Interactive Fly, Drosophila

Motif structure of Hairy-related proteins

Hairy is a member of the Hairy/Enhancer of split family of bHLH proteins that act as negative regulators of neuron system development. Family members share several traits: a characteristic domain adjacent to their HLH domain, two postulated helical regions that lie carboxy-terminal of the bHLH sequence and the extreme carbox-terminal Try-Arg-Pro-Trp (WRPW) motif (Van Doren, 1994).

A remarkable feature of the Cs-Hairy sequence is the change in the conserved carboxyl-terminal tetrapeptide WRPW found in the Hairy family of basic helix-loop-helix transcription factors, which include the Hairy, Deadpan, and Enhancer of split proteins. The WRPW tetrapeptide is changed to WRPF in Cs-H. The WRPW motif is required for interaction with the corepressor Groucho and for transcriptional repression. It is not known whether this 1-aa change in the tetrapeptide affects a putative interaction of Cs-H with Groucho. Runt domain proteins contain a very similar carboxyl-terminal motif, WRPY, which also is required for Groucho-dependent repression in Drosophila (Damen, 2000).

The basic helix-loop-helix (bHLH) gene Hes6, a novel member of the family of mammalian homologs of Drosophila hairy and Enhancer of split has been isolated. Hes6 is expressed by both undifferentiated and differentiated cells, unlike Hes1, which is expressed only by the former cells. Hes6 alone does not bind to the DNA but suppresses Hes1 from repressing transcription. In addition, Hes6 suppresses Hes1 from inhibiting Mash1-E47 heterodimer and thereby enables Mash1 and E47 to upregulate transcription in the presence of Hes1. Furthermore, misexpression of Hes6 with retrovirus in the developing retina promotes rod photoreceptor differentiation, like Mash1, in sharp contrast to Hes1, which inhibits cell differentiation. These results suggest that Hes6 is an inhibitor of Hes1, supports Mash1 activity and promotes cell differentiation. Mutation analysis reveals that Hes1- and Hes6-specific functions are, at least in part, interchangeable by alteration of the loop region, suggesting that the loop is not simply a nonfunctional spacer but plays an important role in the specific functions (Bae, 2000).

XHRT1 is a member of the HRT/Hey protein subfamily whose members are known as Notch effectors. XHRT1 is expressed in the developing floor plate and encodes a basic helix-loop-helix (bHLH) transcription repressor. XHRT1 misexpression in the neural plate inhibits differentiation of neural precursor cells and thus may be important for floor plate cells to prevent them from adopting a neuronal fate. Based on their primary structure, members of the bHLH-Orange (bHLH-O) family are subdivided into four distinct subfamilies, Hairy, E(spl), HRT ( also named Hey, HERP, Hesr, CHF, and in the case of HRT2, Gridlock) and Stra13. Members of these gene subfamilies encode repressors that inhibit target gene expression by acting as direct or indirect DNA-binding-dependent transcriptional repressors or by sequestering positive bHLH factors or their common heterodimer partners. Like other bHLH proteins, bHLH-O proteins have a region of basic amino acids immediately N-terminal to the HLH domain that mediates DNA-binding and are known to form homodimers or heterodimers via their HLH domain. The role of the Orange domain is not well understood. It may confer specificity of function to different family members or play a role in transcriptional repression. Deletion analysis indicates that inhibition of differentiation by XHRT1 requires the DNA-binding bHLH motif and either the Orange domain or the C-terminal region. XHRT1 can efficiently homodimerize and heterodimerize with hairy proteins. Among those hairy genes, Xhairy2b shows extensive overlap of expression with XHRT1 in floor plate precursors and may be a biologically relevant XHRT1 partner. Dimerization is mediated through both the bHLH and downstream sequences, the Orange domain being particularly important for the efficiency of the interaction. Using chimeric constructs between XHRT1 and the ESR9 bHLH-O protein that does not interact with Xhairy1 and Xhairy2b, it was found that both the bHLH domain and downstream sequences of XHRT1 are required for heterodimerization with Xhairy2b, while only the XHRT1 sequences downstream of the Orange domain are required for the interaction with Xhairy1. Together, these results suggest that XHRT1 plays a role in floor plate cell development and highlight the importance of the Orange and downstream sequences in dimerization and in the selection of the bHLH partners (Taelman, 2004).

Ancestral and conserved cis-regulatory architectures in developmental control genes

Among developmental control genes, transcription factor-target gene 'linkages'-- the direct connections between target genes and the factors that control their patterns of expression--can show remarkable evolutionary stability. However, the specific binding sites that mediate and define these regulatory connections are themselves often subject to rapid turnover. This paper describes several instances in which particular transcription factor binding motif combinations have evidently been conserved upstream of orthologous target genes for extraordinarily long evolutionary periods. This occurs against a backdrop in which other binding sites for the same factors are coming and going rapidly. These examples include a particular Dpp Silencer Element upstream of insect brinker genes, in combination with a novel motif referred to as the Downstream Element; combinations of a Suppressor of Hairless Paired Site (SPS) and a specific proneural protein binding site associated with arthropod Notch pathway target genes; and a three-motif combination, also including an SPS, upstream of deuterostome Hes repressor genes, which are also Notch targets. It is proposed that these stable motif architectures have been conserved intact from a deep ancestor, in part because they mediate a special mode of regulation that cannot be supplied by the other, unstable motif instances (Rebeiz, 2012).

Previous studies described the phylogenetically widespread occurrence of single, high-affinity bHLH repressor (R) binding sites (a consensus GGCACGCGCC, with variants in the last two bases) upstream of bilaterian proneural genes (Rebeiz, 2005). The possibility could not be ruled out that only the 'linkage' (direct transcription factor-target gene relationship) has been maintained, and that the binding site itself has been replaced repeatedly in the course of animal evolution. However, several lines of evidence suggest that these R sites have been conserved from a deep common ancestor. These included the stability of the precise 10-bp sequence of the site over very long intervals, and the strong conservation of both the motif and flanking sequences in some instances, clearly suggesting that the sites are indeed orthologous (Rebeiz, 2012).

The present report substantially expands the inventory of such apparently ancient and conserved cis-regulatory motifs in developmental control genes. This study describes five additional cases in which specific motif combinations have evidently been retained over hundreds of millions of years of evolution. With the exception of two novel elements [the insect brk Downstream Element (DE) and the deuterostome Hes XE], these motifs represent high-affinity binding sites for known transcription factors. The retention of these specific motif instances is especially striking when considered against the background of rapid appearance and disappearance of other binding sites for the same factors (Rebeiz, 2012).

The conservation of the distinctive SE + DE motif (SE: GRCGNCN₅GTCTG) combination upstream of insect brk genes extends over perhaps 270-300 My, reflecting the fact that the brk gene itself is found only in insects. A similar (minimum) age can be assigned to the P + SPS architecture found upstream of insect bHLH repressor genes, while the E + SPS + P combination associated with arthropod BFM genes is even older, in excess of 400 My, in view of its occurrence in the crustacean D. pulex. Finally, it is likely that the X + R + SPS ensemble upstream of deuterostome Hes1 genes was present in the common ancestor, over 500 My ago. It is also possible that an SPS element was associated with an ancestral bilaterian Hes repressor gene, which would make this feature close to 600 My old (Rebeiz, 2012).

This analyses do not permit the discerning of the population genetic/microevolutionary processes by which the distinctive cis-regulatory architectures first arose and became fixed in an ancestral population. However, it is believed that some useful insights can be offered into why these architectures have endured over such lengthy timescales (Rebeiz, 2012).

What characteristics of ancient and conserved motifs drive their long-term preservation by selection, even as other binding sites for the same factors come and go rapidly in evolution? An earlier proposal is first reiterated that such deeply conserved motifs mediate abstract or generic regulatory functions of fundamental utility to all or most members of an ancient clade (Rebeiz, 2005). It is certainly plausible that, once established, the capacity to repress brk transcription in response to a Dpp signal remained of great utility to all the descendants of the common insect ancestor, as diverse as they became. Similarly, the abstract ability to activate a Hes repressor gene via Notch signaling would remain of exceptional utility to descendants of a bilaterian (or earlier) ancestor that had evolved it. Finally, a generic capability for autorepression of a Hes bHLH repressor gene might very well be retained by descendants of a deuterostome ancestor (Rebeiz, 2012).

But it is certainly sensible to argue that, to retain such abstract and valuable regulatory capabilities, it would suffice to preserve only the linkage between the appropriate transcription factors and their targets. In this view, individual factor-binding motifs need not be retained; they would be free to turn over during evolution. However, the examples described in this study suggest a second important reason for the long-term evolutionary retention of particular motifs or motif combinations. It is proposed that these conserved sequence elements mediate a distinctive regulatory capability not conferred by other instances of the same motif or motifs. In the case of the SPS element, considerable confident can be had that this perspective is correct. The SPS has been shown to mediate cooperative binding of two Su(H)/Mam/NICD trimers, thus conferring on the associated target gene unusually high sensitivity to Notch signaling. While two 'lone' Su(H) sites are indeed able to contribute to a target gene's response to activated Notch, they would not do so in a cooperative manner. In a similar vein, it seems plausible to suggest that while all SE motifs may be able to participate in signal-dependent repression of brk, the SE + DE combination offers a unique and valuable version of this capability (e.g., greater signal sensitivity), possibly conferring a fitness advantage. It is hypothesized that in both cases, once the specialized motif architecture (SPS or SE + DE) had evolved to confer a distinctive capacity, it would be selectively retained in evolution. As is known, other instances of the SE or Su(H) binding motifs do arise and become fixed in individual clades, but these would not be expected to exhibit the same durability, since (according to the hypothesis) they confer no unique capability. The foregoing interpretation is particularly supported by the frequent observation that if only one element mediating a particular response [either SE or Su(H) site] is present upstream of an orthologous gene in a given species, it is of the 'special' type (SE + DE or SPS). Examples include the SE + DE combination in T. castaneum brk and the SPS motifs in the A. gambiae bHLHR1 gene, the A. mellifera BFM gene, and H. sapiens HES1 (Rebeiz, 2012).

Another factor that may contribute to the long-term evolutionary conservation of the specialized motif architectures this study has considered is their very complexity. Both the SE + DE unit and the SPS represent unusually extended and constrained motif combinations. While in principle this does not prevent them from turning over by duplication/degeneration, they are unlikely to evolve de novo (Rebeiz, 2012).

Finally, an intriguing feature is noted of the conserved motif architectures described in this study that involve the SPS: the apparently conserved order and even orientation of the individual sequence elements. The arthropod BFM genes are associated with a 'lower-strand' E motif followed by an SPS followed by a 'lower-strand' P site; insect Hes repressor genes bear an 'upper-strand' P site followed by an SPS; and deuterostome Hes1 genes have an 'upper-strand' X site followed by an 'upper-strand' R site followed by an SPS, which also has fixed orientation. Inter-site distances are often not conserved; consider the varying separation of the SPS and the P site in the BFM genes, or the different distances between the X + R combination and the SPS in the deuterostome Hes1 genes. Evidently, the motif order and orientation of these architectures have functional significance, consistent with an 'enhanceosome' model for the structure of these regions. Alternatively, these features may suggest the existence of a 'scanning' mechanism for optimal enhancer-promoter interaction. Such a property might be a particular characteristic of promoter-proximal cis-regulatory modules such as these, as contrasted with more distal enhancers. In the latter case, interaction with the promoter by 'looping' may impose fewer architectural constraints (Rebeiz, 2012).

This study has proposed that the distinctive cis-regulatory architectures ancient ones that have been conserved from a deep ancestor. However, it also seems likely that, because of their very complexity, they may not represent the 'original' version of their respective regulatory linkages. These two realizations can be reconciled via the following general evolutionary scenario (Rebeiz, 2012).

