Differential Interactions Between Notch and ID Factors Control

STEM CELLS AND REGENERATION RESEARCH ARTICLE Differential interactions between Notch and ID factors control neurogenesis by modulating Hes factor autoregulation Marcelo Boareto1,2,*, Dagmar Iber1,2,* and Verdon Taylor3,*

ABSTRACT neurogenesis is active in two defined regions: the ventricular- During embryonic and adult neurogenesis, neural stem cells (NSCs) subventricular zone (V-SVZ) of the lateral ventricle wall; and generate the correct number and types of neurons in a temporospatial the dentate gyrus of the hippocampus (Doetsch et al., 1999; fashion. Control of NSC activity and fate is crucial for brain formation Doetsch, 2003; Spalding et al., 2013; Ernst et al., 2014; Fuentealba and homeostasis. Neurogenesis in the embryonic and adult brain et al., 2015; Furutachi et al., 2015). In the adult brain the majority differ considerably, but Notch signaling and inhibitor of DNA-binding of the NSCs are mitotically inactive (qNSC) and infrequently enter (ID) factors are pivotal in both. Notch and ID factors regulate NSC cell cycle, becoming active NSCs (aNSCs) to generate neurons maintenance; however, it has been difficult to evaluate how these before returning to quiescence or differentiating into glial cells pathways potentially interact. Here, we combined mathematical (Fig. 1A) (Lois et al., 1996; Kirschenbaum et al., 1999; Encinas modeling with analysis of single-cell transcriptomic data to elucidate et al., 2011; Ihrie and Alvarez-Buylla, 2011; Shook et al., 2012; unforeseen interactions between the Notch and ID factor pathways. Giachino et al., 2014). Thus, although embryonic and adult During brain development, Notch signaling dominates and directly neurogenesis share some similarities, there are also fundamental regulates Id4 expression, preventing other ID factors from inducing differences and stem cell quiescence is one of them. Currently, it is NSC quiescence. Conversely, during adult neurogenesis, Notch not known why NSCs of the adult brain remain quiescent and the signaling and Id2/3 regulate neurogenesis in a complementary mechanisms that control the transition of NSC to activation are manner and ID factors can induce NSC maintenance and also unclear. However, the balance between activity and quiescence quiescence in the absence of Notch. Our analyses unveil key is crucial not only to maintain the NSC pool for later neuron molecular interactions underlying NSC maintenance and mechanistic production and regeneration but also to prevent overproliferation differences between embryonic and adult neurogenesis. Similar Notch and tumor formation (Lugert et al., 2010; Silva-Vargas et al., 2016). and ID factor interactions may be crucial in other stem cell systems. Thus, understanding the molecular mechanism that regulates maintenance and differentiation of NSCs is not only of theoretical KEY WORDS: Notch signaling, Id transcription factors, Neural stem interest but crucial for understanding disease mechanism and cells, Neurogenesis, Computational biology developing new therapeutic strategies (Lie et al., 2004; Lazarov et al., 2010). INTRODUCTION The processes of NSC maintenance and differentiation are Neurogenesis is the production of neurons from neural stem cells controlled by a core regulatory network of basic helix-loop-helix (NSCs). The correct balance between NSC proliferation and (bHLH) transcription factors (Lee, 1997; Ross et al., 2003; Heng differentiation is essential for embryonic formation of the brain and Guillemot, 2013; Imayoshi and Kageyama, 2014b). Members and to confer regenerative capacities in the adult brain (Doe, 2008). of the bHLH family have two conserved functional domains: a basic Any deviation from the regulated neurogenic program can lead to region for DNA binding and a helix-loop-helix (HLH) region for drastic problems during development, including microcephaly and dimerization. These transcript factors can act as repressors or cognitive impairment. During embryonic development of the activators of gene expression. The hairy and enhancer of split (Hes) central nervous system, NSCs divide frequently and produce proteins Hes1 and Hes5 are central repressors of NSC differentiation neurons either directly or via a committed intermediate progenitor during brain development (Ohtsuka et al., 1999; Kageyama et al., (IP) cell (Fig. 1A). In the peak neurogenic period, a few NSCs exit 2007, 2008), while bHLH factors including Ascl1 and Neurog2 are the cell cycle and become quiescent (qNSCs) (Furutachi et al., activators of neural differentiation and thus referred to as proneural 2013, 2015; Fuentealba et al., 2015). qNSCs are only reactivated in factors (Wilkinson et al., 2013; Imayoshi and Kageyama, 2014b). the adult neurogenic niches. In the adult brain, NSCs remain and Hes proteins in conjunction with TLE factors repress gene expression by binding to N-box and class-C sites in the promoters of target genes. Proneural factors activate gene expression by 1 Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, binding to E-box consensus sequences in the promoters of their Mattenstrasse 26, 4058 Basel, Switzerland. 2Swiss Institute of Bioinformatics, Mattenstrasse 26, 4058 Basel, Switzerland. 3Department of Biomedicine, University targets (Fig. 1B) (Imayoshi and Kageyama, 2014b). Furthermore, of Basel, Mattenstrasse 28, 4058 Basel, Switzerland. the binding affinity of proneural factors to E-boxes can be enhanced

*Authors for correspondence ([email protected]; dagmar.iber@bsse. by the formation of heterodimers with other members of the bHLH ethz.ch; [email protected]) family: the E-proteins Tcf4 and Tcf3 (Massari and Murre, 2000; Bohrer et al., 2015). V.T., 0000-0003-3497-5976 During brain development, Notch signaling activates Hes gene This is an Open Access article distributed under the terms of the Creative Commons Attribution expression, which in turn inhibits NSC differentiation by repressing License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. proneural genes, including Ascl1 and Neurog2 (Lee, 1997; Heng and Guillemot, 2013). In addition, Hes proteins repress expression

Received 23 March 2017; Accepted 14 August 2017 of their own genes, counteracting Notch and leading to oscillations DEVELOPMENT

