Hes genes are mammalian homologues of Drosophila hairy and Enhancer of split, which encode basic helix-loop-helix (bHLH) transcriptional repressors. In the developing central nervous system, Hes1, Hes3 and Hes5 are highly expressed by neural stem cells. Inactivation of these Hes genes leads to upregulation of proneural genes, acceleration of neurogenesis and premature depletion of neural stem cells. Conversely, overexpression of Hes genes leads to inhibition of neurogenesis and maintenance of neural stem cells. At later stages of development, Hes genes promote gliogenesis. Furthermore, Hes genes regulate maintenance of boundaries, which partition the nervous system into many compartments and endow the neighboring compartments with regional identities by secreting morphogens. Boundary cells usually proliferate slowly and do not give rise to neurons, unlike neural stem cells in compartments. Interestingly, these different characteristics between boundary cells and compartmental neural stem cells are regulated by different modes of Hes1 expression, which is variable in neural stem cells in compartments and persistent and high in boundary cells. Thus, Hes genes play an essential role in neural development by regulating proliferation, differentiation and specification of neural stem cells.
In the developing central nervous system, neuroepithelial cells proliferate extensively by symmetric cell division at early stages (Fig. 1). As development proceeds, neuroepithelial cells gradually change to radial glial cells, which have the cell body in the ventricular zone and the radial fiber reaching the pial surface (Fig. 1). Both neuroepithelial cells and radial glial cells are embryonic neural stem cells (Alvarez-Buylla et al. 2001; Fishell & Kriegstein 2003; Fujita 2003; Götz & Huttner 2005). Radial glial cells give rise to many different types of neurons by repeating asymmetric cell division. Each asymmetric cell division generates one new radial glial cell and one neuron or neuronal precursor. Neurons migrate along the radial fibers to the outer layers, while radial glial cells remain in the ventricular zone (Fig. 1). After production of neurons, radial glial cells finally differentiate into glial cells such as astrocytes (Fig. 1). Thus, in the developing nervous system, the following three steps occur sequentially: symmetric proliferation of neural stem cells, neurogenesis and gliogenesis.
During neurogenesis, regional specification is regulated by boundaries, which partition the developing nervous system into many compartments. For example, the isthmus is the boundary between the midbrain and the hindbrain and regulates the specification of the neighboring compartments (midbrain and hindbrain) by secreting Fgf8 (Kiecker & Lumsden 2005). Boundary cells usually proliferate slowly and do not give rise to neurons, although they are morphologically similar to neural stem cells in compartments (Kiecker & Lumsden 2005).
It has been shown that Hes genes, repressor-type basic helix-loop-helix (bHLH) genes, play an essential role in development of both compartment and boundary cells of the central nervous system (Bertrand et al. 2002; Ross et al. 2003; Kageyama et al. 2007). Interestingly, the expression mode of Hes1 seems to be different between the two structures (persistent and high in boundary cells and variable in compartmental neural stem cells), and this different expression mode is involved in different characteristics of compartment and boundary cells. In this review, we describe the expression and functions of Hes genes in compartment and boundary cell formation.
Hes factors are repressors and oscillators
Hes genes are mammalian homologues of Drosophila hairy and Enhancer of split, which negatively regulate neurogenesis by antagonizing proneural genes such as the achaete-scute complex (Akazawa et al. 1992; Sasai et al. 1992). There are seven members in the Hes family, and each member has a conserved bHLH domain (Fig. 2A). Hes factors form homodimers through the HLH region and bind to the N box CACNAG and the class C sites CACG(C/A)G through the basic region with high affinities, unlike most other bHLH factors, which bind to the E box CANNTG with high affinities. In addition to the bHLH domain, Hes factors have another conserved domain, WRPW (Trp-Arg-Pro-Trp), at the C-terminus, which functions as a repression domain by recruiting co-repressors, Groucho homologues (Fig. 2A). Thus, Hes factors repress transcription by binding to the target DNA sequences and by actively recruiting co-repressors (active repression; Fig. 2B). Target genes include Mash1, a mammalian homologue of the Drosophila achaete-scute proneural gene complex, which encodes a bHLH activator and induces neuronal differentiation. Hes1 represses transcription of Mash1 by binding to the class C site in the Mash1 promoter (Chen et al. 1997). Hes factors also form heterodimers with bHLH activators such as Mash1 and E47. However, these heterodimers do not bind to the DNA, thus inhibiting the transcriptional activity of bHLH activators (passive repression; Fig. 2B). Thus, Hes factors antagonize proneural gene activities by at least two different mechanisms.
