Introduction

During vertebrate neurogenesis, the spatiotemporal generation of the precise number and type of cells with neuronal and glial lineage involves the orchestration of various regulatory events at cellular and molecular levels [1–4]. This includes harmony between multiple cues such as growth factors, signal transduction pathways, transcription factors, and epigenetic events along with other environmental factors. However, neural stem/progenitor cells that reside in multiple regions of the developing brain respond differentially both intrinsically and extrinsically and give rise to different neuronal and glial cell types [5, 6]. Neural stem/progenitor cells generated from the neuroepithelial cells in the primary germinal zones of embryonic brain undergoes limited number of proliferative symmetric divisions for their self-renewal and enters differentiation phase through asymmetric division [7–10]. This will generate multipotent neural progenitors with a distinct molecular profile in germinal centers and is well regulated by multiple cues. Notch signaling, one of the most characterized, evolutionarily conserved developmental pathways, plays a pivotal role in the maintenance, proliferation, and differentiation of neural stem/progenitor cells [11–14]. Though the primary role of this pathway is to regulate the maintenance and proliferation of neural stem/progenitor cells through its downstream target genes such as Hes factors, it controls the neuronal differentiation through its cell–cell interactive lateral inhibition [15, 16]. Hence, extensive expansion of radial glial cells and its neurogenic/gliogenic switch is mechanistically controlled by Notch signaling in spite of its ability to imprint the intrinsic molecular profile at the cellular level through its multiple downstream targets such as Hes genes, BLBP etc. [17–19].

Among the Hes genes (Hes1-7), Hes1, in particular, attains prime significance because of its pleiotropic nature and context-dependent regulation at multiple levels. The indispensable role of Hes1 in regulating the unidirectional prevailing path of neuronal differentiation is well evidenced by precocious neuronal differentiation and hypoplasia of the brain as observed in Hes1-null mice [20–22]. Though other Hes factors (Hes3/5) can compensate for Hes1 expression in Hes1-null mice, being a potent repressor of various proneural genes and cell cycle regulators, Hes1 acts as a key regulator of neurogenesis [23, 24]. Hes1 expression is essential for maintenance and proliferation of neural stem/progenitor cells and thereby conferring controlled competence for differentiation [22, 25]. In neural stem/progenitor cells, well-tuned oscillatory expression of Hes1 and its repressive role over the differentiation genes make it a master gene during neurogenesis [26–29]. Apart from its role in progenitor maintenance, it has been evidenced that Hes1 is crucial for boundary formation, astroglial differentiation, and even in neuronal differentiation. Moreover, Hes1 acts as a molecular convergent point for various signaling pathways and this cross talk results in implementation of very specific function in a context-dependent manner during the highly complicated process of neurogenesis.

In this review, we discuss how the master gene, Hes1, regulates the process of neurogenesis in a context-dependent manner. The elusive task of Hes1 in maintaining progenitor compartment with cellular diversity at germinal centers and tissue morphogenesis rely on its response towards multiple regulatory cues that might be different at the molecular, cellular, and/or regional level. Though, Hes1 is a classical Notch target gene, its expression is fine-tuned by all possible processes of gene regulation, which includes either transcriptional/epigenetic or post-transcriptional/translational regulatory mechanisms. Since various regulatory events that mediate Hes1 expression and its repressive role has been extensively described in other cellular and physiological processes, in this review, we mainly focus on the immense significance drawn by this maestro in view of its regulation and functional implications during neurogenesis.

Hes1: functional implications during neurogenesis

Neural stem/progenitor cell maintenance and proliferation

Among the Hes genes, Hes1, in particular, maintains the neural stem/progenitor cells in an undifferentiated and proliferative mode in germinal zones of central nervous system [25, 30]. It has been shown that Hes1 carries out these functions in retinal progenitors [31, 32], cortical progenitors in VZ of telencephalon [22, 33], midbrain dopaminergic progenitors [34], DRG progenitors [35] and CGNs [36]. The bHLH repressor, Hes1 accomplished its pivotal role in the germinal zone of CNS because of its repressive ability and antineurogenic activity over differentiation genes [33]. Precocious neuronal differentiation accompanied with up-regulation of its counterparts such as proneural genes (Ascl1, Ath5, Neurog2 etc.) and cell cycle regulators (p27) in Hes1-null mice revealed its immense importance in maintenance and proliferation of neural progenitors [20–22, 26]. However, depletion of neural progenitors along with premature neuronal differentiation was severe in Hes1; Hes3; Hes5 triple KO mice compared to Hes1-null mice [20, 30]. Though other Hes factors can compensate Hes1 to an extent, differential Hes1 expression, unlike Notch target gene Hes5, and its extensive regulatory mechanisms in neural progenitors deserve prime significance during neurogenesis [23, 30]. Moreover, misexpression of Hes1 maintains neural stem/progenitor cells in an undifferentiated state for a prolonged time period implied its primary functional implications as the neural stem cell/progenitor maintenance factor [22, 37]. Congruently, the inability of neurosphere formation from cortical progenitors derived from Hes1 −/− ; Hes5 −/− mice even in the presence of active Notch indicated the essential role delivered by Hes1 in neural progenitor maintenance [23]. Moreover, neural stem/progenitor cells maintained in an adult hippocampus also require Hes1. Intriguingly, recent reports demonstrated that the enhancement of the stemness and proliferation of human neural stem cells (hNSCs) could be achieved through co-culturing with human bone marrow-derived mesenchymal stromal stem cells (hMSCs). Though this finding augmented the essential role of mesodermal guidance in fate determination, at the molecular level, Hes1 upon Notch activation played an inevitable role to execute this via contact-based hMSC-hNSC interaction [38].

Neuronal differentiation/neuroprotection

Though Hes1 is a well-known potent repressor of proneural genes and precludes neuronal differentiation [26, 27], very few recent reports have unraveled its unexpected role in neuronal differentiation and neuroprotection. It has been demonstrated that Camk2δ-mediated Hes1 phosphorylation upon PDGF treatment is required for Ascl1 activation at the onset of neurogenesis despite its repressive role on pro-neural genes at progenitor state [39]. Induction of Ascl1 expression by covalently modified Hes1 during early phase of neuronal differentiation in cortical cultures supports its role in neurogenic function. Apart from this initial induction of neurogenesis, recent reports have highlighted the role of Hes1 in synaptic plasticity in differentiated rat cortical neuronal culture [40]. Here, Notch-independent, but JNK-mediated Hes1 phosphorylation in differentiated neurons prevents calcium influx evoked by AMPA receptor through suppression of one of its subunit, mGluR1. Moreover, Hes1 also plays an important role in pro-differentiation events such as dendritogenesis in cultured hippocampal pyramidal neurons. Dendritic outgrowth in differentiating neurons is implemented by NGF-mediated NF-κB-dependent sustained expression of Hes1 that makes the preference over the induction of dendritogenesis because of proneural genes such as Ascl1 and Ngn3 [41, 42]. Intriguingly, the same NGF/TGFβ1-NF-κB signaling cascade is indispensable for the neuroprotective role conferred by Hes1 in neurodegenerative Alzheimer’s disease (AD) [43]. This non-canonical Hes1 expression is responsible for alleviating the Aβ-induced neurotoxicity and is concomitant with the survival of hippocampal neurons, increased GABAergic synaptic terminals and dendrite patterning. Moreover, at the molecular level, the neuroprotective role of Osthole (7-methoxy-8-isopentenoxycoumarin) is well implemented through Hes1, thereby enhancing hippocampal neurogenesis and rescues the cognitive impairment in APP/PS1 Tg mice [44].

