to identify the adult-born neurons and not proliferating microglia. These data suggest the mechanism of increased DCX+ immature neurons at 3-weeks post TAM and the final mature adult-born neurons (BrdU+/Neun+) 12-weeks post TAM is at least partially increased via survival of newly born cells at the SGZ as tracked in the BrdU pre-labeling paradigm (Fig. 4E–H) because Alk5 gene knockout was not induced until the newly born cells (BrdU+) were already pre-labeled. This hypothesis is also supported by the increased percentage of BrdU+ cells that survived in the 3-12-week time window in KO vs control mice (50% survived in iKO vs 30% survived in control mice) (Fig. 2). To further test this hypothesis, we further evaluated the number of proliferating cells at 3-, 6-, and 12-weeks post TAM with an endogenous cell proliferation marker at the SGZ (quantification is for Ki67+/IBA1- cells) and found no significant difference between control and Cx3cr1CreER-Alk5fl/fl mice at any of the timepoints examined (Supplementary Fig. 8).

Microglia ablation via PLX5622 does not lead to increased SGZ adult neurogenesis but repopulation of CNS with Alk5 KO microglia after PLX5622 ablation increases adult neurogenesis in the hippocampus

Loss of TGF-β signaling leads to loss of homeostatic signature microglia genes and upregulation of proinflammatory genes such as Tnf and genes that stimulate proliferation and cell survival, such as Igf136. Microglia have been speculated to regulate adult neurogenesis via their secretome or by phagocytosing of apoptotic newborn cells28,29. To investigate whether the observed changes in SGZ adult neurogenesis are due to the loss of a critical homeostatic function of “physiologically normal” microglia, we investigated the SGZ adult neurogenesis in mice that lacked microglia using microglia ablation agent, PLX562236. To label adult neural stem cells and their progeny, we crossed the NestinCreER line59 with the Ai9 reporter line (Fig. 5A) so that upon TAM administration, we can endogenously track a NSC-specific cohort of adult-born tdTomato+ cells in the SGZ. We treated NestinCreER-Ai9 mice with TAM and fed the mice either control or PLX5622 diet for 3 weeks (Fig. 5A), a similar timeline to the loss of Alk5 in microglia in the MG-Alk5 iKO mice. A significant decrease of Iba1+ cells was observed (Fig. 5B, C, G), indicating successful depletion of microglia in the adult mouse brain. However, we observed no significant difference in the number of tdTomato+ (Fig. 5B–D), DCX+ (Fig. 5B, C, E), or tdTomato+/DCX+ cells (Fig. 5B, C, F), suggesting that the increase in DCX+ cells observed in the Cx3cr1CreER-Alk5fl/fl, P2ry12CreER/CreER-Alk5fl/fl, Cx3cr1CreER-Tgfb1fl/fl, and Cx3cr1CreER-Tgfbr2fl/fl mouse models is not due to a loss of “normal” microglia function. In addition to this, Cx3cr1CreER-Alk5WT/WT and Cx3cr1CreER-Alk5fl/fl mice were subjected to a control or PLX5622 diet for 3 weeks and administered TAM post-microglia ablation (Fig. 6A), proceeding the withdrawal of PLX5622 diet to allow the repopulation of these WT or KO microglial cells. Our previous study has shown that repopulating CNS microglia from TGF-β signaling-deficient (via Alk5 gene knockout) microglia after PLX5622 ablation leads to overpopulation of microglia and repopulation of reactive microglia (Fig. 6 and Bedolla et al.36). This experiment allows us to evaluate the effects of repopulating KO microglia on adult SGZ neurogenesis. Repopulated microglia in MG-Alk5 iKO microglia displayed less ramified morphology and downregulation of homeostatic marker Tmem119 and increased CD68 expression (Fig. 6B, C, G, H), consistent with our previous report36. Importantly, consistent with our previous report36, TGF-β deficiency in iKO microglia does not lead to acute neurodegeneration in adult mouse brain (evaluated by unbiased stereology counts of all layers of cortical neurons Fig. 6E and Bedolla, et al.36). Interestingly, with this treatment paradigm after Alk5 KO microglia repopulation, the number of immature neurons in the hippocampus was also significantly increased in MG-Alk5 iKO when compared to the control mice (Fig. 6D, I, J), with a large effect size (Cohen’s d=8.755227 and coefficient r=0.9748874 for Fig. 6I and Cohen’s d=5.4435 and coefficient r=0.9386 for Fig. 6J), albeit a small n number. Taken together, these results suggest that the increased SGZ neurogenesis is not merely due to loss of a “physiologically normal” microglia function, but rather, it suggests that the enhanced neurogenesis in SGZ is likely due to certain gain-of-function mechanisms from the KO microglia that occur after the disruption of Alk5-mediated TGF-β signaling.

Reactive microglia in MG-Alk5 iKO mice enhance SGZ neurogenesis not by upregulation of microglial Igf1 or Tnf but by upregulation of epilepsy-related genes and downregulation of PTEN pathway in the neuroblasts at the hippocampus