The direct linkage of an ancestral Hes gene to Su(H) and the Notch pathway evidently originated in a deep metazoan ancestor, and was very likely mediated by a lone Su(H) binding site or sites. The genome of the demosponge A. queenslandica includes one member of the closely related Hey repressor family, but no Hes genes; this Amphimedon Hey gene has one high-affinity Su(H) site 600 bp upstream of the transcription start site. The placozoan T. adhaerens has one Hey ortholog, one Hey-related gene, and one Hes gene. The Hey ortholog has three high-affinity Su(H) sites in the first 800 bp upstream of the ATG start codon, while the Hes gene includes a single such site within 500 bp of its ATG. The genome of the cnidarian N. vectensis (sea anemone) is endowed with a large paralogous family of 11 Hes genes, many of them with multiple lone Su(H) sites immediately upstream. Likewise, the Nematostella Hey ortholog has two upstream Su(H) sites. The SPS evidently did not appear upstream of a Hey/Hes gene until after the cnidarian-bilaterian divergence, but this association is now widespread among both protostomes and deuterostomes (Rebeiz, 2012).

It is suggested, then, that what appeared first was the simple capacity to regulate a Hey/Hes gene directly by Su(H) (presumably linked to the Notch pathway), via one or more lone Su(H) binding sites. Then, in a bilaterian ancestor, an SPS came into being upstream of an individual Hes gene, making possible a cooperative and thus highly sensitive response to Notch-activated Su(H). Once this novel regulatory capacity was established, it bestowed a sufficient selective advantage to ensure its subsequent retention in a wide variety of bilaterian taxa. Such a scenario can account for the phylogenetic distribution of the SPS-containing cis-regulatory architectures described. More complex histories cannot be ruled out, including the possibility that the SPS arose independently more than once in association with Hes genes (Rebeiz, 2012).

It is important to note the finding that, in the case of target genes that are part of paralogous families (Hes repressor and BFMs), only one particular paralog in a given species is typically associated with the conserved motif architectures described. This is true even if other paralogs make use of the same overall cis-regulatory 'code' (combination of transcription factor binding sites) to direct a similar expression specificity. For example, of the seven unambiguous Hes repressor paralogs in H. sapiens, only HES1 bears the X + R + SPS motif combination, though four others have upstream S sites and two of these also have upstream R sites. Likewise, the D. melanogaster genome includes nine BFM genes, most of which employ the S + P code, but only one, E(spl)m4, is associated with an SPS + P combination. It seems likely that, while the distinctive regulatory capability conferred by an ancient and conserved motif combination is of long-term selective value, it suffices for a single paralog in the genome to retain it (Rebeiz, 2012).

This observation is consistent with a duplication-divergence model for the evolution of Hes and BFM paralogs. The special cis-regulatory architectures this study has described, along with the associated protein coding sequences, comprise functional units that have been conserved from deep common ancestors because of the unique regulatory capabilities they confer. Paralogous genes that arise by duplication within various taxa (this is a widespread phenomenon in the case of Hes genes) would not be subject to the same stringent constraints on their cis-regulatory architecture, since the ancestral gene would be present to provide the distinctive capabilities. The paralogs would thus be free to evolve their cis-regulatory motifs according to other selective pressures or genetic drift, yielding the many variations on a basic theme (e.g., S + P) that is observed within a single species today (Rebeiz, 2012).

Brain factor 1 (BF-1: Drosophila homologs Slp1 and Slp2) is a winged-helix transcriptional repressor that plays important roles in both progenitor cell differentiation and regional patterning in the mammalian telencephalon. The aim of this study was to elucidate the molecular mechanisms underlying BF-1 functions. BF-1 is shown to interact in vivo with global transcriptional corepressors of the Groucho family and also to associate with the histone deacetylase 1 protein. The ability of BF-1 to mediate transcriptional repression is promoted by Groucho and inhibited by the histone deacetylase inhibitor trichostatin A, suggesting that BF-1 recruits Groucho and histone deacetylase activities to repress transcription. These studies also provide the first demonstration that Groucho mediates a specific interaction between BF-1 and the basic helix-loop-helix protein Hes1 and that BF-1 potentiates transcriptional repression by Hes1 in a Groucho-dependent manner. These findings suggest that Groucho participates in the transcriptional functions of BF-1 by acting as both a corepressor and an adapter between BF-1 and Hes1. Taken together with the demonstration that these proteins are coexpressed in telencephalic neural progenitor cells, these results also suggest that complexes of BF-1, Groucho, and Hes factors may be involved in the regulation of progenitor cell differentiation in the telencephalon (Yao, 2001).

These studies have provided the first demonstration that BF-1 and Groucho/TLE proteins can physically interact with each other in vivo. Their interaction appears to be direct, since it was also observed in binding assays using bacterially purified TLE proteins and in vitro-translated BF-1 preparations. Two separate TLE domains, the amino-terminal Q domain and the carboxy-terminal WDR region, are involved in BF-1 binding. This finding is in agreement with previous investigations showing that these two domains mediate protein-protein interactions with a number of other factors, including RUNX, NK-3, and UTY proteins. These observations suggest that the use of multiple protein-protein interaction domains is a strategy regularly utilized by Groucho/TLEs, perhaps to achieve a specificity that may not be provided by each interaction domain alone. A short region of BF-1, located immediately after the winged-helix domain, is involved in TLE binding. This region contains a sequence, YWPMSPFLSLH, that is conserved among all BF-1 family members and is characterized by two adjacent aromatic residues followed by the motif PFLSL. This arrangement of aromatic residues separated by one or two proline residues is reminiscent of the bona fide Groucho/TLE-binding motif WRP(W/Y) found in Hes and RUNX family members. Importantly, a similar sequence, YAFNHPFSINN, is present in the CRII region that mediates the interaction of TLEs with the winged-helix protein hepatic nuclear factor 3ß. Thus, it is possible that these short sequences may perform the common task of mediating the interaction of these winged-helix proteins with TLEs (Yao, 2001).

BF-1 also associates with HDAC1 in mammalian cells. This interaction is not direct and may be mediated by TLE proteins, which can bind to both BF-1 and HDAC1. Importantly, BF-1-mediated transcriptional repression is reduced by an inhibitor of histone deacetylase activities. Thus, it is proposed that BF-1 can recruit TLEs and histone deacetylases to repress transcription, a possibility consistent with previous studies showing that histone deacetylases are involved in transcriptional repression mediated by Groucho/TLE proteins. It remains to be determined, however, whether the recruitment of histone deacetylase activity represents the general mechanism normally utilized by BF-1 to repress transcription or whether other mechanisms may also be utilized. For instance, it will be important to determine whether Groucho/TLE proteins are always involved in repression by BF-1 or whether the latter can also repress transcription independently of the former. Moreover, Groucho/TLEs may contribute to BF-1 mediated repression in ways that may not always involve the recruitment of histone deacetylases (Yao, 2001).

BF-1 is an important regulator of the progenitor-to-neuron transition in the mammalian telencephalon. In the absence of BF-1, telencephalic progenitor cells differentiate prematurely, leading to early depletion of the progenitor population. These findings suggest that BF-1 promotes cell proliferation and/or inhibits cell differentiation in the telencephalon. BF-1 does not appear to have a direct growth-promoting activity, however, since disruption of BF-1 function in BF-1 minus mice has a demonstrable effect on the proliferation of neuroepithelial cells only after embryonic day 10.5, even though BF-1 is expressed in these cells at earlier stages. It is possible that BF-1 may act as a regulator of the activities of growth-regulatory signals. Support for this hypothesis derives from the finding that the loss of BF-1 leads to ectopic expression of BMP4 in the telencephalic neuroepithelium. This observation suggests that BF-1 may, at least in part, facilitate proliferation by inhibiting BMP4 expression, since BMP4 inhibits telencephalic progenitor cell proliferation. In addition, Xenopus XBF-1 may be a direct regulator of the p27^Xic1 gene, the amphibian counterpart of the mammalian cell cycle inhibitor p27^Kip1 (Yao, 2001 and references therein).

It is proposed that BF-1 may control telencephalon development by coordinating the control of cell proliferation with the timing of differentiation in the neuroepithelium. In both invertebrates and vertebrates, Hes and Groucho/TLE proteins act as negative regulators of neuronal differentiation by preventing progenitor cells from differentiating prematurely. The finding that BF-1 interacts with and enhances the transcription repression activity of Hes1 suggests that BF-1 may contribute to the regulation of the timing of neuronal differentiation together with Hes1 and TLE proteins. This possibility is consistent with the demonstration that Hes1 minus mice display a forebrain phenotype very similar to that of BF-1 minus mice, namely, premature differentiation of precursor cells with consequent depletion of the progenitor cell population. In the future, it will be important to determine whether BF-1 is involved in the regulation of the expression of genes that are thought to be targets of the transcriptional inhibitory functions of Hes proteins, like the proneural gene Mash1 (Yao, 2001).

A functional interaction between BF-1 and Hes1 may also help explain the results of studies of Xenopus embryos showing that ectopic expression of high doses of XBF-1 causes suppression of neuronal differentiation in the injected area in a cell autonomous way. It is conceivable that the BF-1-Hes1 interaction may be favored in cells expressing high doses of BF-1. As a result, the inhibitory function of Hes1 during neuronal differentiation may be promoted due to the potentiation of its transcription repression activity, leading to suppression of neuronal differentiation within the areas of high BF-1 expression. It remains to be determined, however, whether a similar situation may occur at lower BF-1 concentrations. Studies in Xenopus have shown that microinjection of low doses of XBF-1 does not cause suppression of neuronal differentiation but instead leads to the formation of supernumerary neurons within the injected area. This observation suggests that at low concentrations, BF-1 may not be able to enhance Hes1 activity but may still be able to suppress the growth-inhibitory function of p27^Kip1 and/or other antiproliferation factors. This would lead to increased proliferation without an antineurogenic effect, eventually resulting in supernumerary neurons when the progenitor cells differentiate. These observations suggest that changes in BF-1 protein levels may have important repercussions during the progenitor-to-neuron transition and underscore the importance of the mechanisms that regulate BF-1 expression and function (Yao, 2001).

Drosophila Runt is the founding member of a family of related transcription factors involved in the regulation of a variety of cell-differentiation events in invertebrates and vertebrates. Runt-related proteins act as both transactivators and transcriptional repressors, suggesting that context-dependent mechanisms modulate their transcriptional properties. The aim of this study was to elucidate the molecular mechanisms that contribute to the regulation of the functions of the mammalian Runt-related protein, Cbfa1. Cbfa1 (as well as the related protein, Cbfa2/AML1) physically interacts with the basic helix loop helix transcription factor, HES-1, a mammalian counterpart of the Drosophila Hairy and Enhancer of split proteins. This interaction is mediated by the carboxyl-terminal domains of Cbfa1 and HES-1, but does not require their respective tetrapeptide motifs, WRPY and WRPW. These studies also show that HES-1 can antagonize the binding of Cbfa1 to mammalian transcriptional corepressors of the Groucho family. Moreover, HES-1 can potentiate Cbfa1-mediated transactivation in transfected cells. Taken together, these findings implicate HES-1 in the transcriptional functions of Cbfa1 and suggest that the concerted activities of Groucho and HES proteins modulate the functions of mammalian Runt-related proteins (McLarren, 2000).

The finding that HES and Cbfa proteins can physically interact with one another is consistent with a number of previous results. (1) Expression studies show that, in both invertebrates and vertebrates, HES and Runt-related proteins are coexpressed in a variety of cell types. (2) Both of these proteins interact with Groucho/TLE family members, suggesting that HES- and Runt-related proteins may come in contact with each other at least during mechanisms involving Groucho/TLEs. (3) Genetic studies in Drosophila show that runt and HES genes participate in common developmental mechanisms involved in the control of sex determination and segmentation. For instance, both Runt and the HES family member, Deadpan, can bind to the promoter of the Sex-lethal gene and regulate its expression. (4) Cbfa1 and HES-1 contribute to the regulation of mammalian osteoblast-specific genes; for instance, they provide antagonistic inputs to the control of the expression of the osteopontin gene. This first demonstration of a direct link between mammalian HES and Cbfa proteins will now facilitate the study of how these factors interact with each other and with TLE proteins. Moreover, it will be important to determine whether invertebrate members of these protein families also interact with each other in similar ways (McLarren, 2000).