Boundary formation

Compartmentalization of the central nervous system by boundary cells in different boundary regions is required for distinct cellular territory and proper structural organization [45, 46]. This boundary region serves as an organizing center for neuronal specification by forming a distinct compartment with a unique molecular profile. It has been shown that Hes1 is expressed in various boundary regions such as the zona limitans intrathalamica (ZLI), a boundary between the thalamus and prethalamus, the isthmus at the midbrain–hindbrain boundary, the interrhombomeric boundaries, and the roof/floor plate in the spinal cord [24]. Unlike the differential Hes1 expression in non-boundary regions, its persistent expression in slow-dividing and non-neurogenic boundary cells are concurrent with the coherent and prevailing role of Hes1 in progenitors. Sustained expression of Hes1 in those regions enhances constitutive repression over proneural genes (Ascl1, Ath1, etc.) and maintains cells in a low proliferative or quiescent mode rather than differentiating. Disruption of boundary regions and ectopic expression of proneural regions in those areas of Hes mutants unraveled the pivotal role of Hes1 in boundary formation and subsequent compartmentalization during the development of the CNS [24]. For instance, it has been shown that Hes1 along with Hes3 is required for the maintenance of isthmic cells in an isthmic organizer [47]. Premature neuronal differentiation of isthmic cells and absence of the midbrain and anterior hindbrain region in double mutant mice indicated the significance of these functionally redundant genes in isthmic organizer activity. Though isthmic organizer activity is not much affected in single mutants, attenuation of isthmic organizer activity in Hes1; Hes3 double mutant mice is revealed by the curtailed expression of Fgf8, Wnt1, and Pax2/5, which is essential for its function [47]. Moreover, normal expression of these factors in Hes3, Hes5 double mutants compared to the severe reduction in Hes1, Hes3, Hes5 triple-null mice indicated an impairment in maintenance of isthmic cells in those boundary regions [24]. Thus, this report suggests that Hes1 plays an important role in neural pattering coupled with well-timed differentiation of boundary cells.

Gliogenesis

Though the primary function of Hes1 is maintenance and proliferation of neural stem/progenitor cells [26], its role in gliogenesis depends on region and developmental stage. In developing telencephalon, Hes1 is not able to promote gliogenesis, rather it maintains neural progenitors or radial glial cells as described earlier [22]. Both the misexpression and knockout studies resulted in the delayed neurogenesis and premature neuronal differentiation, respectively, without affecting gliogenesis in telencephalic development [20, 22]. Conversely, the role of Hes1 in gliogenesis is well documented in retina [48] and PNS [49]. In retina, Hes1 enhances the genesis and development of Müller glia, the only type of glia present in the retina, suggesting its role in gliogenesis [48]. Similarly, in the developing ventral spinal cord, Hes1 promotes astrogenesis by induction of glial precursors along with Id factors, and this promotion also depends on the down regulation of pattering genes such as Pax6 [49]. Moreover, it has been reported that Hes1 induced by Notch1 promotes induction of glial restricted progenitors (GRP), but not neuroepithelial progenitors (NEP), to an astrocyte fate at the expense of oligodendrocyte fate [50]. This result is also consistent with promotion of adult hippocampus-derived progenitors to an astrocyte fate by Hes1 induced by Notch1 and Notch3 [51]. However, any deregulation of this Notch–Hes1 axis results in the progression of glioma, the most common malignant tumors in adult brains [52]. Thus, the functional role of Hes1 in promoting gliogenesis always depends on several other factors such as stage or time, region, molecular profile, and competency of the cell.

Hes1: the ripened repressor of neurogenesis

Generation of the precise number of neural stem/progenitor cells and its temporal differentiation is required for structural integrity of the nervous system [1, 5, 6]. More restricted and well-controlled expression pattern of Hes1 in the neural stem/progenitor cells allows it to deliver very defined functions such as its maintenance and proliferation [26]. This primary role of Hes1 in neural progenitor territory undoubtedly depends on its well-known repressive effect over genes responsible for cell cycle arrest, neuronal induction, and differentiation. Among the seven members of Hes factors (Hes1–7) [53], Hes1 has the most repressive role because of its functional domains that prove inevitable for its action.

Molecular mechanism of repression

The murine Hes1 gene is structurally organized with four exons and intervening introns along with upstream regulatory elements on chromosome 16 (Fig. 1a) [54]. The Hes1 protein, which showed a highly conserved structural relationship with Drosophila hairy and Enhancer of split [E(spl)] basic helix-loop-helix (bHLH) proteins, have distinct domains for its functional implications (Fig. 1b) [55]. This 282-amino-acid-long protein with conserved domains and unique functional significance makes this maestro able to attain multiple outputs, including anti-neurogenic activity, inhibition of differentiation, antero-posterior segmentation, and sex determination etc. [33, 56, 57]. The bHLH domain at the amino terminal region confers its DNA binding ability at the N box (CACNAG) of its target gene’s regulatory sequences [15]. However, Hes1 also shows strong interaction with class C sites (CACGCG), probably with different binding affinity to mediate its repression [58–60]. The invariant proline residues present at the basic region enhances the DNA binding ability of Hes1 protein. The HLH region serves as a dimerization domain and for protein interaction. The orange domain located next to the bHLH domain at the carboxy terminal is functionally required for its protein–protein interaction to recruit its transcriptional co-repressors. The WRPW domain present at its carboxy terminal translates the amino acid sequence Trp–Arg–Pro–Trp and this peptide motif interacts with the co-repressor, TLE/Groucho, thereby mediating its repressive effect [19, 57, 61, 62]. Recruitment of these TLE/Groucho co-repressors further strengthens the direct repression implemented by Hes1 through its homodimerization or heterodimerization with Hey genes (Fig. 1c, d) [54, 63–65]. Apart from the direct repression by binding at N/C-boxes of target genes, Hes1 forms transcriptionally inactive heterodimers with E proteins in order to preclude its interaction with its binding partners, especially with bHLH activators such as Ascl1 (Fig. 1e) [53]. This kind of active and passive repression either through homodimerization or heterodimerization indeed helps Hes1 to deliver its pleiotropic function during neural progenitor propensity. However, the repression by Hes1 in this context is relieved by various factors such as Id proteins [66, 67] and Hes6 (Fig. 1f) [68, 69]. These proteins can directly interact with Hes1 and aid in either neural stem cell maintenance through sustained Hes1 expression by relieving only the negative feedback auto inhibition [67] or neuronal differentiation by antagonizing Hes1 [68, 69].

Fig. 1
figure 1

Genetic structural organization of Hes1 and its mode of repressive action. a Schematic showing the Hes1 gene with promoter, UTRs, exons, and introns. b Schematic of Hes1 protein with its domains and corresponding functions. c, d Schematic of direct binding of Hes1 after homodimerization and heterodimerization at target gene promoters along with co-repressors such as TLE/Groucho. e Schematic of passive repression mediated by Hes1 by forming transcriptionally inactive heterodimers. f Schematic showing the relieving of repression implemented by Hes1 either through interaction with Id proteins or Hes6

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Major Hes1 target genes in neurogenesis

The repressor type bHLH gene, Hes1, and its well-known repressive role over proneural genes maintain the neural progenitors either in quiescent or proliferative mode in all the germinal centers of the brain (Table 1). For instance, most evolutionarily conserved repressive roles of Hes1 were experimentally demonstrated in the case of proneural gene, Ascl1 [25, 55]. It has been shown that Hes1 is able to repress Ascl1 in both ways, i.e., either through direct transcriptional repression or through protein–protein interaction [33]. Hes1-induced active repression of Ascl1 is delivered by direct binding at C box in Ascl1 promoter while the passive repression involves the formation of non-functional heterodimer with E2A/E47, an inducer of E-box-dependent transcription, which is very much required during the heterodimer formation with bHLH activator Ascl1 (Fig. 1c, e) [53, 60, 70]. Hence, the direct DNA binding property and indirect inhibition due to sequestration of co-activators through protein–protein interaction mediated by Hes1 implemented its anti-neurogenic activity in neural stem/progenitor cells [55]. Likewise, the expression dynamics of another proneural gene neurogenin-2 (Neurog2) and the Notch ligand Dll1 at dorsal telencephalon is regulated by Hes1, thereby leading to a well-controlled switch from progenitor compartment to differentiation mode [27]. Here, oscillatory versus sustained expression of Neurog2 and Dll1 in neural progenitors of VZ territory in developing neocortex depends on the well-regulated Hes1 oscillation [71].

