Stem cells in embryos give rise to all the tissues in the body. Adults also have stem cells, but they are fewer in number and they are usually dedicated to repairing and regenerating specific tissues. A region of the brain called the hippocampus, which is involved in learning, memory and mood, has a pool of neural stem cells. These cells can produce new brain cells long into adulthood, but maintaining their regenerative potential is a balancing act. Enough new brain cells need to be made to keep up with the brain’s demands, but if every stem cell matured into a brain cell, the brain’s capacity for repair would be lost. So, some neural stem cells hit a metaphorical snooze button to enter a resting state known as quiescence.

Stem cells in the hippocampus make a protein called Ascl1 that interacts with DNA to switch on quiescent cells so they will divide and mature. Left unchecked, Ascl1 could deplete the stem cell supply, so resting stem cells must have a way to turn Ascl1 off, but it was previously unknown how. Clues point to the E proteins, which interact with Ascl1 to allow it to bind to DNA. If the E proteins are not present, Ascl1 cannot work as a genetic switch. E proteins can also interact with inhibitor of DNA binding/differentiation proteins, known as Id proteins for short. To find out whether Id proteins affect Ascl1 activity, Blomfield et al. looked at stem cells in the hippocampus of adult mice, and at quiescent stem cells grown in the laboratory.

Blomfield et al. showed that all stem cells in the hippocampus make Ascl1, but its levels are much lower when stem cells are resting. This difference was down to an Id protein called Id4. In resting stem cells, Id4 interacted with E proteins, preventing them from binding to Ascl1, and stopping Ascl1 from ‘waking up’ the cells. This not only left Ascl1 unable to activate its target genes, it also made it vulnerable to destruction by the cell's protein recycling system. Mice with no Id4 in their hippocampus stem cells had higher levels of Ascl1, and their stem cells were more active.

The number of stem cells in a resting state increases as we age, and in illnesses like depression, limiting brain cell replacement. Uncovering the signals that switch Id4 on or off could reveal why stem cells rest more with age and illness. This could help us find ways to kick-start the production of new brain cells in adulthood.

Tissue stem cells must maintain their long-term activity while minimising the accumulation of genetic and metabolic damages. In several adult tissues, stem cells can remain inactive for long periods of time in a state of quiescence. Specific stimuli promote the exit from quiescence of different types of adult stem cells, such as hypoxia for stem cells of the carotid body or muscle injury for satellite stem cells (Dumont et al., 2015; Sobrino et al., 2018). Regulation of the transit between quiescent and active compartments is essential to maintain a pool of stem cells able to sustain tissue homeostasis and provide an adequate response to insults over the lifespan of the organism. An excessive retention of stem cells in the quiescent compartment would not produce enough differentiated progeny to maintain functionality, as happens for instance during aging (García-Prat et al., 2016; Leeman et al., 2018). On the other hand, excessive stem cell activity would eventually result in stem cell exhaustion, also leading to loss of functionality (Castilho et al., 2009; Ho et al., 2017). Quiescence is an essential property of cancer stem cells that allows them to evade immune surveillance and results in resistance to treatment (Agudo et al., 2018). Despite their relevance for the fields of tissue repair, aging and cancer biology, the mechanisms regulating quiescence in adult stem cells are still largely unknown.