We next carried out scRNAseq of the dissected whole hippocampus in control, MG-Tgfb1, and MG-Alk5 iKO mice at 3 weeks after TAM treatment (Fig. 7A, B and Supplementary Fig. 9 for QC plots) in the presence of transcription and translation inhibitors60 to avoid microglia profile changes during tissue collection. ScRNAseq data identified different cell clusters, including microglia and neuroblasts/immature neurons (Fig. 7C). Utilizing known cells markers such as Aif1 (Fig. 7D) and Dcx (Fig. 7E) alongside Cell Marker 2.0 or Allen Cell Types Database, we were able to annotate the clusters and determine cell type for further downstream analysis. The microglia cluster showed enriched signature microglia marker genes, such as Aif1 (Fig. 7D), TMEM119, P2ry12 (Fig. 7F and G) but not other cell type markers, such as astrocyte markers (Aldh1l1 or Gfap, Fig. 7H and I). DEG analysis shows differences in the gene expression of 360 genes between MG-Alk5 iKO vs control (Fig. 7J, see Supplementary Fig. 9 for split UMAP for all three groups and complete DEGs listed in Supplementary Data 1). MG-Tgfb1 iKO mice showed differences in the gene expression of 327 genes in the microglia cluster (Supplementary Fig. 9F, complete DEGs listed in Supplementary Data 3 and Supplementary Fig. 9H for 141 overlapping genes between Alk5 and Tgfb1 iKO microglia DEGs). In the neuroblast cluster, the expression of 2324 genes are altered in the MG-Alk5 iKO mice (Fig. 7K, complete DEGs listed in Supplementary Data 2) while 2229 genes are altered in the MG-Tgfb1 iKO (Supplementary Fig. 9G, complete DEGs listed in Supplementary Data 4, with 1779 overlapping of neuroblast DEGs between Alk5 and Tgfb1 iKO mice shown in Supplementary Fig. 9I). Interestingly, out of the overlapping neuroblasts DEGs (Supplementary Fig. 9I), GO:BP gene pathway analysis shows upregulation of multiple biological processes related to neurogenesis, neuron development, generation of neurons, neuron projection morphogenesis, neuron projection development, neuron differentiation and synapse organization (Supplementary Fig. 9J, see full list in Supplementary Data 5) which is consistent with the observed phenotype in altered neurogenesis. Biological processes that were downregulated in the Go:BP gene pathway analysis include regulation of cellular process, localization, and establishment of localization (Supplementary Fig. 9K, see full list in Supplementary Data 6), consistent with aberrant neurogenesis. Additionally, our analysis using LIANA revealed strong cell-cell interactions between microglia and endothelial cells and microglia-DCX+ immature neuron population mediated by predicted specific ligand-receptor interactions (Fig. 7L, see full list in Supplementary Data 7). Enricher pathway analysis (Elsevier pathway) of the differentially expressed genes (DEGs) from neuroblasts isolated from both MG-Alk5 iKO and MG-Tgfb1 iKO mice identified several overlapping top significantly altered disease-related or cellular processes (Fig. 7M, complete DEGs listed in Supplementary Data 8 for MG-Tgfb1 iKO) and specific molecular pathways (Fig. 7N) identified from the neuroblasts derived from either MG-Alk5 iKO or MG-Tgfb1 mice. Among these, we focused on the TNF-α, IGF-1, and PTEN-PI3K/AKT/mTOR signaling pathways (Fig. 7N). IGF1 has been shown to increase adult neurogenesis40,61,62 and oligodendrocyte development63. Interestingly, our data show that there is also increased OLIGO2+ cells and OLIGO2+/NG2+ oligodendrocyte progenitor cells (OPCs) in the hippocampus of MG-Alk5 iKO mice (Supplementary Fig. 10). Peripheral infusion of IGF-1 increased the amount of progenitors present in the GCL62. Additionally, increased levels of IGF-1 in the brain have been found after voluntary exercise, an external method commonly used to increase neurogenesis64. Interestingly, both Tgfb1 and Alk5 gene deletion lead to increased Igf1 expression in microglia (Fig. 1I and Bedolla et al.36). To investigate whether increased Igf1 growth factor from iKO microglia is responsible for the increased adult neurogenesis in the MG-Alk5 iKO mice, we generated Cx3CR1creERT2-Alk5fl/fl/Igf1fl/fl double knockout mouse model (Fig. 8A). Following a similar experimental timeline, control, Alk5 single iKO, and Alk5/Igf1 double iKO mice were harvested at 3 weeks post TAM treatment (Fig. 8A). We compared the Cx3CR1creERT2-Alk5fl/fl/Igf1fl/fl model against the Cx3cr1CreER-Alk5fl/fl model to ensure we had efficient gene deletion of Alk5 in sorted microglia in both the Alk5 single iKO and the Alk5/Igf1 double iKO (dKO) mice (Fig. 8H–J). Additionally, in the Alk5/Igf1 dKO mice, the upregulation of Igf1 in microglia is abolished (Fig. 8K). Interestingly, abolishment of Igf1 gene in the Alk5/Igf1 dKO mice does not rescue the downregulation of homeostatic markers Tmem119 and P2ry12 (Fig. 8B–D). More importantly, despite the successful abolishment of Igf1 upregulation in the dKO mice, the phenotype of increased adult neurogenesis (measured by increased newly born neuroblasts DCX+ cells and dendritic arborization) remained in the Alk5/Igf1 dKO mice (Fig. 8E–G, L, M). Despite previous studies reporting the importance of microglial Igf1 in regulating adult neurogenesis under other contexts and the substantial upregulation of Igf1 in the Cx3cr1CreER-Alk5fl/fl mouse model, our data suggests that at least microglial-derived Igf1 growth factor is not likely the cellular factor that mediate the microglia-NPCs crosstalk in our MG-Alk5 iKO mouse model. Similarly, TNF-α has been reported to have both pro- and anti-neurogenic effects34,65, we therefore generated double MG-Alk5 iKO on a constitutive Tnfa KO mouse background (Supplementary Fig. 11) at followed the same experimental paradigm as our single iKO mouse experiment. Again, to our surprise, Tnf loss of function did not alter the phenotype of heightened SGZ neurogenesis (measured by total DCX+ cells and DCX+ dendritic arborization) in the MG-Alk5 iKO/Tnf dKO mice (Supplementary Fig. 11), suggesting that upregulated Tnf by itself is also not sufficient to explain the phenotype. Previous studies have shown that while blocking of individual cytokines failed to abolish the effects of reactive microglia on stimulating neurogenesis in vitro, combined antibodies that block several of the candidate cytokines had a significant effect, suggesting that synergistic and compensatory effects might be at play between the microglial-NSC crosstalk66. Given our negative results targeting individual cytokines and the reports from previous studies mentioned above, we then turned our focus more towards cellular pathways that could potentially have a convergent effect on immature neurons. We next focused on the PTEN-PI3K/AKT/mTOR pathway. Notably, PTEN, a negative regulator of the mTOR pathway, was downregulated among the DEGs of immature neurons (Fig. 7K). Moreover, there was a significant alteration in the PTEN-dependent cell cycle arrest and apoptosis pathway (Fig. 7N). Loss of PTEN function in neurons is known to induce epilepsy and enhance neurogenesis in the adult hippocampus67. Downregulation of PTEN could lead to enhanced mTOR signaling, which was one of the identified altered pathways in the neuroblast cluster in the iKO mice (Fig. 7N). This hypothesis is further supported by the significant changes observed in the “Proteins involved in Epilepsy” pathway (Fig. 7M) in both MG-Alk5 and MG-Tgfb1 iKO mice. To test whether a TGF-β-deficient microglia profile in the MG-Alk5 iKO mice enhances SGZ neurogenesis by downregulation of PTEN and upregulation of mTOR pathway, we treated the MG-Alk5 iKO mice with rapamycin daily, a well-characterized mTOR inhibitor starting from 4 days after TAM administration (Fig. 9A, B) and harvested mice 3 weeks after TAM administration. Interestingly, Rapamycin treatment reversed the phenotype of increased DCX+ immature neurons and increased dendritic arborization in SGZ of the MG-Alk5 iKO mice (Fig. 9C–F) while having no effects on the total DCX+ cell number in WT mice (Fig. 9C–F). Similarly, dendritic arborization of DCX+ immature neurons was decreased following rapamycin treatment in MG-Alk5 iKO but not in WT mice (Fig. 9C–F). Additionally, rapamycin-treated WT and MG-Alk5 iKO both show the ectopic location of DCX+ neuroblast cells in the outer layer of GCL (Fig. 9C–F) compared to vehicle-treated mice, suggesting either delayed maturation and failure to downregulate DCX in migrated newly-born neurons or enhanced ectopic migration of DCX+ neuroblasts in the GCL by rapamycin treatment. To examine whether MG-Alk5 results in mTOR pathway upregulation in vivo in SGZ neuroblasts in vivo, we analyzed phosphorylated S6 protein (pS6) as a downstream target for the mTOR pathway specifically in DCX+ immature neuroblasts. pS6 levels in DCX+ cells increased in MG-Alk5 iKO SGZ, which is abolished by rapamycin treatment (Fig. 9H and G). Because both DCX+ cells and OPCs increased in the MG-Alk5 iKO mice at 3 weeks post TAM, to further investigate whether microglia directly interact with NSCs during neuronal differentiation, we examined WT primary adult NSCs in 3D neurosphere differentiation when they are co-cultured with either WT or Alk5 KO primary microglia (Fig. 9I and J). When NSCs undergo neuronal differentiation (induced by growth factor withdrawal), compared to those co-cultured with WT microglia, NSCs co-culture with the Alk5 KO microglia show increased neuronal differentiation, measured by TUJI immunoreactivity (Fig. 9K, L). Similar to our in vivo data, rapamycin treatment rescued this phenotype (Fig. 9K, L), supporting a direct interaction of reactive microglia with adult NSCs during differentiation. To further examine whether rapamycin itself blocks the reactivity of microglia in Alk5 iKO mice, we also evaluated the morphology of Alk5 iKO microglia in either the vehicle- or rapamycin-treated group and found similar reactivity index quantified by the total length of processes, ramification index, and the area covered by individual microglia processes. Rapamycin treatment does not block the reactivity of microglia in the MG-Alk5 iKO mice, nor does it affect the morphology of microglia in WT mice (Supplementary Fig. 12). Taken together, our data support a PTEN-mTOR axis that could mediate enhanced adult SGZ neurogenesis caused by reactivity in microglia due to silencing of TGF-β signaling (Fig. 9M).

Disruption of microglial TGF-β signaling decreases anxiety-like behavior in Elevated Plus maze that corresponds to microglia status

Next, we sought to further explore our findings related to the increased survival of these newly-born neurons and how their integration into the neural circuitry may impact behavioral functions. Given that the receptor (Alk5) and ligand (Tgfb1) iKO mice show different properties in the recovery of microglia homeostasis after TAM-induced gene deletion in microglia, we are especially interested in whether the behavioral phenotypes mirror the temporal pattern of changes in adult hippocampal neurogenesis in these two different mouse lines. First, we examine the behavioral phenotype in the MG-Alk5 iKO mice. Cx3cr1CreER-Alk5WT/WT or Cx3cr1CreER-Alk5fl/fl mice (Fig. 10A) were subjected to behavioral assessments at 7-8 weeks post TAM administration (Fig. 10A) to allow sufficient maturity and integration of adult-born SGZ neurons into the circuitry. Notably, there were no motor deficits seen in these young adult mice (Fig. 10) as previously reported by us in the ablation of microglial TGF-β1 ligand in young adult mice36. Adult-born neurons have been implicated in behavioral functions such as anxiety-like and depressive-like behaviors17. Given the observed increases in a cohort of adult-born neurons in the SGZ of the MG-Alk5 iKO mice, we tested the Cx3cr1CreER-Alk5fl/fl or Cx3cr1CreER-Alk5WT/WT control mice in an Elevated Plus maze (EPM, Fig. 10B, C). Interestingly, we did observe both increased time and distance in the open arm of the EPM test, suggesting decreased anxiety-like behavior. Alternatively, this could also be attributed to decreased inhibition in exploratory activities in mice, similar to the behavioral phenotypes observed in multiple Alzheimer’s disease models68,69. In addition to affective functional dysregulation, we also observed spatial memory and learning deficits during the Barnes maze test (Fig. 10F, G) with mild adverse cues (bright light and fan above the maze) to motivate learning. Notably, in the MG-Alk5 iKO mice, the increase in DCX+ neuroblasts returns to baseline levels at 6-12 weeks after TAM. Additionally, adult-born neurons are reported to show a critical time window when their plasticity (measured by long term potentiation- LTP) is increased70. We next investigate whether the phenotype in EPM and BM in the MG-Alk5 iKO mice persists or recovers with microglial profile recovery at 12 weeks post TAM, when there is no longer an increase in DCX+ cells or when the critical window for the increased plasticity has passed for the cohort of newly integrated neurons. For the 12-week time point, we used Elevated Zero Maze (EZM) instead of EPM to avoid repeated exposure to the same maze, as anxiety-like or passive avoidance behavior is sensitive to such repetition71,72. Both phenotypes in the elevated maze and the BM returned to control levels at 12 weeks post TAM injection in the MG-Alk5 iKO mice. (Fig. 10D, E, G).

Next, we examine the behavioral phenotype in the MG-Tgfb1 iKO, in which microglia reactivity persists and the adult hippocampus shows a sustained increase in DCX+ cells both at 3 weeks and at 12 weeks after TAM treatment. In young adult iKO mice, similar to the MG-Alk5 iKO, general motor learning is not affected as measured by the accelerating speed rotarod test (Fig. 10P). Cx3cr1CreER-Tgfb1fl/fl iKO mice showed similar increased activity in the open arm of EPM at 6 weeks post TAM (Fig. 10I–K) and learning/memory deficit in the Barnes maze, as previously reported by our study36 (Fig. 10N). At 12 weeks post TAM, MG-Tgfb1 iKO mice show a consistent increase in time and distance spent in the open arm in the EZM and deficits in the BM, consistent with the sustained microglia reactivity and increased DCX+ cells in this line. These recapitulated behavioral deficits in learning/memory and altered anxiety-like behavior in both the Cx3cr1CreER-Alk5fl/fl and Cx3cr1CreER-Tgfb1fl/fl mice, which correspond to the microglial and adult neurogenesis status, provide further support for the functional consequences of impaired microglial TGF-β signaling in the regulation of adult neurogenesis and neuronal functions.