Table 1 Targets of Hes1 associated with neurogenesis
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Intriguingly, Hes1 binds to its own promoter at putative N-box and represses its own expression [25, 55]. Periodic expression of Hes1 in neural progenitors is partly dependent on auto-negative feedback regulation by direct binding to its own promoter [54]. The highly unstable mRNA and protein of Hes1 make them to oscillate with simultaneous direct repression over its own expression and proneural gene expression in neural progenitors [65, 71]. Moreover, microarray analysis in telencephalic neural progenitors revealed that more than two-fold repression of 40 target genes was achieved by sustained expression of Hes1 [71]. Both quantitative and qualitative analysis indicated Hes1 induced inhibition of Notch ligands (Dll1, Jag1), proneural genes (Ascl1, Neurod4, and Neurog2) and cell cycle regulators (cyclin E2/D1) in neural progenitors. In the developing neocortex, double-negative feedback regulations implemented by mir-9 in Hes1 oscillation partly depend on the repressive role of Hes1 over mir-9 at the transcriptional level [72, 73]. Here, Hes1 maintains the oscillation of relatively unstable pre-mir-9 in neural progenitors through its periodical repression.

Hes1 also regulates the expression of atonal homologue Ath5 that is essential for RGC generation and its subsequent differentiation and optic nerve development during retinogenesis [74]. Though Ath5 along with Hes1 is differentially expressed in RGC precursors during early retinogenesis, unlike Hes1, Ath5 promotes RGC-specific genes later [75]. It has been shown that Hes1 is able to strongly repress Ath5 at the transcriptional level inside the progenitors, but this down-regulation is partly dependent on Neurog2, which is a potent activator of Ath5 during the differentiation stage [76, 77]. This interplay between the bHLH factors in RGC specification is congruent with coherent oscillatory expression seen in cortical neural progenitors [71]. Another example of repressive activity of Hes1 is observed with Serotonin (5-HT) receptor. 5-HT1A is negatively regulated by Hes1 at promoter level by its direct binding at putative N-box [78]. Though another bHLH repressor Hes5 is known to down-regulate 5-HT1A [79], its expression pattern culminates in midbrain and hindbrain regions of Hes1 −/− mice and is not associated with precocious neurogenesis in those regions. This shows that the compensatory increase in Hes5 expression in Hes1 −/− mice in those regions is unable to curtail 5-HT1A receptor expression. These results strongly imply the repressive role of Hes1 during early development of serotonergic raphe cells [78]. However, Prdm (PRDI-BF1 and RIZ homology domain containing), a mammalian homologue of Drosophila Hamlet, is differentially regulated by Hes1 in developing telencephalon [80]. Co-expression of Prdm16 and complementary expression of Prdm8 with Hes1 in the telencephalon surmised its differential regulatory role over Prdm proteins during CNS development. Inverse expression pattern of both Prdm8 in post-mitotic neurons and Prdm16 in neural progenitors in Hes1-null mice also corroborated with these results. However, being a repressor, Hes1 mediated up-regulation of Prdm16 in neural progenitors might not be direct but possibly through its repressive effect over the repressors of Prdm16 [80]. It has been shown that Hes1 regulates the cell cycle progression through its DNA-binding-dependent transcriptional repressive function. Repression of cell cycle regulator, cyclin-dependent kinase (CDK) inhibitor, p21cip1/WAF−1 by Hes1 involves its helix-loop-helix or orange domain-dependent direct binding at the p21 promoter [81]. Inhibition of Hes1 is concomitant with the predominant expression of p21 in terminally differentiated neuronal cells. Hes1-mediated cell cycle progression is also evidenced by the repression of another cyclin-dependent kinase (CDK) inhibitor, p27Kip1 [82]. Though its functional significance in neural progenitor maintenance/proliferation through its regulation over cell cycle progression or neuronal differentiation is well characterized, reports from our lab point towards its role in neurotransmitter/subtype fate specification. The homeodomain transcription factor Tlx3 (HOX11L2) expression begins to wane in ES cell-derived neural progenitors upon Hes1 over-expression favoring preclusion of glutamatergic fate over GABAergic output. Hes1 mediates this effect by suppressing the Tlx3 expression, which leads to the subsequent increase in GABAergic fate [83]. Furthermore, active and passive mode of repression by Hes1 was well documented in differentiated neurons of rat cortical culture [40]. Here, Hes1 repressed GluR1 subunit of AMPA receptor, which plays an important role in synaptic plasticity, either by direct binding to the N-box of GluR1 promoter or by sequestering Ascl1/E47 complex from binding to the E-box. Moreover, it has been shown that repression of GluR1 at the transcriptional level by Hes1 is enhanced by protein stabilization through JNK1 phosphorylation at Ser-263 [40].

Hes1: the rhythmic composer of neurogenesis

The oscillatory behavior of Hes1 was initially well documented during somite segmentation during embryogenesis with a 2 h cycle [84, 85]. Later, serum-induced stimulation of oscillatory expression of Hes1 was experimentally revealed in cultured fibroblast with almost the same time interval [65]. Highly unstable Hes1 protein and mRNA with half-lives of 22.3 ± 3.1 and 24.1 ± 1.7 min, respectively [65], attributed to its auto-negative feedback regulation. However, this ultradian expression pattern and feedback regulation opened up the plausible mechanisms for its oscillatory expression. The negative feedback regulation exerted at the transcriptional level by bHLH repressor protein, Hes1 on its own promoter made the first insight into the molecular mechanism behind the oscillatory nature of this classical Notch target gene [54]. It has been shown that the auto-regulation of Hes1 expression is well associated with the ubiquitin-mediated proteasome degradation of Hes1 protein that in turn relieves the repression on the promoter and results in mRNA synthesis [65]. This result implied that synergistic regulation both at the transcriptional and proteomic level is an inevitable mechanism for maintaining oscillation of Hes1 expression (Fig. 2a).

Fig. 2
figure 2

Molecular mechanisms behind the oscillation of Hes1 in neural progenitors. a Schematic showing the integrative molecular mechanism behind Hes1 expression and its negative auto feedback regulation, which in turn is regulated by its proteasome degradation and post-transcriptional regulation by mir-9. b Schematic showing dynamic oscillatory expression of Hes1, Neurog2, Dll1, and mir-9 in neural progenitors during the course of neurogenesis

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Significance of Hes1 oscillation

The ultradian oscillation of Hes1 expression makes the neural stem cells undergo proper maintenance, proliferation, and differentiation during neurogenesis [71] while the sustained expression of Hes1 in roof plate and floor plate is critical for the quiescent state of neural stem cells or boundary formation [24]. Using a real-time imaging method, it was documented that Hes1 expression oscillates with an average of 2–3 h cycles in neural progenitors along with Notch ligand, Dll1, and proneural gene Neurog2 [71, 86]. Functionally, Hes1 oscillation is very much required for the maintenance of the progenitor pool along with out-of-phase oscillation of Dll1 and Neurog2. Sustained expression of Neurog2 may cause persistent expression of Dll1 in differentiating neurons that make them oscillate out of phase with Hes1 as development progresses. This observation is also consistent with Dll1 up-regulation by Neurog2 at the transcriptional level [87]. Thus, oscillatory expression of Hes1, Neurog2, and Dll1 is also attributed to the inverse correlation between the expression patterns in neural progenitors (Fig. 2b). However, sustained Hes1 expression is required for the repression of genes involved in cell-cycle progression [81, 88] and mediates the maintenance and proliferation of neural stem cells [24]. It has also been found that the 3–5 h periodical expression pattern of Hes1 in embryonic stem cells made them competent for differential output by controlling the Notch signaling pathway [89]. Analysis of the differentiation pattern of cells having differential expression of Hes1 revealed that cells with low Hes1 predominantly prefer a neuroectodermal fate, whereas cells with high Hes1 follow a mesodermal fate. Moreover, this oscillatory expression of Hes1 in ES cells leads to the differential expression of Notch ligand, Dll1, and Gadd45g, a gene required for cell-cycle progression [71, 86, 90].

