Gene Id4 appears to be a key component in controlling stem cell proliferation

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Scientists from Basel have investigated the activity of stem cells in the brain of mice and discovered a key mechanism that controls cell proliferation.

According to the researchers, the gene regulator Id4 controls whether stem cells remain in a state of rest or enter cell division.

The results were published in Cell Reports and may be relevant for treating neurodegenerative disease in human brains.

Whether stem cells also occur in the human brain has long been controversial.

Today, it is considered certain that the brain can form new neurons throughout life.

The stem cells that have been found to be behind this process are restricted to specialized regions in the brain, so-called niches, which provide key signals that regulate stem cell self-renewal and differentiation.

With increasing age, however, the stem cells become increasingly inactive and divide less frequently. They transition into a “quiescent” or dormant state.

Hyperactive signaling pathway inhibits cell division

So far, it was unclear why stem cells in the adult and aged brain fall into a state of rest. A research team led by Prof. Verdon Taylor from the Department of Biomedicine at the University of Basel has now discovered which factors block entry of stem cells into cell division.

They were investigating the so-called Notch signaling pathway in more detail, a pathway central for regulating stem cell activity in the brain.

The study shows that the Notch2 signaling pathway controls the expression of a specific transcription regulator called Id4.

Once expressed, Id4 inhibits the division of stem cells and blocks the production of new neurons in the hippocampus of the adult brain.

Notch2 signaling maintains high levels of Id4 in some neural stem cells, and thereby explains why these stem cells increasingly enter a state of rest in the adult and geriatric brain.

This shows Id4 expression in hippocampal neurons

The image depicts Id4 (blue) and GFAP (black) expression in genetically labeled stem cells and their progeny (magenta) in the hippocampus of the mouse brain.

The image presents an artistic coloration of the spatial and molecular factors in a niche. The image is credited to University of Basel.

As the brain ages, the Notch2-Id4 pathway enters into a state of hyperactivity, presenting a strong molecular brake that inhibits stem cell activation and neuron production.

Conversely, inactivation of this pathway releases the brake and enables the production of new neurons – even in the brain of geriatric mice.

Reversible resting state

The results show that the stem cells in the mammalian brain are in a reversible resting state regulated by signals and factors in the niche.

By manipulating the signaling pathway, the production of new nerve cells can be specifically stimulated.

The study provides important information on the basic mechanisms of neurogenesis in the adult mouse brain.

Since the Notch signaling pathway is widespread and occurs in most organisms, the researchers hope that the findings can also be transferred to humans. In this way, brain damage caused by degenerative and neuropsychiatric diseases could be repaired in the future.


Neurogenesis is the production of neurons from neural stem cells (NSCs).

The correct balance between NSC proliferation and differentiation is essential for embryonic formation of the brain and to confer regenerative capacities in the adult brain (Doe, 2008).

Any deviation from the regulated neurogenic program can lead to drastic problems during development, including microcephaly and cognitive impairment.

During embryonic development of the central nervous system, NSCs divide frequently and produce neurons either directly or via a committed intermediate progenitor (IP) cell (Fig. 1A).

In the peak neurogenic period, a few NSCs exit the cell cycle and become quiescent (qNSCs) (Furutachi et al., 20132015Fuentealba et al., 2015). qNSCs are only reactivated in the adult neurogenic niches. In the adult brain, NSCs remain and neurogenesis is active in two defined regions: the ventricular-subventricular zone (V-SVZ) of the lateral ventricle wall; and the dentate gyrus of the hippocampus (Doetsch et al., 1999Doetsch, 2003Spalding et al., 2013Ernst et al., 2014Fuentealba et al., 2015Furutachi et al., 2015).

In the adult brain the majority of the NSCs are mitotically inactive (qNSC) and infrequently enter cell cycle, becoming active NSCs (aNSCs) to generate neurons before returning to quiescence or differentiating into glial cells (Fig. 1A) (Lois et al., 1996Kirschenbaum et al., 1999Encinas et al., 2011Ihrie and Alvarez-Buylla, 2011Shook et al., 2012Giachino et al., 2014). T

hus, although embryonic and adult neurogenesis share some similarities, there are also fundamental differences and stem cell quiescence is one of them.

Currently, it is not known why NSCs of the adult brain remain quiescent and the mechanisms that control the transition of NSC to activation are also unclear.

However, the balance between activity and quiescence is crucial not only to maintain the NSC pool for later neuron production and regeneration but also to prevent overproliferation and tumor formation (Lugert et al., 2010Silva-Vargas et al., 2016).

Thus, understanding the molecular mechanism that regulates maintenance and differentiation of NSCs is not only of theoretical interest but crucial for understanding disease mechanism and developing new therapeutic strategies (Lie et al., 2004Lazarov et al., 2010).

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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.

The processes of NSC maintenance and differentiation are controlled by a core regulatory network of basic helix-loop-helix (bHLH) transcription factors (Lee, 1997Ross et al., 2003Heng and Guillemot, 2013Imayoshi and Kageyama, 2014b). Members of the bHLH family have two conserved functional domains: a basic region for DNA binding and a helix-loop-helix (HLH) region for dimerization.

