The transcriptional co‐activator Yap1 promotes adult hippocampal neural stem cell activation

Abstract Most adult hippocampal neural stem cells (NSCs) remain quiescent, with only a minor portion undergoing active proliferation and neurogenesis. The molecular mechanisms that trigger the transition from quiescence to activation are still poorly understood. Here, we found the activity of the transcriptional co‐activator Yap1 to be enriched in active NSCs. Genetic deletion of Yap1 led to a significant reduction in the relative proportion of active NSCs, supporting a physiological role of Yap1 in regulating the transition from quiescence to activation. Overexpression of wild‐type Yap1 in adult NSCs did not induce NSC activation, suggesting tight upstream control mechanisms, but overexpression of a gain‐of‐function mutant (Yap1‐5SA) elicited cell cycle entry in NSCs and hilar astrocytes. Consistent with a role of Yap1 in NSC activation, single cell RNA sequencing revealed a partial induction of an activated NSC gene expression program. Furthermore, Yap1‐5SA expression also induced expression of Taz and other key components of the Yap/Taz regulon that were previously identified in glioblastoma stem cell‐like cells. Consequently, dysregulated Yap1 activity led to repression of hippocampal neurogenesis, aberrant cell differentiation, and partial acquisition of a glioblastoma stem cell‐like signature.


Introduction
During embryonic development, neural stem cells (NSCs) migrate from the dentate epithelium to settle in the dentate gyrus anlage of the hippocampus. Some of these will then eventually give rise to NSCs in the adult dentate gyrus (Berg et al, 2019). Located in the subgranular zone (SGZ), many of these Sox2-positive NSCs are characterized by a glial fibrillary acidic protein (GFAP)-positive radial process that extends across the granule cell layer towards the molecular layer, lending them a radial glia-like appearance (Seri et al, 2001;Suh et al, 2007). Alongside these radial glia-like NSCs, the SGZ harbors also Sox2-positive nonradial neural progenitor cells shown to be derived from dividing radial glia-like cells and themselves capable of undergoing either symmetric neurogenic divisions or asymmetric divisions, yielding one renewed nonradial cell and one neuronal daughter cell (Pilz et al, 2018). A key process in the establishment of the adult NSC pool is the progressive entry of NSCs into a quiescent state in which many will persist before eventually becoming re-activated during adulthood and re-entering cell cycle (Berg et al, 2019). The regulation of both transitions, that is, the transition from actively dividing to quiescent and the converse, the de novo activation is one of the key mysteries of adult NSC biology (Urb an et al, 2019). Quiescence is defined as "reversible cell cycle arrest" and is believed to protect stem cells against damage and premature exhaustion of the stem cell pool (Urb an et al, 2019). Intriguingly, the maintenance of quiescence is dynamically regulated in that, during aging, the rate of activation decreases with more NSCs persisting in a quiescent state (Dulken et al, 2019;Kalamakis et al, 2019;Harris et al, 2021;Ibrayeva et al, 2021). Several signaling pathways have been implicated in the bidirectional traffic between quiescence and activation, including BMP (Mira et al, 2010), Notch (Kawai et al, 2017;Engler et al, 2018;Zhang et al, 2019;Harada et al, 2021), VEGFR3 (Han et al, 2015), and Wnt signaling (Lie et al, 2005;Wexler et al, 2009) but also include circuit-specific mechanisms (Song et al, 2012). At a transcriptional level, previous work has shown that in the adult hippocampus expression of Ascl1 is a prerequisite for activation of quiescent NSCs (Andersen et al, 2014), while degradation of Ascl1 protein is an important step in controlling return to quiescence (Urb an et al, 2016). However, the full spectrum of transcriptional mechanisms underpinning NSC activation is still incompletely understood.
Accruing evidence points to the importance of the Hippo signaling pathway in regulating stem cell renewal and reactivation during regenerative processes in various tissues (Mo et al, 2014;Moya & Halder, 2019). The Hippo pathway is an evolutionarily conserved kinase cascade that regulates the activity of its effector proteins, the transcriptional activators Yap1 (Yes-associated protein 1) and Taz, through phosphorylation, thereby causing their cytoplasmic retention and degradation (Yu & Guan, 2013). Several mechanisms converge on this kinase cascade, including mechanical stimuli, cell polarity, and other signaling pathways (Yu & Guan, 2013).
Recent work has implicated the Hippo effector protein Yorkie (Yki) in the control of activation of quiescent NSCs in the brain of Drosophila larvae (Ding et al, 2016;Gil-Ranedo et al, 2019). For instance, loss of Yki has been shown to result in the failure of larval NSCs to grow in response to hormone stimulation and to re-enter cell cycle (Ding et al, 2016). The Hippo pathway and the Yki orthologue Yap1 have been shown to be highly active in the developing mammalian forebrain and to regulate radial glial cell proliferation (Cappello et al, 2013;Lavado et al, 2018Lavado et al, , 2021Kostic et al, 2019;Han et al, 2020;Mukhtar et al, 2020;Najas et al, 2020), but its role in the functional state of NSCs in adult neurogenic niches has not been addressed so far.
Thus, we wondered whether Yap1 activation is an evolutionarily conserved mechanism in the regulation of NSC activity. Towards this, here we have re-analyzed published single cell RNA-sequencing data , which revealed that Yap1 is highly enriched in activated NSCs of the adult hippocampus. This has prompted us to perform loss-and gain-of-function studies in the adult hippocampus, which provide evidence for an important role of this transcriptional coactivator in the regulation of adult hippocampal NSC activity.