Pathways involved in oscillation

Abolishment of Hes1 oscillation was observed in neural progenitors in the presence of γ-secretase inhibitor (DAPT), which implied that Notch signaling has a very prominent role in dynamic expression of Hes1 [71]. Interestingly, oscillation of Hes1 in ES cells is not regulated by Notch signaling, but is mediated by BMP and LIF signaling [90]. In addition to the Notch signaling pathway, experiments with inhibitors of Jak2-Stat3 signaling pathways in neural progenitors resulted in the repression of oscillation as with DAPT treatment, indicating that Jak2-Stat3 signaling also regulates Hes1 oscillation [71]. Earlier, it has been shown that serum stimulation in fibroblasts leads to the phosphorylation of Stat3 by Jak that in turn induced the expression of Socs3 [91]. However, Socs3 is able to antagonize the phosphorylation of Stat3 by Jak. This negative feedback regulation subsequently maintains the oscillation between the phosphorylated Stat3 (p-Stat3) and Socs3. This oscillation is required for the Hes1 oscillation to happen, and any disturbance leads to its termination and shift from oscillatory to sustained expression [91]. Moreover, it has recently been shown that metabolic intermediates such as reactive oxygen species (ROS) can modulate Hes1 expression and its oscillation via intracellular calcium signaling [92]. Overall, oscillatory Hes1 expression is necessary for the neural progenitor maintenance and proliferation, and is well regulated at multiple levels by signaling pathways such as Notch [71], BMP, LIF [90], and Jak-Stat [91], at the transcriptional level, by its own repression and at proteomic level [54], by the ubiquitin-mediated proteasome degradation [65].

Termination of oscillation

The negative feedback regulation of Hes1 causes an equilibrium that in turn makes it to oscillate with a precise time interval. Alteration of this molecular regulation either at the mRNA or protein level is very much required for the dynamic expression pattern with functional significance in cellular level for its progression from quiescent or maintenance state to differentiation mode. Hence, dampening of Hes1 oscillation should be employed permanently to push the neural progenitors from a maintenance/proliferative stage to a differentiation stage. The pioneering work by Bonev et al. demonstrated that post-transcriptional regulation by non-coding RNA, mir-9 terminates the oscillation of Hes1 at the mRNA level, thereby inducing the differentiation process during neocortical development [72, 93]. Mechanistically, the double-negative feedback loop formed between Hes1 and mir-9 wherein Hes1 mRNA stability is negatively regulated by mature mir-9 in one way at the post-transcriptional level while mir-9 transcription is abolished by repressive Hes1 at transcriptional level (Fig. 2a). This molecular mechanism is also well correlated with the inverse expression profile of these molecules since Hes1 is abundant in neural progenitors and boundary cells [24, 94]. It is also observed that mir-9 exhibits complementary expression as in differentiating neurons where Hes1 is down-regulated [95, 96]. Interestingly, oscillation of Hes1 and pre-mir-9 in neural progenitors leads to the accumulation of highly stable mature mir-9 over time, which eventually dampen the translation of Hes1 mRNA to protein and leads to the cell-cycle exit and differentiation process (Fig. 2b) [72, 73, 93]. Hence, the fine-tuning of cyclical and dynamic oscillatory behavior of Hes1 makes it a rhythmic composer in neural progenitors and synchronizes its maintenance, proliferation, and differentiation during neocortical development.

Hes1: the Notch target gene, but not always

Notch-dependent Hes1 expression

Hes1 expression in neural progenitors is primarily regulated by evolutionarily conserved Notch signaling pathway transduced through cell–cell interaction [17, 23, 25]. This juxtacrine signaling pathway constitutes highly conserved transmembrane Notch receptors (Notch1-4) that interact with either of their ligands (Delta1-3/Jagged1-2) present in the neighboring cell to implement its versatility in various developmental programs [11–13, 18, 97]. Even though the ligand–receptor interaction is specific with respect to its functional significance, molecular mechanisms behind this juxtacrine signaling pathway remain more or less the same (Fig. 3a) [14, 98]. Ligand-induced activation of Notch receptor leads to proteolytic cleavage at its extracellular and intracellular domains by an ADAM family protease, TACE, and presenilin–secretase complex, respectively [99]. The cleaved Notch intracellular domain (NICD) is translocated into the nucleus and interacts with RBP-Jκ/CBF-1, the mammalian homologue of Drosophila, Suppressor of Hairless [Su(H)]. Interaction of NICD with RBP-Jκ/CBF-1 leads to the conversion of RBP-Jκ/CBF-1 from a transcriptional repressor to activator complex, which in turn induces Hes gene expression [100]. The Notch signaling pathway as an upstream activator of Hes1 has been well evidenced by its diminished expression and associated phenotype in Notch knockouts [101, 102] and in neural progenitors treated with γ-secretase inhibitors (DAPT, GSI, etc.) [103]. Premature neuronal differentiation accompanied by a decrease in the neural progenitor pool resembles the knockout of its downstream target, Hes genes [20, 30, 104].

Fig. 3
figure 3

Differential mode of Hes1 expression through canonical and non-canonical pathways. Schematic showing Hes1 activation by canonical Notch signaling (a) and other pathways/factors in a Notch-independent manner (b)

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Notch-independent Hes1 expression

Even though Hes1 is one of the immediate downstream effectors of the Notch signaling pathway [15, 23], recent reports highlight differential Hes1 expression that can be regulated through either of these dependent or independent Notch/CBF1 interactions (Fig. 3b) [105–107]. This kind of non-canonical Hes1 expression was reported in hematopoietic progenitors [108], T/B cell precursors [109], human endothelial cells [110], and cancer cells [111, 112]. Since this review mainly focuses on the regulation and functional implications of Hes1 associated with neurogenesis, we have emphasized those aspects. This kind of differential Hes1 expression was described in human neuroblastoma cells [113], neocortical progenitors [114, 115], hippocampal neurons [41–43], and retinal progenitors [116–118] along with our previous reports that clearly demonstrated FGF2-JNK-ATF2-Hes1 axis in ES-cell-derived neural progenitors [103].

The context-dependent differential expressions of Notch target genes that own diverse upstream modulators were categorized based upon the requirement of either NICD or CBF1. This differential activation can be type I (NICD dependent and CBF1 independent), type II (NICD independent and CBF1 independent) or type III (NICD independent and CBF1 dependent) [105]. Notch/CBF1-independent (type II) non-canonical activation of Hes1 was induced by Shh in retinal progenitors [118]. Differential expression of Hes1 selectively enhances the maintenance and proliferation of retinal progenitors and confers a Müller glial and bipolar fate at the expense of rod photoreceptors. The reduction in Müller glia and bipolar cells upon inhibition of Shh/Gli pathway also supports the significance of non-canonical activation of Hes1 in those progenitors [118]. Moreover, the growth factors such as VEGF [117] and CNTF [116] have been shown to mediate differential Hes1 expression during retinal progenitor proliferation and fate specification. In addition to this, differential Hes1 expression was reported during mid-neurogenesis both in granule neurons of cerebellum [36] and neocortex [115]. Though Notch and Shh co-operate to culminate at Hes1 expression in developing neocortex, Notch/CBF1-independent (type II) but Shh-dependent Hes1 expression is present only in a small population of the progenitor pool in neocortex [115]. It has also been shown that Hes1 expression in neocortical progenitors depends on FGF-FRS2α-Erk-AP1 axis, but independent of Notch/CBF1 [114]. Moreover, in developing murine neocortex, discrepancy between Hes1 expression and Notch pathway components in early neuroepithelial development from the ectoderm also suggested Notch/CBF1-independent Hes1 expression [102]. Also, NGF [41, 42] or TGFβ1-mediated [43] sustained expression of Hes1 in a non-canonical manner was reported in hippocampal pyramidal neurons. This differential Hes1 expression through NF-κB is functionally attributed to either neuroprotection [41, 43] or dendritogenesis [42]. Correspondingly, transactivation of Hes1 is reported in human endothelial cells by JNK [110]. Our report has also shown that FGF2 can activate JNK through the Cdc42-Ras pathway and the activated JNK resulted in the phosphorylation of its downstream effector ATF2 [103]. In turn, binding of phospho-ATF2 on Hes1 promoter triggered Hes1 expression in a Notch/CBF1-independent manner. This molecular mechanism that confers the non-canonical Hes1 expression in a Notch/CBF1-independent manner functionally attains relevant significance in the maintenance or proliferation of a subset of ES cell-derived neural progenitors. Similarly, type II activation has been well documented in differential expression of Hes1 in PC12 cells upon induction with secreted signaling factor Wnt1 [119]. Wnt1 induces the transcriptional activation of Hes1 through the Wnt1 responsive elements in the Hes1 promoter in a Notch/CBF1-independent manner. CBF1-dependent, but Notch-independent (type III) Hes1 activation in human neuroblastoma cells (SK-N-BE(2)c) was triggered by TGFα through EGF receptor/Ras [113]. Additionally, SK-N-BE(2)c cells are maintained by Hes1 activation, which happens through the MEK-ERK pathway in the absence of externally administrated TGFα. This signal transduction phosphorylates ERK1/2 and the activated phospho-ERK induces the expression of Hes1 in those cells [113].