In the adult brain, NSC populations in the ventricular-subventricular zone (V-SVZ) of the lateral ventricles and in the dentate gyrus (DG) of the hippocampus, generate new neurons and glia that integrate into pre-existing neuronal networks (Bond et al., 2015; Lim and Alvarez-Buylla, 2016). In both regions, a large fraction of stem cells is quiescent. Extensive work has led to the identification of extracellular signals present in the V-SVZ and DG niches that regulate quiescent and active states (Choe et al., 2016; Silva-Vargas et al., 2013). Notch, BMP4 and the neurotransmitter GABA have been shown to maintain stem cell quiescence while Wnt, Shh and the neurotransmitter glutamate are thought to promote stem cell activity (Bao et al., 2017; Choe et al., 2016; Engler et al., 2018; Imayoshi et al., 2010; Lie et al., 2005; Mira et al., 2010; Petrova et al., 2013; Qu et al., 2010; Song et al., 2012; Yeh et al., 2018). In contrast, little is known of the cell intrinsic machinery that NSCs employ to adjust their activity to the different signals received from the niche. Ascl1 is one of the few intrinsic regulators of NSC quiescence described so far. Ascl1 is a basic-helix-loop-helix (bHLH) transcription factor that is present in a fraction of dividing stem cells and intermediate progenitors in the adult hippocampus. Loss of Ascl1 completely blocks the activation of adult hippocampal stem cells, inhibits the generation of new neurons and prevents the depletion of the stem cell pool over time (Andersen et al., 2014). Ascl1 may therefore determine the balance between quiescence and activity of hippocampal NSCs. Indeed, stabilization of Ascl1 protein by inactivation of the E3 ubiquitin ligase Huwe1 results in over-proliferation of hippocampal stem cells and prevents their return to quiescence (Urbán et al., 2016). However, Huwe1 inactivation is not sufficient to trigger the large-scale activation of quiescent stem cells, indicating that additional mechanisms maintain the quiescent state of hippocampal stem cells.

The Id (Inhibitor of differentiation/DNA binding) proteins are known inhibitors of bHLH transcription factors such as Ascl1 (Imayoshi and Kageyama, 2014; Ling et al., 2014). Id proteins contain a conserved HLH domain with which they dimerize with some bHLH proteins. However, they lack a DNA binding domain and therefore prevent bHLHs with which they interact from binding DNA and other bHLH factors (Benezra et al., 1990). For instance, Id proteins have previously been shown to sequester E proteins, the dimerization partners of Ascl1. The resulting monomeric form of Ascl1 can no longer bind DNA and is furthermore rapidly degraded by the proteasome (Shou et al., 1999; Viñals et al., 2004). In mammals, the Id family comprises four genes, Id1-4. The Id genes are expressed in multiple tissues during development and in adult stem cell niches, and have been shown to promote stemness and proliferation in different systems, including in hematopoietic stem cells and in stem cells of the adult SVZ (Niola et al., 2012; Singh et al., 2018). However single cell transcriptome analysis has also shown that expression of Id3 and Id4 in particular, is highly enriched in quiescent hippocampal NSCs in vivo, thus linking Id genes with NSC quiescence (Hochgerner et al., 2018; Shin et al., 2015).

Here we show that Ascl1 mRNA is expressed by hippocampal stem cells independently of their proliferative or quiescent states, but that only active stem cells reach significant levels of Ascl1 protein. This non-transcriptional regulation of Ascl1 is recapitulated in hippocampal stem cell cultures in vitro, where the quiescence-inducing factor BMP4 has no effect on Ascl1 mRNA expression but is sufficient to reduce Ascl1 protein levels. We performed a gene expression screen in these cells and found that Id4 is strongly induced in quiescent NSCs. Accordingly, analysis of the expression of Id1-4 proteins in the hippocampus also showed that Id4 is expressed by the highest percentage of NSCs. We demonstrated that Id4 sequesters the Ascl1 heterodimerization partner E47 and that the resulting Ascl1 monomers are rapidly degraded by the proteasome. Therefore, Id4 blocks the pro-activation transcriptional program driven by Ascl1 and keeps stem cells quiescent. Indeed, elimination of Id4 from the adult brain results in increased Ascl1 protein levels in stem cells of the hippocampus and in their rapid entry into the cell cycle, and also leads to an increase in Id3 expression that might partially compensate for the loss of Id4 and suppresses Ascl1 protein in the absence of that factor.

In this study, we show that the repressor protein Id4 promotes the maintenance of adult hippocampal stem cells (RGLs) in a quiescent state. The function of Id4 in maintenance of RGL quiescence is in remarkable contrast with its role in promoting the proliferation of progenitor cells in the embryonic cerebral cortex (Bedford et al., 2005; Yun et al., 2004). This difference may reflect the different role of the bHLH proteins that are Id4 targets in embryonic versus adult neural lineages. In the embryonic forebrain, NSCs are in a proliferative state and proneural bHLH proteins mostly act to promote neuronal differentiation. Their inactivation by Id4 during development therefore results in a block of differentiation and extended proliferation. In contrast, RGLs in the adult hippocampus are mostly quiescent, Ascl1 is required to promote their activity, and inactivation of Ascl1 by Id4 results in their failure to proliferate.