3465 STEM CELLS AND REGENERATION Development (2017) 144, 3465-3474 doi:10.1242/dev.152520

Fig. 1. The NSC differentiation processes in the embryonic and adult brain and its regulatory network. (A) NSC fate in the embryonic and adult brain is dependent on the levels of proneural transcription factor expression. During embryonic neurogenesis, the majority of the NSCs are in a mitotically active state (aNSC) while a few will enter quiescence (qNSC) and remain inactive until adulthood. In the adult neurogenic niches, most NSCs are mitotically inactive (qNSC) and rarely transit to the mitotically active, neurogenic state (aNSC). In aNSCs, low levels of proneural activity drive cell cycle progression but is insufficient to induce differentiation. In the absence of proneural transcription factor activity, NSCs are quiescent (qNSC) and high proneural transcription factor activity drives neural differentiation (Diff). (B) The Notch-Hes-Proneural transcription factor interaction network. Notch signaling through the DNA-binding protein Rbpj activates expression of Hes genes. Hes protein homodimers repress proneural gene expression, including Ascl1 and Neurog2 via N-box and class-C sites, and their own expression by binding to N- box sites in their promotor regions. Proneural transcription factors activate cell cycle progression and differentiation via E-box sites. (C) The current known Notch-Hes-IDs-proneural interactions. IDs form heterodimers with Hes transcription factors, which are unable to bind to N-box sites but can bind to class-C sites, although with lower efficiency than Hes homodimers. IDs also form heterodimers with proneural factors that are unable to activate the differentiation and cell cycle progression genes.

in their expression (Fig. 1B) (Hirata et al., 2002). This dynamic 2014b). Hence, IDs potentially regulate neurogenesis at multiple Hes gene activity has been suggested to result in low-level levels, including enhancing Hes expression and blockage of expression of proneural factors, including Ascl1, and this low proneural factor activity (Fig. 1C). expression drives cell cycle progression but is not sufficient to Owing to the complex and reciprocal interplay between Notch- induce NSC differentiation (Castro et al., 2011; Imayoshi et al., Hes and IDs, it has been challenging to access the consequences of 2013; Andersen et al., 2014). In contrast, high and sustained their interactions experimentally and their respective roles in the expression of Hes proteins drives complete repression of Ascl1, control of NSC activity. As a first step to address this problem, we leading to cell cycle exit and NSC entry into a quiescent state (Baek developed a specific theoretical framework that takes into account et al., 2006; Castro et al., 2011; Imayoshi et al., 2013; Andersen the interactions between Notch, IDs and the members of the bHLH et al., 2014). NSCs in the adult brain niches are predominantly family of transcriptional factors. Our theoretical framework is in line quiescent, a state not observed frequently in the developing brain. with previous models of Hes (Lewis, 2003; Monk, 2003; Novák and How regulation of the Hes-proneural gene axis is differentially Tyson, 2008; Wang et al., 2011; Pfeuty, 2015) and explicitly controlled in NSCs during development and in the adult brain is incorporates Notch-mediated activation of Hes gene expression, unknown. However, previous observations suggest that different Hes-mediated repression of proneural expression, Hes auto- levels of proneural activity in the NSCs lead to three possible output repression and homodimer formation. In order to recapitulate the states in the NSCs: NSC quiescence, proliferation and different effects of IDs, we incorporated the possibility of Hes-ID differentiation when proneural activity is absent, intermediate/low and proneural-ID heterodimer formation into the model. We and high, respectively (Fig. 1A). explored computationally the properties of this gene regulatory In the adult brain, NSCs quiescence has been linked to the network and the conditions required to obtain NSC quiescence, expression of inhibitor of DNA-binding factors (IDs) (Nam and maintenance of activated NSCs and differentiation. Once we had Benezra, 2009). IDs also have a HLH domain, which enables the established a robust model that fulfilled these criteria, we challenged formation of heterodimers with other bHLH factors, but lack the and validated our predictions by analyzing the gene expression of basic domain and for this reason cannot efficiently bind to DNA NSCs at the single-cell level. Finally, by evaluating the differences (Tzeng, 2003; Heng and Guillemot, 2013). Therefore, IDs act as in the single-cell expression profile of adult and embryonic NSCs, inhibitors of the activity of bHLH factors. Experimentally, IDs have we uncovered key differences between embryonic and adult been shown to form dimers with Hes proteins and these neurogenesis. heterodimers are unable to bind to the N-box-binding motifs in the Hes promoter and thus relieve Hes auto-repression (Bai et al., RESULTS 2007). Interestingly, Hes-ID heterodimers can still repress target Notch signaling alone cannot completely repress proneural genes, including Ascl1, via class-C binding sites, albeit with lower activity and drive NSC quiescence efficiency than Hes homodimers (Bai et al., 2007). Thus, IDs are The balance between Notch signal activity and proneural factor able to segregate auto-repressive and downstream target gene expression is pivotal in the regulation of NSC activity and neurogenic repression functions of Hes factors. In addition, IDs also form differentiation. Proneural factors, including Ascl1, are important for ineffective heterodimers with proneural factors, including Ascl1, neuronal differentiation but at lower transient levels also induce NSC reducing their potential to drive differentiation by blocking their cell cycle entry. Complete repression of Hes expression is a binding to E-boxes in target genes (Imayoshi and Kageyama, prerequisite for proneural gene expression to levels that induce DEVELOPMENT