Hes1: the instrumental gene with multiple regulatory keys

Though Hes1 is the classical Notch target gene, its expression is finely modulated in a context-dependent manner as we discussed earlier. Spatiotemporal expression of Hes1 at the cellular or regional level definitely deserves this kind of fine-tuning for the proper orchestration of cell genesis and differentiation. The regulatory keys for distinct translation of its functional knot involve all the manifestations associated with gene regulation. This involves the transactivation or repression by diverse upstream or downstream factors such as growth factors/mitogens, epigenetic marks, transcriptional factors, miRNAs, and other factors such as pharmacological cues and environmental factors (Table 2).

Table 2 Regulators of Hes1
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Growth factors/mitogens

Even though there is no direct evidence at the transcriptional level for the molecular mechanisms, several reports highlight the involvement of various growth factors or cytokines behind the Hes1 expression during neuronal development. Fibroblast growth factor (FGF), one of the most extensively characterized mitogens in various systems [120–123], is reported to regulate Hes1 expression primarily for neural stem/progenitor cell maintenance and proliferation [103]. Dependence of Hes1 expression on FGF was well depicted in midbrain [124] and cortical neural progenitors [114]. Abrogation of Hes1 expression in VZ of developing midbrain in Fgfr1cko;Fgfr2cko mutants along with the simultaneous up-regulation of neurogenic Hes1 targets such as Dll1 and Neurog2 demonstrated the role of FGF signaling in activation of Hes1 [124, 125]. Moreover, the requirement of FGF signaling for Hes1 expression is well demonstrated by FGF-Frs2α-ERK pathway in developing neocortex wherein Notch/CBF1-independent (type II) Hes1 expression is required for self-renewal and proliferation of neural stem cells or progenitors [114]. Both in vitro and in vivo experiments with GOF and LOF analysis of FRS2α clearly emphasize the involvement of FGF signaling in Hes1 expression during neocortical development.

Notch/CBF1-independent Hes1 expression by FGF2-JNK-ATF2 signaling cascade was reported from our lab in ES cell-derived neural progenitors and this differential Hes1 expression functionally attributed to the maintenance and proliferation of a subset of neural progenitors [103]. Also, activated Hes1 expression responsible for the cell cycle progression, which in turn makes the oligodendrocyte progenitor pool in proliferative mode, was regulated by the Mek-Erk pathway and PDGF+FGF2 treatment [126]. This growth factor-induced Hes1 activation maintains the progenitors and prevents early maturation or differentiation of oligodendrocytes. Hence, the cumulative effect of FGF2 along with PDGF on Hes1 expression leads to the maintenance of lineage-restricted progenitors [126]. Intriguingly, PDGF treatment in cortical progenitors leads to the post translation modification of Hes1, which in turn promotes the neuronal differentiation by activating Ascl1, an unexpected role of this bHLH repressor [39].

In response to various growth factors, differential Hes1 expression and its divergent role in cell fate determination were well documented during early retinal neurogenesis. Elevated Hes1 expression through the VEGF-FLK1-MEK-ERK pathway in retinal progenitors partially contributed to proliferation during early retinogenesis [117]. Intriguingly, Notch/CBF1-independent (type II) VEGF-induced Hes1 expression suppresses retinal ganglion cell (RGC) fate without the requirement of the MEK-ERK pathway. Hence, in response to extrinsic factors such as VEGF, Hes1 expression is modulated in a non-canonical way by opting divergent upstream signaling cascade inside retinal progenitors [117]. Hence, the divergent intracellular transduction machineries implemented by VEGF ends up in a converging point Hes1, which in turn regulate the balance between the number of retinal progenitors and RGC fate specification. Evidence for growth factor/cytokine-mediated Hes1 activation in retinal progenitors is extended towards the role of ciliary neurotrophic factor (CNTF) in proliferation of retinal progenitors in explant culture [116]. Here, CNTF induces signal transducer and activator of transcription factors (STATs) such as STAT3, which in turn lead to the persistent expression of Hes1 in outer nuclear layer of the retina.

Interestingly, neurotrophin-mediated dendritic growth in hippocampal pyramidal neurons is regulated by sustained expression of Hes1 [41, 42]. This sustained Hes1 expression is facilitated by the neurotrophin NGF, which binds to the neurotrophin receptor p75NTR, and this interaction leads to the accumulation of NF-κB inside the cell, which in turn up-regulates Hes1. Any perturbation of this signaling cascade is reflected in the initiation of dendritogenesis rather than the dendritic growth. Indeed, initiation of dendritogenesis is well regulated by proneural genes Ascl1 and Ngn3, but their expression pattern is inversely proportional to the NGF-mediated NF-κB-dependent Hes1 expression. Moreover, these genes, responsible for the induction of dendrites in the neurons, are prime candidates for the bHLH repressor Hes1 [53]. Hence, the pro-differentiation event such as dendritogenesis is well regulated by Notch target gene Hes1 in neural progenitors where they suppress Ascl1 and Ngn3, thereby preventing stimulation of dendritic outgrowth while sustained expression of Hes1 in a non-canonical way by NGF promotes the dendritic length in cultured hippocampal neurons [42]. However, the expression pattern of p75NTR, Hes1, Notch, and its ligands (Delta-1 and Jagged-1) in CA3 pyramidal neurons of the hippocampus indicated the interplay between these two signaling pathways and their convergence at Hes1 unraveled its unexpected role in dendritogenesis [42]. Apart from dendritogenesis, the neuroprotective role of Hes1 also stands in line with the NF-κB-dependent Hes1 activation by NGF [41]. In neurodegenerative diseases such as Alzheimer’s disease (AD), amyloid beta (Aβ) competes with NGF to bind to p75NTR [127, 128], which ultimately impairs the NF-κB-dependent Hes1 expression. Dampening of this signaling cascade subsequently translated in amyloid beta (Aβ) neurotoxicity and defects in the survival of hippocampal neurons, dendritic patterning, and GABAergic input. Alternatively, another growth factor, transforming growth factor β1 (TGFβ1) was able to promote Hes1 expression in the same way as NGF did through NF-κB [43]. This non-canonical Hes1 expression by TGFβ1 counteracted the effects of amyloid beta (Aβ) and confers the resistance to Aβ neurotoxicity along with enhancement in cell survival, increased GABAergic synaptic terminals and dendrite patterning [43]. Also, Hes1 activation in a Notch-independent manner was reported by another but closely related mitogen, TGFα, in human neuroblastoma cells through MAP kinase ERK pathway [113].