Another important target for Hes1 is Hes1 itself. Hes1 represses its own expression by directly binding to the N box sequences in the Hes1 promoter (Fig. 3; Takebayashi et al. 1994). This negative feedback leads to disappearance of both Hes1 mRNA and Hes1 protein, because they are extremely unstable, which allows the next round of expression. In this way, Hes1 autonomously starts oscillatory expression with a periodicity of about 2 h (Fig. 3; Hirata et al. 2002). This oscillation is observed in many cell types such as fibroblasts, presomitic mesoderm (PSM) cells and neural stem cells (Jouve et al. 2000; Hirata et al. 2002; Masamizu et al. 2006; Shimojo et al. 2008). In the PSM, Hes7 expression also oscillates by negative feedback with a periodicity of about 2 h, and this oscillation regulates the somite segmentation, which occurs every 2 h in mouse embryos (Bessho et al. 2001, 2003). In the absence of Hes7, other oscillator molecules such as Lunatic fringe and Dusp4 stop oscillatory expression and somites fuse severely (Bessho et al. 2001; Niwa et al. 2007). It is thus likely that Hes7 regulates the timing of somite segmentation while Hes1 regulates the timing of other events as biological clocks, although the precise role of Hes1 oscillation remains to be determined.
Maintenance of neural stem cells by Hes genes
At the neural plate stage, Hes1 and Hes3 are widely expressed by neuroepithelial cells along the entire neuraxis, but Hes5 is not expressed at this stage (Fig. 1; Hatakeyama et al. 2004). As neuroepithelial cells gradually change to radial glial cells, Hes3 expression is downregulated, and instead Hes5 expression occurs in the developing nervous system (Fig. 1; Hatakeyama et al. 2004). At the neural tube stage, Hes3 expression is gradually restricted to the isthmus, whereas Hes5 expression is expanded and becomes mostly complementary to Hes1 expression, and Hes1 and Hes5 expression cover almost all regions of the developing nervous system of mouse embryos around embryonic day 10.5 (E10.5) (Hirata et al. 2001; Hatakeyama et al. 2004). Later, Hes1 and Hes5 expression are restricted to the ventricular zone, which contains the cell bodies of radial glia (Akazawa et al. 1992; Sasai et al. 1992).
Overexpression of Hes genes in mouse embryos inhibits neurogenesis and maintains neural stem cells (Ishibashi et al. 1994; Ohtsuka et al. 2001). Conversely, in Hes1 knock-out (KO) mice, expression of proneural genes such as Mash1 is upregulated and neurogenesis is accelerated (Ishibashi et al. 1995). However, because Hes5 expression is upregulated, the defects are relatively mild in Hes1 KO mice. In Hes1;Hes5 double KO mice, the defects are severer than in Hes1 KO mice, but there are still many neural stem cells in the developing nervous system, suggesting that Hes3 compensates the defects to some extent (Ohtsuka et al. 1999). In contrast, in Hes1;Hes3;Hes5 triple KO mice, although neuroepithelial cells form normally around E8, they are not properly maintained and prematurely differentiate into neurons at the neural plate stage (Hatakeyama et al. 2004). In these mutant mice, virtually all cells become neurons in the developing spinal cord by E10, indicating that Hes1, Hes3 and Hes5 are responsible for maintenance of all neural stem cells in this region (Hatakeyama et al. 2004). Thus, Hes genes are required for maintenance of, but not for the initial formation of, neural stem cells. Which genes regulate the initial formation of neural stem cells remains to be determined. In the absence of Hes genes, neural stem cells differentiate into early-born neurons only and become depleted before generating later-born cell types such as astrocytes. Neural stem cells cannot change their competency rapidly, and it takes time for them to become competent to generate later-born cell types. Thus, Hes genes are essential for generation of a full diversity of cell types by maintaining neural stem cells until later stages.
Hes1 and Hes5 expression regulated by Notch signaling
Notch, a transmembrane protein, is activated by its ligands such as Delta (Fig. 4). Upon activation, the intracellular domain (ICD) is released from the membrane region and transferred into the nucleus, where the ICD forms a complex with the DNA-binding protein RBP-J. Without ICD, RBP-J represses Hes1 and Hes5 expression by binding to their promoters. However, the complex of RBP-J and the ICD acts as a transcriptional activator. Thus, activation of Notch signaling leads to induction of Hes1 and Hes5 expression (Fig. 4; Honjo 1996; Artavanis-Tsakonas et al. 1999). ICD maintains neural stem cells by inhibiting neurogenesis (Furukawa et al. 2000; Gaiano et al. 2000). However, in the absence of Hes1 and Hes5, ICD is unable to inhibit neurogenesis, indicating that Hes1 and Hes5 are essential effectors of Notch signaling (Fig. 4; Ohtsuka et al. 1999).