3466 STEM CELLS AND REGENERATION Development (2017) 144, 3465-3474 doi:10.1242/dev.152520 progenitor cell commitment and differentiation. By contrast, binary fates: high Notch leads to low-intermediate proneural activity proneural gene expression is completely repressed in qNSCs and is that induces proliferation and active NSCs; low Notch leads to high necessary for NSCs to exit cell cycle (Castro et al., 2011; Imayoshi proneural activity that results in differentiation (Fig. 2D). Therefore, et al., 2013; Andersen et al., 2014). our computational results suggest that, in the absence of any extra Therefore, we initially studied the dynamics of the Notch/Hes factor, Notch signaling cannot completely repress proneural activity regulatory module in NSCs in the absence of any extra factor and thereby induce NSC quiescence. Interestingly, this situation is (Fig. 1B). To be consistent with previous theoretical and seen during embryonic development where most NSCs of the experimental evidence (Hirata et al., 2002; Monk, 2003; Wang developing brain are mitotically active. et al., 2011), we adjusted our model such that Hes gene expression oscillates with a periodicity of 2-3 h in the presence of a Notch Hes-negative feedback, and not oscillations, leads to the signal (see Materials and Methods; Fig. 2A,B). We then evaluated accumulation of the minimal proneural activity required for mathematically the effect of Notch activity on the levels of NSC proliferation proneural gene expression. In agreement with experimental data, As quiescence is the major state of NSC in the adult brain, we our model recapitulated that increasing Notch signaling decreases investigated the conditions required for a complete repression of proneural gene expression (Fig. 2C). However, Notch signaling proneural gene expression. It has been suggested that the oscillatory alone was unable to completely suppress proneural gene expression expression of Hes leads to low levels of proneural factor expression or even reduce it to the levels necessary for cell cycle exit (NSC that enable and drive NSC proliferation while being insufficient for quiescence) (Fig. 2C). We evaluated whether complete repression of differentiation (Kageyama et al., 2009; Imayoshi et al., 2013; proneural gene activity could be achieved by increasing the basal Imayoshi and Kageyama, 2014a). In order to investigate the impact production rate of Hes and maximal levels of Hes by increasing of oscillations on proneural expression, we explored the regions of the Notch activity (Fig. 2D). Surprisingly, an increase in Hes parameter space where Hes expression no longer oscillates (Fig. S1). production had little effect on the levels of proneural gene Rapid Hes protein degradation, long intronic delay and Hes auto- expression. Moreover, changes in Notch/Hes signaling leads to repression have all been shown to be required for oscillatory Hes expression (Bai et al., 2007; Yoshiura et al., 2007; Takashima et al., 2011). Analytical studies of Hes oscillations have identified intronic delay and protein degradation as key parameters that modulate Hes dynamics (Ay et al., 2013, 2014; Wang et al., 2014). Therefore, we explored how changes in these parameters would affect the expression level of the downstream proneural genes. We initially evaluated the effects of changing Hes protein half- life on the oscillatory behavior of Hes gene expression (Fig. 3A). Increasing Hes half-life substantially above the experimentally determined 22 min resulted in loss of oscillations and sustained Hes mRNA expression levels (Fig. 3A). Similarly, reducing Hes protein half-life shortened the periodicity of the oscillations (Fig. 3A). These findings are consistent with experimental evidence suggesting that both increases or decreases in Hes protein degradation rate stops or dampens Hes mRNA oscillations, and validates our mathematical framework (Yoshiura et al., 2007; Harima et al., 2014). Interestingly, however, we found that the average levels of proneural mRNA expression do not change with changes in Hes protein degradation rate and does not lead to a complete block in proneural gene levels (Fig. 3B). Hence, Fig. 2. Notch-Hes-Proneural regulatory module can regulate NSC regulation of Hes protein degradation in NSCs cannot account for differentiation but cannot drive NSCs into quiescence. (A) Hes mRNA cell cycle exit and quiescence. ∼ levels oscillate with a periodicity of 140 min. (B) A minimum activation of gene Therefore, we examined the effect of intronic delay of transcript expression is required for oscillatory behavior, which is induced in response to a large range of Notch signal activation. Upper and lower curves represent the maturation rate on oscillatory Hes gene expression, proneural gene maximum and minimum expression levels of Hes mRNA during the oscillations activity and NSC fate. It has been shown that Hes genes have three (as indicated by the red and green circles, respectively). Dashed curve long introns that regulate transcription and splicing of the primary represents the temporal average expression of Hes mRNA. The mean RNA transcript and maturation of the Hes7 mRNA to ∼19 min expression level of Hes mRNA does not change dramatically in the oscillatory (Harima et al., 2014). Removal of one or two of the Hes gene introns region of expression, irrespective of the levels of Notch signal activity. increases Hes transcript maturation rate and dampens or completely (C) Expression level of proneural genes as a function of Notch signaling activity. Proneural gene expression becomes oscillatory at the same level of abolishes oscillations in expression (Takashima et al., 2011; Harima Notch signal due to the oscillatory expression of Hes factors. (D) Predictions of et al., 2014). Consistent with this, in our model we observed that the proneural gene expression and NSC fate at different levels of Notch signal and oscillatory behavior of the Hes genes is absent and expression is Hes mRNA production rate. Changes in Hes basal production rate have little sustained with rapid transcript production and maturation. effect on proneural gene expression (blue to red heat map) at any given level of Conversely, a longer delay in Hes mRNA maturation leads to an Notch signal. Low Notch signal activity leads to high proneural expression (red) increase in the periodicity of oscillations at most levels of Notch and this results in NSC differentiation (Diff). In contrast, high Notch activity leads to low-intermediate levels of proneural expression (light blue) leading to signal activity (Fig. 3C). Our model also predicts that proneural active, proliferative NSCs (aNSC). Production rates are in mRNA/min and the expression is not affected by changes in Hes transcript maturation yellow circle indicates the standard value determined experimentally and used (production) rate and that it has little or no effect on NSC fate in all simulations (see Material and Methods). (Fig. 3D). Therefore, we examined the effect of Hes auto-repression DEVELOPMENT

3467 STEM CELLS AND REGENERATION Development (2017) 144, 3465-3474 doi:10.1242/dev.152520

se, is crucial for the small accumulations in proneural expression required for NSC proliferation and differentiation.