Methods
Animals
Laboratory Animal Medical Services (LAMS), Animal Care and Use Program (ACUP) and the Institutional Animal Care and Use Committee (IACUC) are housed at the University of Cincinnati. All animal protocols were approved by IACUC. Approved animal protocol number is 24-02-27-01. Within LAMS, mice are held to a 14 h light/10 h dark cycle. NestinCreER (JAX:016261)59 and Ai9R26-CAG-tdTomato (JAX:007909)105 were used to endogenously label and track NSCs. We generated cell-type-specific inducible knockout models of critical TGF-β signaling components using Cx3cr1CreER(Jung) (JAX:020940)53, Cx3cr1CreER(Littman) (JAX:021160)54, and P2ry12CreER (JAX:034727)58, to delete Alk5, Tgfbr2, Igf1 in microglia or Tnfα constitutive knockout. Alk5 (JAX:028701)106 floxed or Tgfbr2 (JAX:012603)107 floxed mice, and Tnfα KO (JAX:005540)108 and Igf1 floxed (JAX:016831)109 mice were crossed with Cx3cr1CreER(Jung), all purchased from the Jackson Laboratory. We crossed the above microglia CreER lines with ROSA26-YFP (JAX:006148)110 in some of the experiments to facilitate microglia sorting. All animals were euthanized by administration of avertin (2.5%) followed by transcardial perfusion with 4% paraformaldehyde (PFA) (for IHC) or 1x HBSS (for FACS, RNAseq, and qRT-PCR) as previously described36. Mice were bred on a C57BL/6J background and both sexes were used in all experiments. All animals were used unless tissue quality was poor as a result of subpar perfusions or inadequate cryoprotection. For all experiments, bromodeoxyuridine (BrdU) was administered intraperitoneally at 100mg/kg per injection (10µl/g bodyweight, Invitrogen #000103) during the indicated time frame in each experimental timeline and harvested between 3-12 weeks post-TAM.

Tamoxifen administration
3 or 5 consecutive gavage injections of TAM (specified in each experiment/figure) solution were given to 6-16-week-old male and female mice, administered at a dosage of 180mg/kg. 900µL of sunflower seed oil and 100µL of ethanol were sonicated with 30mg of TAM powder protected from light36. To evaluate the effect of microglia-mediated TGF-β signaling silencing in adult neurogenesis, young adult mice were harvested at 3, 6-, or 12-weeks post-TAM to evaluate microglia phenotype and SGZ neurogenesis.

Microglia ablation PLX5622
Mice were given either the control diet (Plexxikon) or PLX5622 diet (Plexxikon) as previously described36,111 for the duration of the study. All animals had unrestricted access to diet and water. For studying the phenotype of homeostatic microglia on baseline adult neurogenesis, we administered TAM to NestinCreER-Ai9 mice to label a NSCs and their progeny, followed by 3 weeks of control or PLX5622 diet. Mice were harvested 3 weeks post-TAM and brains were harvested for IHC staining. For assessing the role of repopulating Alk5 KO microglia in the stimulation of adult neurogenesis, mice were treated with a control PLX5622 diet for 3 weeks, followed by TAM treatment during the last 3 days. These animals were then switched to a standard chow diet to allow the repopulation of WT or Alk5 KO microglia over the course of 3 weeks, when they were harvested by administration of avertin (2.5%) followed by transcardial perfusion with 4% paraformaldehyde (PFA). Brain tissue was harvested for IHC staining.

Fluorescent activated cell sorting (FACS)
Mice were harvested by administration of avertin (2.5%) followed by transcardial perfusion with cold 1x HBSS for 3–4 mins before the brain was extracted. The brains were mechanically dissociated via a scalpel before enzymatically dissociated with the papain dissociation system (9001-73-4, Worthington Biochemical Corporation) while all of the above processes are carried out in the presence of transcriptional and translational inhibitors to prevent microglial gene expression changes during the isolation protocol60. Once the brain is sufficiently dissociated, 37% percoll solution was used to clear any excess myelin or debris. Cells were resuspended in FACS buffer (0.5% BSA in PBS) for staining and sorting using a BD/FACSAria II (BD Biosciences, Franklin Lakes, NJ). CD11b, CD45, and GFP reporter were used to develop a gating strategy to enrich microglia. Sorted microglial cells were used for qRT-PCR or bulk RNAseq for gene expression analysis as described below.

qRT-PCR
Using the RNAqueous-Micro Total RNA isolation kit (AM1931, Thermo Fisher Scientific), RNA was isolated from sorted microglia as previously described36. Supercript III reverse transcriptase (18080044, Thermo Fisher Scientific) or iScript cDNA synthesis kit (1708890, BioRad) was used to generate cDNA. Following cDNA synthesis, qRT-PCR probes Hmbs1 (hydroxymethylbilane synthase), Hprt1 (hypoxanthine phosphoribosyltransferase 1), Aif1, Alk5, Igf1, Tnf, Tmem119, and P2ry12 were used to analyze cDNA levels using a Roche LightCycler II 480. Relative gene expression was calculated using the ΔΔCt method compared to Hmbs1 as a reference gene and expressed as fold change compared to the average of control cells for each individual gene.

Immunohistochemistry
Mice were anesthetized with avertin (2.5%), then perfused with 4% PFA. The brains were harvested and post-fixed in 4% PFA overnight. In the following days, brains were switched to 20% and 30% sucrose respectively until brains sank. Each brain was sectioned at 30µM thickness on a cryostat and used for immunohistochemistry (IHC) staining as previously described36. Antibodies for BrdU (1:500, Abcam), DCX (1:1000, Cell Signaling), Ki67 (1:500, Invitrogen), IBA1 (1:1000, Abcam), TMEM119 (1:2000, GeneTex), P2RY12 (1:200, Biolegend), GFAP (1:1000, Sigma), pSMAD3 (1:200, Abcam), beta-3 tubulin (1:2000, Promega), pS6 (1:800, Cell Signaling), Olig2 (1:500, R&D Systems), MBP (1:250, Biorad), NG2 (1:500, Millipore) were used. P2RY12 clone: P2YM 17A12112 (1:500), and P2RY12 clone: P2YM 1E5112 (1:500) was generated and validated as previously described. Brain sections were blocked for 1h at room temperature in 4% BSA/0.3% Triton-x100 and incubated in primary antibody solution (diluted in blocking buffer) on a shaker overnight at 4°C. Secondary antibodies conjugated with Alexa fluorescence 488, 555, 647 or 790 (1:500) were incubated with the brain sections for 4 hours at room temperature. Confocal imaging of stained brain sections was carried out using a Leica Stellaris 8. Quantification of cell counts was carried out using ImageJ (1.53) software. At least 3 coronal sections with the respective region of interest were quantified for each mouse and averaged to be used as a single data point.

BrdU Immunohistochemistry
Animals injected with BrdU labeling reagent (10µl/g bodyweight, Invitrogen #000103) at various time points after injection were harvested following avertin (2.5%) injection and PB/PFA perfusion. As described previously113, coronal or sagittal cryosections were washed 3x at RT and incubated in pre-warmed freshly made DNA denaturation solution (50% formamide, 50% 2x SSC buffer) for 2 hours at 60°C with shaking. After being washed 3x in 2x SSC at 60°C with shaking, the tissue was incubated in 2N HCl at 37°C with shaking. Then, the sections were incubated 1x in borate buffer (pH 8.5) at RT with shaking for 10 min and washed 1x in PB at RT. The tissue was blocked in blocking buffer for 1h at RT and the standard immunofluorescent protocol was followed (see Immunohistochemistry above).

scRNAseq
Single-cell RNA sequencing was performed at Single Cell Discoveries (https://www.scdiscoveries.com/) according to the 10x Genomics Single Cell Gene Expression Flex solution protocol. Mice were anesthetized with avertin (2.5%), then perfused with 1x PB before whole hippocampal pieces were micro-dissected from the brain and flash-frozen on dry ice. Samples were shipped on dry ice to Single Cell Discoveries where all Processing of samples was performed by Single Cell sequencing experts. For each sample, 8000–14,000 cells were loaded and sequencing libraries were prepared following a standard 10x Genomics protocol.
As similarly described by others114, sample reads were aligned to the mouse genome GRCm39 (Datasets - 10x Genomics) using Cell Ranger 9.0.0 Flex pipeline (Flex with Cell Ranger multi-Official 10x Genomics Support). The data from all samples were loaded in R (version 4.3.2) and processed using the Seurat package (5.1.0). Cells expressing least 200 genes and less than 20% mitochondrial gene content were retained for analysis. The data of all 10x libraries was merged and processed together and normalized for sequencing depth per cell and log transformed. After data cleaning and filtering, datasets were integrated using Harmony115. Following this, clustering was done using the FindNeighbors function (dims=1:20) at a resolution of 0.12. To identify cell types in separate clusters, signature genes for each cell type were used based on the Cell Marker 2.0 database for mouse species in brain tissue. To analyze differentially expressed genes (DEGs) between control and MG-Alk5 or MG-Tgfb1 iKO samples, we used FindMarkers to filter for genes that were expressed in at least 10% of cells in either of the conditions and had an adjusted p-value of <0.05. DEGs from either neuroblast or microglia cluster were plotted onto a volcano plot using R packages DESeq2 and EnhancedVolcano. These DEGs were input into EnrichR for pathway enrichment analysis. Intercellular signaling was profiled using LIANA (v0.2.0). LIANA aggregated predictions from multiple computational methods (including CellPhoneDB, NATMI, SCA and LogFc) and ranked interactions based on the consensus mean rank score. The Mouse Consensus ligand–receptor database was used as the reference resource. Visualization of significant ligand–receptor pairs was generated using ggplot2 based on LIANA’s aggregated output.