These studies have also shown that the carboxyl-terminal domains of Cbfa1 and HES-1 are involved in these proteins' interactions . Specifically, the last 60 amino acids of Cbfa1 can interact with the last 88 residues of HES-1. Importantly, the same carboxyl-terminal region of Cbfa1 involved in HES-1 binding also contains binding sites for TLE proteins, raising the possibility that HES-1 and TLE proteins may compete with each other for Cbfa1 binding (McLarren, 2000).

The observation that the carboxyl-terminal region of Cbfa1 is involved in TLE binding is consistent with the identification of a transcriptional repressor function within this domain and suggests that this repressor activity is due to the recruitment of the TLE corepressors. Interestingly, the region of Cbfa1 containing residues 443-516 is ~70% identical to amino acids 366 through 438 of mouse Cbfa2. Since this domain of Cbfa2 also harbors a transcription repression function, it is possible that TLE-binding sites are present within this carboxyl-terminal region of Cbfa2 (McLarren, 2000).

Binding of TLE proteins to Cbfa1 is not dependent on the presence of a carboxyl-terminal WRPY motif. This result is in agreement with the fact that the binding of TLE1 to AML1 occurs even in the absence of the WRPY motif. Moreover, these findings are consistent with transcription studies in transfected mammalian cells showing that TLE overexpression reduces transactivation by both Cbfa1 and a truncated Cbfa1 form lacking the WRPY motif (albeit not as effectively in the latter case). These combined results differ from the previous report that binding of Drosophila Groucho to Runt requires the carboxyl-terminal WRPY motif of the latter. However, those same studies show a weak Groucho/Runt interaction when a truncated form of Runt lacking solely the WRPY motif is used. Only when additional sequences are deleted together with the WRPY motif does Runt fail to bind to Groucho, suggesting that other elements in addition to the WRPY tetrapeptide may mediate this interaction. It is also possible that the difference between the investigations in Drosophila and mammals may reflect differences between Drosophila Runt and its mammalian counterparts or may derive from the use of different experimental protocols (McLarren, 2000).

Cbfa1 can interact with two separate TLE domains located within either the amino-terminal Q region or the carboxyl-terminal WDR domain, both of which are highly conserved among all Groucho/TLE family members. The identification of the WDR domain of Groucho/TLEs as a protein-protein interaction element is not surprising given the demonstrated involvement of WD40 repeats in molecular interactions and the demonstration that the WDR domain of Drosophila Groucho is involved in the interaction with the HES protein, Hairy. The amino-terminal Q domain of TLE proteins has also been shown to mediate protein-protein interactions, including those with the PRDI-BF1/Blimp-1 (see Drosophila Blimp-1) and UTY proteins. Moreover, in agreement with these results, Cbfa1 has recently been shown to interact with the product of the Grg5 gene, which encodes a roughly 200-amino acid protein homologous to the amino-terminal Q domain of Groucho/TLEs but lacking the carboxyl-terminal SP and WDR regions. Thus, it appears that TLE proteins utilize both of their recognized protein-protein interaction domains to interact with Cbfa family members. Although the specific contributions of these separate TLE domains to the interaction with Cbfa proteins remain to be determined, it is worth mentioning that TLEs also utilize both the amino-terminal Q domain and the carboxyl-terminal WDR domain to associate with specific members of the family of winged-helix DNA-binding proteins. This suggests that the use of separate protein-protein interaction domains may be a feature underlying the association of Groucho/TLE proteins with distinct DNA-binding factors (McLarren, 2000).

The present demonstration that both Cbfa1 and AML1 can interact with HES-1 suggests that members of these two protein families can regulate each other's transcriptional functions. In agreement with this possibility, HES-1 can potentiate Cbfa1-mediated transactivation in transfected cells. A number of observations suggest that HES-1 may perform this function by binding directly to Cbfa1 and inhibiting the interaction between Cbfa1 and endogenous TLE proteins, thereby reducing/inhibiting the repressive effect that TLEs can exert on the transactivating function of Cbfa1. (1) Cbfa1 and HES-1 can directly bind to each other. (2) Binding sites for both HES-1 and TLE proteins are present within the same carboxyl-terminal domain of Cbfa1. (3) HES-1 can interfere with the Cbfa1/TLE interaction in in vitro binding assays. (4) Cbfa1-mediated transactivation can be potentiated by a truncated form of HES-1 that does not interact with TLEs due to HES-1's loss of the carboxyl-terminal WRPW motif but is still competent to bind to Cbfa proteins. Together, these observations suggest that the positive effect of HES-1 on the transcriptional activity of Cbfa1 may involve an active competition with TLEs for direct binding to Cbfa1, rather than a situation in which HES-1 simply titrates away TLEs from Cbfa1 but does not associate with the latter (McLarren, 2000).

Alternative mechanisms can also be proposed. In particular, HES factors may mediate transcriptional activation, instead of repression, when they are associated with Cbfa proteins rather than with TLEs. Although a number of previous studies have shown that invertebrate and vertebrate HES proteins generally act as transcriptional repressors, recent investigations in Xenopus have implicated certain HES family members in both negative and positive feedback loop mechanisms that either repress or maintain the expression of genes of the Notch signaling pathway during embryonic somitogenesis. Together with the present observations, this finding suggests that, perhaps under appropriate conditions in which they escape interactions with Groucho/TLE proteins, HES factors may contribute to the transcriptional activity of other transcription factors (McLarren, 2000).

The possibility that HES-1 may interfere with the Cbfa1/TLE interaction and, vice versa, that Cbfa1 may interfere with the HES-1/TLE interaction may help to explain the finding that HES-1 can repress the expression of the osteopontin gene in osteoblasts, whereas Cbfa1 can activate osteopontin expression. It is possible that HES-1·TLE complexes keep the osteopontin promoter silent and that, by becoming recruited to the promoter, Cbfa1 may contribute to gene activation both directly -- by providing a transactivating function -- and indirectly, by interfering with the TLE/HES-1 interaction. These combined functions may mediate a shift from transcriptional repression mediated by DNA-bound HES-1·TLE complexes to transcriptional activation mediated by Cbfa1. In this model, the direct interaction between Cbfa1 and HES-1 may provide a way to prevent the interaction of Cbfa1 with TLEs. Specifically, by interacting with HES-1, Cbfa1 may become unavailable to TLE proteins and thus protect its transactivation ability from the repressive effect of the TLEs. This situation may provide a molecular explanation for the ability of Cbfa1 to promote transactivation of osteopontin and other osteoblast-specific genes even in the presence of TLE proteins. This would likely not be possible if Cbfa1 were simply titrating away TLEs from HES-1, because the resulting Cbfa1·TLE complexes would probably not be able to promote transactivation (McLarren, 2000).

This model is also consistent with the involvement of Drosophila Runt and Deadpan in the regulation of the Sex-lethal gene. Deadpan mediates repression of Sex-lethal and Groucho is required for this function. Conversely, Runt can bind to the Sex-lethal promoter and stimulate its activation. It is possible that in males, where Runt dosage is one-half of that in females, Deadpan binds to the Sex-lethal promoter and, together with Groucho, mediates transcriptional repression. In females, Runt may be able to antagonize the Deadpan/Groucho-mediated repression by interacting with Deadpan and disrupting the repressive complexes of Deadpan and Groucho. The ensuing Runt-Deadpan complexes may then be able to promote transcription. This model would thus provide a way to regulate the Runt/Groucho interaction through the formation of Runt-Deadpan complexes, a situation that might help to explain the apparent paradox that Runt can activate Sex-lethal expression while at the same time mediating repression of other target genes in the same cells (McLarren, 2000).

Transcriptional regulation of Hairy-related proteins: Hairy-related proteins and the Notch pathway

The Notch receptor is involved in many cell fate determination events in vertebrates and invertebrates. It has been shown in Drosophila that Delta-dependent Notch signaling activates the transcription factor Suppressor of Hairless, leading to an increased expression of the Enhancer of Split genes. Genetic evidence has also implicated the Kuzbanian gene, which encodes a disintegrin metalloprotease, in the Notch signaling pathway. A two-cell coculture assay has shown that vertebrate Dl-1 activates the Notch-1 cascade. Consistent with previous data obtained with active forms of Notch-1, a HES-1-derived promoter construct is transactivated in cells expressing Notch-1 in response to Dl-1 stimulation. Impairing the proteolytic maturation of the full-length receptor leads to a decrease in HES-1 transactivation, further supporting the hypothesis that only mature processed Notch is expressed at the cell surface and activated by its ligand. Dl-1-induced HES-1 transactivation is dependent both on Kuzbanian and RBP-J activities, consistent with the involvement of these two proteins in Notch signaling in Drosophila. Exposure of Notch-1-expressing cells to Dl-1 results in an increased level of endogenous HES-1 mRNA. Finally, coculture of Dl-1-expressing cells with myogenic C2 cells suppresses differentiation of C2 cells into myotubes, as previously demonstrated for Jagged-1 and Jagged-2, and also leads to an increased level of endogenous HES-1 mRNA. Thus, Dl-1 behaves as a functional ligand for Notch-1 and has the same ability to suppress cell differentiation as do the Jagged proteins (Jarriault, 1998).

The Notch signaling pathway is important for cellular differentiation. The current view is that the Notch receptor is cleaved intracellularly upon ligand activation. The intracellular Notch domain then translocates to the nucleus, binds to Suppressor of Hairless (RBP-Jk in mammals), and acts as a transactivator of Enhancer of Split (HES in mammals) gene expression. The Notch 3 intracellular domain (IC), in contrast to all other analyzed Notch ICs, is a poor activator, and in fact acts as a repressor by blocking the ability of the Notch 1 IC to activate expression through the HES-1 and HES-5 promoters. A model is presented in which Notch 3 IC interferes with Notch 1 IC-mediated activation at two levels. (1) Notch 3 IC competes with Notch 1 IC for access to RBP-Jk and does not activate transcription when positioned close to a promoter. (2) Notch 3 IC appears to compete with Notch 1 IC for a common coactivator present in limiting amounts. Further support for the existence of a coactivator comes from the finding that the Notch 3 ankyrin repeat construct, which lacks the strong RBP-Jk-binding RAM23 domain, is still able to repress Notch 1 IC-mediated activation. The common coactivator is most likely not required for all transcriptional complexes, since activation via the GAL4/VP16 fusion protein is not inhibited by Notch 3 IC. In keeping with this, cotransfection of the general coactivators TIF2, SRC1 and p300, does not neutralize Notch 3 ICs repressor activity. In conclusion, this is the first example of a Notch IC that functions as a repressor in Enhancer of Split/HES upregulation, and shows that mammalian Notch receptors have acquired distinct functions during evolution (Beatus, 1999).

What is the structural basis for the difference in transactivating capacity between Notch 1 IC and Notch 3 IC? All Notch receptors, including Notch 3, are highly structurally related in the intracellular domains, in particular in the ankyrin repeat region. The ankyrin repeat region is important for the transactivating activity in Drosophila Notch, LIN-12 and Notch1. The high degree of conservation between Notch 1 IC and Notch 3 IC in this domain may at first seem paradoxical. It should however be noted that relatively subtle mutations in the ankyrin repeat region can dramatically alter its transactivation competence. The RAM23 region is conserved to a somewhat lesser extent, but apparently the conservation is sufficient for both Notch 1 IC and Notch 3 IC to bind to RBP-Jk. The most obvious differences between Notch 1 IC and Notch 3 IC are found at the C-terminal end, where Notch 3 IC is shorter and lacks the OPA repeats found in other Notch homologs. It remains to be tested, however, whether this region plays a role in transactivation (Beatus, 1999).