IDs potentiate Notch/Hes activity and drive complete repression of proneural expression Our mathematical model confirms the crucial importance of regulating Hes auto-repression in order to enable a complete repression of proneural expression and drive NSC quiescence. This is an important finding as it provides a mode for modulating NSC behavior to achieve the transition from NSC quiescence to activation and differentiation. Although IDs have been shown to regulate neurogenesis and NSC activity as well as alter Hes and proneural factor activity, it remains unclear how the combination of IDs, Hes and proneural factor activity achieve the effects observed in genetic manipulation experiments (Baek et al., 2006; Nam and Benezra, 2009). We then incorporated IDs into our theoretical framework and observed that by reducing Hes auto-repression, IDs lead to an increased and sustained expression of Hes genes (Fig. 4A). Thus, Hes expression levels are uncoupled from Notch signal intensity by simply increasing expression of ID proteins, which also results in non-oscillatory expression and high levels of Hes (Fig. 4A). In addition, by evaluating the effects of Notch and IDs on Hes, we found that Hes gene expression only reaches maximum levels in the presence of both Notch signaling and IDs (Fig. 4B). Fig. 3. Release of Hes auto-repression drives complete repression of Furthermore, high levels of IDs are able to completely repress proneural expression. (A) Period of oscillations of Hes expression for proneural gene activity, on the one side by increasing Hes levels to different values of Notch signal and Hes protein half-life. White area indicates repress transcription either via Hes homodimers or ID-Hes the region where Hes does not oscillate (sustained expression). Yellow circle indicates the standard value used in all simulations. (B) Proneural expression heterodimers, and on the other side by directly interacting with levels for different values of Notch signal and Hes protein half-life. Low Notch proneural proteins to inhibit target gene activation (Figs 1C and 4C). activity leads to NSC differentiation (Diff), whereas high Notch activity By combining Notch signals and ID activity, our model then maintains NSCs active/proliferative (aNSC). Changes in Hes protein predicts that proneural activity segregates into three expression degradation have little effect on proneural expression. (C) Period of oscillations states that directly translate into different stem cell states: low/absent of Hes expression and (D) proneural expression levels for different values of Notch signal and intronic delay. Low Notch activity leads to NSC differentiation (Diff), whereas high Notch activity maintains NSCs active/proliferative (aNSC). Changes in intronic delay have little effect on proneural expression. (E) Period of oscillations of Hes expression and (F) proneural expression levels for different values of Notch signal and Hes auto-repression. Release of Hes auto- repression, together with high Notch activity, can drive complete repression of proneural activity, leading to NSC quiescence (qNCS). on Hes mRNA oscillatory period, proneural gene expression and NSC fate. We considered an auto-repression factor value of 0.0 to represent a complete repression of Hes gene expression by the Hes proteins. Conversely, an auto-repression factor value of 0.05 represents Hes gene repression of 95% with a residual transcription of 5% (Fig. 3E). Under these conditions, a small relief of Hes auto- repression is enough to completely abolish Hes mRNA oscillations and induce sustained Hes gene expression over most of the range of Notch signal (Fig. 3E). This also leads to a substantial decrease in the expression of the proneural genes (Fig. 3F). So our modeling of the Notch network revealed a mechanism by which relatively small Fig. 4. Combined effect of Notch and IDs can drive NSC differentiation, reductions in the efficiency of Hes gene auto-repression leads to proliferation and quiescence. (A) Expression level of Hes mRNA for different dramatic changes in proneural gene expression. Furthermore, the levels of ID expression in the presence of constant Notch activity (I=500 modulation of Hes auto-repression predicts a relative transcriptional molecules). (B) Expression level of Hes mRNA relative to different levels of ID profile of Notch, Hes and proneural factor genes that determines expression and Notch activity. (C) Expression level of proneural factor mRNAs NSC differentiation, quiescence and active NSC maintenance. for different values of ID expression in the presence of constant Notch activity. Together, these results suggest that the Hes auto-repression restricts (D) Levels of proneural factor activity (protein level) for different levels of Notch activity and ID expression. IDs potentiate the effect of Notch signaling by the effect of Notch signal on proneural expression, leading to the releasing Hes auto-repression and can act in concert with Notch, forming a maintenance of a minimum level of proneural activity even in three-way switch that segregates NSCs into quiescent (qNSC) (high IDs), the presence of high Notch signaling. Therefore, the efficiency proliferative/active (aNSC) (low IDs, high Notch/Hes) or differentiated (Diff) of the Hes-negative feedback, and not Hes oscillatory expression per (low IDs, low Notch/Hes). The oscillatory region is presented in Fig. S2. DEVELOPMENT

3468 STEM CELLS AND REGENERATION Development (2017) 144, 3465-3474 doi:10.1242/dev.152520 proneural expression leading to NSC quiescence (qNSCs); low/ NSC markers than Id3+Hes−,Id3+Hes+ and Id3−Hes+ NSCs (Fig. 5A,B). intermediate proneural levels in the absence of IDs in cells with Interestingly, Id3+Hes+ and Id3+Hes− NSCs expressed low levels of active Notch signaling, resulting in NSCs being mitotically active active NSC/progenitor markers, while Id3−Hes+ cells had high levels (aNSCs); and high proneural activity in the absence of both Notch of active NSC/progenitor markers. As TAPs are mitotically active and signaling and IDs, which drives NSC into differentiation (Diff) Id3+ NSCs do not express genes associated with an active mitotic state, (Fig. 4D and Fig. S2). This model establishes then clear and sharp these findings indicate that Id3+ cells are mostly quiescent (blue in boundaries in NSC fate that present a feasible explanation for the Fig. 5A,B), whereas most Id3−Hes+ cells also express markers of experimental data linking IDs and Notch signaling to NSC proliferation and TAPs (red in Fig. 5B). We come to similar activation and differentiation. conclusions when we consider the expression of Id2 in place of Id3, or even when considering the expression of all Id genes together Notch and IDs regulate adult neurogenesis in a (Figs S5 and S6), and when we use alternative NSC and proliferation complementary manner markers (Figs S7 and S8). The analysis of these experimental data To test and validate our model and its predictions, we analyzed suggest that IDs are sufficient to drive NSC quiescence, which lends published expression profiles of single cells isolated from the SVZ direct support to our theoretical model indicating that ID expression of adult mouse forebrain that were classified as NSCs (130 cells; and not Notch signaling itself is crucial for NSC quiescence (Fig. 5C) Slc1a3, Nr2e1, Sox9, Vcam1+) and transit amplifying progenitors (Nam and Benezra, 2009). Or, put another way, ID expression (TAPs, 27 cells; Ascl1, Fos, Egr1, Sox4, Sox11+) (Llorens- represses adult NSCs from entering the mitotically active state by Bobadilla et al., 2015). We inferred Notch activity from the blocking proneural gene expression and activity. Mechanistically, our levels of Hes gene family expression in the data set. Of the four ID results suggest that downregulation of IDs by qNSCs releases the genes (Id1, Id2, Id3 and Id4), Id2 and Id3 were expressed in most complete repression of proneural activity induced by sustained high- cells and strongly correlated with NSC marker expression (Figs S3 level Hes expression, and promotes cell cycle entry, at least in part, by and S4). Most NSCs (85 out 130) expressed either Hes or Id3 enabling proneural factor expression. Our data analysis also indicates mRNA, and were clearly divided into three subpopulations: that active NSCs express the Notch ligand Delta (Fig. 5B). Notch- Id3+Hes+ (50 cells), Id3+Hes− (10 cells) and Id3−Hes+ cells (25 Delta signaling leads to lateral inhibition between neighboring cells, cells) (Fig. 5A). segregating them into two populations: receiver cells with high Notch/ We then compared the expression levels of NSC markers (Slc1a3, Hes and low Delta expression; and sender cells with low Notch/Hes Nr2e1, Sox9, Vcam1) (Llorens-Bobadilla et al., 2015) and active and higher Delta expression. Sender cells further differentiate into NSC/progenitor markers (Ascl1, Fos, Egr1, Sox4, Sox11) (Llorens- TAPs while receiver cells remain as active NSCs and can go through Bobadilla et al., 2015) by these progenitor subpopulations. As another round of proliferation (Fig. 5C). According to our model, NSC expected, TAPs (HeslowIDlow) expressed relatively lower levels of the differentiation could be controlled by IDs even in the absence of Notch