Evidence for Yap1 activity in active neural stem cells of the adult hippocampus
To obtain first evidence for ongoing Yap1 activity in the lineage of adult hippocampal neural stem cells (NSCs), we first re-analyzed a published dataset of single cell transcriptomes comprising NSCs and their early-stage progeny isolated from the dentate gyrus of the adult hippocampus (Data ref . This identified a cell population enriched in a reference signature of active NSCs (Shin et al, 2015), originally classified as neuronal intermediate progenitor cells (nIPCs) (Appendix Fig S1). We next assessed which of these cell populations, if any, exhibited expression of a reference signature of Yap1 activity (Cordenonsi et al, 2011). This revealed a specific enrichment of Yap1 activity signature in the same cell population enriched for the active NSC signature (Appendix Fig S1). This analysis, thus, suggests that activation of NSC may involve Yap1mediated transcription.
Based on this analysis, we next examined whether we could detect Yap1 protein expression within the NSC lineage (Fig 1). We found Yap1 specifically expressed in Sox2-positive cells within the subgranular zone (SGZ) ( Fig 1A) and hilar astrocytes (identified by GFP expression driven from human GFAP [hGFAP] gene regulatory elements and hilar localization; Fig 1B) but virtually absent in doublecortin (DCX)-positive young neurons (Fig 1C) or Olig2positive cells (i.e., comprising cells of oligodendroglial lineage; Fig 1D). While in hilar astrocytes Yap1 immunoreactivity appeared to be largely cytoplasmic, within the SGZ, some of the Yap1 immunoreactivity co-localized with Sox2 expression, demarcating the nuclei of these Sox2-positive cells (Fig 1A, inset). These data suggest that Sox2-positive SGZ cells (i.e., NSCs and intermediate progenitor cells) are enriched in Yap1 expression and that Yap1 may have undergone nuclear translocation in a subpopulation of these cells. To further characterize Yap1 expression dynamics during the lineage progression from quiescent NSCs to early neuronally-restricted progenitor cells, we assessed nuclear Yap1 co-localization with the quiescent NSC marker Hopx, the activated NSC marker Ascl1 and the neuronal progenitor marker Tbr2, respectively ( Fig 1E). This indicated a trend towards increased nuclear Yap1 expression following NSC activation and a significant drop in nuclear Yap1 following neuronal commitment (Fig 1E and F).
In order to confirm that NSC activation is associated with nuclear enrichment of Yap1, we took advantage of primary adult hippocampal NSC cultures (Babu et al, 2011;Fig EV1A), which have been successfully used to study the transition between NSC states of activation and quiescence (Martynoga et al, 2013;Fig EV1B-D). Immunofluorescence analysis for Yap1 location revealed a clear enrichment in nuclear Yap1 in active versus quiescent NSCs (Fig EV1E and F). Conversely, when previously activated adult hippocampal NSC cultures were treated with BMP4 for a brief period to induce the transition towards quiescence, we observed nuclear Yap1 enrichment in those cells still in cell cycle (i.e., Ki67-positive), while Ki67-negative cells exhibited a significant reduction in nuclear Yap1 (Fig EV1G and H). These data support the notion that NSC activation is accompanied by the transfer of Yap1 from the cytoplasm to the nucleus, while the reverse process takes place during the return to quiescence.

Long-term Yap1 loss-of-function compromises NSC activation
To study the role of Yap1 in regulating the NSC transition from quiescence to activation, we next analyzed the impact of Yap1 loss-offunction on the ratio of quiescent and active NSCs. Towards this, we deleted Yap1 specifically in NSC (and astrocytes) and their lineage by crossing Yap1 conditional knockout mice (Yap1fl/fl) (Zhang et al, 2010) with Glast-CreERT2 and CAG-CAT-EGFP mice (Mori et al, 2006;Nakamura et al, 2006) followed by treatment with tamoxifen (Fig 2A). Immunofluorescence for Yap1 revealed a reduction in expression by day 7 following induction of recombination, indicating successful Yap1 deletion (Fig 2B and C). To assess the proportion of actively dividing cells among NSCs 7 days after induction of recombination, we first determined the number of recombination reporterpositive (GFP-positive) cells among radial glia-like cells (RGLs, i.e., the major NSC population, identified by their localization in the SGZ, GFAP immunoreactivity, and the presence of a radially-oriented process) and then quantified those RGLs that were engaged in cell cycle (minichromosome maintenance complex component-2, Mcm2-positive  Ó 2023 The Authors The EMBO Journal 42: e110384 | 2023 cells; Fig 2D). Quantitative, double-blind analysis revealed that the proportion of active versus quiescent (Mcm2-positive versus Mcm2negative) RGLs had not changed on day 7 following the deletion of Yap1 as compared to controls (Fig 2E and F). Likewise, when we performed the same double-blind analysis 30 days after inducing the deletion of Yap1, we did not observe significant changes in active versus quiescent RGLs (Fig EV2A and B). By contrast, on day 60 following the deletion of Yap1 (Fig EV2G), we noted a significant drop in the number of active RGLs as compared to controls (Fig 2G-I). This set of experiments indicates that loss of Yap1 does not immediately impact on the levels of NSC transition from quiescence to activation but that long-term Yap1 loss-of-function results in decreased NSC activation, evidencing an important physiological role of Yap1 in this process. One possible explanation for this delayed manifestation of Yap1 lossof-function maybe dependence on other mechanisms for NSC activation. Given the fact that nuclear expression of Yap1 was even more conspicuous in hippocampal NSCs treated with EGF and FGF2 in vitro, we tested the impact of Yap1 deletion on NSC proliferation in culture by assessing the incorporation of the thymidine analog EdU ( Fig EV2H). This revealed a drastic drop in DNA synthesis following Yap1 loss-of-function ( Fig EV2I). These in vitro data qualitatively support our observations in vivo, while at the same time suggesting quantitative differences in the dependence of NSC activation on Yap1 function. Interestingly, the deletion of Yap1 did not negatively impact lineage progression towards neurogenesis in vivo as Yap1-deleted NSCs gave rise to DCX-positive neurons to a similar extent as control NSCs when assessed at either 30 or 60 days following tamoxifen administration (Fig EV2C-F).