Epigenetic factors

The deciding cues for epigenetic regulation always rely on various intrinsic or extrinsic factors, which confer to the identity and environmental niche where the cell exists. Epigenetic regulation of Hes1 mostly lies in its regulatory regions located upstream of the coding region. This primarily involves chromatin remodeling events and the transactivation/repression by downstream effectors of various signaling pathways. Hes1 expression is modulated by either of these regulatory events, which are executed alone or simultaneously. The chromatin remodeling by folic acid on Hes1 promoter was well described in neural stem cell proliferation and neural tube development [129], dorsal root ganglion cell proliferation [35], and hippocampal neurogenesis [130]. It has been shown that folic acid activates DNA methyltransferase (DNMT), which in turn cause the hypomethylation of H3K27 in Hes1 promoter. In ND7 cell line derived from dorsal root ganglion (DRG) cells, increased H3K9 and H3K19 acetylation and decreased H3K27 methylation by folic acid at Hes1 promoter promotes cell proliferation while sensory neuron differentiation requires the same epigenetic marks at Neurog2 promoter and association of Neurog2 with NeuroD1 promoter [35]. Though folate does not support sensory neuron differentiation, it promotes DRG cell proliferation by inducing epigenetic changes at Hes1 promoter, which is indispensable for maintenance and proliferation. Correspondingly, increased expression of Hes1 and associated adult hippocampal neurogenesis by folic acid in the rat model of cerebral ischemia supports the importance of fine-tuning of gene expression at the epigenetic level [130]. Moreover, enhancement of Notch signaling cascade and Hes1 expression upon treatment with well-known HDAC inhibitor, valproic acid in human neuroblastoma cells stands in line with the previous reports.

Moreover, premature sensory neurogenesis in paired box gene transcription factor, Pax3 mutant Splotch (Sp/Sp) embryo is associated with down-regulation of Hes1 expression [129]. Both chromatin immunoprecipitation assays and luciferase assay confirmed that Pax3 can bind directly to cis-regulatory elements within Hes1 promoter and regulation of Hes1 expression maintains neural crest stem cells in an undifferentiated state [131]. However, these plausible molecular mechanisms that happen during caudal neural tube development also depend on post-translational modifications of Pax3 and epigenetic marks on Hes1 promoter [132]. Acetylation of Pax3 at lysine residue, K437, and subsequent binding at Hes1 promoter leads to its down-regulation while the recruitment of histone deacetylase, SIRT1 facilitates Pax3 deacetylation thereby exerting the reverse effect as increased Hes1 expression, which in turn enhances neural crest stem cell maintenance and proliferation. Hence, the coupling of post-translational modification of a transcription factor (Pax3 acetylation) [132] and associated epigenetic changes regulate the master gene, Hes1 during caudal neural tube development by implementing the switch from stem cell proliferation to differentiation.

Transcription factors

Among all the regulatory factors, transcription factors deserve the most diversity in modulating gene expression [133–136]. The distinct molecular profile of each cell type generated during neurogenesis depends on the spatio-temporal expression of various transcription factors [2, 3, 70, 137]. Though these nuclear proteins are transcribed in response to particular upstream pathways or interaction among them, activation or repression of its downstream targets specifies the molecular identity of a cell. Despite the fact that Hes1 is the direct target of Notch, its expression is modulated by diverse transcription factors independently or in concert with Notch. Being the maestro in neurogenesis, Hes1 expression is executed in a well-defined manner by these choirs of nuclear proteins during neurogenesis.

Ubiquitously expressed nuclear protein, RBP-Jκ/CBF1 is bound to Hes1 promoter along with other co-repressors, thereby shutting down its expression [16, 138, 139]. However, interaction of NICD with RBP-Jκ/CBF1 results in the conversion of the transcriptional repressor to activator complex that in turn drives the Hes1 expression [16, 100]. Moreover, the downstream effectors of other signaling pathways such as Shh, Wnt, and JNK are also reported to bind at its putative consensus binding site and activate Hes1 expression in a Notch/CBF1-dependent or -independent manner [103, 118, 140]. The maintenance and proliferation of retinal progenitors and its Müller glial or bipolar fate specification is mediated by differential Hes1 expression through Shh pathway and its downstream effector, Gli2. It has been demonstrated that Gli2 can bind to Hes1 promoter and enhance its expression in retinal progenitors [118]. This could be the possible molecular mechanism behind the differential Hes1 expression by Shh in cortical [115] and cerebellar neural progenitors as well [36]. Similarly, FGF2-induced JNK phosphorylates its downstream effector, ATF2, which in turn binds to the Hes1 promoter, thereby driving its expression in ES cell-derived neural progenitors [103]. Moreover, it has been shown that the signaling cross talk among FGF2, Wnt, and Notch signaling enhances the Hes1 expression in neural progenitors [140]. Activation of phosphatidylinositol 3 kinase (PI3K) by FGF2 induces the nuclear accumulation of β-catenin, which interacts with NICD and enhances Hes1 expression in a Notch/CBF1-dependent manner. Recently, it has been shown that Hes1 expression in neocortical progenitors requires LIM-homeodomain transcription factor Lhx2 [141]. Though Lhx2 is well known as a cortical organizer in determining the fate choice between hem and neocortex [142, 143], depletion of neural progenitors accompanied by precocious neuronal differentiation in Nestin:Cre Lhx2 cKO mouse is primarily due to the down-regulation of Hes1. Even though Lhx2 regulates multiple factors for fate specification in cortical neurogenesis, its regulatory role over Hes1 is well evidenced by cortical hypoplasia, increase in cell cycle exit, and abundant Neurog2 expression, as seen in Hes1-null mice [20, 102]. Diminished Hes1 expression in Lhx2 cKO (but not Hes5) indicated differential expression of Hes1 by Lhx2 [141]. Moreover, its regulatory role over Hes1 is also well documented during retinal gliogenesis [144]. Intriguingly, regulation of Hes1 by classical axonal guidance receptor Robo1/2 was documented in neural progenitors of developing neocortex [145]. Reduction in apical progenitors at VZ and the high number of intermediate neural progenitors at SVZ in double mutants of both Robo1 and Robo2 or Slit1 and Slit2 also point towards its regulatory role over Hes1, which is essential for apical neural progenitor maintenance and proliferation. During the early phase of neurogenesis, the Slit/Robo signaling pathway modulates Hes1 expression at the transcriptional level either independently or synergistic action with Notch in progenitor cells. Though transcriptional activity of Robo1/2 depends on its specific cytoplasmic domains [146, 147], the exact molecular mechanism remains to be elucidated. Up-regulation of Hes1 by a homeobox gene, rax, was reported in retinal progenitors [48]. Though there is no evidence for the direct binding of rax in Hes1 promoter, both the reporter assay (CAT) and the presence of putative rax binding sites in the upstream regulatory regions of Hes1 strengthen the notion that rax-mediated transcriptional activation of Hes1 is crucial for Müller glial development in retina [48].