Fig. 5. Expression pattern of adult NSCs at the single-cell level and mechanistic interplay between Notch/Hes and IDs. (A) Single cells from adult murine brain are represented based on their levels of Hes (Hes1, Hes5) and Id3 (Llorens-Bobadilla et al., 2015). Color code represents three subpopulations of NSCs: Id3+Hes−, Id3+Hes+ and Id3−Hes+; and transient amplified progenitors (TAP) cells. (B) Expression level of NSC markers (Slc1a3, Nr2e1, Sox9, Vcam1), active NSC/progenitor markers (Ascl1, Fos, Egr1, Sox4, Sox11) and Delta ligands (Dll1, Dll3, Dll4) for different populations of NSCs and TAP cells. NSC markers and active NSC/progenitor markers were chosen based on the analysis of the expression profile of adult NSCs (Llorens-Bobadilla et al., 2015). (C) Schematic representation of adult neurogenesis based on the modeling presented in Fig. 4D and on experimental results presented in B. High levels of IDs drive NSC quiescence. By decreasing IDs, the NSC become proliferative and stimulate the expression of the Notch ligand Delta. Notch-Delta lateral inhibition segregates neighboring active NSCs into high and low Notch signal. While the NSC with low Notch differentiates into a TAP cell, the NSCs with high Notch remain proliferative and can go another round of differentiation. Similar results are found using Id2 instead of Id3, or considering all IDs (Id1-Id4) together, and for an alternative choice of NSC and proliferative markers (Figs S5-8). DEVELOPMENT

3469 STEM CELLS AND REGENERATION Development (2017) 144, 3465-3474 doi:10.1242/dev.152520 signaling. By lowering the levels of IDs in qNSCs, they can become Id1-3. Thus, the predominance of Id4 over Id1-3 during embryonic active/proliferative. A further decrease in the levels of IDs would then neurogenesis is a key difference between embryonic and adult lead to differentiation and ultimately to depletion of the NSC NSCs. This suggests that the expression of Id4 induced by population. This is consistent with experimental evidence showing Notch signaling prevents Id1-3-modulated repression of Hes that inhibition of Notch signaling in the adult niche leads to the loss of autoregulation, thus preventing the sustained high level expression stem cell population and highlights the crucial role of Notch signaling of Hes genes that is required to block proneural gene expression in controlling NSC maintenance and differentiation (Chapouton et al., and drive cell cycle exit. In addition, the inhibitory effect of Id4 on 2010; Imayoshi et al., 2010; Basak et al., 2012; Kawaguchi et al., the other ID proteins also blocks Id1-3-mediated inhibition of the 2013). proliferation. Consistent with this mechanism, neural progenitor cells in Id4 mutant mice show prolonged G1-S transition during brain Differences between ID-Notch regulation of embryonic and development (Yun et al., 2004). Moreover, Id4-mutant neural adult neurogenesis progenitor cells also show precocious differentiation, suggesting that Our modeling results show a feasible mechanism by which Notch Id4 also plays a role in regulating NSC differentiation, likely via and IDs collaborate to regulate neurogenesis in a complementary sequestration of proneural factors (Yun et al., 2004). manner. However, during embryonic stages of brain development, most NSCs are mitotically active although they express IDs (Yun DISCUSSION et al., 2004). Therefore, we addressed the differences in Notch/Hes The regulation of stem cell fate is highly complex. In the nervous and ID interactions in the embryo and adult NSCs, and the reason system, an ever-increasing number of factors that can change NSC why Notch-Hes-ID induce quiescence of NSCs in the adult V-SVZ activity and fate are being uncovered. Signaling pathways but not during embryonic development. We analyzed publically downstream of many of these factors have either been shown to available expression datasets of single embryonic progenitor cells in converge, synergize or even counteract each other. Notch signaling is the ventricular zone of both murine and human brain (Kawaguchi a central regulator of NSC fate and plays key roles in regulating et al., 2008; Pollen et al., 2015). We found that the cells could be maintenance, proliferation and differentiation (Nyfeler et al., 2005; divided into two populations: proliferative NSCs that have high levels Ehm et al., 2010; Imayoshi et al., 2010; Lugert et al., 2010; Basak of both Hes and radial glial markers (embryonic NSCs; Slc1a3, Pax6, et al., 2012). The best known mode of Notch activity is to suppress Sox2, Pdgfd and Gli3), and basal intermediate progenitors that expression of the proneural genes through expression of Hes factors express low levels of these radial glial markers and high levels of (Lutolf et al., 2002). Hence, deletion of the core DNA binding intermediate progenitors markers (Tbr2, Elavl4, Neurog1, Neurod1, component of the Notch pathway, Rbpj, or the effectors Hes1 and Neurod4, Ppp1r17, Penk) (Fig. 6A,C and Fig. S9). We then evaluated Hes5, leads to NSC activation and precocious differentiation (Ehm the expression profile of IDs (Id1-4) in these different cells. We found et al., 2010; Imayoshi et al., 2010; Lugert et al., 2010; Basak et al., that Id2 and Id4 were expressed by a significant fraction of cells and 2012). Similarly, ID proteins control NSC maintenance and that, in contrast to the situation in adult NSCs, Id4 but not Id2 activation in the developing nervous system and control NSC expression overlapped with the expression of Hes genes and radial quiescence and fate in the adult brain (Nam and Benezra, 2009). glia markers (Fig. 6B,D). IDs are downstream components of the TGFβ pathway but share The function of Id4 has been identified as a paradigm shift bHLH transcriptional regulators of the Hes and proneural family as compared with Id1-3 in different tissues during development and in common targets with the Notch pathway (Viñals et al., 2004; Yun cancer (Patel et al., 2015). Id4 but not Id1, Id2 and Id3, is a target of et al., 2004; Bai et al., 2007; Nam and Benezra, 2009). IDs form Notch/Rbpj signaling (Li et al., 2012). Consistent with this, we heterodimers with bHLH factors, which produces inactive complexes observed an overlap in expression between Id4 and Hes genes in with most partners. However, heterodimers of Hes and IDs retain embryonic progenitors (Fig. 6). In addition, it has been suggested that some activity, particularly at the promoters of proneural factor targets Id4 can inhibit the function of other IDs (Sharma et al., 2015). This (Bai et al., 2007). suggests that Notch regulates the expression of both Hes and Id4 in It has been a major challenge to understand how Notch signaling embryonic NSCs, and that Id4 blocks the inhibitory function of controls fate. Until recently Notch signaling was considered to be a