What is the role of Notch3 IIC in vivo? A partial reduction of HES-5 expression in the rhombomere region was observed in a nestinp/Notch 3 IC transgenic mouse embryo with a distinct CNS phenotype. This suggests that Notch 3 IC also acts as a repressor of HES expression in vivo. Downregulation of HES-5 is evident around the rhombic lip and in the myelencephalic region, but not in more anterior and posterior CNS regions. Interestingly, this is reminiscent of the situation in RBP-Jk and Notch 1 -/- mice, in which HES-5 expression is also reduced in this region. This further supports a role of Notch 3 IC as a repressor of Notch 1-signaling, but also suggests that HES-5 expression is, at least in part, regulated by other factors in other regions of the CNS. A role for Notch 3 as a repressor of HES expression in vivo receives further support from comparisons of the phenotypes resulting from targeting of HES-1 and overexpression of Notch 3 IC in transgenic mice. Expression of Notch 3 IC in the developing CNS of transgenic mouse embryos produces an embryonically lethal phenotype. The transgenic embryos have an undulating spinal cord, fail to close the anterior neural pore and exhibit protrusions of neural tissue from the anterior neural pore region. Although initially interpreted differently, the latter phenotype may be a consequence of the open neural pore, in particular considering that the transgenic embryos show a relatively modest increase in proliferative rate in the CNS. Embryos lacking the HES-1 gene die just after birth, and show a kinked neural tube, open anterior neural pore and an everted neuroepithelium. Thus, the HES-1 -/- phenotype shows clear similarities to those observed in embryos overexpressing Notch 3 IC in the early CNS (Beatus, 1999 and references).

The finding that Notch 3 IC acts as a negative modulator of HES expression will be important for understanding of the CADASIL (Cerebral Autosomal Dominant with Arteriopathy and Subcortical Infarcts with Leukoencephalopathy). CADASIL is a familial disease which leads to migraine, subcortical brain infarcts and dementia and is caused by missense mutations in the EGF-repeat region of the human Notch 3 gene. CADASIL is a dominant disease, but it is not yet known whether the mutations in Notch 3 lead to haploinsufficiency (a condition whereby normal function is impaired by loss of one functional allele), or if the CADASIL mutations result in gain-of-function receptors. Since Notch 1, 2 and 3, and HES genes are expressed in the adult brain, it is conceivable that the function of Notch 3 as a negative modulator of HES expression may be affected in the disease. Thus, if CADASIL mutations produce gain-of-function Notch 3 receptors, this would result in decreased HES expression. Conversely, in the haploinsufficiency scenario, repression of HES expression would be reduced (Beatus, 1999 and references).

Notch receptors are involved in cell-fate determination in organisms as diverse as flies, frogs and humans. In Drosophila, loss-of-function mutations of Notch produce a 'neurogenic' phenotype in which cells destined to become epidermis switch fate and differentiate to neural cells. Upon ligand activation, the intracellular domain of Notch (ICN) translocates to the nucleus, and interacts directly with the DNA-binding protein Suppressor of hairless [Su(H)] in flies, or recombination signal binding protein Jkappa (RBP-Jkappa) in mammals, to activate gene transcription. But the precise mechanisms of Notch-induced gene expression are not completely understood. The gene mastermind has been identified in multiple genetic screens for modifiers of Notch mutations in Drosophila. MAML1, a human homolog of the Drosophila gene Mastermind, has been cloned; it encodes a protein of 130 kD localizing to nuclear bodies. MAML1 binds to the ankyrin repeat domain of all four mammalian NOTCH receptors, forms a DNA-binding complex with ICN and RBP-Jkappa, and amplifies NOTCH-induced transcription of HES1. These studies provide a molecular mechanism to explain the genetic links between mastermind and Notch in Drosophila and indicate that MAML1 functions as a transcriptional co-activator for NOTCH signaling (Wu, 2000).

Notch signal transduction is mediated by proteolysis of the receptor and translocation of the intracellular domain (IC) into the nucleus, where it functions as a regulator of HES gene expression after binding to the DNA-binding protein RBP-Jk. The mammalian Notch receptors are structurally very similar, but have distinct functions. Most notably, Notch 1 IC is a potent activator of the HES promoter, while Notch 3 IC is a much weaker activator and can repress Notch 1 IC-mediated HES activation in certain contexts. This report explores the molecular basis for this functional difference between Notch 1 and Notch 3 IC. Notch 3 IC, like Notch 1 IC, can bind the SKIP and PCAF proteins. Furthermore, both Notch 1 and Notch 3 ICs displace the co-repressor SMRT from the DNA-binding protein RBP-Jk on the HES promoter. The latter observation suggests that both Notch 3 IC and Notch 1 IC can access RBP-Jk in vivo, and that the difference in activation capacity instead stems from structural differences in the two ICs when positioned on RBP-Jk. Two distinct regions in the Notch IC are critical for the difference between the Notch 1 and Notch 3 IC; (1) the origin of the ankyrin repeat region is important, i.e. only chimeric ICs containing a Notch 1-derived ankyrin repeat region are potent activators; (2) a novel important region has been identified in the Notch IC. This region, named the RE/AC region (for repression/activation), is located immediately C-terminal to the ankyrin repeat region, and is required for Notch 1 IC's ability to activate and for Notch 3 IC's ability to repress a HES promoter. The interplay between the RE/AC region and the ankyrin repeat region provides a basis to understand the difference in HES activation between structurally similar Notch receptors (Beatus, 2001).

Previous work on Notch ICs has focused on three different domains: the RAM, ankyrin repeat and C-terminal regions. Another region in the Notch IC, the RE/AC region, is described in this study and has been shown to be important for regulation of HES promoters. Several lines of evidence support the importance of the RE/AC region, which is 120 amino acid residues long, located immediately C-terminal to the ankyrin repeat region. In Notch 1 IC, deletion constructs in which only the RE/AC region is removed fail to activate transcription, while more C-terminal deletions result in a much less dramatic decrease in activation. Similarly, removal of the RE/AC region from Notch 1 IC fused to GAL4 DB (G4-1101) largely abolishes activation from a GAL4 responsive promoter. In Notch 3 IC, the RE/AC region is also important, but here it plays a critical role in repression in trans of Notch 1 IC-mediated activation of the HES-1 promoter. Deletion constructs of Notch 3 IC, where the RE/AC region is removed, largely fail to repress Notch 1 IC-mediated activation on HES promoters. Moreover, a small region encompassing the Notch 3 IC RE/AC region, together with the last four ankyrin repeats, is sufficient to mediate substantial repression. The importance of this region has been shown in ESR-1 activation in Xenopus, in activation of the TP-1 promoter and for cellular transformation. Furthermore, it has been shown that Notch 1 IC and Notch 2 IC inhibit myeloid differentiation in response to different cytokines and that a region corresponding to RE/AC is involved in mediating the cytokine specificity of Notch 1 IC and Notch 2 IC. The finding that the RE/AC region is crucial for HES promoter regulation, oncogenesis and cytokine specificity underlines its importance for Notch function in various contexts (Beatus, 2001).

It has been proposed that the Notch 3 IC-mediated repression of Notch 1 IC activation is caused both by competition for access to RBP-Jk and by competition for a nuclear factor present in limiting amounts. The most parsimonial explanation for the role of the RE/AC region is to postulate that it constitutes a binding domain for the putative factor present in limiting amounts. This line of reasoning receives support from the finding that the Notch 3 IC RE/AC region can replace the Notch 1 IC RE/ AC region in the full IC context. It is interesting to note that there are local stretches of very high amino acid sequence similarity when the RE/AC regions of Notch 1 and Notch 3 ICs are compared. However, these highly conserved domains are not binding sites for known transcription factors. The C-terminal region of Notch 1 IC functions as a transcriptional activator on a multimerized RBP-Jk binding site, and has consequently also been referred to as TAD (transcription activation domain). The C-terminal region, alone or with the RE/AC region, is a potent activator also on the GAL4 responsive promoter. In contrast, the C-terminal region of Notch 1 IC is dispensable for potent activation on an endogenous 250 bp HES promoter. This indicates that the C-terminal region is crucial only on the multimerized RBP-Jk binding sites and in the GAL4 context, but not on the endogenous HES promoter (Beatus, 2001).

While the presence of a RE/AC region is required for activation from Notch 1 IC and repression from Notch 3 IC, the origin of the RE/AC region is not important for activation, since it can be exchanged between the two ICs without altering the effect. Therefore, the RE/AC region per se, does not explain why Notch 1 IC is a good activator and why Notch 3 IC is not. To address whether the origin of other regions is important, a set of chimeric Notch 1 IC/Notch 3 IC molecules was tested for activation and repression. It is concluded that the origin of the ankyrin repeat region is the most critical determinant for activation from HES promoters. This is based on the observation that only chimeric ICs containing the ankyrin repeat region from Notch 1 are good activators, while Notch 3 ankyrin repeat-containing constructs are not. The origin of the ankyrin repeat region is also most important for repression in trans. Only chimeric ICs harboring Notch 3 IC-derived ankyrin repeats are repressors of the same magnitude as Notch 3 itself, while proteins with ankyrin repeats derived from Notch 1 are considerably less potent repressors (Beatus, 2001).

How can the data presented here be incorporated into a model explaining the difference in HES promoter activation by Notch 1 and Notch 3? There could be two principally different explanations for Notch 3 IC's poor activation capacity; (1) Notch 3 IC may not get access to RBP-Jk on the HES promoter in vivo or (2) something in the structure of Notch 3 IC makes it an inferior activator once positioned on the HES promoter. The first explanation appears less likely, based on the data from the SMRT and SKIP experiments. Notch 3 IC, like Notch 1 IC, is capable of displacing VP16-SMRT from RBP-Jk fused to GAL4 in a two-hybrid assay. Furthermore, addition of SMRT to both Notch 1 IC and Notch 3 IC results in repression of activation on the HES promoter; in the case of Notch 1 IC a dramatic decrease, and in the case of Notch 3 IC a decrease from a very low level of activation. It is also observed that both Notch 1 IC and Notch 3 IC can bind to SKIP, which facilitates Notch function and has been shown to bind Notch 1 IC and SMRT in a mutually exclusive manner. In conclusion, it therefore appears reasonable to assume that Notch 3 IC can access RBP-Jk on a HES promoter in a manner similar to Notch 1 IC, i.e. by displacing SMRT from binding to SKIP/RBP-Jk. Assuming that both Notch 1 IC and Notch 3 IC have access to RBP-Jk in vivo, three different models are proposed to explain the differences in activation. These models take into account that the origin of the ankyrin repeat regions is important and that a Notch IC requires the presence of a RE/AC region, which binds a factor, present in limiting amounts. In the first model, the ankyrin repeat region would be important for the conformation of the Notch IC/factor complex, and the factor binding to the RE/AC region would only be optimally presented to the transcription machinery when the ankyrin repeat region is of Notch 1 IC origin. In the second model, the ankyrin repeat region of Notch 1 IC serves as a docking site for a second co-activator, which can not bind or binds less well to the ankyrin repeat region of Notch 3 IC. Only the cooperative binding of the co-activator on the Notch 1 IC ankyrin repeats and the factor binding to the RE/AC region would lead to potent activation. PCAF binds less well to Notch 3 IC than to Notch 1 IC, and could thus be a candidate factor. PCAF has indeed been shown to bind both to the ankyrin repeats and the C-terminal region in Notch 1 IC. The less efficient PCAF binding to Notch 3 IC could result in a more compacted chromatin structure at the promoter, as compared to when Notch 1 IC- PCAF is present. In the third model, the presence of an additional factor is also postulated, but in this model the co-factor would be a co-repressor specifically recruited to the Notch 3 IC ankyrin repeat region. This could quench the activity of the factor binding to the RE/AC region, thus rendering Notch 3 IC incapable of activating transcription. Irrespective of the finer details of how different factors work together to fine-tune transcriptional regulation, the discovery of the novel RE/AC region in the Notch IC is important for a more complete understanding of Notch signal transduction. The RE/AC region, combined with the observation that the origin of the ankyrin repeat region is important for activation, helps to explain why different Notch ICs are endowed with different activation properties on downstream HES promoters (Beatus, 2001).

Different cell types that occupy the midline of vertebrate embryos originate within the Spemann-Mangold or gastrula organizer. One such cell type is hypochord, which lies ventral to notochord in anamniote embryos. Hypochord precursors arise from the lateral edges of the organizer in zebrafish. During gastrulation, hypochord precursors are closely associated with the Brachyury homolog no tail-expressing midline precursors and paraxial mesoderm; these mesoderm cells also express deltaC and deltaD. Loss-of-function experiments have revealed that deltaC and deltaD are required for her4 expression in presumptive hypochord precursors and for hypochord development. Conversely, ectopic, unregulated Notch activity blocks no tail expression and promotes her4 expression. It is proposed that Delta signaling from paraxial mesoderm diversifies midline cell fate by inducing a subset of neighboring midline precursors to develop as hypochord, rather than as notochord (Latimer, 2002).