Active Yap1 promotes cell cycle entry of quiescent hippocampal NSCs
We next investigated the effect of Yap1 gain-of-function on the activation state of adult hippocampal NSCs. Towards this, we targeted adult NSCs (as well as hilar astrocytes) using lentiviruses driving transgene expression under the control of hGFAP regulatory elements (LV-hGFAP-IRES-EGFP, LV-hGFAP-Yap1WT-IRES-EGFP, LV-hGFAP-Yap1-5SA-IRES-EGFP; Appendix Fig S2). Interestingly, 7 days following lentivirus-mediated expression of the wild-type form of Yap1 (Yap1 WT) in adult hippocampal SGZ, no significant change in the number of proliferative Sox2-positive progenitors (i.e., lentivirus-transduced Sox2-positive cells in the SGZ) was observed (Fig 3A-C). By contrast, lentivirus-mediated expression of a mutant form of Yap1, encoding a protein in which the serines of the five HXRXXS Lats motifs had been replaced by alanine (Yap1-5SA), rendering the protein insensitive to phosphorylationdependent inhibition by Lats and thereby constitutively active (Zhao et al, 2007), caused a dramatic rise in the number of lentivirustransduced Ki67-positive/Sox2-positive cells (Fig 3B and C). Finally, we also observed an overall increase in the density of Sox2-positive cells in the SGZ (Fig 3B and D). These data show that active Yap1 is sufficient to drive quiescent NSCs towards cell cycle entry. It also supports the notion that Yap1 activity is under strict control as a forced expression of Yap1 WT fails to induce NSC activation, and only a regulation-deficient form of Yap1 overcomes this control. In line with the Yap1-mediated activation of hippocampal NSC in vivo, we found that lentivirus-mediated expression of Yap1-5SA promoted dramatic cell cycle entry in primary cultures of hippocampal NSCs kept in quiescence conditions as assessed by phosphohistone-H3 staining and EdU incorporation (Fig EV3A-D).
Finally, in these injections, some astrocytes in the hilus of the dentate gyrus were targeted by the lentiviruses encoding GFP only or Yap1-5SA and GFP. While we did not observe any Ki67-positive astrocytes following control virus injection, many astrocytes were Ki67-positive following the expression of Yap1-5SA. This indicates that Yap1-5SA also stimulates cell cycle re-entry in postmitotic astrocytes (Fig EV3E and F).
Constitutively active Yap1 induces an aberrant active NSC-like signature in adult hippocampal NSCs and/or astrocytes Given our observation that forced expression of Yap1-5SA appears to activate quiescent NSCs, we finally addressed how this molecular perturbation alters gene expression using single cell RNAsequencing. Yap1-5SA was targeted to adult hippocampal NSCs and hilar astrocytes by injection of lentiviruses encoding Yap1-5SA and EGFP or EGFP alone for control, under hGFAP regulatory elements, into the SGZ of the adult dentate gyrus. We performed fluorescenceactivated cell sorting (FACS) to isolate EGFP-positive cells on day 3 and 7 following lentivirus transduction (Appendix Fig S3). We selected isolation on day 3 as this was the earliest time point when we could detect reliable EGFP expression, while day 7 corresponds to the time point when we had previously observed massive activation of quiescent NSCs (Fig 3). Single cell transcriptomes were measured following library preparation using Smart-seq2 methodology ( Fig 4A) (Picelli et al, 2014). This yielded 121 (day-3) and 258 (day-7) control and 273 (day-3) and 320 (day-7) Yap1-5SA-expressing single cell transcriptomes. Using Scanpy (Wolf et al, 2018) we identified 13 distinct clusters (Appendix Fig S4A) and employed forcedirected graph embedding for visualization. Clusters expressing astrocyte, quiescent NSC, neuron, oligodendrocyte precursor cell (OPC), oligodendrocyte, and microglia markers consisted exclusively of control cells (Fig EV4A-H). Conspicuously, none of these clusters contained cells expressing Yap1-5SA, suggesting that this perturbation exerts a drastic effect on the targeted cells forcing them to alter their cell state within a 3-day time window. Among the remaining clusters, several contained both control and Yap1-5SAexpressing cells, while a final set of clusters consisted of Yap1-5SA cells only. Consistent with Yap1-5SA being a transcriptional coactivator (Zhao et al, 2007), clusters containing Yap1-5SAexpressing cells exhibited an increased number of transcripts compared with control cell clusters ( Fig EV4B). To further reveal the identity of Yap1-5SA-expressing cells and their lineage relationship to quiescent NSC and astrocytes, we performed re-clustering after removal of microglia, OPCs, oligodendrocytes, as well as a cluster enriched in cell death-related genes (Mdm2, Trp53), while the cluster identified with neurons was retained as the main natural progeny of the adult hippocampal NSC lineage (Fig 4B-F). This reanalysis yielded 12 clusters comprising astrocytes, quiescent NSCs, neurons, none of which contained Yap1-5SA cells, and nine clusters entirely consisting of Yap1-5SA-expressing cells ( Fig 4D). Moreover, only few cells were enriched in gene expression related to an activated NSC state (activated NSC signature; Shin et al, 2015), consistent with the fact that most adult hippocampal NSCs are quiescent (quiescent NSC signature (Shin et al, 2015), Fig 4D and Appendix Fig S4). By contrast, following Yap1 gain-of-function, we observed an enrichment in Yap1 signature across all Yap1-5SA-expressing clusters (Fig 4D). A Schematic diagram of the experimental design. Adult (8-week-old) Yap1 cKO mice or Control mice were injected with Tamoxifen for 5 consecutive days and