Fig. 6. Hes and IDs expression profile in mouse and human NSCs during embryonic brain development. (A) PCA representation of single cells from the murine embryo (Kawaguchi et al., 2008). Color represents the expression levels of Hes genes (Hes1, Hes5) and radial glia (embryonic NSC) markers (Slc1a3, Pax6, Sox2, Pdgfd, Gli3). (B) Color represents the expression level of Id1-Id4 genes (log2 scale). (C) PCA representation of single cells from the ventricular zone of the human embryo (Pollen et al., 2015). Color represents the expression levels of Hes genes (Hes1, Hes5) and radial glia markers (Slc1a3, Pax6, Sox2, Pdgfd, Gli3). (D) Color represents the expression level of Id1-Id4 genes (log2 scale). DEVELOPMENT

3470 STEM CELLS AND REGENERATION Development (2017) 144, 3465-3474 doi:10.1242/dev.152520 molecular switch, activated by lateral signaling between neighboring form ineffective heterodimers with Hes proteins and reduce the auto- cells. More recently, it was found that, rather than being a switch, repressive feedback on the Hes promoters in qNSCs. This results in Notch signaling is highly dynamic; in addition, in-built oscillatory increased Hes protein expression and complete repression of the expression of the Notch target genes Hes1 and Hes5 is crucial to proneural genes. Hence, although Id2/3 can repress proneural factor Notch function. The dynamic expression of Hes proteins projects onto activity on their target genes, our model indicates that their major role the proneural genes whose expression oscillates out of phase with the in inducing NSC quiescence is to relieve Hes auto-repression. Hes genes. During embryonic neurogenesis, Notch signaling and a In summary, computational modeling has enabled us to reconcile dynamic expression of Hes genes and proneural genes is the different experimental findings and data and provide a solid predominant mechanism regulating NSC maintenance and hypothesis for how Notch and IDs work together to regulate differentiation; both show a salt-and-pepper pattern in NSCs of the neurogenesis in the embryonic and adult brain. It is astonishing that VZ. This dynamic Notch activity coincides with most NSCs being the predictions of our model were supported by gene expression data mitotically active, whereas neurogenic differentiation is blocked by both from mouse and human. Furthermore, our model demonstrates repressing proneural gene expression to low levels and preventing key differences in Notch and ID activity in embryonic and adult accumulation of these neurogenic factors to levels sufficient for NSCs, and provides an explanation for the quiescent state observed in differentiation. The relationship between Hes oscillations and NSC adult NSC. Why adult NSCs are predominantly quiescent although proliferation is intriguing and raises the possibility that NSC fate is they remain sensitive to Notch signaling is a central question in the also based on the dynamic behavior of Hes proteins. Our results adult neurogenesis field. Hence, the use of mathematically modeling suggest that the low proneural factor activity required for NSC and validation by analysis of unbiased sequence data allowed us to proliferation, can be generated independently of Hes oscillations, unravel a complex cross-regulatory network mechanism that has been although oscillations might provide a robust way of keeping proneural difficult to address experimentally. However, as our model now factor levels low. However, we do not exclude the possibility that uncovers the multiple potential modes of action of ID proteins in the Hes oscillations lead to an alternative mechanism independent of regulation of Notch activity and NSC fate, it will be important to proneural factor expression through which NSC fate can be controlled. address these complex Hes-ID interactions and their functions using Here, we show that this dominance of Notch signaling in embryonic cell biological and genetic experiments in vivo. Because IDs can NSCs is due to synergy between Notch signaling and Id4. Id4 potentially modulate any bHLH factor, it is expected that these expression correlates strongly with Hes gene expression (and Notch factors play different and even opposite roles in different systems. activity) in both human and mouse progenitors isolated from the Therefore, understanding the role of ID interactions with other factors embryonic brain. Furthermore, both Hes gene and Id4 expression are will require development of specific theoretical frameworks. This enriched in NSCs rather than TAPs and more committed cells. These will allow novel hypotheses of complex biological processes to be findings parallel data indicating that Id4 is a transcriptional target of developed that can be examined and validated with currently Notch signaling (Li et al., 2012). Therefore, in embryonic NSCs Id4 available data or specifically designed experimental approaches. blocks proneural activity and target gene activation while enabling and supporting Hes auto-repression and maintaining oscillations in MATERIALS AND METHODS Hes gene expression. We propose that this is a key requirement during Theoretical framework embryogenesis, as the dynamics in Notch and proneural activity Our model is inspired by a reductionist approach proposed by Julian Lewis enable a rapid transition of the NSCs to differentiation during brain in 2003 to study the oscillatory dynamics of Hes/her genes (Lewis, 2003). development. In addition, our model proposes that Id2 and Id3, Lewis introduced a simple model that captures many key features of Hes/ although expressed by NSCs and committed progenitors during Her oscillatory dynamics during somitogenesis and served as a theoretical foundation of many other mathematical models that was further developed embryonic development are outcompeted for common partners by to elucidate Hes/Her oscillatory dynamics in different tissues (Lewis, 2003; Id4, preventing the formation of inactive Hes-Id2/3 heterodimers Monk, 2003; Novák and Tyson, 2008; Wang et al., 2011; Pfeuty, 2015). (Sharmaetal.,2015). Therefore, we considered the dynamics of Hes mRNA (m) and Hes protein Conversely, in the adult nervous system, most NSCs are ( p) to be regulated by auto-inhibition with a transcriptional delay (Eqns 1,2). mitotically inactive and, in contrast to embryonic NSCs, Notch As we observe that the expression levels of the Hes genes can reach a signaling does not oscillate and proneural gene expression is absent in maximum of 212 reads per million (RPM) (Fig. 5A). We assumed that one most NSCs, except those that activate and enter cell cycle (Imayoshi cell expresses a total of 0.5 million mRNA molecules and thus a maximum 11 and Kageyama, 2014a). The mechanism that inhibits the oscillatory of 2 (∼2000) Hes transcripts per cell. We used the following model of expression of the Notch effectors Hes1 and Hes5 has been elusive. mRNA regulation: However, it has been demonstrated that high level Notch signaling and maintained expression of Hes1 are required to induce NSC dm ¼ m b; quiescence and this correlates with ID expression (Hirata et al., 2002; dt 0 Nam and Benezra, 2009; Imayoshi et al., 2013). How both processes are regulated and how Notch switches from promoting quiescence to where m0 is the mRNA transcription rate and b is the degradation rate. In the blocking differentiation of aNSCs is unclear (Chapouton et al., 2010; equilibrium dm/dt=0, therefore, m=m0/b. The Hes mRNA half-life has been Imayoshi et al., 2010; Lugert et al., 2010; Basak et al., 2012). We measured to be in the order of 20 min, resulting in b being ∼1/20=0.05 per show computationally for the first time that the synergistic and minute. Hence, from the equation m0=bm, in order to obtain an expression level (m) of 2000 transcripts per cell, the production rate m0 would be antagonistic interactions between Hes factors and IDs are central to ∼ both of these mechanisms during adult neurogenesis. We modeled (m0=0.05×2000) 100 mRNAs per minute. Our choice of an m0=200 mRNA per minute sets the absolute upper limit for Hes mRNA gene computationally different paradigms to test how the oscillatory expression. The effective transcription rate in Eqn 1 depends not only on the expression of Hes could be modulated and found that reducing the transcription rate (m0), but also on the positive regulation of the Notch signal + − auto-repression on the Hes promoter was more effective in stabilizing [H (I)] and negative regulation via the auto-repression [H (p2[t-ti])]. Hes gene expression than changing Hes protein stability or even Because the values of the Hill functions are always <1, this leads to a increasing its expression levels. Our results suggest that Id2 and Id3 decrease in the effective transcript production rate. In the presence of typical DEVELOPMENT