Though different transcription factors act as activators of Hes1, accumulating evidence points toward its repressive role over this well-known bHLH repressor. Here, these nuclear proteins aid the transition from progenitor state to differentiation by suppressing the master gene, Hes1. Intriguingly, negative feedback regulation of Hes1 by its own repression is crucial for its rhythmic oscillatory expression in neural progenitors [54]. Periodic binding of Hes1 at its own promoter coupled with its proteomic degradation makes it oscillate both at the mRNA and protein level [65, 71]. Hes6, another Hes gene functionally well known for its role in neuronal differentiation, exerts its repressive effect on anti-neurogenic activity of Hes1 in neural progenitors [68, 69]. Multiple molecular mechanisms imparted by Hes6 for its neurogenic activity primarily lies on its precluding passion over repressive Hes1. Hes6 promotes the proteolytic degradation of Hes1 and prevents its interaction with transcriptional co-repressors, Groucho and Transducin-like Enhancer of split (TLE), which in turn ultimately leads to suppression of Hes1-mediated repression of proneural genes [69]. The pleiotropic nature of the Notch signaling pathway during neocortical development is implemented through the differential activation of its target genes [13, 14, 148]. Similar to Hes1, another Notch target gene nuclear factor IA (NFIA) is very essential for neocortical and spinal cord development [149, 150]. However, the interplay between these two target genes regulates the maintenance and proliferation of neural progenitors and subsequent neurogenic or gliogenic switch in telencephalic development. Recently, it was demonstrated that NF1A is able to curtail Hes1 expression both in dorsal telencephalon and hippocampus [151]. NFIA occupancy at highly conserved putative sequences in Hes1 promoter and its abundant expression in neural progenitor territory of both hippocampus and telencephalon in NFIA mutant unravel the repressive effect of NFIA over Hes1 [151]. Delay in astrocyte development in NFIA mutant revealed that NFIA-mediated Hes1 repression is required for gliogenic switch from the progenitor compartment in developing neocortex. Recently, it has been demonstrated that antineurogenic activity of Hes1 in midbrain dopaminergic progenitors (mDA) is abolished by LIM homeodomain transcription factors, Lmx1a and Lmx1b, thereby promoting its differentiation [34]. Even though the molecular mechanism behind this repression is unknown in midbrain dopaminergic progenitors, up-regulation of Hes1 in Lmx1a/b double mutant (Lmx1a dr/dr ;Shh Cre/+ ;Lmx1b f/f ) surmised the repressive role of these determinants of mDA neuron development. These results are also concomitant with the up-regulation of Hes1 targets such as Neurog2 and cell cycle regulator p27Kip1in mDA development [34]. It has been surmised that Runt-related (Runx) transcription factor, Runx1, negatively regulates Hes1 expression in DRG cells [152]. Both the complementary expression of Runx1 and Hes1 in DRG cells and increase in the number of Hes1+ve cells in Runx1 −/− E12.5 embryos proposed that Runx1 may down-regulate Hes1 expression there by controlling the neuronal differentiation of DRG cells during the early embryonic period. Similarly, human homolog of Yeast Eco1/Ctf7, Esco2-mediated Hes1 down-regulation in C17.2 neural progenitor cells regulates neuronal differentiation by inhibiting the transactivation of NICD on Hes1 promoter [153]. Here, sequestering of NICD by Esco2 precludes its interaction with CBF1 to drive Hes1 expression. Thus, Esco2 down-regulates Hes1 expression by antagonizing Notch signaling in neural progenitors [153]. It has also been shown that one of the closely related Hey factors, HeyL, induced by BMP4 in neural progenitors, is able to down-regulate Hes1 [154]. Even though the repression by HeyL on Hes1 promoter is weak, it induces the neuronal differentiation by activating the Hes1 counterpart Neurog2.

Post-transcriptional modification

Being a master regulator of neurogenesis, post-transcriptional regulation of Hes1 by small non-coding RNAs was postulated because of its inevitable role in brain development [93, 155–158]. The hypotrophy of neocortex in Dicer knockouts also revealed the same [159]. However, the classical work from Bonev et al. unraveled the fine-tuning of neuronal differentiation regulated by highly conserved micro RNA, mir-9 [72], one of the most abundant micro RNAs present in CNS having pro-differentiation function [94, 160, 161]. The mutually exclusive and complementary expression pattern of mir-9 and Hes1 in a spatio-temporal manner in developing CNS highlighted the possible interaction, which may happen for neuronal or brain development [95]. As discussed before, the oscillatory expression of Hes1 is well controlled mechanistically by mir-9 in neural progenitors [72, 73, 93]. The double-negative feedback loop coupled with Hes1 and pre-mir-9 resulted in the accumulation of more stable mir-9 within neural progenitors. From these observations, it appears that mir-9 controls the expression efficiency and stability of Hes1 mRNA in neural progenitors [72]. As the neural progenitors exit the cell cycle, highly expressed mir-9 dampens the Hes1 oscillation, which leads the cell to differentiate into a neuron. This kind of post-transcriptional regulation at Hes1 mRNA levels by mir-9 causes the termination of its oscillation and provides the plausible explanation that how a neural progenitor having oscillatory expression of Hes1 adopts a neuronal fate in a well-controlled manner. Another non-coding RNA, mir-124, is reported in regulating Hes1 in P19 cells [162]. Abundant accumulation of Hes1 upon LNA-mediated inhibition of mir-124 resulted in the hindrance of retinoic acid-induced neuronal differentiation of P19 cells. Since mir-124 is one of the abundantly expressed miRNAs in CNS and well known as a micromanager of neurogenesis [163], this could be also involved in Hes1 oscillation, as described above with mir-9. Apart from these post-transcriptional regulations during neurogenesis, it has also been shown that anchorage-independent growth and proliferation rate of medulloblastoma cells were reduced because of impairment in cancer stem cell (CD133+) population, which in turn mediated by the down-regulation of Notch effector gene, Hes1 by mir-199b-5p [164]. This tight regulation of Hes1 at the post-transcriptional level by miRNA in medulloblastomas also resembles the phenotype shown by Hes1-null mice. mir-524–5p, a brain-specific tumor suppressor microRNA, has been shown to suppress Hes1 in glioma. This regulation is well evidenced by their complementary expression pattern both in normal and ectopic expression studies, thereby mediating the proliferation and invasion of glioma [165]. Moreover, identification of this kind of fine-tuning of master gene Hes1 by miRNA widen the plausible mechanisms, which might help in raising the molecular markers for diagnosis or treatment at the clinical level for diseases such as malignant brain tumor and medulloblastoma.

Post-translational modification

A post-translational modification such as phosphorylation [166] and ubiquitination [167, 168] events has been proven to be crucial in fine-tuning of gene regulation. Being a very dynamic protein, Hes1 also owns specific arms for various kinases to be modified covalently either for stabilization or to confer particular functions during neurogenesis [57]. Although few reports indicated its functional role during neural development in vivo, very few coherent studies in in vitro models emphasize the importance of Hes1 phosphorylation in neural development. Anti-neurogenic repression exerted by Hes1 along with TLE1-mediated repression complex having PARP-1 is well known [169] and is indispensable for neural progenitor maintenance and proliferation. However, it has been well demonstrated that PDGF-induced neuronal differentiation resulted in de-repression over proneural gene, Ascl1 [39]. The molecular mechanism behind the de-repression involves the activation of calmodulin kinases delta isoform, Camk2δ by PDGF. Camk2δ simultaneously activate PARP-1, which removes TLE1 co-repressor complex and phosphorylation of Hes1 at specific sites thereby allowing Hes1 to promote neurogenesis through co-activator recruitment [39]. This post-translational modification of Hes1 has proven to be indispensable for Ascl1 activation that is known to positively regulate neuronal differentiation. This kind of multiple regulatory event on Hes1 such as Notch signaling for repression of proneural genes at the progenitor state, and PDGF induced Camk2δ followed by the covalent modification for de-repression of proneural genes at the onset of neuronal differentiation makes it a prime candidate for normal development. Moreover, a member of the MAPK family, c-Jun N-terminal kinase (JNK), has been shown to stabilize Hes1 protein by phosphorylation at Ser-263 in rat cortical culture [40]. Here, both JNK1 and JNK2 phosphorylate Hes1 at Ser-263, which culminate at the repression mediated by Hes1 in GluR1 expression at the transcriptional level. Hence, the down-regulation of AMPA receptor subunit, mGluR1, by JNK-mediated Hes1 phosphorylation subsequently alleviates the calcium influx in differentiated neurons, thereby highlighting the involvement of Hes1 in synaptic plasticity [40]. Conversely, it has been reported that phosphorylation of specific serine residues at DNA binding basic region of Hes1 by Protein kinase C (PKC) attenuates its antineurogenic activity in PC12 cells [63]. Nerve growth factor (NGF) mediated induction of Protein kinase C phosphorylate Hes1, and this precludes its binding at proneural target genes. This post-translational modification of Hes1 provides the possible mechanism behind the enhancement of neurite outgrowth in PC12 cells upon NGF treatment [63]. However, these results indicated the importance of post-translational modification of Hes1 for fine-tuning of neural development at least in in vitro system.