We also show that Id4 promotes the degradation of the pro-activation factor Ascl1 in RGLs. As Ascl1 protein is only detectable in proliferating RGLs (Andersen et al., 2014), we were not expecting the Ascl1 gene to be transcribed by most RGLs including many quiescent cells. We found that despite Ascl1 mRNA being expressed and translated, Ascl1 protein does not accumulate in quiescent RGLs due to its rapid degradation. This surprising finding could be the reason why single cell transcriptomic analysis of hippocampal cells did not identify Ascl1 among the genes differentially expressed between quiescent and active stem cells (Artegiani et al., 2017; Hochgerner et al., 2018; Shin et al., 2015). This non-transcriptional control of a key activation factor is also found, for instance, in satellite stem cells where the bHLH factor MyoD is transcribed in quiescent cells but its translation is inhibited by an RNA-binding protein to prevent stem cell activation (de Morrée et al., 2017).

It is well established that Id proteins, including Id4, form non-functional heterodimers with E proteins, which are dimerization partners of tissue-specific bHLH transcription factors such as Ascl1 (Imayoshi and Kageyama, 2014; Ling et al., 2014; Patel et al., 2015; Sharma et al., 2015). The three genes encoding for E proteins in mice are all expressed by RGLs, and since they are thought to have redundant functions, it is difficult to investigate their specific contributions to RGL behaviour. Nevertheless, high levels of Ids are expected to result in the sequestration of all E proteins away from functional dimers with Ascl1. Non-dimerized Ascl1 is not able to bind DNA, and this alone could explain why Ascl1 target genes are downregulated in NSCs upon Id4 overexpression or BMP treatment, which increases the expression of all Id proteins. But how does Id4 prevent Ascl1 protein accumulation? Exposure of different cell types to BMPs has been shown to trigger the proteolytic degradation of Ascl1 (Shou et al., 1999; Viñals et al., 2004). In lung carcinoma cells, the formation of heterodimers with E47 stabilizes Ascl1, and induction of Id1 by BMP2 sequesters E47, resulting in degradation of the unstable monomeric form of Ascl1 (Viñals et al., 2004). We show that Ascl1 is more unstable in quiescent than proliferating hippocampal NSCs and that Ascl1-E47 dimers are disrupted by Id4. Therefore, we propose that a similar mechanism to that in lung carcinoma cells promotes the degradation of Ascl1 when quiescent hippocampal stem cells express high levels of Id4.

To interfere with the function of Id4 in NSCs and circumvent the compensation by other Id proteins, we have overexpressed E47, which is expected to interact with and titrate all Id proteins. We found that this is indeed sufficient to stabilize Ascl1 protein and promote cell cycle re-entry of BMP-treated NSCs. However, we realize that E47 overexpression might interfere with other factors than Id proteins and Ascl1. Silencing Id4 in quiescent NSCs in culture was confounded by the functional compensation of the other Id proteins, which are able to suppress Ascl1 protein levels when overexpressed. Loss of Id4 in hippocampal RGLs also resulted in increased Id1 and Id3 protein levels, suggesting that Id4 may suppress these proteins in vivo, and that its loss may be partially compensated by the increase in their expression. Since deletion of Id4 has the limitation of functional compensation by other Ids, more refined tools will be required in the future to dissect the specific mechanisms by which Id4 upregulation leads to Ascl1 degradation.

Our transcriptomic analysis suggests that Id4 alone contributes to a large extent to the gene expression program induced by BMP to promote NSC quiescence. Overexpression of Id4 in the absence of BMP induces many of the genes that BMP4 induces, and suppresses many of the genes suppressed by BMP4. Among the genes suppressed by both BMP4 treatment and Id4 overexpression, an important fraction corresponds to cell cycle regulators, including many Ascl1 targets.