Rapamycin treatment
To evaluate the contributions of mTOR signaling on adult neurogenesis after deletion of microglia-mediated ALK5 signaling, control or MG-Alk5 iKO mice received TAM for 5 days to delete Alk5 in microglia. Daily intraperitoneal (I.P.) injections of rapamycin were given starting from the 4th day of TAM for 21 days to 4-6-month-old male and female mice, administered at a dosage of 10mg/kg similar to previous studies96,98. 5% PEG 400 and 5% Tween 80 was diluted into MQH2O with 4% rapamycin (diluted in EtOH) or 4% EtOH to generate a working solution of 1mg/mL. Mice were harvested 3 weeks post-TAM by administering avertin (2.5%) and then transcardially perfused with 4% paraformaldehyde (PFA).

Tissue culture
Isolation of primary adult neural stem cells (aNSCs) from C57B6 WT mice is established as previously described5. Mice (6-week-old) were culled by cervical dislocation, and the SVZ was microdissected and dissociated into single cells. Adult NSCs are cultured with neurobasal media containing heparin, EGF, bFGF, and B-27 as described in our previous studies5,116,117. Single cells are cultured as neurospheres in the media above. Neurospheres between passages 5 and 7 were used for in vitro experiments where the disassociated single cells were plated at 3×10^3 cells/well in a ultra-low attachment 96-well round-bottom plate. These aNSCs were then given 5 days to form a sphere and proliferate before co-cultured with primary mouse microglia (mMG). The mMG used was obtained from P3 pups with a P2ry12CreERT/CreERT2-Alk5WT/WT or P2ry12CreERT2/CreERT2-Alk5fl/fl genotype via a mixed glial culture of the whole brain (excluding the olfactory bulb and cerebellum). The tissue collected and processed were put into a poly-D-lysine coated T75 flask with one flask per pup in glial media (DMEM/F-12, 10% FBS, 1% 100X L-Glutamate, 1% Pen/Strep). Feeds consisting of glial media were given every 3 days. On day 11, the flasks were given 1μM 4-OH TAM diluted in ethanol. On day 14, the flasks were then fed with glial media and shaken on an orbital shaker at 180 rpm for 3 h to collect WT or KO mMG. Alk5 mRNA decrease in the KO mMG was confirmed by qRT-PCR. The collected WT or KO mMG (3000 cells per sphere) was added to the NSC neutrospheres to establish the 3D co-culture. 3 days after the microglia were added to the neurospheres, the media were changed to differentiation media consisting of neurobasal media without growth factors but with microglia serum (from ScienCell’s microglia medium) to help maintain the microglia while differentiating the aNSCs. Additionally, 100 nM rapamycin or vehicle (ethanol) was added to the differentiation media. The co-culture was then differentiated for 4 days before fixation via 4% PFA. The fixed co-culture spheres were then stained by permeabilizing them via 0.3% 100X Triton in DPBS for 10 minutes, blocked with blocking buffer (4% BSA/0.3% 100X Triton) for 1 hour, incubated in primary antibody solution (βIII-Tubulin [1:2000, Promega] and GFAP [1:1000, BioLegend]) for 4 days at 4C, washed with DPBS 4x, incubated in secondary antibodies (Donkey anti-Mouse 647 [1:500, Jackson ImmunoResearch] and Donkey anti-rabbit 488+ [1:1000, Invitrogen]) and DAPI diluted in blocking buffer for 3 days at 4C, and washed 4x for 5 mins each. Neurospheres were then imaged using a Leica Stellaris 8 confocal microscope and quantified in ImageJ for % area of immunoreactivity.

Behavioral tests
EPM/EZM. Elevated Plus Maze (EPM) or Elevated Zero Maze (EZM) was used to assess anxiety-like behavior in mice. The EPM or EZM is elevated ~60 cm off the floor. The EPM consists of a cross-shaped platform that is 5cm wide and 30cm long with two opposing arms containing dark walls that are 15cm high. The EZM is an O-ring-shaped platform that is 60cm in diameter with a 5cm wide platform. Two sections of the O-shaped apparatus are enclosed by dark walls 15cm high. Black-out curtains surround the testing area to help maintain a consistent lighting of 18 lux via LED strips45. Animals are habituated to the room for at least 20 minutes before testing. In between each animal, the apparatus is cleaned with warm water and 10% Conflikt (Decon Labs). Each animal is allowed to freely explore the apparatus for 5 minutes before being returned to their home cage. EPM is the first behavioral test, and for the second anxiety test, EZM is used to prevent repeated exposure to the same maze. Data was automatically scored using AnyMaze (Stoelting Company, Wood Dale, IL).

Barnes maze. To evaluate spatial learning and memory in mice, the Barnes Maze apparatus (Stoelting Company, Wood Dale, IL) was used as previously described36. Using a short and challenging paradigm, we trained the animals in two separate sessions 4h apart where the animals were motivated to locate the escape hole during each session with light and a fan. Animals were tested the following day. In between each animal, the apparatus was cleaned with warm water followed by 20% ethanol and allowed to dry. Data was automatically scored using AnyMaze (Stoelting Company, Wood Dale, IL).

Rotarod. Motor coordination and motor learning were assessed in animals using the Roto-rod Series 8 apparatus (IITC Life Science Inc., Woodland Hills, CA) with an acceleration paradigm starting at 5 rpm and reaching a maximum of 20 rpm over 300 seconds. Once each animal reached the end of the 300 seconds or fell off the rotating rod, they were returned to their cages after 3 consecutive trials with rest in between. The Roto-rod apparatus was sanitized using 70% ethanol in between each animal.