sacrificed 7, 30, or 60 days later for immunohistological analysis. B Immunofluorescence for GFP (recombination reporter), GFAP, and Yap1 in control and Yap1 cKO RGLs at 7 days after tamoxifen administration. Arrowhead indicates nonrecombined RGL, and arrow indicates recombined RGL. C Quantification of Yap1 intensity in (B). Yap1 levels are significantly decreased in Yap1 cKO RGLs at 7dpi. n = 4 (50 cells, four mice, biological replicates) in the control group, n = 3 (37 cells, three mice, biological replicates) in the Yap1 cKO group. D Immunofluorescence for GFP, GFAP, and Mcm2 in control and Yap1 cKO RGLs at 7 days after tamoxifen administration. Yellow arrow indicates proliferating adult RGL, and white arrows indicate quiescent adult RGLs. E, F Quantification of the data in (D). Loss of Yap1 does not induce any changes neither in the number of proliferative RGLs nor the overall number of RGLs. n = 4 mice (biological replicates) for control group, n = 3 mice (biological replicates) for Yap1 cKO group.  A Schematic diagram of experimental design. P60 mice were analyzed at 7 days after lentivirus injection of control, wild-type Yap1 and Yap1-5SA. B Immunofluorescence for GFP, Sox2 (to identify NSCs, arrows), and Ki67 (to identify proliferative cells) in control conditions or overexpression of wild-type Yap1 or Yap1-5SA 7 days after lentivirus injection. Insets show a magnified view of the areas outlined by the dashed lines. C, D Quantification of proliferative NSCs. Yap1-5SA induces the proliferation of adult NSCs and increases the number of Sox2-positive cells. n = 3 mice (biological replicates) for each group.
Data information: Data are represented as mean AE SEM. One-way ANOVA. Scale bars: 50 lm. Source data are available online for this figure.
6 of 14 The EMBO Journal 42: e110384 | 2023 Ó 2023 The Authors Yap1-5SA-expressing clusters transcriptionally closer to controltransduced NSCs exhibited elevated cell cycle signature (Figs 4D and EV4J). Accordingly, these clusters (2,4-7) downregulated genes enriched in quiescent NSCs (Fig 4D), while up-regulating an activated NSC signature (Fig 4D; Appendix Fig S4). These data are thus consistent with the notion that Yap1 gain-of-function can elicit activation of quiescent NSCs and cell cycle entry, as observed in our in vivo and in vitro experiments (Figs 3 and EV2). One intriguing observation was the absence of significant Ascl1 expression in Yap1-5SA-expressing cells, in sharp contrast to control NSCs, consistent with the fact that the former cells failed to follow a trajectory leading to adult neurogenesis. While Yap1-5SA-expressing did not contribute to neuronal fate (Fig EV4A), we observed at least two additional RNA velocity vector endpoints (clusters 9 and 11) comprising exclusively Yap1-5SA-expressing cells and characterized by reduced cell cycle and activated NSC signatures (Figs 4B and D,and EV4A). Intriguingly, these clusters were typified by high levels of Scg2 (cluster 11) and Pmp22 (cluster 9) expression, respectively ( Fig 4E). However, these cells also expressed an arsenal of nonneural-specific genes, suggestive of aberrant differentiation (Appendix Fig S4A). Also, while less segregated in pseudotime, Yap1-5SAexpressing cluster 1 also showed a reduced cell cycle signature and exhibited a prominent expression of mesenchymal cell-type-related genes (e.g., Col1a1, Fig 4D and E and Appendix Fig S4A). Given the strong implication of Yap1 in brain tumor initiation, we finally scored control and Yap1-5SA-expressing cells for their expression of glioblastoma stem cell-related genes (glioblastoma stem cell-like signature; Castellan et al, 2021), such as the Yap1 paralogue transcriptional regulator Taz and some of their primary downstream transcriptional regulators (Fig 4F). While Yap1-5SA-expressing cells were overall enriched in glioblastoma stem cell-like signature across clusters, clusters 9 and 11 showed lower enrichment consistent with their partial, albeit aberrant differentiation. Finally, we noted that most Yap1-5SA-expressing clusters comprised cells isolated on days 3 or 7 but enrichment of day-7 cells in clusters 2 and 1 ( Fig 4B). This may suggest that progression towards a more mesenchymal phenotype may involve prolonged Yap1 activity. In sum, our scRNAseq data indicate that Yap1 gain-of-function in adult hippocampal NSC and astrocytes can induce molecular hallmarks of an activated NSC. However, prolonged Yap1 activity disrupts the physiological molecular trajectory towards neurogenesis and instead induces a gene expression signature akin to glioblastoma stem cells and can lead to aberrant differentiation.
To address the long-term consequences of Yap1 gain-of-function, we finally analyzed dentate gyri of animals 30 days after lentivirusmediated control GFP, wild-type Yap1, or Yap1-5SA expression (Fig EV5A). While even after 30 days, wild-type Yap1 did not cause any visible alterations in the cytoarchitecture of the dentate gyrus, the latter was massively disrupted following Yap1-5SA gain-offunction (Fig EV5B), accompanied by a drastic increase in the number of Sox2-positive cells (Fig EV5B and C). Moreover, whereas control reporter-or wild-type Yap1-expressing cells were capable of undergoing neurogenesis (as assessed by morphology and location in the granule cell layer), Yap1-5SA-expressing cells maintained Sox2 expression (Fig EV5C) and failed to acquire NeuN expression ( Fig EV5D). Likewise, Yap1-5SA-expressing cells remained negative for the astrocytic protein S100ß indicating that these cells also did not differentiate into astrocytes (Fig EV5E).