Ubiquitination and deubiquitination processes are another post-translational modification that plays a pivotal role in protein stability and function [167, 168]. The ultradian oscillation of Hes1 expression [71] partially relies on negative auto-feedback regulation mediated by this bHLH repressor [54] concomitant with its ubiquitin-mediated proteasome degradation by specific ubiquitin ligases (E3 ligases) [65]. However, it has been well documented that Hes1 physically interacts with deubiquitinases (DUBs), which in turn extend the protein stability and half-life, thereby regulating the neural stem cell propensity [170]. Ubiquitin-specific protease 27x (Usp27x) and its homologs Usp22 and Usp51 are the major candidates to execute this function during oscillation. Among them, Usp22 deserves a prime role in modulating Hes1 stability in developing mouse brain and is well evidenced by dampening of oscillation and enhanced neuronal differentiation upon Usp22 knockdown [170].

Pharmacological cues

Apart from these endogenous intrinsic and extrinsic factors, which enhance Hes1 expression, other pharmacological drugs such as fluoxetine [171], venlafaxine, imipramine [172], and atorvastatin [173] have been reported to activate Hes1 during neurogenesis. The serotonin reuptake inhibitor fluoxetine, which is widely used as an antidepressant drug, is able to up-regulate Notch signaling cascade and Hes1 during hippocampal neurogenesis [171]. Cell survival, proliferation, and differentiation associated with hippocampal neurogenesis along with increased Hes1 expression were observed upon chronic fluoxetine administration. In addition, anti-depressant imipramine treatment in human astrocyte culture showed induction of astrocytes to differentiate into cells with the neuronal phenotype accompanied by a significant increase in Hes1 mRNA level [172]. However, these results implied that Hes1 is a key molecule in antidepressant-induced neurogenesis. Another drug, atorvastatin, widely used for hypercholesterolemia, increased component of γ-secretase enzyme, presenilin-1 (PS1) and thereby up-regulates Hes1 downstream of the Notch signaling pathway in neurospheres derived from SVZ. This facilitates proliferation of neural progenitors in SVZ neurosphere culture [173]. Conversely, a new member of the broad-spectrum glycylcycline antibiotic, tigecycline, has been reported to induce cell-cycle arrest in malignant glioma cells [174]. This therapeutic effect is executed through the up-regulation of mir-199b-5p, which subsequently down-regulate Hes1-mediated cell proliferation [164, 174].

Other factors

Apart from these regulators of Hes1 more or less at molecular level, recent evidence provides physiological and therapeutical significance towards the repressive regulation over this maestro during neuronal development. For instance, omega-3 polyunsaturated fatty acids (omega-3PUFAs) such as docosahexaenoic acid (DHA) is reported to repress Hes1 in neural stem cells and also lead to cell cycle exit by interfering in the expression of p27kip1 [175], a well-known Hes1 target and inhibitor of the cell cycle [82]. This provides molecular insight into the role of omega-3 polyunsaturated fatty acids in promoting neuronal differentiation. In agreement with this result, another PUFA, eicosapentaenoic acid (EPA), also induces neuronal differentiation through an indirect repression of Hes1 through Hes6 [176]. Osthole (7-methoxy-8-isopentenoxycoumarin), one of the natural coumarin derivatives isolated from medicinal plants, is well known for its neuroprotective ability in pathogenesis of Alzheimer’s disease. However, the cellular/molecular mechanism underlying this involves promoted adult neural stem cell proliferation caused due to the enhanced Hes1 expression upon Notch activation [44]. Another noteworthy report illustrated that medical treatment such as electro acupuncture has been shown to down-regulate Hes1 mRNA in the hippocampus of a Aβ injected rat model, thereby alleviating the Alzheimer’s disease by enhancing the learning and memory function [177]. The molecular basis behind this enhancement along with the reduction in neuronal apoptosis and stimulated synaptic connectivity by this supplementary therapeutic treatment is its ability to quell to avert the persistent expression of Hes1 by Aβ in an Alzheimer’s disease model. Conversely, it has been shown that acupuncture enhances expression of Hes1 in the rat model of traumatic brain injury [178]. These intriguing results indicated that the effect of acupuncture on Hes1 might be associated with altered environmental niche or molecular machinery in two different contexts. Moreover, the molecular mechanism underlying neuronal induction property of Baicalin, a flavonoid from Scutellaria baicalensis G, is also attributed to its repressive effect on Notch target gene Hes1 [179]. Neurogenic switch in the expense of gliogenesis by baicalin in neural progenitors is associated with its repression over Hes1 and up-regulation of neuronal markers such as NeuroD, MAP2, etc. Conversely, Hes1 expression is up-regulated by plasma membrane protein caveolin-1 during astroglial differentiation of neural progenitor cells [180]. Decreased expression of astroglial markers such as GFAP and S100β concomitant with low expression of Hes1 in Caveolin-1 knockout mice also strengthen this notion. It has recently been shown that the cell-adhesion molecule, contactin-associated protein (Caspr) expressed by radial glial cells, can regulate Hes1 indirectly by attenuating transduction of Notch signaling [181]. This indirect repression by Caspr over Hes1 expression maintains the cortical neurogenesis in a temporal manner. Another very intriguing (but physiological significant) hormone-mediated Hes1 expression was documented in lateral and rostral regions of the cortex. The influence of maternal status of thyroid hormone on Hes1 expression, thereby regulating corticogenesis, harbors prime significance [182]. However, this report shed light into the requirement of maternal factors required for the activation of critical genes such as Hes1 in fetal prenatal brain development. Equally, another report stands in line with the physiological significance emphasizing the role of Hes1 in regulating NSC proliferation depending on glucose concentration [183]. The molecular mechanism behind this metabolic regulation of neurogenesis relies on the antagonism between two cellular nutrient sensors, CREB (cyclic AMP responsive element binding protein) and Sirt-1 (Sirtuin 1) in regulating Hes1 promoter in response to changes in glucose concentration and can be possibly linked to the association between diabetes and neurodegenerative diseases.

Conclusions and future perspectives

In this review, we summarized how a single dynamic gene, Hes1, is regulated and involved in the process of neurogenesis at multiple levels in a context-dependent manner both in vitro and in vivo. Though both activator and repressor bHLH proteins play a crucial role in regulating intact development and cytoarchitecture of nervous system, Hes1 deserves the role at most, despite its molecular redundancy in some contexts. As discussed, Hes1 activation is primarily activated by Notch-dependent mechanisms in neural progenitors, but signaling cascade constituted as a result of combinatorial complexity of different Notch ligands and receptors may deliver differential output at the cellular or functional level. Also, many reports stand in line regarding the Notch/CBF1-independent activation even in a single context. Hence, it remains to be dissected how and why this kind of discrete regulation happens with distinct function. This might be attributed to the differential regulation that relies on either extrinsic factors, such as signaling pathways, growth factors, etc., or intrinsic factors, such as downstream effectors of signaling pathways, epigenetic status, etc. The apparent ability of dynamic Hes1 in delivering functions such as neural progenitor maintenance, proliferation, gliogenesis, and boundary formation/compartmentalization is always dependent on its oscillatory versus sustained spatiotemporal expression. However, further molecular studies on the mechanism for temporal changes of Hes1 expression will be required.

Though various Hes1 target genes have been identified and characterized despite numerous unknown targets, the molecular mechanisms behind the direct or indirect repression executed by this bHLH repressor need to be further unraveled with respect to its feedback regulations and binding partners. Moreover, simultaneous repression over proneural genes and cell cycle regulators during the progression of progenitor lineage still remains elusive when we consider the indispensable role of Hes1 in neural progenitor maintenance and proliferation. The observations found in in vitro systems especially related to neuroprotection and differentiation should extend towards the in vivo model for a better understanding of its indispensable role in adult neurogenesis. Apart from these coherent studies and insights into the endogenous regulatory molecular machinery, more attention should be paid to the physiological and therapeutical significance of Hes1 in response to the most possible candidates of pharmacological cues and nutritional food supplements. Such analysis will prove to be helpful in understanding the plausible mechanism of brain functions and various neurodegenerative diseases. Though our review highlights only the involvement of this maestro in neurogenic aspects, various relevant findings, which were reported in other fields of biology with respect to Hes1, should be taken into consideration to extend further our knowledge about its unraveled role in CNS development and therapeutic applications.