Quantification and statistical analysis
All data were analyzed using SigmaPlot (12.0) for statistical significance. Results are expressed by mean±SEM of the indicated number of experiments. Statistical analysis was performed using the two-sided Student’s t-test, two-sided Welch’s t-test, and two-sided one- or two-way analysis of variance (ANOVA), as appropriate, with Tukey’s post hoc tests. A p-value to or less than 0.05 was considered significant. When the data did not meet the assumptions of normality or equal variance, the corresponding nonparametric tests were used. The significance level was set at α=0.05. Number of animals used in each experiment plotted as single data point and are indicated in Figure legends. Graphs were made in GraphPad Prism (10.6.1) and portions of figures were created with BioRender.com. The notation for p-values was as follows: n.s – p≥0.05, * – p<0.05, ** – p<0.01, *** – p<0.001, **** – p<0.0001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Ware and colleagues investigated the role of microglia in subgranular zone neurogenesis in adult microglia-specific Tgfb1- and Tgfbr1-deficient mice. The authors convincingly demonstrated that Tgfb1- and Tgfbr1-deficient mice develop dyshomeostatic microglia (as previously shown by the same group) resulting in transiently increased neurogenesis in the hippocampal subgranular zone which was accompanied by altered anxiety-like behaviour. Using elegant transgenic models and BrdU injections, the authors revealed that enhanced survival and not proliferation of DCX+ cells is essential for the observed increased adult neurogenis in the presence of Alk5-deficient microglia. Depletion of microglia did not result in increased neurogenesis but repopulation with Alk5-deficient microglia increased adult neurogenesis. Moreover, using scRNAseq, the authors identified reduced PTEN signaling in new born neuroblasts and reversed the transiently heightened neurogenesis by rapamicycin-induced mTOR signaling inhibition. Overall, the study is well designed, well structured, and the results are presented with high quality figures. Together, the study provides valuable data which further broadens the knowledge of microglia-regulated hippocampal adult neurogenesis and highlights the essential role of microglial TGF-beta signaling.\n\nThe authors should address the following points to further strengthen the overall message of the current manuscript.\n- The authors should demonstrate that Cre-mediated Alk5 ablation permanently affects microglia and recovers in peripheral monocytes/macrophages. Are BAMS affected in the same way as microglia?\n- Igf1 upregulation was linked to adult neurogenesis. Igf1-positive microglia have also been described to drive oligodendrocyte differentiation during postnatal development. Has the number of mature and immature oligodendrocytes been analysed? Is the myelination affected in Alk5 deficient mice?\n- The authors observed increases in DCX+ cells at 3 weeks but not by 6 and 12 weeks post tamoxifen treatment. However, the microglia phenotypes have only been analysed after 3 weeks (Fig.1). Do microglia at 6 and 12 weeks show the same phenotype at 6 and 12 weeks?\n- Experimental schemes in all figures should by consistent (see Fig.1A vs. Fig.3A)\n- DCX+ cells as well as the dendrite arborization has been analysed only in P2ry12Cre/Alk5 hippocampi. Why was the dendrite arborization not addressed in Cx3cr1Cre/Alk5 mice?\n- The overall story would extremely benefit from data showing spine density (Golgi staining) and/or levels of pre and postsynaptic markers such as synapsin and PSD95 (via IHC or western blot)\n- During the depletion experiments (Fig.5) DCX+ cells were analyzed after 12 weeks. According to Fig.2, numbers of DCX+ cells were only increased after 3 weeks in Alk5-deficient mice. Why were the numbers of DCX+ cells analysed 3 weeks and 6 weeks after microglia depletion? Without this information it is difficult to state that microglia depletion has no effect on adult neurogenesis.\n- Although the authors have convincingly demonstrated that Igf1 as well as Tnfa (as evidenced by substantial supplementary material including Alk5/Igf1 and Alk5/Tnfa dKO mice data) are not the key players driving increased adult neurogenesis, other neurotrophic factors could be involved and should be part of the discussion.\n- For the behaviour experiments, mice received a different TAM regime as in all other parts of the study. Why has that approach been chosen and not applied to all other experimental parts?\n- Elevated zero maze data are not presented for Alk5-defeicient mice. Why? Moroever, why is the number of mice analyzed for EZM so low compared to all other tests?\n\nOverall, the manuscript contains very interesting data describing increased adult neurogenesis in microglia-specific TGFb-signalling and TGFb1 deficient mice. Next to the abovementioned points, the story would substantially improve if data on synaptic densities, microglial synapric pruning will be added. Spine density (Golgi staining) and/or levels of pre and postsynaptic markers such as synapsin and PSD95 (via IHC or western blot) as well uptake of synaptic material by microglia (either IHC or flow cytomtrey) should be added.", "reviewer_2": "Summary\nThis potentially interesting study investigates the role of TGF-β signaling in microglia and its impact on adult hippocampal neurogenesis and behavior. The authors use inducible knockout models targeting various TGF-β pathway components (e.g., Alk5, Tgfbr2, Tgfb1) specifically in microglia. They report that disruption of TGF-β signaling leads to microglial dyshomeostasis, increased proliferation and survival of adult-born neurons in the subgranular zone, and altered anxiety-like and cognitive behavior. scRNA-seq and pharmacological interventions suggest this effect is mediated via downregulation of PTEN and activation of the mTOR pathway in neuroblasts. The findings suggest that activated microglia can stimulate adult neurogenesis and modulate affective and cognitive behavior, but they do not demonstrate this directly. Additional experiments are needed to establish a causal link. Furthermore, more thorough analyses of the histological data, the animal models, and sex of mice are needed.\n\nMajor Comments:\n1. Although both Cx3cr1CreER and P2ry12CreER drivers are used, the reliance on Cx3cr1CreER raises concerns about off-target recombination in BAMs and peripheral myeloid cells. Additional validation using BAM-specific markers or region-specific analysis (e.g., meninges, choroid plexus) would strengthen the microglia-specific conclusions.\n2. The degree of Alk5 deletion and recombination efficiency at the protein level is not shown. Demonstrating this with IHC or Western blot for ALK5 in sorted microglia would reinforce the mechanistic link to TGF-β signaling loss.\n3. The study concludes that TNF-α and IGF-1 are not responsible for the observed increase in neurogenesis. However, given known redundancy and interplay in cytokine signaling, additional discussion or testing of combinatorial cytokine blockade would be important.\n4. Rapamycin only partially rescues the adult neurogenesis phenotype. This suggests that alternative or parallel pathways might be involved. Additional discussion is needed to address the possibility of other downstream signals in activated microglia.\n5. While the manuscript demonstrates increased adult neurogenesis following TGF-β signaling disruption in microglia, the causal molecular mechanism remains underdeveloped. The study eliminates IGF-1 and TNF-α as single factors and implicates the PTEN/mTOR, it does not explore whether the changes in PTEN/mTOR in neuroblasts are a direct result of microglial-derived cues (e.g., cytokines).\n6. The authors report reduced avoidance behavior in the EPM and diminished cognitive performance on the Barnes maze in microglial TGF-β iKO mice but cannot attribute this directly to increased adult neurogenesis. Without this important connection, it is impossible to interpret the microglia effects as working through changes in adult born neurons. This possibility could be assessed by blocking adult neurogenesis in the MG-Alk5-iKO during the behavioral testing window.\n7. The cell count data are presented per section or per image field. This type of analysis can lead to inaccurate comparisons if the brain regions of different treatment groups differ in size/volume. The data should be presented as overall counts in a given brain region or as cell densities (number of cells per volume).\n8. The n size varies greatly across experiments. What is the justification for this? In cases where just 3 mice are analyzed, it is questionable whether reliable results will be obtained. Also, some of the data are presented with points representing individual cells. This is misleading and the data should be shown as points for individual mice.\n9. The distribution of males and females is not even across the studies. Did the authors investigate whether trends toward sex differences exist? If not, it is difficult to compare across measures when some of them have with mixed sex mice and others have only one sex (e.g., Figure 4H).\n10. Avoidance behavior on the EPM has been linked to ventral hippocampus, not dorsal hippocampus. The images are all from dorsal hippocampus and it is not clear whether the entire hippocampus was analyzed or just the dorsal part. Ventral hippocampus should be included.\n11. Terms like \"microglial activation,\" \"dyshomeostasis,\" and \"reactivity\" are used interchangeably. Consider defining and using them more consistently for clarity. It is not clear at all what microglial dyshomeostasis is. Unless there is something different from microglial reactivity, it should be eliminated from the text. If there is something different, it should be defined as such.\n12. The authors note that increased adult neurogenesis is transient (DCX+ peaks at 3 weeks). Discussion on whether this reflects compensatory regulatory feedback, or microglia reversion to a homeostatic state might be helpful. Whether changes in neurogenesis correlate with sustained changes in microglial profile (e.g., do activated microglia persist at 6–12 weeks?).", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This manuscript investigates the consequences of microglia-specific TGF-β signaling inhibition on hippocampal adult neurogenesis and behavior. Using multiple inducible knockout models (targeting TGF-β1, ALK5, and TβRII), the authors demonstrate that loss of microglial homeostatic signaling leads to a transient increase in SGZ neurogenesis and a lasting increase in mature neurons. Single-cell RNA sequencing and pharmacological inhibition experiments support a mechanistic link via downregulation of PTEN and upregulation of the mTOR pathway in neuroblasts. Behavioral testing indicates reduced anxiety-like behavior but no alteration in forgetting.\n\nThis work is conceptually innovative, methodologically rigorous, and highly relevant for the fields of neurogenesis, microglia-neuron crosstalk, and neuroinflammation. However, clarification and strengthening of mechanistic interpretation, figure presentation, and behavioral conclusions would improve the manuscript.\n\nMajor Comments\n1. The finding that activated microglia can stimulate, rather than inhibit, adult neurogenesis in the absence of injury contradicts longstanding assumptions and presents a paradigm shift.\n2. The integration of transcriptomic and behavioral data makes this study of broad relevance to neuroscience and regenerative biology.\n3. The authors argue that the observed transient increase in DCX+ neuroblasts results in a long-term gain in mature neurons. While this is supported by BrdU/NeuN data, the term \"transient\" may be misleading. Please clarify this distinction and emphasize that the transient increase refers to proliferation/differentiation, not integration.\n4. The PTEN-mTOR link is well-supported; however, the authors should discuss whether other converging pathways from their DEG data (e.g., synaptic organization, apoptotic signaling) might also contribute.