Although Notch signaling regulates Hes expression, the initial expression of Hes1 and Hes3 occurs in neuroepithelial cells before Notch signaling components are expressed, indicating that the initiation of Hes1 and Hes3 expression is independent of Notch signaling (Hatakeyama et al. 2004). However, the subsequent Hes5 expression occurs together with the expression of Notch signaling components, suggesting that initiation of Hes5 expression is dependent on Notch signaling. In addition, Hes1 expression also later overlaps with the expression of Notch signaling components, suggesting that the later Hes1 expression is regulated by Notch signaling. Which factors are responsible for the initial Hes1 and Hes3 expression in neuroepithelial cells remains to be determined.
Proneural genes such as Mash1 and Neurogenin2 (Ngn2) not only activate the neuronal differentiation program but also induce Delta expression, thereby activating Notch signaling and inhibiting neuronal differentiation of neighboring cells (Castro et al. 2006). This process is called “lateral inhibition” (Honjo 1996; Artavanis-Tsakonas et al. 1999). Lateral inhibition prevents all neural stem cells from responding to the same way, allowing only subsets of cells to differentiate into neurons and keeping others as neural stem cells. Those remaining neural stem cells later differentiate into different cell types. Thus, lateral inhibition is essential for maintenance of neural stem cells and generation of a full diversity of cell types.
Promotion of gliogenesis by Hes genes
Overexpression of Hes genes in the developing brain maintains neural stem cells at earlier stages, as mentioned above, but promotes astrocyte formation at later stages (Fig. 1), thus indicating that Hes genes have dual activities depending on the developmental stages (Ohtsuka et al. 2001). Similarly, overexpression of Hes genes in the postnatal retina promotes generation of Müller glia, the only glia formed in the retina (Furukawa et al. 2000; Hojo et al. 2000). These phenotypes are similar to those of inactivation of proneural genes. In the absence of proneural genes such as Mash1 and Math3, neuronal differentiation is inhibited, and neural stem cells are maintained at earlier stages, whereas at later stages formation of astrocytes and Müller glial cells is enhanced, suggesting that proneural genes have dual activities: promotion of neuronal differentiation and inhibition of astrocyte and Müller glia formation (Tomita et al. 2000; Hatakeyama et al. 2001; Nieto et al. 2001). In agreement with this notion, Ngn1 upregulates neuronal-specific gene expression and represses astrocyte-specific gene expression by sequestering the co-activator p300/CBP (Sun et al. 2001). Thus, it is likely that promotion of astrocyte and Müller glia formation by He s genes is due to repression of proneural genes.
Neural stem cells are unable to differentiate into glial cells at early stages, and it takes time for these cells to acquire the gliogenic competency. The molecular basis underlying the gliogenic competency remains to be determined, but the methylation status of the promoter regions of astrocyte-specific genes may contribute to this competency. At earlier stages, the promoter regions of astrocyte-specific genes are hyper-methylated and thus resistant for gene activation, whereas they are hypo-methylated and ready for expression at later stages (Takizawa et al. 2001). Furthermore, demethylation treatment of neural stem cells at earlier stages accelerates astrocyte differentiation (Takizawa et al. 2001). These results suggest that the methylation status of the promoter regions is important for the gliogenic competency. Another mechanism is the presence of partner factors such as the homeodomain factor Pax6. For example, Hes1 induces the neural stem cell fate in cells expressing the homeodomain factor Pax6 but the astrocytic fate in Pax6-negative cells in the developing spinal cord (Sugimori et al. 2007).
Maintenance of boundary cells by Hes genes
The developing central nervous system is partitioned into many compartments by boundaries, which function as the organizing centers (Fig. 5A). Boundary cells usually proliferate slowly and do not give rise to neurons. Cells in the roof plate and the floor plate have similar features to boundary cells. In these regions, Hes1 protein is persistently expressed at high levels (Fig. 5B). Hes3 is mainly expressed in the isthmus, whereas Hes5 is not expressed in boundaries. However, ectopic Hes5 expression occurs in boundary regions of Hes1 KO mice, compensating for their maintenance. Thus, all three Hes genes are involved in boundary cells (Hirata et al. 2001; Baek et al. 2006). In boundaries, proneural genes are not expressed, suggesting that persistent Hes expression constitutively represses expression of proneural genes. In agreement with this notion, in Hes1;Hes3;Hes5 triple KO mice, proneural genes are ectopically expressed and neurogenesis occurs in boundaries. As a result, all boundaries are not maintained or formed, and the regional specification is severely affected (Hirata et al. 2001; Baek et al. 2006).