How might Delta signals induce hypochord development? One key might be regulation of ntl expression. ntl mutant embryos lack notochord and rostral hypochord and have excess floor plate. It has been proposed that ntl regulates a midline precursor fate decision by promoting notochord and inhibiting floor-plate development. It is further proposed that modulation of ntl expression within midline precursors by Delta-Notch signaling is required for hypochord development. In this model, ntl promotes formation of a population of midline precursors that have the potential to develop either as notochord or hypochord. Activation of Notch in a subset of precursors by Delta ligands expressed by neighboring paraxial mesoderm cells induces her4 and represses ntl expression. Consistent with this, constitutive Notch activity can cell-autonomously drive ectopic her4 expression. In the absence of Notch activity, her4 expression is not induced, and excess midline cells express ntl. Thus, Notch activity diverts midline precursors from notochord to hypochord fate (Latimer, 2002).

Notch receptors play various roles for cell fate decisions in developing organs, although their functions at the cell level are poorly understood. Notch1 and its ligand are each expressed in juxtaposed cell compartments in the follicles of the bursa of Fabricius, the central organ for chicken B cell development. To examine the function of Notch1 in B cells, a constitutively active form of chicken Notch1 was expressed in a chicken B cell line, DT40, by a Cre/loxP-mediated inducible expression system. Remarkably, the active Notch1 causes growth suppression of the cells, accompanied by a cell cycle inhibition at the G(1) phase and apoptosis. The expression of Hairy1, a gene product up-regulated by the Notch1 signaling, also induces the apoptosis, but no cell cycle inhibition. Thus, Notch1 signaling induces apoptosis of the B cells through Hairy1, and the G(1) cell cycle arrest through other pathways. This novel function of Notch1 may account for the recent observations indicating the selective inhibition of early B cell development in mice by Notch1 (Morimura, 2001).

her3 encodes a zebrafish bHLH protein of the Hairy-E(Spl) family. During embryogenesis, the gene is transcribed exclusively in the developing central nervous system, according to a fairly simple pattern that includes territories in the mesencephalon/rhombencephalon and the spinal cord. In all territories, the her3 transcription domain encompasses regions in which neurogenin 1 (neurog1) is not transcribed, suggesting regulatory interactions between the two genes. Indeed, injection of her3 mRNA leads to repression of neurog1 and to a reduction in the number of primary neurons, whereas her3 morpholino oligonucleotides cause ectopic expression of neurog1 in the rhombencephalon. Fusions of Her3 to the transactivation domain of VP16 and to the repression domain of Engrailed show that Her3 is indeed a transcriptional repressor. Dissection of the Her3 protein reveals two possible mechanisms for transcriptional repression: one mediated by the bHLH domain and the C-terminal WRPW tetrapeptide; and the other involving the N-terminal domain and the orange domain. Gel retardation assays suggest that the repression of neurog1 transcription occurs by binding of Her3 to specific DNA sequences in the neurog1 promoter. Interrelationships of her3 with members of the Notch signalling pathway have been examined by the Gal4-UAS technique and mRNA injections. The results indicate that Her3 represses neurog1 and, probably as a consequence of the neurog1 repression, deltaA, deltaD and her4. Moreover, Her3 represses its own transcription as well. Surprisingly, and in sharp contrast to other members of the E(spl) gene family, transcription of her3 is repressed rather than activated by Notch signalling (Hans, 2004).

Histone deacetylases (Hdacs) are widely implicated as key components of transcriptional silencing mechanisms. hdac1 is specifically required in the zebrafish embryonic CNS to maintain neurogenesis. In hdac1 mutant embryos, the Notch-responsive E(spl)-related neurogenic gene her6 is ectopically expressed at distinct sites within the developing CNS and proneural gene expression is correspondingly reduced or eliminated. Using an hdac1-specific morpholino, this requirement for hdac1 is shown to be epistatic to the requirement for Notch signalling. Consequently, hdac1-deficient embryos exhibit several defects of neuronal specification and patterning, including a dramatic deficit of hedgehog-dependent branchiomotor neurons that is refractory to elevated levels of hedgehog signalling. Thus, in the zebrafish embryo, hdac1 is an essential component of the transcriptional silencing machinery that supports the formation and subsequent differentiation of neuronal precursors (Cunliffe, 2004).

The early Xenopus organiser contains cells equally potent to give rise to notochord or floor plate, and Notch signalling triggers a binary decision, favouring the floor plate fate at the expense of the notochord. Evidence has been found that Delta1 is the ligand that triggers the binary switch, which is executed through the Notch-mediated activation of hairy2a in the surrounding cells within the organiser, impeding their involution through the blastopore and promoting their incorporation into the hairy2a+ notoplate precursors (future floor-plate cells) in the dorsal non-involuting marginal zone (Lopez, 2005).

Much of the patterning of early C. elegans embryos involves a series of Notch interactions that occur in rapid succession and have distinct outcomes; however, none of the targets for these interactions have been identified. The REF-1 family of bHLH transcription factors is a major target of Notch signaling in all these interactions and most examples of Notch-mediated transcriptional repression can be attributed to REF-1 activities. The REF-1 family is expressed and has similar functions in both Notch-dependent and Notch-independent pathways, and this dual mode of deployment is used repeatedly to pattern the embryo. REF-1 proteins are unusual in that they contain two different bHLH domains and lack the distinguishing characteristics of Hairy/Enhancer of Split (HES) bHLH proteins that are Notch targets in other systems. These results show that the highly divergent REF-1 proteins are nonetheless HES-like bHLH effectors of Notch signaling (Neves, 2005).

Most of the 39 bHLH genes in C. elegans can be grouped within Drosophila and vertebrate families of bHLH genes. However, there are six related orphan genes that are unique to nematodes; these are referred to as the ref-1 family after the first described member, ref-1. The ref-1 genes encode unusual bHLH proteins that each contain two distinct bHLH domains, a configuration thus far described only for a rice protein. The basic regions of the REF-1 proteins have moderate similarity to the basic regions of E(spl) or HERP proteins. Otherwise, the proteins are highly divergent and they lack the Orange domain, the conserved proline/glycine in the basic domain, and the terminal WRPW sequence. Only ref-1 has been studied genetically, and it was found to be required for cell fusion events during larval development that are not known to involve Notch signaling. This report provides evidence that the ref-1 family is a major target of Notch signaling in nematodes and that these genes function in all six embryonic Notch interactions examined. REF-1 and at least one additional family member appear to utilize the corepressor UNC-37/Groucho, thus linking UNC-37 with Notch-mediated repression in C. elegans. These results provide insight into the network of Notch signaling events in the embryo and suggest that the ref-1 and E(spl) genes may be highly diverged relatives of the same ancestral bHLH target of Notch signaling (Neves, 2005).

The HES family of bHLH repressors plays a key role in regulating the differentiation of neural precursors in the vertebrate embryo. Members of the HES gene family are expressed in neural precursors as targets of the Notch signaling pathway, but how this occurs in the context of neurogenesis is not known. This issue is addressed by identifying enhancers driving Notch-dependent gene expression of two Hes5-like genes expressed in Xenopus called Esr1 and Esr10. Using frog transgenesis, enhancer elements were identified driving expression of Esr1 and Esr10 in neural precursors or in response to ectopic expression of the proneural protein, Xngnr1. Using deletion and mutation analysis, motifs required for enhancer activity of both genes were defined, namely Notch-responsive elements and, in the case of Esr10, E-box motifs. Esr1 and Esr10 are differentially regulated both in terms of Notch input and its interaction with heterologous factors. These studies reveal inputs required for proneural expression of genes encoding bHLH repressors in the developing vertebrate nervous system (Lamar, 2005).

The data indicates that proneural bHLH input to the Esr10 enhancer is both indirect (through Notch) and direct. The Notch intracellular domain (ICD) and Xngnr1 synergistically upregulate transcription in transfection assays, Xngnr1 binds to the Esr10 downstream E-box in vitro, and the Esr10 proneural enhancer with mutant E-boxes shows marked loss of activity in vivo, which cannot be rescued by exogenous Xngnr1. These findings extend observations in Drosophila that proneural proteins synergize with Notch in activating E(spl) genes in larval discs. The data also support analysis of the Drosophila E(spl) gene m8. In that case, E boxes and Su(H) sites only in the configuration of a classical SPS enabled synergy between ICD and bHLH proteins, and enhancer activity was lost when one Su(H) site was mutant or oriented incorrectly. The SPS motif is a bipartite binding site for the Suppressor of hairless protein. The binding sites are separated by 30 or 29 nucleotides in the promoters of E(spl) genes of Drosophila melanogaster and higher vertebrates, respectively. One of the binding sites occurs in a reverse orientation to the other. Furthermore, a hexamer motif, which lies between or within the motifs, has a functional aspect. The Esr10 proneural enhancer behaves similarly in transgenics and provides the first example of such a required architecture among vertebrate Notch targets (Lamar, 2005).

By contrast, Esr1 is not directly regulated by proneural proteins. Although Esr1/RV has three E-boxes, E3 is not conserved in X. tropicalis, E1 is not conserved in the proneural enhancer of the closely related Esr7 gene, and neither E1 nor E3 fits the RCAGSTG consensus required for high-affinity binding of Drosophila proneural proteins to E-boxes. However, the CACCTG motif seen in E2 is targeted by Drosophila proneural proteins, a CACCTG E-box is required for retinal expression of Xenopus Ath5, and CACCTG binds MyoD in vitro and in vivo. Furthermore, E2 is embedded in a 13-base homology extending beyond the E-box in numerous Hes5 orthologs, although it is not seen in the Esr10 promoter. E2 was mutated using two strategies and no effect was seen on transgene expression in vivo. Further mutation may be required to evaluate the contribution of this motif to Esr1 expression. Nonetheless that E2 is contained within the Esr1 enhancer rules out the possibility that any factor binding to E2 is sufficient (with Notch acting through S1) to activate robust enhancer activity (Lamar, 2005).

Sites required for proneural Esr1 expression other than Su(H) sites have not been identified. Su(H) sites could be sufficient to activate Esr1, and tissue-specific responses to Notch might be due either to tissue-specific repressors or to the spacing of Su(H) sites providing a distinct platform for co-activators. Alternatively, Su(H) sites in the Esr1 enhancer could synergize with heterologous (non-bHLH) factors induced by Xngnr1, which, unlike direct bHLH input to either Esr10 or m8, interact with Notch through an S1-S4 configuration of Su(H) sites. Finally, enhancer activity could require input from both Notch (dependent on Xngnr1) and neural factors not dependent on Xngnr1. Although all three scenarios are possible, observation of attenuated but spatially appropriate GFP expression driven by the Esr1 enhancer argues against Su(H) site spacing as the sole determinant of specificity and suggests rather that tissue specific input to Esr1 requires sequences downstream of Hin3 (Lamar, 2005).

The hope of developing new transplantation therapies for degenerative diseases is limited by inefficient stem cell growth and immunological incompatibility with the host. This study shows that Notch receptor activation induces the expression of the specific target genes hairy and enhancer of split 3 (Hes3) and Sonic hedgehog (Shh) through rapid activation of cytoplasmic signals, including the serine/threonine kinase Akt, the transcription factor STAT3 and mammalian target of rapamycin, and thereby promotes the survival of neural stem cells. In both murine somatic and human embryonic stem cells, these positive signals are opposed by a control mechanism that involves the p38 mitogen-activated protein kinase. Transient administration of Notch ligands to the brain of adult rats increases the numbers of newly generated precursor cells and improves motor skills after ischaemic injury. These data indicate that stem cell expansion in vitro and in vivo, two central goals of regenerative medicine, may be achieved by Notch ligands through a pathway that is fundamental to development and cancer (Androutsellis-Theotokis, 2006).