\n5. Behavioral phenotypes (reduced anxiety-like behavior and spatial learning deficits) are presented as consequences of increased neurogenesis. However, causality remains correlative. While the authors acknowledge limitations, the discussion would benefit from more elaboration on future strategies to test this directly (e.g., combining NSC-targeted inhibition with microglial KO background).\n6. The fear-conditioning “forgetting” paradigm might not be ideally timed. Consider clarifying whether pre-treatment with TAM would be more suitable to test the influence of neurogenesis on memory clearance.\n7. The Barnes Maze was used to assess spatial learning, but reversal learning—a component of cognitive flexibility strongly linked to adult hippocampal neurogenesis—was not tested. Given prior work by Kempermann and others demonstrating that reversal learning in tasks like the Morris Water Maze is sensitive to neurogenesis status, the authors should briefly discuss this limitation or provide justification for the behavioral test battery selected.\n8. The scRNA-seq data are rich, but dense. Please consider a summary table or schematic of key pathway-level changes (PTEN/mTOR, IGF1, TNF-α) for clarity.\n9. Improve visual clarity of UMAPs and volcano plots (Figure 7), including font sizes, axis labels, and color keys.\n10. Some experiments (e.g., PLX5622 repopulation) use n=3–4 animals. These data should be interpreted cautiously, and limitations of statistical power noted.\n11. Sex as a variable and tamoxifen effects: The inclusion of both sexes and annotation of data points by sex across figures is commendable. However, no sex-specific analysis is reported. If the study was not powered or intended to detect sex differences, please state this explicitly in the Methods or Discussion sections.\nAdditionally, tamoxifen is a selective estrogen receptor modulator (SERM), and estrogen signaling differs between males and females—particularly in the hippocampus where it influences both neurogenesis and microglial function. Although tamoxifen is used here solely for Cre activation, the potential for sex-dependent off-target effects should be acknowledged, especially when interpreting behavioral and neurogenic outcomes.\n\nMinor Comments\n12. Figure 7K–N: Highlight DCX+ cells migrating out of SGZ more clearly with arrows or labels.\n13. Clarify whether the scRNA-seq was done on the whole hippocampus or DG-specific regions.\n14. Refer consistently to “dyshomeostatic microglia” or “TGF-β-deficient microglia” rather than broadly as “activated microglia.”"}, "author_response": "Reply to the reviewers’ comments\nWe would like to thank the reviewers for their valuable comments and suggestions. Based on\nthese comments, we have carried out additional experiments that have helped us substantially\nimprove our manuscript further. Below is our point-by-point response to the reviewers’\ncomments. Since we made many substantial changes to the manuscript (9 new figures, major\nrevisions in some previous figures, and major revisions in the texts), we only marked major\nrevisions with a yellow highlight in the text.\nReviewer #1 (Remarks to the Author):\nThe manuscript by Ware and colleagues investigated the role of microglia in subgranular zone\nneurogenesis in adult microglia-specific Tgfb1- and Tgfbr1-deficient mice. The authors\nconvincingly demonstrated that Tgfb1- and Tgfbr1-deficient mice develop dyshomeostatic\nmicroglia (as previously shown by the same group) resulting in transiently increased\nneurogenesis in the hippocampal subgranular zone which was accompanied by altered anxiety-\nlike behaviour. Using elegant transgenic models and BrdU injections, the authors revealed that\nenhanced survival and not proliferation of DCX+ cells is essential for the observed increased\nadult neurogenis in the presence of Alk5-deficient microglia. Depletion of microglia did not\nresult in increased neurogenesis but repopulation with Alk5-deficient microglia increased adult\nneurogenesis. Moreover, using scRNAseq, the authors identified reduced PTEN signaling in new\nborn neuroblasts and reversed the transiently heightened neurogenesis by rapamicycin-induced\nmTOR signaling inhibition. Overall, the study is well designed, well structured, and the results\nare presented with high quality figures. Together, the study provides valuable data which further\nbroadens the knowledge of microglia-regulated hippocampal adult neurogenesis and highlights\nthe essential role of microglial TGF-beta signaling.\nThe authors should address the following points to further strengthen the overall message of the\ncurrent manuscript.\n1. The authors should demonstrate that Cre-mediated Alk5 ablation permanently affects\nmicroglia and recovers in peripheral monocytes/macrophages. Are BAMS affected in the\nsame way as microglia?\n- We thank Reviewer 1 for this important feedback, which has led to additional interesting\ninsights into this study.\nFirst, we now show that peripheral monocytes/macrophages are not affected in Alk5, Tnf\nor Igf1 gene expression at 3 weeks post TAM measure by qRT-PCR in the\nCx3cr1CreERAlk5 iKO mice, likely due to their replenishment by wildtype cells (new\nSupplemental Fig. 1). Second, using pSMAD3 as a readout for ALK5-mediated\ndownstream TGF-β signaling, we now show that BAMs are affected in the brain of the\nCx3cr1CreER Alk5fl/fl line (New Supplementary Fig 7). This result is consistent with the\nrobust recombination of the ROSA26-YFP reporter in the BAMs in this line, reported in\nour previous study (PMID: 38217856). Third, we now show that in the Cx3cr1CreER\nAlk5fl/fl mice, both microglia morphology, downregulation of homeostatic marker (P2ry12\nexpression), and a decrease in pSMAD3 show a robust phenotype at 3 weeks post\ntamoxifen treatment; however, this is followed by gradual recovery at 6 weeks and 12\nweeks post tamoxifen (New Supplemental Fig 3).\nThis is consistent with an increase of DCX+ immature neuroblasts at 3 weeks post TAM,\nbut a reversal of DCX+ cell increase at 6-12 weeks after TAM. Interestingly, Cx3cr1CreER\nTgfb1fl/fl mice do not show this recovery in microglia phenotype up to 12 weeks post\nTAM (PMID: 38906887 and new supplemental Fig 6). Consistently, DCX+ cell numbers\nremain increased compared to WT mice at least up to 12 weeks post TAM (New\nsupplemental Fig. 5). The mechanism for the difference in recovery in the microglia\nphenotype in the Alk5 and Tgfb1 iKO mice is an interesting topic, which is beyond the\nscope of this study and should be further investigated in future studies. Important to this\nstudy, the recovery of microglia in the Alk5 iKO mice, accompanied by the recovery of\nthe phenotype in the DCX+ immature neuroblasts, strongly supports that the observed\nphenotype of increased neurogenesis is caused by the phenotype in microglia.\nConversely, when the microglia do not recover in the MG-Tgfb1 iKO mice, the\nphenotype in the neurogenesis also persists up to 12 weeks after TAM.\n2. Igf1 upregulation was linked to adult neurogenesis. Igf1-positive microglia have also been\ndescribed to drive oligodendrocyte differentiation during postnatal development. Has the\nnumber of mature and immature oligodendrocytes been analysed? Is the myelination affected\nin Alk5 deficient mice?\n- We carried out quantification of MBP, Olig2+ and NG2+ cells in the hippocampus of WT\nor Cx3cr1CreER Alk5fl/fl iKO mice at 3 weeks post TAM. Our data show that, indeed, total\nOlig2+ cells and OPCs (Olig2+/NG2+) cells are increased in the DG of Cx3cr1CreER\nAlk5fl/fl iKO mice at 3 weeks post TAM. However, MBP immunoreactivity is not altered.\nThis data is now added to the new Supplemental Fig. 10 and the results.\n3. The authors observed increases in DCX+ cells at 3 weeks but not by 6 and 12 weeks post\ntamoxifen treatment. However, the microglia phenotypes have only been analysed after 3\nweeks (Fig.1). Do microglia at 6 and 12 weeks show the same phenotype at 6 and 12 weeks?\n- Please see the response to point #1 above. Essentially, we show that microglia phenotype\nand pSMAD3 signal recover partially at 6 weeks and nearly fully at 12 weeks in the MG-\nAlk5 iKO line (new supplemental Fig 3). MG-Tgfb1 iKO mice do not recover in\nmicroglia phenotype (New supplemental Fig. 6) which is accompanied by a sustained\nincrease in DCX+ cells at least up to 12 weeks after TAM (New Supplemental Fig 5).\nThese data strongly support that the neurogenesis phenotype is causally linked to the\nmicroglia phenotype.\n4. Experimental schemes in all figures should by consistent (see Fig.1A vs. Fig.3A)\n- This has now been corrected.\n5. DCX+ cells as well as the dendrite arborization has been analysed only in P2ry12Cre/Alk5\nhippocampi. Why was the dendrite arborization not addressed in Cx3cr1Cre/Alk5 mice?\n- We have now added the quantification of dendritic arborization in Fig 2I which is\nconsistent with the results of the DCX+ cell number increase.\n6. The overall story would extremely benefit from data showing spine density (Golgi staining)\nand/or levels of pre and postsynaptic markers such as synapsin and PSD95 (via IHC or\nwestern blot)\n- We are not experienced in Golgi staining; therefore, we performed a Western blot for\nPSD95 (postsynaptic) and synaptophysin 1 (presynaptic) from CA3, to which DG\nneurons project at 6 weeks after TAM when the cohort of increased adult neurons is\nexpected to mature and integrate. However, we did not observe any significant\ndifferences for either of the synaptic markers in the CA3 (see figure below). This is likely\ndue to the small number of newly born neurons added to DG during the short period (It is\nestimated that in 6 days, about 0.3% of new neurons are added to the DG, PMID:\n9294224, which only makes about 1% of new neurons added in 3 weeks). This may not\nbe detected as an increase in the total synaptic density at the population level, albeit\nfunctionally, these newly added adult-born neurons might still play important roles. It is\nproposed that although constituting a small percentage of neurons compared to the total\npopulation, adult-born neurons demonstrate increased plasticity (LTP) in a critical\nwindow, which is critical for aiding cognitive flexibility or affective functions (PMID:\n17521569). Alternatively, we could label newly born neurons using retrovirus to analyze\ndendritic spine density; however, intracerebral injection activates microglia, which will\nintroduce additional confounds to this study. We will add these data as a Supplementary\nfigure if the Reviewer determines it to be necessary.\n7. During the depletion experiments (Fig.5) DCX+ cells were analyzed after 12 weeks.\nAccording to Fig.2, numbers of DCX+ cells were only increased after 3 weeks in Alk5-\ndeficient mice. Why were the numbers of DCX+ cells analysed 3 weeks and 6 weeks after\nmicroglia depletion? Without this information it is difficult to state that microglia depletion\nhas no effect on adult neurogenesis.\n- For the PLX experiment, the analysis was carried out in 12-week-old mice which are at 3\nweeks after TAM treatment (Fig 6).\n8. Although the authors have convincingly demonstrated that Igf1 as well as Tnfa (as evidenced\nby substantial supplementary material including Alk5/Igf1 and Alk5/Tnfa dKO mice data) are\nnot the key players driving increased adult neurogenesis, other neurotrophic factors could be\ninvolved and should be part of the discussion.\n- We have now added a discussion on other neurotrophic factors that could be involved\n(page 24 at the end and page 25, paragraph 1).\n9. For the behaviour experiments, mice received a different TAM regime as in all other parts of\nthe study. Why has that approach been chosen and not applied to all other experimental\nparts?