Besides regulating the activity of tissue-specific bHLH transcriptional activators such as Ascl1, Id proteins also interact with bHLH transcriptional repressors of the Hes family (Bai et al., 2007). Direct interaction of Id2 with Hes1 blocks the autorepressive activity of Hes1 protein resulting in its stable expression at a high level. Therefore, Id proteins promote a switch of the expression pattern of Hes proteins from oscillating, resulting in oscillatory expression of target genes such as Ascl1, to stably high, resulting in constant repression of these targets (Bai et al., 2007; Boareto et al., 2017; Sueda et al., 2019). However, we find that Ascl1 is transcribed in most quiescent RGLs, indicating that Hes proteins do not repress Ascl1 transcription in these cells. Id4 has been shown to inhibit the action of Id1-3 proteins by interacting with stronger affinity with them than with other binding partners (Patel et al., 2015; Sharma et al., 2015). This non-canonical role of Id4 has been proposed to explain that Id4 promotes the proliferation of embryonic neural progenitors by blocking the interaction of Id1-3 with Hes proteins and thus promoting oscillations of Hes proteins – and consequently of Ascl1 – and progenitor proliferation (Bedford et al., 2005; Boareto et al., 2017; Yun et al., 2004). Id4 might therefore maintain Ascl1 transcription – separately from its role in eliminating Ascl1 protein – by interfering with the role of other Id proteins in stabilising Hes protein expression. In support of this model, Id4 deletion in RGLs in vivo results not only in upregulation of other Id proteins but also in a reduction of Ascl1 transcript levels. The function that we have identified for Id4 in the hippocampus is also distinct from the role reported for other Id factors in the adult V-SVZ, where Id1 and Id3 promote stem cell self-renewal (Nam and Benezra, 2009) and Id1-3 maintain stem cell function by keeping stem cells adherent to their niche environment (Niola et al., 2012).

Id4 is not the only Id protein expressed in hippocampal RGLs, as Id1 is also expressed in nearly half of them. Nevertheless, Id4’s role in the regulation of stem cell quiescence is clearly different from that of Id1. While Id4 expression is restricted to RGLs that are quiescent and express low levels or no Ascl1 protein, Id1 protein is found in proliferating RGLs, many of which also express Ascl1 protein, suggesting that contrary to Id4, Id1 at the level it is expressed in RGLs in homeostasis does not promote stem cell quiescence or the degradation of Ascl1, although the same factor may have the potential to promote Ascl1 degradation when expressed at higher levels, that is when overexpressed in cultured NSCs. In agreement with this, Id1 has recently been shown to have a role in the activation of hematopoietic stem cells upon stress signals (Singh et al., 2018). We have addressed the effect of loss of Id1 from RGLs by examining Smad4cKO mice, where Id1 expression in RGLs is greatly reduced while Id4 expression is unaffected (Blomfield et al., 2018). Loss of Smad4 did not affect RGLs, indicating that Id1 is not required to suppress Ascl1 expression or RGL proliferation. It is unclear why Id1, which has been shown to dimerize with E proteins and promote Ascl1 degradation in another cell type (Viñals et al., 2004) has no such effect in hippocampal stem cells, but this may be the result of its relative expression levels in RGLs. Id3 expression, on the other hand, is mostly restricted to quiescent RGLs and is therefore a better candidate to compensate for the loss of Id4 in quiescent cells.

While Id4 is expressed in the vast majority of quiescent RGLs, Id4 deletion results in loss of quiescence of only a fraction of them at both 5d and 30d post-deletion, suggesting that compensatory mechanisms operate to blunt the Id4^cKO phenotype in RGLs. Thus, other Ids - in particular Id3, because of its expression pattern in quiescent RGLs - or unrelated factors might compensate partially for the loss of Id4. An increase in the repression of Ascl1 transcription, due to increased Hes-Id protein interactions and Hes protein stabilization, might also contribute to the blunting of the Id4^cKO phenotype. In support of this, Ascl1 transcription was lower in Id4^cKO mice as early as 5 days after Id4 deletion. We have previously shown that the ubiquitin ligase Huwe1 degrades Ascl1 in proliferating RGLs and allows a fraction of these cells to return to quiescence (Urbán et al., 2016). Huwe1 is expressed in quiescent RGLs (Urbán et al., 2016) and although Id4 might mask its role in degrading Ascl1 in these cells, it might be able, in the absence of Id4, to eliminate excess Ascl1 and maintain RGL quiescence.