In contrast to boundary cells, neural stem cells in compartments express variable levels of Hes1 (Baek et al. 2006). This variability could reflect gradual downregulation of Hes1 expression in differentiating cells (Fig. 5B). Another possibility is that Hes1 expression oscillates in neural stem cells, as in fibroblasts (Fig. 5B). Our recent data by a real-time imaging method (Masamizu et al. 2006) indicates that the latter is the case (Shimojo et al. 2008). Interestingly, the Mash1 level is high in cells expressing low levels of Hes1 and vice versa, suggesting that Mash1 expression also oscillates in the opposite phase to Hes1 (Fig. 5B; Baek et al. 2006). It is possible that neural stem cells in compartments are ready for neuronal differentiation by periodically expressing Mash1, whereas boundary cells are resistant to neuronal differentiation by constitutively repressing proneural genes. To support this notion, forced expression of Hes1 in a sustained manner in compartment cells leads to inhibition of neuronal differentiation. Furthermore, this procedure reduces the cell proliferation rate by inducing retardation at G1 phase of the cell cycle (Baek et al. 2006). Thus, sustained Hes1 expression leads to inhibition of both cell proliferation and differentiation, two important features of boundary cells. These results suggest that the oscillatory and sustained Hes1 expression modes regulate compartment and boundary cell characteristics, respectively.
Regulation of Hes1 oscillation by Stat3-Socs3 oscillations
The precise mechanism of how variable/oscillatory versus sustained Hes1 expression is regulated in the developing nervous system is not known, but it has been shown that Stat3-Socs3 oscillations are important for Hes1 oscillation in fibroblasts (Yoshiura et al. 2007). Jak2 activates Stat3 by phosphorylation, and phosphorylated Stat3 (pStat3) is transferred from the cytoplasm to the nucleus and upregulates expression of target genes (Fig. 6). One of them is Socs3, which antagonizes Jak2-dependent activation of Stat3, thereby forming the Jak2-Stat3-Socs3 negative feedback loop (Fig. 6; Levy & Darnell 2002; Yu & Jove 2004). By this negative feedback, the levels of pStat3 and Socs3 oscillate with a periodicity of about 2 h (Yoshiura et al. 2007). Interestingly, blockade of these oscillations inhibits Hes1 oscillation, making Hes1 protein level persistent (Yoshiura et al. 2007). Thus, oscillatory versus persistent Hes1 expression is regulated by pStat3-Socs3 oscillations. The mechanism by which pStat3-Socs3 oscillations regulate Hes1 oscillation remains to be determined, but pStat3 leads to instability of Hes1 protein, which is important for oscillatory expression (Fig. 6). Previously, it was shown that Hes1 promotes Jak2-dependent activation of Stat3 by physically interacting with each other in neural stem cells (Kamakura et al. 2004). Thus, it is likely that Hes1 oscillation and pStat3-Socs3 oscillations depend on each other in these cells (Fig. 6).
It has been shown that bHLH genes play an essential role in neural development. Particularly, antagonistic regulation between activator-type and repressor-type bHLH genes is important for maintenance of neural stem cells and proper timings of neurogenesis and gliogenesis. Hes genes maintain neural stem cells by antagonizing activator-type bHLH genes until later stages, lead to generation of a full diversity of cell types and thereby regulate the size, the shape and the cytoarchitecture of the nervous system. Furthermore, variable Hes1 expression is important for proliferation and differentiation of compartmental neural stem cells, while persistent and high Hes1 expression is required for maintenance of slowly proliferating and non-neurogenic boundary cells. It is likely that this variable versus persistent Hes1 expression is controlled by pStat3-Socs3 oscillations, although the exact mechanism remains to be analyzed. It was also shown that the bHLH genes Hes1 and Mash1 are expressed in the adult brain (Parras et al. 2004; Ohtsuka et al. 2006), suggesting that the genes regulating embryonic neural development also regulate adult neurogenesis. Adult neurogenesis is known to be important for learning and memory, and its inhibition seems to result in depression. Future research should thus reveal whether or not these bHLH genes are involved in adult neurogenesis. Such analysis will be useful to understand the mechanism of brain functions and diseases.
This work was supported by the Genome Network Project, and by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Conflict of Interest
No conflict of interest has been declared by R. Kageyama, T. Ohtsuka or T. Kobayashi.