\n- 3 or 5 injections of TAM give similar results regarding microglia and DCX+ phenotype\nin the Cx3cr1CreER Alk5 iKO mouse line. P2ry12CreER/CreER mouse driver has weaker\nrecombination efficiency; therefore, we use only the 5-day TAM regimen for the\nP2ry12CreER/CreER driver line. To keep treatment consistent, we used a 5-day TAM regimen\nin all later experiments except Figures 1 and 2 samples. We have now corrected all\ntimeline schematics to reflect the accurate regimen for each figure.\n10. Elevated zero maze data are not presented for Alk5-defeicient mice. Why? Moroever, why is\nthe number of mice analyzed for EZM so low compared to all other tests?\n- For the anxiety test, we only test the same animal once in EPM since subsequent tests in\nthe same maze will interfere with the anxiety phenotype (PMID: 7855188 and PMID:\n1975449). For the second anxiety test, we use a different maze (EZM). For the Tgfb1-\ndeficient mice, in the previous figure, only a subset of the mice were tested at 12 weeks\nbecause some mice had been harvested for other experiments before the 12-week time\npoints. Nevertheless, we have now added new experiments to increase the n number in\nEZM for Tgfb1-deficient mice at 12 wks post TAM. We also carried out EZM for the\nAlk5-deficient mice at 12 wk post TAM as a comparison to the earlier EPM test.\nInterestingly, the EZM results from Tgfb1 iKO show a persistent behavioral phenotype of\nincreased time and distance in open arms at 12 wks post TAM, consistent with the\nsustained microglia phenotype, while the Alk5 iKO mice show an absence of a behavioral\nphenotype at 12 wks post TAM, which is also consistent with a recovered microglia\nphenotype in this mouse line. These results are now included in the revised Figure 10.\nThis evidence again strongly suggests that microglia phenotype might be driving the\nbehavioral phenotype in these iKO mice.\nOverall, the manuscript contains very interesting data describing increased adult\nneurogenesis in microglia-specific TGFb-signalling and TGFb1 deficient mice. Next to the\nabovementioned points, the story would substantially improve if data on synaptic densities,\nmicroglial synapric pruning will be added. Spine density (Golgi staining) and/or levels of pre\nand postsynaptic markers such as synapsin and PSD95 (via IHC or western blot) as well\nuptake of synaptic material by microglia (either IHC or flow cytomtrey) should be added.\n- We thank the reviewer for their very valuable suggestions. Please see the above point-by-\npoint responses.\nReviewer #2 (Remarks to the Author):\nSummary\nThis potentially interesting study investigates the role of TGF-β signaling in microglia and its\nimpact on adult hippocampal neurogenesis and behavior. The authors use inducible knockout\nmodels targeting various TGF-β pathway components (e.g., Alk5, Tgfbr2, Tgfb1) specifically in\nmicroglia. They report that disruption of TGF-β signaling leads to microglial dyshomeostasis,\nincreased proliferation and survival of adult-born neurons in the subgranular zone, and altered\nanxiety-like and cognitive behavior. scRNA-seq and pharmacological interventions suggest this\neffect is mediated via downregulation of PTEN and activation of the mTOR pathway in\nneuroblasts. The findings suggest that activated microglia can stimulate adult neurogenesis and\nmodulate affective and cognitive behavior, but they do not demonstrate this directly. Additional\nexperiments are needed to establish a causal link. Furthermore, more thorough analyses of the\nhistological data, the animal models, and sex of mice are needed.\nMajor Comments:\n1. Although both Cx3cr1CreER and P2ry12CreER drivers are used, the reliance on\nCx3cr1CreER raises concerns about off-target recombination in BAMs and peripheral myeloid\ncells. Additional validation using BAM-specific markers or region-specific analysis (e.g.,\nmeninges, choroid plexus) would strengthen the microglia-specific conclusions.\n- We have now added data to show that peripheral myeloid cells are not affected in the\nCx3cr1CreER Alk5 iKO mice (New Supplemental Fig 1), consistent with our previous\nreport in the Cx3cr1CreER Tgfb1 iKO mice (PMID: 38906887). Additionally, we\nquantified the pSMAD signal in microglia, perivascular macrophages, and choroid plexus\nmacrophages in both the Cx3cr1CreER and P2ry12CreER/CreER Alk5 iKO mice (New\nSupplemental Fig 7). Interestingly, both perivascular macrophages and choroid plexus\nmacrophages show reduced pSMAD3 signaling in the Cx3cr1CreER mice. In the P2ry12\nCreER Alk5 iKO mice, microglia show partial pSMAD3 loss compared to Cx3cr1CreER\nAlk5 iKO microglia, consistent with the lower recombination efficiency of this mouse\nline. Perivascular macrophages show similar pSMAD3 levels, and choroid plexus\nmacrophages show reduced pSMAD3 levels, consistent with the previous report of the\nlack of reporter recombination in perivascular macrophages but presence of\nrecombination in choroid plexus macrophages (PMID 32573436 and 38217856). Based\non this data, we have now revised the results and discussion to reflect that the phenotype\nof increased DCX+ cells might be due to changes in parenchymal microglia and/or\nchoroid plexus macrophages.\n2. The degree of Alk5 deletion and recombination efficiency at the protein level is not shown.\nDemonstrating this with IHC or Western blot for ALK5 in sorted microglia would reinforce the\nmechanistic link to TGF-β signaling loss.\n- We were not able to validate any good antibody for ALK5 via Western blot or\nimmunostaining (using WT and constitutive Alk5 KO tissue), therefore, we used the\ndownstream signaling effector of the Alk5 receptor, phosphorylation of SMAD3\n(pSMAD3) as a readout for ALK5-mediated TGF-β signaling. We have now provided\ndata on pSMAD3 staining in the Alk5 and Tgfb1iKO lines at 3, 6 and 12 weeks post TAM\n(New Supplemental Fig 3 and 6).\n3. The study concludes that TNF-α and IGF-1 are not responsible for the observed increase in\nneurogenesis. However, given known redundancy and interplay in cytokine signaling, additional\ndiscussion or testing of combinatorial cytokine blockade would be important.\n- We have revised our conclusion to be that increased TNF-α or IGF1 in the iKO microglia\nis not necessary for the observed increase in neurogenesis. The results on Igf1 KO are\nconsistent with a recent publication showing that in humans, microglia-derived IGF1 is\ncritical for interneuron neurogenesis, which is not recapitulated in mice (PMID\n40770097). There might be species differences at play here. Nevertheless, we have now\nadded more discussion on combinatorial effects of growth factors as well as redundancy\nand interplay in cytokine signaling (pages 17, 24-25).\n4. Rapamycin only partially rescues the adult neurogenesis phenotype. This suggests that\nalternative or parallel pathways might be involved. Additional discussion is needed to address\nthe possibility of other downstream signals in activated microglia.\n- Rapamycin in vivo treatment completely rescues the adult neurogenesis phenotype in the\nmeasurements of DCX+ cells and dendrite arborization (no difference was observed\nbetween the WT and KO in the rapamycin-treated group). Rapamycin shows an effect of\nits own on the migration of these cells in the DG. Our new in vitro experiments of the 3D\nco-culture of WT NSC with WT or Alk5 KO microglia show a similar phenotype in\ndifferentiated immature neurons, which is also reversed by Rapamycin treatment (New\nFig. 9).\n5. While the manuscript demonstrates increased adult neurogenesis following TGF-β signaling\ndisruption in microglia, the causal molecular mechanism remains underdeveloped. The study\neliminates IGF-1 and TNF-α as single factors and implicates the PTEN/mTOR, it does not\nexplore whether the changes in PTEN/mTOR in neuroblasts are a direct result of microglial-\nderived cues (e.g., cytokines).\n- We now show increased mTOR signaling (measured by downstream signaling pS6) in\nDCX+ neuroblasts in vivo in the MG Alk5 iKO mice, and Rapamycin treatment abolishes\nthis increase in iKO mice (New Fig 9). We also show directly that coculture of KO\nmicroglia with WT neuroblasts increased the neuronal differentiation in NSC 3D co-\nculture system compared to co-culture with WT MG. This phenotype is also rescued by\nrapamycin treatment in the iKO coculture, while neuronal differentiation is not affected\nin the WT coculture (New Fig 9), demonstrating that microglia-derived cues acting on the\nmTOR pathway are causally linked to this phenotype.\n6. The authors report reduced avoidance behavior in the EPM and diminished cognitive\nperformance on the Barnes maze in microglial TGF-β iKO mice but cannot attribute this directly\nto increased adult neurogenesis. Without this important connection, it is impossible to interpret\nthe microglia effects as working through changes in adult born neurons. This possibility could be\nassessed by blocking adult neurogenesis in the MG-Alk5-iKO during the behavioral testing\nwindow.\n- We now report that microglia morphology and pSMAD3 signal recover in the MG-Alk5\niKO mice (New Supplemental Fig 3), which is accompanied by reversal of the\nneurogenesis phenotype and recovery (Fig 2) in both EPM/EZM and BM behavioral\nphenotypes (Fig 10). Conversely, MG-Tgfb1 iKO microglia do not recover (New\nSupplemental Fig 6), and the increase in neurogenesis in Tgfb1 is sustained for at least 12\nweeks (New Supplemental Fig 5), which is accompanied by consistent behavioral\nphenotypes at this time point (Fig 10). This supports the causal contribution of microglia\nto neurogenesis phenotype and behavioral outcome. It is technically challenging to\nspecifically block neurogenesis without affecting the microglia phenotype in our mouse\nmodel because microglia undergo cell division in iKO mice. Therefore, any agents that\ntarget proliferating cells would also affect microglia phenotype in the iKO mice. We also\ntested retroviral targeting of NSCs in HC in our microglial iKO mice; however, the\ntransduction efficiency is low, and the intracerebral injection activated microglia in the\nhippocampus, which introduces additional confounds. Therefore, we are not confident\nthat intracerebral injection of anti-mitotic agents or retroviral-mediated DREADDs\ninhibition will be ideal methods for directly testing this.\n7. The cell count data are presented per section or per image field. This type of analysis can lead\nto inaccurate comparisons if the brain regions of different treatment groups differ in size/volume.\nThe data should be presented as overall counts in a given brain region or as cell densities\n(number of cells per volume).\n- We apologize for this misrepresentation. For DG, we pick 6 brain sections at the same\nanterior/posterior location where DG has a similar shape and length, and the cell count\nfor DCX+ cells is normalized to DG length. This is now reflected in revised figures.\nMicroglia and astrocyte counts were normalized to volume using unbiased stereological\ncounts.\n8. The n size varies greatly across experiments. What is the justification for this? In cases where\njust 3 mice are analyzed, it is questionable whether reliable results will be obtained. Also, some\nof the data are presented with points representing individual cells. This is misleading and the\ndata should be shown as points for individual mice.\n- Most experiments have a sample size of at least 4 and animals from at least two different\nlitters, which already reached statistical significance. For behavioral tests, we calculated\nn based on the pilot study and power calculation. The only figure that labels individual\ncells is Figures 6G and 6H, with the intention to show the distribution of microglia in\neach mouse. However, the statistical analysis was carried out with the average from each\nanimal as a single n (this is now clearly stated in figure legend and raw data file). DCX+\ncells were presented as the average from each animal as a single data point, which also\nshows statistical significance since the phenotype is very robust. We have now calculated\nthe effect size for Figure 6 (with a large effect size; Cohen’s d=8.755227 and coefficient\nr=0.9748874 for 6I and Cohen’s d=5.4435 and coefficient r=0.9386 for 6J). Nevertheless,\nwe now also pointed out the limitation of the small sample size in the revised manuscript.