3471 STEM CELLS AND REGENERATION Development (2017) 144, 3465-3474 doi:10.1242/dev.152520 levels of Notch signal and Hes auto-repression, this leads to an effective Fig. S1. transcription rate of a few dozens transcripts per minute. In order to incorporate the effect of IDs, we expanded this model by dma ma ¼ ma0H ð p2 þ epidÞ ð7Þ incorporating the formation of Hes-Hes homo-dimers ( p2) and Hes-ID dt ga hetero-dimers ( p ) (Eqns 3,4): id da a ¼ a0ma kaða þ idÞ ð8Þ dm m dt ga ¼ m H þðIÞHð p ½t t Þ ð1Þ dt 0 2 i g da a m 2 ¼ kaa 2 ð9Þ dt ga dp ¼ ð þ Þ p ð Þ p0m kp p id 2 daid aid dt gp ¼ kaid ð10Þ dt ga dp p 2 ¼ kpp 2 ð3Þ We assumed that the levels of proneural factors, represented by the variables dt g p ma (expression level) and a2 (activity level), controls the fate of the NSCs, irrespective of whether Hes gene dynamics are oscillatory or sustained. dpid pid Whether the oscillatory expression of Hes genes leads to NSC proliferation ¼ kpid ð4Þ dt gp via a mechanism besides small accumulations of proneural factor activity remains to be determined. Here, we assumed that the mean high, low/ where the positive and negative Hill functions are given, respectively, by: intermediate and low/absent levels of proneural factors drive NSC differentiation, proliferation and quiescence, respectively. It should be ð x Þn noted that our results are qualitative than quantitative in nature. H þðxÞ¼ H þðx; x ; nÞ¼ x0 ð5Þ 0 1 þðx Þn We considered the amount of IDs available to interact with Hes and x0 proneural factors to be constant. However, ID gene expression can be highly dynamic and has been shown to oscillate in other tissues (William et al., 1 H ðxÞ¼ H ðx; x0; nÞ¼ ð6Þ 2007). To discuss the dynamics of IDs and their effects on proneural gene 1 þðx Þn x0 expression, we expanded our model by incorporating one extra equation describing the dynamics of the IDs (supplementary Materials and Methods, and the parameters x and n are the Hill factor and Hill coefficient, 0 Figs S11-13). respectively. To consider the release of Hes gene-mediated auto-repression (Fig. 3E,F), We considered that Hes mRNA is produced by a rate m and can be 0 we replaced the negative Hill function in Eqn 1 with a shifted Hill function modulated positively by an external input I (Notch signal), as represented by − HS(x)=H (x)+fH+ (x), where f represents the auto-repression factor or fold a positive Hill function (H+), and negatively by Hes homo-dimer ( p ), as 2 change of repression and f=0 represents a complete repression while f=1 represented by a negative Hill function (H−). Similarly, Hes protein is represent no repressive effect. produced by a translation rate of p . Based on experimental measurements, 0 Parameter values used in the simulations are shown in Table 1 unless we considered that the half-life of Hes mRNA (g ) and Hes protein (g )are m p indicated otherwise. The same Hill factor (h ) was used in all Hill functions. 24 and 22 min, respectively (Hirata et al., 2002). For simplicity we assumed 0 that the degradation rate of Hes monomer, Hes-Hes homo-dimer and Hes-ID Single cell transcriptomics datasets hetero-dimer are the same (gp). We also assumed a high affinity between Hes monomers, and between Hes and ID monomers, represented by the We used recently published datasets in order to validate our model variable k. The maturation of Hes mRNA has been shown to be delayed by predictions. Two datasets were used to evaluate embryonic NSCs. The first 19 min due to intronic processing (Harima et al., 2014). For simplicity, we dataset consists of cells extracted from the ventricular zone (VZ) and subventricular zone (SVZ) of human embryos at gestation week 16-18 considered that this is the only delay involved in the process (ti=19 min), although extra delays are expected due to transport of mRNA from the (GW16-18) (Llorens-Bobadilla et al., 2015). By selecting only the cells nucleus to cytoplasm, protein production and dimer formation. Analytical from the VZ and with more than 1 million reads, we analyzed 179 cells. The studies have shown that the oscillatory behavior of Hes genes is highly second dataset consists of cells from the embryonic murine brain at E14.5 dependent on the intronic delay and Hill coefficient (cooperativity (Kawaguchi et al., 2008; Pollen et al., 2015). We also evaluated the profile coefficient) (Bernard et al., 2006). The delay and Hill coefficient have of single cells from the adult murine brain between 8 and 12 weeks of age contrasting effects on Hes dynamics: longer delays require lower levels of (Llorens-Bobadilla et al., 2015). We selected all the cells annotated as NSCs cooperativity in order to maintain oscillations (Bernard et al., 2006). (total of 130 cells) and as TAP (total of 27 cells). Cells from the mice with Therefore, by considering only the intronic delay, a high cooperativity was ischemia were not used. required to obtain oscillations (n=5). Similar dynamics were obtained by Expression levels are represented by the number of reads per million considering a delay t =25 min and Hill coefficient equal to n=4 (Fig. S10). (RPM) in the log2 scale: expression level=log2(RPM+1). Radial glia, NSCs i and active NSCs markers were chosen based on the markers suggested by the The translation rate ( p0) was chosen so that protein values were in a biological range and oscillations maintained in the order of 2-3 h. original manuscript that introduces the database (Llorens-Bobadilla et al., We also took into account the dynamics of a target bHLH gene a, which 2015; Pollen et al., 2015). All analysis can be reproduced by following the represents a proneural gene such as Ascl1. We considered that this gene is activated at a rate of ma0 and degraded a rate of ga. Experimental evidence Table 1. Parameter values used in the simulations suggests that IDs can release Hes gene-mediated auto-repression via N- Parameter Value Unit boxes, but cannot release Hes gene-mediated repression on proneural genes via class-C sites (Bai et al., 2007). Therefore, we considered that this gene is gm, gp, ga 24, 22, 50 min t repressed by both Hes-Hes homo-dimers ( p2) and Hes-ID hetero-dimers i 19 min h (Hill factor) 1000 molecules ( pid), in contrast to Hes genes, which are repressed only by Hes-Hes homo- 0 dimers. We also assumed that Hes-ID heterodimers are less efficient than m0, ma0 200, 2.0 molecules/min Hes-Hes homodimers, where ε represents the relative strength of repression p0, a0 2.0, 0.1 molecules/(min*mRNA) of Hes-ID when compared with Hes-Hes. We assumed that ε=0.5 (twice the id, I 0, 500 molecules Ε concentration of Hes-ID is required to have the same repressive effect as 0.5 K 0.2 1/min Hes-Hes). A sensitivity analysis evaluating the effect of each parameter of nI, n, na (Hill coefficient) 1, 5, 2 Hes circuit on the levels of both Hes and proneural factors is presented in DEVELOPMENT