Discussion
In this study, we examined the hypothesis that the transcriptional co-activator Yap1 plays a role in the transition of adult hippocampal NSCs from quiescence to activation. Consistent with this hypothesis, we find that Yap1 expression is enriched in activated NSCs and YAP1 gain-of-function can trigger cell cycle entry in quiescent NSCs. Conversely, Yap1 gene deletion in adult hippocampal NSCs results in a reduction in the ratio of activated vs. quiescent NSCs. Finally, prolonged Yap1 activity in adult hippocampal NSCs disrupts physiological neurogenesis promoting aberrant cell differentiation and partial acquisition of a glioblastoma stem cell-like signature.
Our study set out from the re-analysis of published scRNAseq data (Data ref  showing that Yap1 activity is highly enriched in activated NSCs, suggesting that Yap1 may play an important role as a regulator of the transition of adult hippocampal NSCs from a quiescent to an activated state. A key step in the regulation of Yap1 activity consists of its translocation from the cytoplasm to the nucleus. Indeed, we did not only find that Yap1 is selectively expressed in adult hippocampal NSCs in vivo, but a hippocampal NSC culture allowed us to reveal that activation of quiescent NSCs is accompanied by a marked nuclear translocation of Yap1 protein. Intriguingly, in vivo overexpression of wild-type Yap1 did not elicit overt NSC activation. These data indicate that in our in vivo experiments, regulatory mechanisms upstream of Yap1 were not overwhelmed by the elevated Yap1 protein levels, suggesting tight control over wild-type Yap1 activity in adult hippocampal NSCs under normal conditions. Interestingly, when quiescent NSCs in culture are experimentally activated by removal of BMP4 and treatment with EGF/FGF2 (Martynoga et al, 2013), NSCs are relieved from this tight control, as illustrated by the drastic increase in nuclear Yap1 protein. In contrast to wild-type Yap1, overexpression of a Yap1 mutant defective for phosphorylation-mediated inhibition (Yap1-5SA; Zhao et al, 2007) induced cell cycle entry in adult hippocampal NSCs. This clearly shows the potency of Yap1 in promoting NSC activation once relieved from inhibitory control. Likewise, we found that hilar astrocytes commence to proliferate in response to Yap1-5SA overexpression.
In the present study, we did not address the upstream mechanisms responsible for the effective control of Yap1 activity in NSCs or astrocytes. In the mouse retina, M€ uller glia have been shown to exit quiescence and commence proliferation upon deletion of Lats1 and 2, that is, key components of the canonical Hippo pathway, indicating that Hippo signaling exerts inhibitory control under physiological conditions (Hamon et al, 2019;Rueda et al, 2019). While it is likely that similar mechanisms also operate in the adult NSC niche, it remains to be shown what molecular signals converge onto the Hippo pathway to maintain Yap1 activity at bay. Conversely, it is likely that multiple extracellular and intracellular signaling mechanisms converge to activate Yap1 in adult NSCs. Yap1 and Taz have been shown to act as signal transducers of tyrosine kinase receptor pathways such as the VEGF-VEGFR2 pathway in angiogenesis (Wang et al, 2017), and it is conceivable that related tyrosine kinase pathways exert their effects on adult NSCs via Yap1 (Han et al, 2015).
Given the apparent tight control of Yap1 in adult hippocampal NSCs, we were somewhat surprised to see the initially only very