The mechanisms controlling the transition from neurogenesis to gliogenesis in the vertebrate CNS are incompletely understood. A family of transcription factors, called NFI genes (Drosophila homolog: Nuclear factor I), was identified that are induced throughout the spinal cord ventricular zone (VZ) concomitantly with the induction of GLAST, an early marker of gliogenesis. NFIA is both necessary and sufficient for GLAST induction in the VZ. Unexpectedly, NFIA is also essential for the continued inhibition of neurogenesis in VZ progenitors. This function is mediated by the requirement of NFIA for the expression of HES5, a Notch effector. However, Notch effectors are unable to promote glial-fate specification in the absence of NFIA. Thus, NFIA links the abrogation of neurogenesis to a generic program of gliogenesis, in both astrocyte and oligodendrocyte VZ progenitors. At later stages, NFIA promotes migration and differentiation of astrocyte precursors, a function that is antagonized in oligodendrocyte precursors by Olig2 (Deneen, 2006).

Cooperative assembly of higher-order Notch complexes functions as a switch to induce transcription

Notch receptors control differentiation and contribute to pathologic states such as cancer by interacting directly with a transcription factor called CSL (for CBF-1/Suppressor of Hairless/Lag-1) to induce expression of target genes. A number of Notch-regulated targets, including genes of the hairy/enhancer-of-split family in organisms ranging from Drosophila to humans, are characterized by paired CSL-binding sites in a characteristic head-to-head arrangement. Using a combination of structural and molecular approaches, it has been establish that cooperative formation of dimeric Notch transcription complexes on promoters with paired sites is required to activate transcription. These findings identify a mechanistic step that can account for the exquisite sensitivity of Notch target genes to variation in signal strength and developmental context, enable new strategies for sensitive and reliable identification of Notch target genes, and lay the groundwork for the development of Notch pathway inhibitors that are active on target genes containing paired sites (Nam, 2007).

Cocrystals of a human Notch transcriptional activation complex (NTC) core, which consists of an N-terminal MAML-1 peptide, the ANK domain of human Notch1, and CSL on a DNA duplex derived from the HES-1 promoter, contain contacts between the convex surfaces of ANK domains from adjacent unit cells that also are seen in crystals of the ANK domain solved in isolation in several different crystallization conditions. These contacts lie near a twofold symmetry axis in the crystals, such that the interacting complexes are positioned head-to-head at a distance roughly equal to that needed to occupy both recognition elements of an SPS. Primary sequence alignment of Notch ANK domains from different homologs shows that the key contacts are evolutionarily conserved. These conserved residues are not engaged in contacts within an individual MAML1/ANK/CSL/DNA complex, suggesting that the observed conservation reflects functional importance in mediating dimerization at SPS sites. The conservation among the four mammalian Notch receptors also predicts that each receptor should be capable of making interactions like those between the adjacent Notch1 complexes (Nam, 2007).

The ANK-ANK contacts primarily are electrostatic and lie in the second and third ankyrin repeats. Key interactions consist of contacts between the guanidino group of Arg-1985 and at least three backbone carbonyl oxygen atoms, as well as interactions between Glu-1950 and Lys-1946. Arg-1983 also forms hydrogen bonds with Ser-1952 and a backbone carbonyl. In addition to homotypic interactions between the ANK domains, unmodeled electron density in the MAML-1/ANK/CSL/DNA complex also suggests the existence of interactions between the ANK domain of one complex and the N-terminal end of MAML-1 in the second complex. Based on the architecture of the complex, and the evolutionary conservation of SPSs and the crystal contact residues, it is postulated that the ANK domains of Notch receptors mediate dimerization of ternary complexes on SPSs found in Notch target gene promoters (Nam, 2007).

To test whether residues engaged in ANK-ANK contacts in the crystal contribute to transcriptional activation of SPS-bearing promoters, the ability of different forms of ICN to induce transcription of a luciferase gene under control of the HES-1 promoter, which has a functionally important SPS element, was tested. In contrast to normal ICN1, mutations that disrupt the predicted dimerization interface either abrogate (R1985A) or diminish (K1946E and E1950K) the ability of ICN1 to induce expression of the HES-1 reporter gene. Combining the K1946E and E1950K mutations in cis, however, rescues the defect in transcriptional activation, indicating that the putative dimerization interface is functionally important in regulating transcriptional activity at a promoter that contains an SPS. In addition, when coexpressed with ICN1, the R1985A mutation dominantly interferes with activation of the HES-1 promoter element by normal ICN1. Importantly, when these mutants are scored on an artificial reporter that contains four CSL-binding sites oriented in the same direction and in tandem, there is no change in the ability of the mutants to activate transcription. Moreover, in cotransfected cells, all ICN1 polypeptides with mutations that disrupt the predicted dimerization interface are expressed at similar levels to normal ICN1, and they coimmunoprecipitate in similar amounts with CSL and MAML-1. Together, these findings indicate that the ability to form monomeric ternary complexes with MAML-1 and CSL is not affected by these mutations (Nam, 2007).

To establish directly whether NTCs (consisting of one molecule each of MAML-1, ICN, and CSL) can cooperatively dimerize on DNA, electrophoretic mobility shift assays (EMSAs) were carried on an oligonucleotide probe containing the HES-1 promoter SPS. Without Notch or MAML-1, CSL binds to each of the two sites independently. When present in excess, most probes bind a single CSL molecule, a finding consistent with previous studies showing that CSL binds its recognition element as a monomer without detectable cooperativity at paired sites. Adding RAMANK from Notch1 does not change the stoichiometric distribution of complexes bound per probe molecule. However, when MAML-1 is added, the stoichiometric distribution of the complexes changes dramatically: all of the probe is either free or bound by NTC dimers, indicating that NTC loading at one site leads to cooperative loading of the second site. As predicted, cooperative loading is abrogated by the R1985A mutation, which instead produces a smear corresponding to an ensemble of species that likely results from a weak residual tendency to self-associate. In contrast, the R1985A mutation does not detectably affect ternary complex formation on a probe containing only a single CSL-binding site, indicating that the R1985A mutation is a cooperativity mutant that specifically interferes with dimerization. The partial loss of activity of the K1946E and E1950K mutants in the HES-1 reporter assays is echoed in EMSA titrations, where the proteins undergo a cooperative transition to form dimers at a concentration ~4-fold greater than normal ICN1 or the K1946E/E1950K double mutant (Nam, 2007).

To test whether higher-order complexes exhibit specificity for the SPS architecture, additional EMSA assays were carried out on variant DNA sequences that eliminate the integrity of one of the SPS sites, flip the site orientation, or alter the spacing between the sites by a half-turn of helix. When either site A or site B is mutated so that it no longer corresponds to a CSL consensus site (YGTGDGAA), cooperative assembly of the dimer is no longer observed. Moreover, cooperative dimerization is no longer detected when the second site is inverted, and it is dramatically diminished when the second site is moved by a 5-base insertion. Because the intrinsic affinity of a single ternary complex for DNA is not altered under the conditions of inversion or insertion, these studies show that the proper spatial arrangement of the two individual binding sites is needed for cooperative dimerization to occur (Nam, 2007).

It was next asked what range of spacer lengths between sites is compatible with cooperative loading of dimeric complexes. The optimal spacing between consensus sites for cooperative dimerization is 16 bp, but cooperative dimerization still can occur on templates with spacers varying from 15 to 19 bp, implying that two NTCs can adjust their positions relative to each other to accommodate a modest range of spacer lengths between sites. This inferred flexibility is consistent with the different conformations of CSL seen in the crystal structures of the Notch ternary complexes formed with the human and worm proteins and with the enrichment of adenosine and thymidine in the spacer between the paired sites (Nam, 2007).

To determine whether the assembly of NTCs and their cooperative dimers is general among the human Notch homologues, the ability of the RAMANK domains of Notch1-4 to form complexes on single and sequence-paired sites was tested. Despite qualitative differences in mobility on the EMSA, all four purified RAMANK polypeptides bind to CSL independent of MAML-1 and then recruit MAML-1 to ternary complexes on a single site probe. When the longer, paired site probe is provided, all RAMANK polypeptides mediate cooperative dimerization, as predicted from the conservation in primary sequence at the dimerization interface. Thus, a similar series of events takes place to assemble single and dimeric NTCs in all four mammalian Notch homologues (Nam, 2007).

Id sustains Hes1 expression to inhibit precocious neurogenesis by releasing negative autoregulation of Hes1

Negative bHLH transcription factor Hes1 can inhibit neural stem cells (NSCs) from precocious neurogenesis through repressing proneural gene expression; therefore, sustenance of Hes1 expression is crucial for NSC pool maintenance. Ids, the dominant-negative regulators of proneural proteins, are expressed prior to proneural genes and share an overlapping expression pattern with Hes1 in the early neural tube of chick embryos. Overexpression of Id2 in the chick hindbrain upregulates Hes1 expression and inhibits proneural gene expression and neuronal differentiation. By contrast, Hes1 expression decreases, proneural gene expression expands, and neurogenesis occurs precociously in Id1;Id3 double knockout mice and in Id1-3 RNAi-electroporated chick embryos. Mechanistic studies show that Id proteins interact directly with Hes1 and release the negative feedback autoregulation of Hes1 without interfering with its ability to affect other target genes. These results indicate that Id proteins participate in NSC maintenance through sustaining Hes1 expression in early embryos (Bai, 2007).

Oscillations in notch signaling regulate maintenance of neural progenitor

Expression of the Notch effector gene Hes1 is required for maintenance of neural progenitors in the embryonic brain, but persistent and high levels of Hes1 expression inhibit proliferation and differentiation of these cells. By using a real-time imaging method this study found that Hes1 expression dynamically oscillates in neural progenitors. Furthermore, sustained overexpression of Hes1 downregulates expression of proneural genes, Notch ligands, and cell cycle regulators, suggesting that their proper expression depends on Hes1 oscillation. Surprisingly, the proneural gene Neurogenin2 (Ngn2) and the Notch ligand Delta-like1 (Dll1) are also expressed in an oscillatory manner by neural progenitors, and inhibition of Notch signaling, a condition known to induce neuronal differentiation, leads to downregulation of Hes1 and sustained upregulation of Ngn2 and Dll1. These results suggest that Hes1 oscillation regulates Ngn2 and Dll1 oscillations, which in turn lead to maintenance of neural progenitors by mutual activation of Notch signaling (Shimojo, 2008).

Thermodynamic and structural insights into CSL-DNA complexes

The Notch pathway is an intercellular signaling mechanism that plays important roles in cell fates decisions throughout the developing and adult organism. Extracellular complexation of Notch receptors with ligands ultimately results in changes in gene expression, which is regulated by the nuclear effector of the pathway, CSL [C-promoter binding factor 1 (CBF-1), suppressor of hairless (Su(H)), lin-12 and glp-1 (Lag-1)]. CSL is a DNA binding protein that is involved in both repression and activation of transcription from genes that are responsive to Notch signaling. One well-characterized Notch target gene is hairy and enhancer of split-1 (HES-1), which is regulated by a promoter element consisting of two CSL binding sites oriented in a head-to-head arrangement. Although previous studies have identified in vivo and consensus binding sites for CSL, and crystal structures of these complexes have been determined, to date, a quantitative description of the energetics that underlie CSL-DNA binding is unknown. This study provides a thermodynamic and structural analysis of the interaction between CSL and the two individual sites that comprise the HES-1 promoter element. Comprehensive studies that analyze binding as a function of temperature, salt, and pH reveal moderate, but distinct, differences in the affinities of CSL for the two HES-1 binding sites. Similarly, structural results indicate that overall CSL binds both DNA sites in a similar manner; however, minor changes are observed in both the conformation of CSL and DNA. Taken together, these results provide a quantitative and biophysical basis for understanding how CSL interacts with DNA sites in vivo (Friedmann, 2010).

Given the small differences in affinity that were observed for CSL binding the 5'consensus and 3' nonconsensus sites of the HES1 SPS, it was interested to know why previous studies that identified the consensus binding site for CSL revealed a strong preference for a G/C base step at this position (-C/tGTGGGAA-), as opposed to A/T, C/G, and T/A base steps. Previous structures have shown that in some, but not all CSL-DNA complexes, the side chain of an absolutely conserved glutamine residue makes a water-mediated contact with the guanine base in the major groove, providing some explanation for the specificity and tolerance for purine bases at this position. Despite these structural results, there is relatively strong conservation for a T/A base step, that is, pyrimidine base, at this position in the HES-1 SPS found in mammals, Xenopus, and Zebrafish. Although the T→A mutation of the nonconsensus site (-CGTGTGAA-) actually enhanced binding similar to the consensus site, strikingly, the T→C mutation had a profound reduction in binding. Taken together, these results suggest that the identity of this base step is important for the affinity and specificity of CSL binding; however, a satisfactory molecular explanation for the observed differences in binding are still lacking (Friedmann, 2010).