Given the important role of Id4 in maintaining RGL quiescence, it seems likely that its expression is regulated by niche signals to control RGL activity. Id genes, including Id4, are well known effectors of BMP signalling in neural cells and other cell types (Ling et al., 2014; Patel et al., 2015; Samanta and Kessler, 2004). Smad4 deletion strongly reduces Id1 but not Id4 levels in RGLs, indicating that Id4 diverges from other Id proteins not only in its activity but also in the regulation of its expression (Blomfield et al., 2018). Id4 has been shown to be directly regulated by Notch signalling in embryonic neural progenitors (Li et al., 2012) and in adult hippocampal stem cells (Zhang et al., 2019) but we found that Id4 expression persists in RGLs lacking the essential Notch signalling effector RBPJk (Blomfield et al., 2018). Id4 expression is only mildly affected in RGLs lacking both Smad4 and RBPJk, indicating that additional pathways beside BMP-Smad4 and Notch-Rbpjk promote RGL quiescence by maintaining Id4 expression.

Id4 is expressed in most quiescent RGLs but it is sharply downregulated in active RGLs. Indeed, it is one of the most differentially expressed genes in quiescent versus active stem cells, both in vivo and in NSC cultures (Shin et al., 2015 and this paper). Down-regulation of Id4 is crucial for RGLs to produce sufficient levels of Ascl1 protein to leave the quiescent state and become active, emphasizing the importance of this gene in the maintenance of RGL quiescence. An important aim of future research will be to identify the niche signals that induce Id4 expression in quiescent RGLs and reduce its expression in active cells.

Contact for reagent and resource sharing: François Guillemot (Francois.guillemot@crick.ac.uk).

Sequencing data have been deposited in GEO under accession code GSE116997.

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Author details

1. Isabelle Maria Blomfield

   The Francis Crick Institute, London, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4412-0226

2. Brenda Rocamonde

   Institut du Cerveau et de la Moelle Epinière, ICM, Inserm U 1127, CNRS UMR 7225, Sorbonne Université, Paris, France

   Contribution

   Resources, Collaborative discussion of data

   Competing interests

   No competing interests declared

3. Maria del Mar Masdeu

   The Francis Crick Institute, London, United Kingdom

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization

   Competing interests

   No competing interests declared

4. Eskeatnaf Mulugeta

   Department of Cell Biology, Erasmus MC, Rotterdam, Netherlands

   Contribution

   Software, Formal analysis, Visualization

   Competing interests

   No competing interests declared

5. Stefania Vaga

   The Francis Crick Institute, London, United Kingdom

   Contribution

   Software, Formal analysis

   Competing interests

   No competing interests declared

6. Debbie LC van den Berg

   Department of Cell Biology, Erasmus MC, Rotterdam, Netherlands

   Contribution

   Software, Formal analysis, Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6026-8808

7. Emmanuelle Huillard

   Institut du Cerveau et de la Moelle Epinière, ICM, Inserm U 1127, CNRS UMR 7225, Sorbonne Université, Paris, France

   Contribution

   Resources, Collaborative discussion of data

   Competing interests

   No competing interests declared

8. François Guillemot

   The Francis Crick Institute, London, United Kingdom

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   francois.guillemot@crick.ac.uk

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0003-0432-5067

9. Noelia Urbán

   1. The Francis Crick Institute, London, United Kingdom
   2. Institute of Molecular Biotechnology (IMBA), Vienna Biocenter Campus (VBC), Vienna, Austria

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   noelia.urban@imba.oeaw.ac.at

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4813-9274

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