\n9. The distribution of males and females is not even across the studies. Did the authors\ninvestigate whether trends toward sex differences exist? If not, it is difficult to compare across\nmeasures when some of them have with mixed sex mice and others have only one sex (e.g.,\nFigure 4H).\n- We did not observe an overt sex effect on our phenotype, consistent with the previous\nreport of a lack of sex effects on hippocampal adult neurogenesis (PMID: 18003841) or\nour previous study on microglia in the TGF-β signaling-deficient mice (PMID:\n38906887). Most experiments have both sexes, with the only exception being Figure 4H.\nWe have acknowledged that our study is not intended or powered to analyze sex\ndifferences in the discussion.\n10. Avoidance behavior on the EPM has been linked to ventral hippocampus, not dorsal\nhippocampus. The images are all from dorsal hippocampus and it is not clear whether the entire\nhippocampus was analyzed or just the dorsal part. Ventral hippocampus should be included.\n- We have now included analysis of the ventral hippocampus, and data show that the\nventral hippocampus has increased DCX+ cells and dendrite arborization at 3 weeks but\nnot at 6 weeks or 12 weeks after TAM treatment, consistent with results from the dorsal\nhippocampus (New supplementary Figure 2). Clarification on the analyzed hippocampal\nregion has been added to the figures.\n11. Terms like \"microglial activation,\" \"dyshomeostasis,\" and \"reactivity\" are used\ninterchangeably. Consider defining and using them more consistently for clarity. It is not clear at\nall what microglial dyshomeostasis is. Unless there is something different from microglial\nreactivity, it should be eliminated from the text. If there is something different, it should be\ndefined as such.\n- We have now changed it to either reactive microglia or TGF-β-deficient microglia.\nThank you for the suggestion.\n12. The authors note that increased adult neurogenesis is transient (DCX+ peaks at 3 weeks).\nDiscussion on whether this reflects compensatory regulatory feedback, or microglia reversion to\na homeostatic state might be helpful. Whether changes in neurogenesis correlate with sustained\nchanges in microglial profile (e.g., do activated microglia persist at 6–12 weeks?).\n- We thank the reviewer for raising this important point. Please see the response to\nReviewer 1's comment #1 regarding the recovery of the microglia profile.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\n- Thank you for providing valuable comments and suggestions for our manuscript.\nReviewer #4 (Remarks to the Author):\nThis manuscript investigates the consequences of microglia-specific TGF-β signaling inhibition\non hippocampal adult neurogenesis and behavior. Using multiple inducible knockout models\n(targeting TGF-β1, ALK5, and TβRII), the authors demonstrate that loss of microglial\nhomeostatic signaling leads to a transient increase in SGZ neurogenesis and a lasting increase in\nmature neurons. Single-cell RNA sequencing and pharmacological inhibition experiments\nsupport a mechanistic link via downregulation of PTEN and upregulation of the mTOR pathway\nin neuroblasts. Behavioral testing indicates reduced anxiety-like behavior but no alteration in\nforgetting.\nThis work is conceptually innovative, methodologically rigorous, and highly relevant for the\nfields of neurogenesis, microglia-neuron crosstalk, and neuroinflammation. However,\nclarification and strengthening of mechanistic interpretation, figure presentation, and behavioral\nconclusions would improve the manuscript.\nMajor Comments\n1. The finding that activated microglia can stimulate, rather than inhibit, adult neurogenesis in\nthe absence of injury contradicts longstanding assumptions and presents a paradigm shift.\n- We thank Reviewer 4 for their positive comments and for acknowledging the novelty and\nsignificance of our study.\n2. The integration of transcriptomic and behavioral data makes this study of broad relevance to\nneuroscience and regenerative biology.\n- We thank Reviewer 4 for their positive comments and for acknowledging the potential\nbroad significance of our study.\n3. The authors argue that the observed transient increase in DCX+ neuroblasts results in a long-\nterm gain in mature neurons. While this is supported by BrdU/NeuN data, the term \"transient\"\nmay be misleading. Please clarify this distinction and emphasize that the transient increase\nrefers to proliferation/differentiation, not integration.\n- We have now revised the manuscript to reflect that the increase in DCX+ is in a cohort of\nnewly born neuroblasts, which later matured into neurons and integrated into the\ncircuitry.\n4.The PTEN-mTOR link is well-supported; however, the authors should discuss whether other\nconverging pathways from their DEG data (e.g., synaptic organization, apoptotic signaling)\nmight also contribute.\n- We have added a discussion to address this comment (page 26).\n5. Behavioral phenotypes (reduced anxiety-like behavior and spatial learning deficits) are\npresented as consequences of increased neurogenesis. However, causality remains correlative.\nWhile the authors acknowledge limitations, the discussion would benefit from more elaboration\non future strategies to test this directly (e.g., combining NSC-targeted inhibition with microglial\nKO background).\n- We acknowledge this limitation due to technical challenges to target NSCs in our mouse\nmodel without affecting proliferating microglia. We have now expanded the discussion\non this limitation and added a discussion on future strategies to test this directly (in\n“Limitation of our study” at the end of the discussion).\n6. The fear-conditioning “forgetting” paradigm might not be ideally timed. Consider clarifying\nwhether pre-treatment with TAM would be more suitable to test the influence of neurogenesis on\nmemory clearance.\n- We agree with the reviewer, and to keep the focus of the current study, we have now\nremoved this data.\n7. The Barnes Maze was used to assess spatial learning, but reversal learning—a component of\ncognitive flexibility strongly linked to adult hippocampal neurogenesis—was not tested. Given\nprior work by Kempermann and others demonstrating that reversal learning in tasks like the\nMorris Water Maze is sensitive to neurogenesis status, the authors should briefly discuss this\nlimitation or provide justification for the behavioral test battery selected.\n- We agree with the reviewer on this point. In our hands, reversal learning does not work\nwell with the Barnes Maze, potentially due to the long-time course needed to train mice\non this task and the less robust motivation (unlike the water maze, we use mild aversive\nconditions, such as light and an overhead fan to motivate animals to find the target hole)\nMice usually are not motivated to find target hole in the later days of the reverse learning\nphase of the test. We chose BM as a general learning/memory test to be consistent with\nour previously published study (PMID: 38906887) to compare the results in Alk5 and\nTgfb1 iKO mice. We have added a discussion to acknowledge this limitation (pages 27-\n28).\n8. The scRNA-seq data are rich, but dense. Please consider a summary table or schematic of key\npathway-level changes (PTEN/mTOR, IGF1, TNF-α) for clarity.\n- We have now revised the scRNA-seq figure. Key pathway changes are included in the\nSupplementary data tables and Figure 7 M, N.\n9. Improve visual clarity of UMAPs and volcano plots (Figure 7), including font sizes, axis\nlabels, and color keys.\n- We have now revised the scRNAseq figure to increase the size of plots to improve visual\nclarity and moved the PTEN-mTOR data into a new figure.\n10. Some experiments (e.g., PLX5622 repopulation) use n=3–4 animals. These data should be\ninterpreted cautiously, and limitations of statistical power noted.\n- We have added a sentence in the result section to note the low n number in this\nexperiment.\n11. Sex as a variable and tamoxifen effects: The inclusion of both sexes and annotation of data\npoints by sex across figures is commendable. However, no sex-specific analysis is reported. If the\nstudy was not powered or intended to detect sex differences, please state this explicitly in the\nMethods or Discussion sections.\n- This has been added to the discussion section.\nAdditionally, tamoxifen is a selective estrogen receptor modulator (SERM), and estrogen\nsignaling differs between males and females—particularly in the hippocampus where it\ninfluences both neurogenesis and microglial function. Although tamoxifen is used here solely for\nCre activation, the potential for sex-dependent off-target effects should be acknowledged,\nespecially when interpreting behavioral and neurogenic outcomes.\n- We acknowledge the effects of TAM on potential sex-dependent off-target effects;\nhowever, we use control mice that are also subjected to the same TAM treatment to\nminimize these effects. Our previous study has also shown that TAM treatment itself does\nnot cause behavior phenotype in Control (Cx3cr1CreER non floxed) mice (PMID:\n38906887).\nMinor Comments\n12. Figure 7K–N: Highlight DCX+ cells migrating out of SGZ more clearly with arrows or\nlabels.\n- Thank you for the suggestion. Migrated DCX+ cells are now marked by an arrowhead in\nthe figure.\n13. Clarify whether the scRNA-seq was done on the whole hippocampus or DG-specific regions.\n- scRNAseq was done on the whole hippocampus. This is now clarified in the method\nsection.\n14. Refer consistently to “dyshomeostatic microglia” or “TGF-β-deficient microglia” rather\nthan broadly as “activated microglia.”\n- We thank the reviewer for this suggestion. Based on this suggestion and that of Reviewer\n#2, we have now changed it to either reactive microglia or TGF-β-deficient microglia."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all comments raised by the reviewer and answered all questions sufficiently. The manuscript has improved and several new aspects have been added to the overall story as reflected by additional supplementary figures and files.\n\nI have no further points and recommend acceptance of the manuscript in its current form.", "reviewer_2": "The authors have addressed all of my concerns. The manuscript is now substantially improved and the claims are well-supported by the data.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "All concerns —methodological, conceptual, and presentational — were addressed by new experiments, data clarification or normalization changes, or expanded discussions and acknowledgments of limitations."}, "author_response": "Response to Reviewers’ Comments\nWe would like to thank the reviewers for their valuable comments and suggestions that have helped us\nsubstantially improve our manuscript further. Below is our point-by-point response to the reviewers’\ncomments.\nReviewer #1 (Remarks to the Author):\nThe authors have addressed all comments raised by the reviewer and answered all questions\nsufficiently. The manuscript has improved and several new aspects have been added to the overall\nstory as reflected by additional supplementary figures and files.\nI have no further points and recommend acceptance of the manuscript in its current form.\nNo response necessary.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed all of my concerns. The manuscript is now substantially improved and\nthe claims are well-supported by the data.\nNo response necessary.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nNo response necessary.\nReviewer #4 (Remarks to the Author):\nAll concerns —methodological, conceptual, and presentational — were addressed by new\nexperiments, data clarification or normalization changes, or expanded discussions and\nacknowledgments of limitations.\nNo response necessary."}]

Decision letter