3472 STEM CELLS AND REGENERATION Development (2017) 144, 3465-3474 doi:10.1242/dev.152520 tutorial source code available on GitHub: http://github.com/mboareto/ Doe, C. Q. (2008). Neural stem cells: balancing self-renewal with differentiation. InterplayNotchID_neurogenesis. Development 135, 1575-1587. Doetsch, F. (2003). The glial identity of neural stem cells. Nat. Neurosci. 6, 1127-1134. Acknowledgements Doetsch, F., Caillé, I., Lim, D. A., Garcıa-Verdugo,́ J. M. and Alvarez-Buylla, A. The authors thank members of Taylor lab for comments. M.B. thanks T. A. Amor for (1999). Subventricular zone astrocytes are neural stem cells in the adult helping with the figures. mammalian brain. Cell 97, 703-716. Ehm, O., Goritz, C., Covic, M., Schaffner, I., Schwarz, T. J., Karaca, E., Competing interests Kempkes, B., Kremmer, E., Pfrieger, F. W., Espinosa, L. et al. (2010). The authors declare no competing or financial interests. RBPJkappa-dependent signaling is essential for long-term maintenance of neural stem cells in the adult hippocampus. J. Neurosci. 30, 13794-13807. Author contributions Encinas, J. M., Michurina, T. V., Peunova, N., Park, J.-H., Tordo, J., Peterson, D. A., Fishell, G., Koulakov, A. and Enikolopov, G. (2011). Division-coupled Conceptualization: M.B., D.I., V.T.; Methodology: M.B., D.I.; Validation: V.T.; Formal astrocytic differentiation and age-related depletion of neural stem cells in the adult analysis: M.B., D.I., V.T.; Investigation: M.B., D.I.; Data curation: M.B.; Writing - hippocampus. Cell Stem Cell 8, 566-579. original draft: M.B., D.I., V.T.; Writing - review & editing: M.B., D.I., V.T.; Supervision: Ernst, A., Alkass, K., Bernard, S., Salehpour, M., Perl, S., Tisdale, J., Possnert, D.I., V.T.; Project administration: D.I., V.T.; Funding acquisition: D.I., V.T. G., Druid, H. and Frisén, J. (2014). Neurogenesis in the striatum of the adult human brain. Cell 156, 1072-1083. Funding Flunkert, V. and Schoell, E. (2009). Pydelay - a python tool for solving delay This work was supported by the SystemX.ch of the Schweizerischer Nationalfonds differential equations. bioRxiv http://arxiv.org/abs/0911.1633. zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Fuentealba, L. C., Rompani, S. B., Parraguez, J. I., Obernier, K., Romero, R., Foundation) and the NeuroStemX project to V.T. and D.I.; by the Schweizerischer Cepko, C. L. and Alvarez-Buylla, A. (2015). Embryonic origin of postnatal neural Nationalfonds zur Förderung der Wissenschaftlichen Forschung (310030_143767 stem cells. Cell 161, 1644-1655. to V.T.); and by Universität Basel (V.T.). Deposited in PMC for immediate release. Furutachi, S., Matsumoto, A., Nakayama, K. I. and Gotoh, Y. (2013). p57 controls adult neural stem cell quiescence and modulates the pace of lifelong Data availability neurogenesis. EMBO J. 32, 970-981. The data used in this manuscript are all publicly available in GEO under GSE67833 Furutachi, S., Miya, H., Watanabe, T., Kawai, H., Yamasaki, N., Harada, Y., Imayoshi, I., Nelson, M., Nakayama, K. I., Hirabayashi, Y. et al. (2015). Slowly and GSE10881 and in the database of Genotypes and Phenotypes (dbGaP) under dividing neural progenitors are an embryonic origin of adult neural stem cells. Nat. phs000989.v1.p1 (Kawaguchi et al., 2008; Llorens-Bobadilla et al., 2015; Pollen Neurosci. 18, 657-665. et al., 2015). Giachino, C., Basak, O., Lugert, S., Knuckles, P., Obernier, K., Fiorelli, R., The simulations for the delayed differential equations were evaluated in Python Frank, S., Raineteau, O., Alvarez-Buylla, A. and Taylor, V. (2014). Molecular using Pydelay version 0.1.1 (Flunkert and Schoell, 2009 preprint). All source codes diversity subdivides the adult forebrain neural stem cell population. 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