Hairy-related proteins - Transcriptional targets

A transcriptional silencer has been identified that is critical for proper expression of the CD4 gene during T-cell development. The Hairy/Enhancer of Split homolog HES-1, a transcription factor in the lin12/Notch signaling pathway, binds to an important functional site in the CD4 silencer. Overexpression of HES-1 leads to the silencer site-dependent repression of CD4 promoter and enhancer function as well as the downregulation of endogenous CD4 expression in CD4(+) CD8(-) TH cells. Interestingly, overexpression of an activated form of Notch1 (NotchIC) leads to the repression of CD4 promoter and enhancer function both in the presence and absence of the silencer. NotchIC-mediated CD4 silencer function is not affected by the deletion of the HES-1-binding site, indicating that multiple factors binding to CD4 transcriptional control elements are responsive to signaling from this pathway, including other silencer-binding factors. Taken together, these data are consistent with the hypothesis that the lin12/Notch signaling pathway is important in thymic development and provide a molecular mechanism via the control of CD4 gene expression in which the lin12/Notch pathway affects T-cell developmental fate (Kim, 1998).

Activated Notch-Delta signaling was shown to inhibit myogenesis, but whether and how it regulates myogenic gene expression is not clear. The implication of Xenopus hairy-1 (Xhairy-1), a member of the hairy and enhancer-of-split [E(spl)] family that may function as a nuclear effector of the Notch signaling pathway in regulating XMyoD gene expression at the initial step of myogenesis was examined. Xhairy-1 transcripts are expressed soon after mid-blastula transition and Xhairy-1 exhibits overlapping expression with Notch pathway genes such as Delta-1 in the posterior somitic mesoderm. Overexpression of Xhairy-1 blocks the expression of XMyoD in early gastrula ectodermal cells treated with the mesoderm-inducing factor activin, and in the mesoderm tissues of early embryos. It inhibits myogenesis and produces trunk defects at later stages. Xhairy-1 also inhibits the expression of the pan-mesodermal marker Xbra, but expression of other early mesoderm markers such as goosecoid and chordin is not affected. These effects require the basic helix-loop-helix (bHLH) domain, as well as a synergy between the central Orange domain and the C-terminus WRPW-Groucho-interacting domain. Furthermore, overexpression in ectodermal cells of Xhairy-1/VP16, in which Xhairy-1 repressor domain is replaced by the activator domain of the viral protein VP16, induces the expression of XMyoD in the absence of protein synthesis. Interestingly, Xhairy-1/VP16 does not induce the expression of Xbra and XMyf5 in the same condition. During neurulation, the expression of XMyoD induced by Xhairy-1/VP16 declines and the expression of muscle actin gene was never detected. These results suggest that Notch signaling through hairy-related genes may specifically regulate XMyoD (Umbhauer, 2001).

Activating the PARP-1 sensor component of the Groucho/ TLE1 corepressor complex mediates a CaMKinase II-dependent neurogenic gene activation pathway: Regulatory mechanisms impinging on HES1 regulated promoters

Switching specific patterns of gene repression and activation in response to precise temporal/spatial signals is critical for normal development. This study reports a pathway in which induction of CaMKII triggers an unexpected switch in the function of the HES1 transcription factor from a TLE-dependent repressor to an activator required for neuronal differentiation. These events are based on activation of the poly(ADP-ribose) polymerase1 (PARP-1) sensor component of the groucho/TLE-corepressor complex mediating dismissal of the corepressor complex from HES1-regulated promoters. In parallel, CaMKII mediates a required phosphorylation of HES1 to permit neurogenic gene activation, revealing the ability of a specific signaling pathway to modulate both the derepression and the subsequent coactivator recruitment events required for transcriptional activation of a neurogenic program. The identification of PARP-1 as a regulated promoter-specific exchange factor required for activation of specific neurogenic gene programs is likely to be prototypic of similar molecular mechanisms (Ju, 2004).

A covalent modification recently linked to transcription is poly(ADP-ribosyl)ation of proteins mediated by the poly(ADP-ribose) polymerase1 (PARP-1) enzyme. PARP-1 catalyzes the transfer of ADP-ribose chains onto glutamic acid residues of acceptor proteins, including itself (automodification), histones, transcription factors, and DNA repair proteins using NAD+ as a substrate involved in chromatin decondensation, DNA replication, and DNA repair. Therefore, poly(ADP-ribosyl)ation by PARP-1 affects cellular processes such as apoptosis, necrosis, cellular differentiation, malignant transformation, and modulations activities of transcription factors. While it has been recently reported that PARP influences both the expression and silencing at diverse times during Drosophila development (Tulin, 2002), it has been demonstrated that high PARP enzymatic activity is observed in areas of high transcriptional activity and chromatin decondensation on the polytene chromatin (Tulin, 2003). Together these observations suggest that PARP-1 may exert its function in transcription through direct binding to the gene-regulating sequences and through modification of transcription factors by poly(ADP-ribosyl)ation (Ju, 2004).

This study finds that HES1-dependent repression of MASH1 is dependent upon the actions of the TLE1 corepressor complex. Not only are additional insights provided into the molecular mechanisms of TLE1-mediated repression but also the molecular mechanism of the switch to activation function has been uncovered. The composition of this TLE1 complex is distinct from those of other reported corepressor complexes such as N-CoR/SMRT and CtBP. Interestingly, roles for transcriptional regulation and chromatin remodeling activities have been described for most of the components of the TLE1 complex identified, but no component alone is indispensable for at least some level of TLE1-mediated repression (Ju, 2004).

Consistent with the observation that the enzymatic activity of PARP-1 is not required for HES1-mediated MASH1 repression, these data favors a model predicting that in the TLE1 holorepressor complex, the enzymatic activity of PARP-1 is inhibited. Exposure to a signal inducing neuronal differentiation causes activation of CaMKIIdelta, which is proven to be required for activation of neurogenic genes. Once sufficient levels of CaMKIIdelta are achieved (5-7 hr), it will, directly or indirectly, mediate phosphorylation and activation of PARP-1, which then catalyzes poly(ADP-ribosyl)ation of TLE1 and most of the other components to the corepressor complex. This is consistent with the observation that calcium signaling evoked by extrinsic and intrinsic cues can induce auto-poly(ADP-ribosyl)ation of PARP-1; however, CaMKIIdelta may also be activated in a calcium-independent fashion. This covalent modification is suggested to result in their dismissal from the biochemical complex and derepression of the MASH1 gene. The role of PARP-1 in derepression of MASH1 and its retention on the activated MASH1 promoter is quite consistent with reports that poly(ADP-ribosyl)ation of chromatin-associated proteins induce major changes in chromosomal architecture. However, in the case of MASH1, it was found that derepression alone is insufficient for induction. This is in accord with the findings for other regulated transcription factors. For example, the loss of the N-CoR corepressor is not alone sufficient to activate most AP-1-regulated genes, and only in a subset of RAR target genes does derepression result in a signal-independent 'default' activation of gene targets.

These data also indicate that, in addition to Ca²⁺-CaMKII-dependent dissociation of the TLE1 corepressor from HES1, a covalent modification of HES1 itself is required to permit activation of MASH1. Thus, activation of MASH1 is linked to sequential CaMKIIdelta-dependent activation of PARP-1 enzymatic activity, which was previously inhibited in the TLE1 holocorepressor complex, permitting dismissal of the TLE1 complex, derepression and phosphorylation of HES1, and recruitment of specific coactivators and thus causing and maintaining derepression of genes mediating neuronal differentiation. The actions of PARP-1 in the TLE1-mediated events are thus analogous to the effects of covalent modifications by phosphorylation and acetylation, as mediators of switches from repressor to activator function (Ju, 2004).

While HES1 is recognized to regulate tissue morphogenesis by maintaining undifferentiated cells and preventing differentiation, continued occupancy of the MASH1 promoter by HES1 in the differentiating neural stem cells has surprisingly proven to be required to initiate MASH1 activation events. Indeed, previous, seemingly contradictory reports of transient transfection assays in which HES1 can inhibit acid beta-glucosidase genes in HepG2 cells but cause activation in fibroblasts are likely to be explained by findings of signal-dependent HES1 switching events (Ju, 2004).

This cortical progenitor culture system has permitted identification of a regulatory pathway that may be, at least in part, partially compensated in vivo because many nestin-positive neural stem cells in the subventricular zone proliferate without losing the multipotentiality to differentiate into neurons in HES1 mutant or even HES1/HES5 double mutant mice. It is suggested that there may be additional HES1-like repressors or unidentified protein partners, including HERP and other E box binding proteins, that are also potentially involved in MASH1 gene activation. The identification of this unexpected mechanism of HES1 action in cortical progenitor cell cultures suggest that, in this system, the other molecules that could assume a similar function were either not expressed or required. The finding of a requirement of HES1 in activation of neurogenic genes is consistent with suggestions that HES1 might promote differentiation, in addition to its role in maintenance of the undifferentiated state, at multiple steps of neural stem cell development (Ju, 2004).

In summary, a pathway is suggested in which a PARP-1-containing TLE1 complex is recruited by the Notch-induced bHLH factor, HES1, initially mediating repression of MASH1 in the proliferating neural stem cells. The data suggest that signals that induce neuronal differentiation, such as PDGF in neural stem cells, act to induce the CaMKIIdelta isoform, which, in turn, is required for HES1-mediated MASH1 activation . The temporal aspects of CaMKIIdelta induction appear to account for the delay in derepression and activation of MASH1 expression following critical PDGF signaling. CaMKIIdelta-induced phosphorylation of a specific serine residue in the orange domain of HES1 permits it to recruit coactivators, including CBP, and has proven to be required for activation of the MASH1. The conserved relationship of the HES1 orange domain with the HLH domain raises the possibility of an additional role in protein interactions that include dimerization (Ju, 2004).

In a sense, the observation that a component of the TLE1-mediated repression complex, PARP-1, is also required for derepression events and maintenance of activation, parallels the requirement of TBL1/TBLR1 complex for ligand-dependent exchange of N-CoR corepressor complexes for coactivators in the switch of nuclear receptor function from repression to activation. TBLR1 is required for recruitment of the ubiquitylation/19S proteosome complex to prevent N-CoR/SMRT-dependent maintenance of a repression checkpoint. It is suggested that PARP-1 may achieve the same effects by a distinct modification strategy and serves as a regulated sensor of neuron-inducing signals based on the actions of CaMKIIdelta induced by the initial stimulus to neuronal differentiation. Therefore, it is tempting to speculate that PARP-1 and TBLR1 may be critical for the exchange events required to overcome the repression checkpoint for TLE- and N-CoR-regulated repressors, respectively (Ju, 2004).

The dual functions of PARP-1 and HES1 in the progression of neural stem cells along a neuronal pathway indicate that while the initial Notch signal causes repression of neurogenic genes by induction of HES1, it simultaneously arms the response to subsequent Ca²⁺-CaMKII signals that permit MASH1 gene activation events. The data illustrate how a single signaling pathway can mediate a sequential, two-step derepression/activation process required for development in gene activation. Induction of a Ca²⁺/CaMKII-dependent program initiates both PARP-1 activation, which is required for dismissal of the TLE1 corepressor complex, and a second event, covalent modification of HES1, which is required for target gene activation. The requirement for both a derepression and independently mediated activation event is likely to be prototypic of many similar functions of PARP-1 factors in development. The sequential calcium-regulated PARP-1-dependent switch from repression to derepression to activation function of HES1 is clearly an effective strategy to maximize the amplitude of the transcriptional response of neurogenic gene expression to signals and to permit temporally precise patterns of cellular response (Ju, 2004).