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The EMBO Journal 42: e110384 | 2023 Ó 2023 The Authors function may cause a gradual but indirect decline in NSC activation by promoting an inflammatory response in local astrocytes or NSCs themselves (Huang et al, 2016b), which in turn could promote a progressive increase in NSC quiescence as shown during aging (Kalamakis et al, 2019). In any case, the physiological requirement of Yap1 in adult NSC activation observed in our study is highly reminiscent of the impaired reactivation of M€ uller glia in the injured adult mouse retina following Yap1 gene deletion (Hamon et al, 2019, Rueda et al, 2019. Also, given its proliferationpromoting effect on hilar astrocytes, it will be interesting to decipher the physiological contribution of Yap1 to the, albeit limited, proliferative activity of parenchymal astrocytes during reactive gliosis. Intriguingly, in the early postnatal cerebral cortex Yap1 loss-offunction was suggested to reduce astrocyte proliferation while at the same time promoting an activated phenotype due to loss of suppressor of cytokine signaling 3, SOCS3, and subsequently increased JAK-STAT signaling in astrocytes (Huang et al, 2016b). Thus, reactive gliosis may require dynamic regulation of Yap1 activity to allow for temporally limited cell cycle entry. Compared with its role in regulating adult NSC activity, Yap1 activity appears to play an even more prominent role during embryonic neurogenesis (Cappello et al, 2013;Lavado et al, 2018Lavado et al, , 2021Kostic et al, 2019;Han et al, 2020;Mukhtar et al, 2020;Najas et al, 2020). Using Yap1 loss-of-function approaches, Kostic et al observed a significant reduction in the proliferation of basal progenitors in ferret and human cortex. Intriguingly, there seems to be an increase in nuclear Yap1 levels alongside an increase in outer radial glia (ORG) from lissencephalic (mouse) to gyrencephalic (ferret and human) brains, implicating differential regulation of Yap1 activity in brain evolution. Recent data suggest a close relationship between ORG and adult hippocampal NSCs (Berg et al, 2019). Future studies may address whether differential Yap1 activity contributes to differences in initial pool size, maintenance, activation rates, and age-dependent exhaustion of adult hippocampal NSCs across mammalian species.
Consistent with a role of Yap1 in inducing NSC activation, scRNAseq from Yap1-5SA-expressing NSCs and astrocytes revealed a reduction in the expression of genes normally transcribed in quiescent NSCs and a concomitant increase in genes related to cell cycle and NSC activation. However, we noted that both on day 3 and day 7, transcriptomes of Yap1-5SA-expressing cells deviated substantially from the trajectory leading to neurons, indicative of a more profound and most likely aberrant reprogramming of gene expression. One particularly intriguing observation was the conspicuous absence of Ascl1 expression in Yap1-5SA-expresing cells. The onset of Ascl1 expression is a hallmark of adult hippocampal NSC activation and its genetic deletion results in NSC activation failure (Andersen et al, 2014;Urb an et al, 2016). While our data indicate that Ascl1 expression may not be an absolute prerequisite for entering cell cycle, it is likely critical for endowing proliferative NSCs with a neurogenic program. It would be interesting to learn in future how Yap1 gain-of-function results in the suppression of Ascl1 induction. ASCL1 expression was reported to be negatively correlated with YAP and TAZ expression in large-cell neuroendocrine carcinoma and small-cell lung cancer cells (Horie et al, 2016). Conversely, proneural factors such as Neurog2 and Ascl1 inhibit Yap1 nuclear localization by triggering phosphorylation of Yap1 at S112 (Zhang et al, 2012). This may suggest that Yap1 and Ascl1 exert a crossinhibitory effect on each other. Intriguingly, instead of neurogenesis, Yap1-5SA-expressing cells may engage with various alternative differentiation programs as indicated by pseudotime analysis incorporating RNA splicing information. Partial differentiation of Yap1-5SAexpressing cells is further supported by the decrease in the expression of cell cycle-relevant genes despite an elevated Yap1 activity signature. One peculiar observation is that Yap1-5SA-expressing cells can adopt distinct differentiated states, highlighted by the differential expression of Scg2 and Pmp22 in subpopulations of Yap1-5SA-expressing cells. Also, cluster 1 appeared to be another (less clearly segregated) endpoint in pseudotime, enriched in collagen gene expression, suggestive of the acquisition of a more mesenchymal phenotype. However, none of these states could be directly identified with existing cell types. Whatever, the differentiation programs induced in Yap1-5SA-expressing cells, canonical lineage progression towards neurogenesis was severely disrupted. This suggests that sustained Yap1 activity occludes a canonical neurogenic program, which is in line with the fact that Yap1 activity signature and Yap1 protein expression were downregulated in differentiating immature neurons. Moreover, prolonged Yap1 gainof-function in astrocytes seems to drive these cells away from their native lineage. While entirely conceivable, our scRNAseq analysis did not allow us to distinguish whether specific Yap1 gain-offunction cell clusters were preferentially or selectively generated from adult hippocampal NSCs or hilar astrocytes.
The highly aberrant transcriptional programs elicited by Yap1 gain-of-function may reflect processes that occur during glioblastoma formation. Work by Castellan et al (2021) recently showed that Yap1 and Taz are master regulators of stemness in glioblastoma stem-like cells (GSCs) and their gain-of-function represent a roadblock to GSC differentiation. Indeed, several of the genes identified by Castellan et al as components of the Yap/Taz regulon in GSCs were also induced following Yap1-5SA expression in hippocampal NSCs and/or astrocytes in our study (Fig 4F). This included the Yap1 paralogue Taz (Wwtr1) itself, as well as FoxO1 and Fos. Consistent with these findings, Yap1-5SA-expressing cells exhibited significant levels of expression of glioblastoma stem cell-related genes ( Fig 4F). Future studies may reveal whether dysregulation of normally tightly regulated Yap1 activity induces the conversion of adult NSCs or postmitotic astrocytes into deadly GSCs.