These transcript factors can act as repressors or activators of gene expression. The hairy and enhancer of split (Hes) proteins Hes1 and Hes5 are central repressors of NSC differentiation during brain development (Ohtsuka et al., 1999Kageyama et al., 20072008), while bHLH factors including Ascl1 and Neurog2 are activators of neural differentiation and thus referred to as proneural factors (Wilkinson et al., 2013Imayoshi and Kageyama, 2014b). 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 binding to E-box consensus sequences in the promoters of their targets (Fig. 1B) (Imayoshi and Kageyama, 2014b). Furthermore, the binding affinity of proneural factors to E-boxes can be enhanced by the formation of heterodimers with other members of the bHLH family: the E-proteins Tcf4 and Tcf3 (Massari and Murre, 2000Bohrer et al., 2015).

During brain development, Notch signaling activates Hes gene expression, which in turn inhibits NSC differentiation by repressing proneural genes, including Ascl1 and Neurog2 (Lee, 1997Heng and Guillemot, 2013). In addition, Hes proteins repress expression of their own genes, counteracting Notch and leading to oscillations in their expression (Fig. 1B) (Hirata et al., 2002).

This dynamic Hes gene activity has been suggested to result in low-level expression of proneural factors, including Ascl1, and this low expression drives cell cycle progression but is not sufficient to induce NSC differentiation (Castro et al., 2011Imayoshi et al., 2013Andersen et al., 2014).

In contrast, high and sustained expression of Hes proteins drives complete repression of Ascl1, leading to cell cycle exit and NSC entry into a quiescent state (Baek et al., 2006Castro et al., 2011Imayoshi et al., 2013Andersen et al., 2014). NSCs in the adult brain niches are predominantly quiescent, a state not observed frequently in the developing brain.

How regulation of the Hes-proneural gene axis is differentially controlled in NSCs during development and in the adult brain is unknown.

However, previous observations suggest that different levels of proneural activity in the NSCs lead to three possible output states in the NSCs: NSC quiescence, proliferation and differentiation when proneural activity is absent, intermediate/low and high, respectively (Fig. 1A).

In the adult brain, NSCs quiescence has been linked to the expression of inhibitor of DNA-binding factors (IDs) (Nam and Benezra, 2009).

IDs also have a HLH domain, which enables the formation of heterodimers with other bHLH factors, but lack the basic domain and for this reason cannot efficiently bind to DNA (Tzeng, 2003Heng and Guillemot, 2013).

Therefore, IDs act as inhibitors of the activity of bHLH factors. Experimentally, IDs have been shown to form dimers with Hes proteins and these heterodimers are unable to bind to the N-box-binding motifs in the Hes promoter and thus relieve Hes auto-repression (Bai et al., 2007). Interestingly, Hes-ID heterodimers can still repress target genes, including Ascl1, via class-C binding sites, albeit with lower efficiency than Hes homodimers (Bai et al., 2007).

Thus, IDs are able to segregate auto-repressive and downstream target gene repression functions of Hes factors. In addition, IDs also form ineffective heterodimers with proneural factors, including Ascl1, reducing their potential to drive differentiation by blocking their binding to E-boxes in target genes (Imayoshi and Kageyama, 2014b). Hence, IDs potentially regulate neurogenesis at multiple levels, including enhancing Hes expression and blockage of proneural factor activity (Fig. 1C).

Owing to the complex and reciprocal interplay between Notch-Hes and IDs, it has been challenging to access the consequences of their interactions experimentally and their respective roles in the control of NSC activity.

As a first step to address this problem, we developed a specific theoretical framework that takes into account the interactions between Notch, IDs and the members of the bHLH family of transcriptional factors. Our theoretical framework is in line with previous models of Hes (Lewis, 2003Monk, 2003Novák and Tyson, 2008Wang et al., 2011Pfeuty, 2015) and explicitly incorporates Notch-mediated activation of Hes gene expression, Hes-mediated repression of proneural expression, Hes auto-repression and homodimer formation. In order to recapitulate the different effects of IDs, we incorporated the possibility of Hes-ID and proneural-ID heterodimer formation into the model.

We explored computationally the properties of this gene regulatory network and the conditions required to obtain NSC quiescence, maintenance of activated NSCs and differentiation. Once we had established a robust model that fulfilled these criteria, we challenged and validated our predictions by analyzing the gene expression of NSCs at the single-cell level. Finally, by evaluating the differences in the single-cell expression profile of adult and embryonic NSCs, we uncovered key differences between embryonic and adult neurogenesis.


Source:
University of Basel
Media Contacts: 
Verdon Taylor – University of Basel
Image Source:
The image is credited to University of Basel.

Original Research: Open access
“Id4 Downstream of Notch2 Maintains Neural Stem Cell Quiescence in the Adult Hippocampus”.Verdon Taylor et al.
Cell Reports. doi:10.1016/j.celrep.2019.07.014

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