Animals and tamoxifen treatment
The study was performed in accordance with the guidelines of the German Animal Welfare Act and the European Directive 2010/63/EU for the protection of animals used for scientific purposes and was approved by the Rhineland-Palatinate State Authority (permit number 23177 07-G15-1-031 and 23177-07-G22-1-007). Mice were housed on a 12-h light/dark cycle in standard cages with free access to food and water. The study was conducted in both male and female mice. Wild-type C57BL/6J mice were purchased from Janvier at 8 weeks of age (Janvier Labs). Yap1 conditional knockout mice (GlastCre ERT2 ; Yap1 flox/flox ; CAG-CAT-EGFP, referred to as Yap1 cKO) were generated by crossing the GlastCre ERT2 (kind gift from Magdalena Götz, Helmholtz Zentrum M€ unchen) with Yap1 flox/flox (Jackson laboratory Stock NO. 027929) and CAG-CAT-EGFP mouse lines.

Ó 2023 The Authors
The EMBO Journal 42: e110384 | 2023 Tamoxifen (Sigma, T5648) was dissolved in corn oil with 10% ethanol to prepare a 20 mg/ml solution and administered once daily to both control (GlastCre ERT2 ; Yap1 wt/wt ; CAG-CAT-EGFP) and Yap1 cKO mice via intraperitoneal injections with 100 mg/kg for 5 consecutive days. Mice were sacrificed at indicated times as described in this article. Quantifications of radial glia-like cells (RGLs) in Figs 2E, F, H, and I, and EV3B were performed double-blind.
Lentivirus production was performed as described previously (Tiscornia et al, 2006). Second-generation lentiviral packaging plasmids psPAX and VSV-G envelope expressing plasmid pMD2.G (kind gifts from Didier Trono, Addgene plasmid #12260 and #12259) were used for the virus production. Viral pellets were resuspended in phosphate-buffered saline (PBS) and stored at À80°C.

Lentivirus injection
Stereotactic injections of lentivirus were performed in 8-week-old C57BL/6J mice. Prior to stereotactic injections and on the day following the surgery, mice were treated with analgesics (Rimadyl â , Zoetis, 10 mg/kg of body weight in 0.9% NaCl) by subcutaneous injection and then anesthetized with a combination of 0.5 mg/kg Medetomidine (Pfizer), 5 mg/kg Midazolam (Hameln), and 0.05 mg/kg Fentanyl (Albrecht) (in 0.9% NaCl) by intraperitoneal injection. Mice were placed in a stereotactic frame and kept on an animal heating pad to control body temperature during surgery. A small craniotomy was performed and 1 ll of the lentivirus was slowly injected using a pulled glass capillary (Hirschmann, 9600105) into the dentate gyrus at the following coordinates relative to Bregma: À2.0 AP (anterior-posterior), 1.4 ML (medio-lateral), À2.0 DV (dorso-ventral). The capillary was left in place for another 5 min before withdrawal to allow diffusion of the virus. Then, anesthesia was antagonized by intraperitoneal injection of 2.5 mg/kg Atipamezol (Pfizer), 0.5 mg/kg Flumazenil (Hameln), and 0.1 mg/ kg Buprenorphine (RB Pharmaceuticals) (in 0.9% NaCl). Animals were allowed to recover, returned to their home cages, and their physical condition was monitored daily. Rimadyl â , Zoetis 15 mg/kg was administered via drinking water for 2 days after surgery.

Adherent adult neural stem cell culture
Adult neural stem cell cultures were prepared from the DG of young adult (8-week-old) mice as previously reported with minor modifications (Babu et al, 2011). Briefly, DG was dissected from an adult mouse brain in calcium and magnesium-free Hank's Balanced Salt Solution (HBSS, ThermoFisher Scientific, cat# 14170112), and the tissue was enzymatically dissociated using the Neural Tissue Dissociation Kit (Miltenyi Biotec,Germany, according to the manufacturer's instructions. For the fluorescence-activated cell sorting (FACS), the cell pellet was resuspended with PBS containing 5% fetal bovine serum (FBS, Life Technology, cat# 10270-106).
Images of immunostained cells in culture were acquired with an upright ZEISS Axio Imager.M2 epifluorescent microscope equipped with 40× dry objective (NA 0.75) and an ApoTome.2 system (Zeiss GmbH). 4-5 images were selected randomly based on the presence of DAPI staining from each independent experiment for quantification. Images were analyzed using Image J software (NIH).

Flow cytometry
For flow cytometry of lentivirus-transduced cells, animals were sacrificed by cervical dislocation and their brains immediately placed on ice. The DG was microdissected and the tissue was enzymatically dissociated using the Neural Tissue Dissociation Kit (Miltenyi Biotec, Germany, cat# 130-092-628) according to the manufacturer's instructions. Sytox Blue (1/1000, Life Technologies, cat# S34857) was added to the cell suspension prior to sorting to exclude dead cells. Gates and compensations were set before every experiment using control samples without any fluorochromes. Single cell suspensions were sorted in a FACSAria II SORP (BD Bioscience) using a 100 lm nozzle at 20 psi. EGFP-positive cells from control and Yap1-5SA groups were directly collected into ice-cold lysis buffer (0.2%Triton X-100, containing RNAse inhibitors, Clontech, cat# 635013) in 96-well plates (Eppendorf, cat# 0030128680), and frozen at À80°C until library preparation.

Library preparation and single cell RNA sequencing
An adapted protocol from the Smart-seq2 method (Picelli et al, 2014) was used. Upon thawing the plates on ice, reverse transcription was performed using an oligo(dT) primer and a locked nucleic acid (LNA)-containing template-switching oligonucleotide (IDT; custom oligo). For all samples, we included ERCC spike-in controls (Ambion, cat#4456740) at a 1:10 6 dilution. Full-length cDNAs were amplified by 18 cycles of PCR using KAPA HiFi DNA polymerase (Roche, cat#7958935001). Amplified cDNAs for a set of randomly selected samples were quantified and run on a High Sensitivity Bioanalyzer chip (Agilent, cat# 5067-4626) on a 2,100 Bioanalyzer (Agilent).
Libraries were prepared using the Illumina Nextera XT DNA sample preparation kit (Illumina, cat#FC-131-1096) with a minor modification. The modification is based on the tagmentation protocol from Fluidigm, which uses 25% of the reagents per tagmentation reaction with a starting amount of 100-125 pg of cDNA. Libraries were amplified in 12 PCR cycles. Three microliters of each library were pooled into a single tube and the pool was subsequently beadpurified using Agencout AMPure XP beads (Beckman Coulter GmbH, cat# A63882). The purified pool was profiled in a High Sensitivity DNA chip on a 2100 Bioanalyzer (Agilent) and quantified using the Qubit dsDNA HS Assay Kit (Invitrogen, cat#Q32854) in a Qubit 2.0 Fluorometer (Life technologies). Four pools (each one from one of four 96-well plates) were pooled together at equal concentrations and sequenced on a single NextSeq 500 High Output Flow Cell with single reads for 1 × 75 cycles for read 1 plus 2 × 8 cycles for each index read.
Single cell RNA-sequencing data processing All downstream analysis was performed using the open-source R software accessed via RStudio server (R version 3.6.3). Smart-Seq2 raw data demultiplexing were performed using Illumina's bcl2fastq conversion software v.2.19.1 and overall sequence quality was assessed with FastQC v0.11.5. STAR v.2.5.2b with default parameters (except -outFilterMismatchNoverLmax 0.04) was used to align reads to the mouse reference genome GRCm38 (mm10), supplemented with ERCC RNA Spike-In Mix (ThermoFischer) control sequences and considering the GTF gene annotation from Ensembl release 88. Read summarization at the gene level was performed using Subread featureCounts v.1.6.2 with default parameters.
The quality control and normalization pipeline were applied as previously described (Amezquita et al, 2020). Briefly, cells with logtransformed library sizes and number of genes below 3 median absolute deviations (MADs) from the median of the respective distributions were removed. In addition, we removed cells in which the proportion of mitochondrial genes and spike-in RNAs was 3 MADs above the respective median proportions. Lastly, genes with average counts across samples below a threshold of 1 were filtered out.

Single cell transcriptome analysis
Using python (3.8.1) and standard python libraries (ipykernel 5.3.0. matplotlib 3.2.2, numpy 1.17.3, pandas 1.0.5, scipy 1.4.1), we employed scanpy (1.5.1), anndata (0.7.3), and anndata2ri (1.0.2) (Wolf et al, 2018) to perform single cell analysis. We filtered out cells with < 500 or more than 6,000 genes detected and cells with < 5% mitochondrial genes. Moreover, we filtered out genes that are expressed in < 10 cells. After normalization and log transformation, we regressed out differences in cell cycle scores, percent mitochondrial genes, number of transcripts per cell, and percentage of spikein. We identified highly variable genes using default parameters, which were further used for exploratory analysis via principal component analysis and force-directed graph embedding (python-igraph 0.8.2). Clustering of cells was done using the Leiden algorithm (0.8.1) implementation within scanpy. Differential gene expression analysis between Leiden clusters was performed using function sc.tl.rank_genes_groups with Wilcoxon testing. For quantification of RNA splicing from the raw sequencing data, we used velocyto (0.17) (La Manno et al, 2018) with default smartseq2 parameters. Subsequently, we employed scvelo (0.2.3) (Bergen et al, 2020) for RNA velocity estimations using the stochastic algorithm with default parameters after recovering the dynamics and using latent time. For the estimation of a trajectory pseudotime, we employed the diffusion pseudotime algorithm implementation in scvelo.
The analysis of adult hippocampal dataset (Data ref  was performed using the open-source R software accessed via RStudio server (R version 3.6.3). Briefly, normalization was performed using SCTransform with default parameters in Seurat (v3.1). We selected different cut-offs of the number of PCs and empirically found that downstream clustering analyses were optimized when using a 15-PC cutoff. The first 15 PCs were selected and used for two-dimension Uniform Manifold Approximation and Projection (UMAP), implemented by the Seurat software with default parameters. Based on the UMAP map, 5 clusters were identified using the function FindClusters in Seurat with the resolution parameter of 0.5. Radial glia-like cells, astrocytes, nIPCs, and neuroblasts were identified using FindAllMakers with default parameters. Differentially expressed genes that were expressed in at least 25% of cells within the cluster and with a fold change of more than 0.5 (log scale) were considered to be marker genes. To calculate Yap signature and active signature, AddModuleScore function was used in Seurat with default parameters.

Statistics
Statistical analysis was performed by a two-tailed unpaired Student's t-test using GraphPad Prism version 7 (GraphPad Software, Inc.). Data are represented as mean AE SEM. P-values are indicated in the graphs.

Data availability
Sequencing data have been deposited at GEO accession GSE144967. Source data are provided with this paper. Additional data related to this paper can be requested from the authors.

Disclosure and competing interests statement
The authors declare that they have no conflict of interest.