C. elegans Hairy-related proteins

In C. elegans, six lateral epidermal stem cells, the seam cells V1-V6, are located in a row along the anterior-posterior (A/P) body axis. Anterior seam cells (V1-V4) undergo a fairly simple sequence of stem cell divisions and generate only epidermal cells. Posterior seam cells (V5 and V6) undergo a more complicated sequence of cell divisions that include additional rounds of stem cell proliferation and the production of neural as well as epidermal cells. In the wild type, activity of the gene lin-22 allows V1-V4 to generate their normal epidermal lineages rather than V5-like lineages. lin-22 activity is also required to prevent additional neurons from being produced by one branch of the V5 lineage. The lin-22 gene exhibits homology to the Drosophila gene hairy. lin-22 activity represses neural development within the V5 lineage by blocking expression of the posterior-specific Hox gene mab-5, an antennapedia homolog, in specific cells. In order to prevent anterior V cells from generating V5-like lineages, wild-type lin-22 gene activity must inhibit (directly or indirectly) at least five downstream regulatory gene activities. In anterior body regions, lin-22(+) inhibits expression of the Hox gene mab-5. It also inhibits the activity of the achaete-scute homolog lin-32 and an unidentified gene that is postulated to regulates stem cell division. Each of these three genes is required for the expression of a different piece of the ectopic V5-like lineages generated in lin-22 mutants. In addition, lin-22 activity prevents two other Hox genes, lin-39 and egl-5, from acquiring new activities within their normal domains of function along the A/P body axis. It appears that the caudal homolog pal-1 allows V6 to ignore lin-22 activity. pal-1 is required for V6 to develop differently from V(1-4). Some, but not all, of the patterning activities of lin-22 in C. elegans resemble those of hairy in Drosophila. However, it is likely that lin-22's role in global A/P patterning is novel. In Drosophila, hairy plays a role in segmentation, but does not similarly promote more global A/P patterning (Wrischnik, 1997).

Hox genes control the choice of cell fates along the anteroposterior (AP) body axis of many organisms. In C. elegans, two Hox genes, lin-39 and mab-5, control the cell fusion decision of the 12 ventrally located Pn.p cells. Specific Pn.p cells fuse with an epidermal syncytium, hyp7, in a sexually dimorphic pattern. In hermaphrodites, Pn.p cells in the mid-body region remain unfused whereas in males, Pn.p cells adopt an alternating pattern of syncytial and unfused fates. The complexity of these fusion patterns arises because the activities of these two Hox proteins are regulated in a sex-specific manner. MAB-5 activity is inhibited in hermaphrodite Pn.p cells and thus MAB-5 normally only affects the male Pn.p fusion pattern. A gene has been identified, ref-1, that regulates the hermaphrodite Pn.p cell fusion pattern largely by regulating MAB-5 activity in these cells. Mutation of ref-1 also affects the fate of other epidermal cells in distinct AP body regions. ref-1 encodes a protein with two basic helix-loop-helix domains distantly related to those of the hairy/Enhancer of split family. ref-1, and another hairy homolog, lin-22, regulate similar cell fate decisions in different body regions along the C. elegans AP body axis (Alper, 2001).

Much of the C. elegans epidermal layer, the hypodermis, is composed of several multinucleate cells (syncytia) that are formed by the fusion of mononucleate cells throughout embryonic and postembryonic development. One such syncytium, hyp7, extends over most of the length of the worm and contains 133 nuclei, close to 15% of all somatic nuclei in the worm. How is the fusion of all these cells coordinately regulated to allow formation of hyp7? To understand how the hyp7 syncytium is generated, the regulation of the fusion decision of one group of cells called the Pn.p cells that line the ventral surface of the worm during the first larval stage (L1) has been studied. Pn.p cell fusion is regulated by two genes of the C. elegans Hox gene cluster. The Hox cluster consists of six genes: ceh-13, lin-39 and mab-5, homologs of Drosophila labial, Sex combs reduced and Antennapedia, respectively, and egl-5, php-3 and nob-1, three Abdominal-B homologs. In C. elegans, as in other organisms, the Hox genes regulate the choice of cell fates along the AP body axis. However, the simple Hox gene expression pattern in C. elegans is insufficient to explain the complex Pn.p cell fusion pattern. This is due to the sex-specific, post-translational regulation of two Hox genes, lin-39 and mab-5. In hermaphrodites, MAB-5 is inactive and only LIN-39 influences Pn.p cell fusion fate. In males, both LIN-39 and MAB-5 are active, but the two proteins interact in an unusual way to control cell fusion. It is quite likely that in most species, Hox proteins interact with each other and with other factors to generate more complexity than their expression patterns alone would allow (Alper, 2001).

Understanding how these interactions modulate Hox protein activity is therefore necessary to understand fully how an animal body plan is laid out. At the end of the first larval stage, some of the 12 Pn.p cells fuse with the hyp7 syncytium in a sex-specific pattern. In hermaphrodites, anterior (P1.p and P2.p) and posterior P(9-11).p cells fuse with the hyp7 syncytium while the six central cells P(3-8).p remain unfused. These six unfused cells, the vulval precursor cells, remain competent to develop further, and some of these cells generate the hermaphrodite vulva later in development. The Pn.p cell fusion pattern is different in males, with P1.p, P2.p, P7.p and P8.p fusing with hyp7 and P(3-6).p and P(9-11).p remaining unfused. The posterior unfused cells generate male-specific copulatory structures later in development (Alper, 2001).

Two Hox genes, lin-39 and mab-5, are known to influence Pn.p cell fusion. lin-39 is expressed in P(3-8).p in both hermaphrodites and males. In hermaphrodites, lin-39 prevents fusion of those Pn.p cells in which it is expressed and therefore P(3-8).p remain unfused. Thus, in a lin-39 mutant, all hermaphrodite Pn.p cells fuse with the hyp7 syncytium and are unable to generate a vulva. The regulation of Pn.p cell fusion in males is more complex because both lin-39 and mab-5 can affect the fusion decision. mab-5 is expressed in P(7-11).p in both sexes, but only functions in males. Acting alone, either Hox gene is able to prevent fusion of those cells within which it is expressed: P(3-6).p for lin-39 and P(9- 11).p for mab-5. However, when cells express both Hox genes (P7.p and P8.p), those cells fuse with hyp7, much like cells that contain neither Hox gene (P1.p and P2.p). The ability of these two Hox genes to negate each other's effects in males occurs post-translationally; that is, LIN-39 and MAB-5 proteins can somehow inhibit each other's activity when both proteins are present in the same cell. Moreover, the relative levels of the two proteins do not matter because the two proteins are still capable of inhibiting each other when one of the Hox genes is strongly overexpressed. This result argues against a model in which the two Hox proteins simply sequester each other and, as a consequence, titrate each other's activity. Instead, something else appears to be limiting in this cell fate decision. One possibility is that both proteins bind to regulatory sites in the same target gene, which in turn encodes a protein that directly affects cell fusion. In this model, the binding of either protein alone influences the activity of the fusion gene, whereas the binding of both Hox proteins together does not (Alper, 2001).

In summary, Hox protein activity is regulated in two key ways to control the Pn.p cell fusion decision. (1) MAB-5 is present in the same cells in both sexes but only functions in male Pn.p cells. Thus, something keeps MAB-5 inactive in the hermaphrodite Pn.p cells. (2) Both Hox proteins can interact to inhibit each other when present in the same Pn.p cell in males. To identify genes that affect Pn.p cell fusion by regulating Hox protein activity, mutations were isolated that alter the Pn.p cell fusion pattern. One such mutation, ref-1(mu220) (REgulator of Fusion-1) prevents fusion of posterior Pn.p cells in hermaphrodites, largely, but not completely, by affecting the sex-specific activity of MAB-5. ref-1 mutants also exhibit a defect in the specification of the fate of a hypodermal cell located on the lateral surface of the worm in this same posterior body region as well as other defects in the anterior part of the worm. ref-1 has been cloned and it encodes a transcription factor with two basic helix-loop-helix (bHLH) domains, both of which are distantly related to the hairy/Enhancer of split [E(spl)] subfamily of such proteins (Alper, 2001).

ref-1 encodes a protein with an unusual structure: it contains two predicted bHLH domains. A family of such predicted proteins is present in the C. elegans genome. Interestingly, the first and second bHLH domains within a given protein tend to be less similar to each other than to the corresponding bHLH domain in other family members. This suggests that this family may have arisen from the duplication of a single gene in which the bHLH domain had already been duplicated. These bHLH domains are most similar to those of the hairy/E(spl) family bHLH domains, although they are substantially diverged. The bHLH domains in ref-1 are only about 30% identical to the bHLH domains of other hairy family members; in contrast, the bHLH domain of lin-22, another C. elegans hairy homolog, is 60% identical to other family members. ref-1 contains a FRPWE domain at its COOH terminus, a variant of the conserved WRPW domain found at the COOH terminus of hairy family members. This domain is used by Hairy to recruit the corepressor protein Groucho. The C. elegans groucho homolog, unc-37, is also a corepressor in other cell fate decisions, although unc-37(e262) does not affect hermaphrodite Pn.p cell fusion. While this is not a null allele of unc-37 (null alleles are lethal), it raises the possibility that the FRPWE motif in REF-1 may no longer function in recruiting groucho (Alper, 2001).

Why does ref-1 contain two bHLH domains? bHLH proteins typically function as dimers with the HLH domains acting as a dimerization region and the basic region contacting DNA. Such dimers could be homodimers or heterodimers between different bHLH proteins. Some proteins lacking the basic domain act as repressors by sequestering partner bHLH proteins in a complex that does not bind DNA. hairy family members, in contrast, are often active repressors that recruit Groucho to inhibit transcription. Since the mu220 allele affects a conserved residue in the first basic region, it is likely that ref-1 functions by binding DNA and that at least the first bHLH domain is required for this interaction. The second bHLH domain, if functional, could interact with the first bHLH domain to regulate it or could interact with other bHLH proteins. The other C. elegans genes that encode proteins with two bHLH domains are candidate genes for this interaction (Alper, 2001).

The molecular identity of ref-1 is intriguing in light of the other ref-1 mutant phenotypes. In Drosophila, hairy acts as both a primary pair rule gene that specifies the fate of alternate segments and also as a regulator of neuron formation later in development. Two C. elegans hairy homologs, ref-1 and lin-22, also affect cell fate and neuron formation; moreover, they both do so in distinct AP body regions. For example, ref-1 mutants have a hermaphrodite Pn.p cell fusion defect in the posterior Pn.p cells (although ref-1 can influence Pn.p cell fate in more anterior Pn.p cells as revealed in a lin-39 or mab-5 background). In contrast, lin-22 mutants have a male-specific Pn.p cell fusion defect in more anterior Pn.p cells. In lin-22 mutants, P7.p and P8.p remain unfused during the first larval stage. In addition, P(3-8).p all continue to remain unfused inappropriately and divide later in development. Like flies carrying a mutation in hairy, ref-1 and lin-22 mutants also generate ectopic neuroblasts. Specifically, ectopic sensory structurs termed postdeirids are generated by the posterior V6 lateral seam cell in ref-1 mutants and by the anterior V1-V4 seam cells in lin-22 mutants. These phenotypes are consistent with a partial (in the case of ref-1) or complete (in the case of lin-22) transformation of the respective V cells into a V5-like cell fate. No other single mutation can cause the transformation of V6 to a V5-like fate, although the double mutant combination of lin-22 and pal-1 (a Caudal homeobox transcription factor homolog) does so. No synergy is observed between lin-22 and ref-1. ref-1;lin-22 double mutant worms have phenotypes that are simply the summation of the phenotypes of the individual mutants. This observation is consistent with the hypothesis that while ref-1 and lin-22 have similar functions, they act in distinct AP body regions (Alper, 2001).

ref-1 mutants have defects not found in lin-22 in still another AP body region: the head. ref-1 mutants exhibit a misshapen head defect and also have defects in the specification of the proper fate of the anterior H1 seam cell. While hairy acts as a pair rule gene affecting the fate of every other segment in Drosophila, in some sense, these two C. elegans hairy homologs are behaving more like gap genes in that they are required for the specification of cell fates in distinct AP body domains. However, since these mutations affect a limited repertoire of phenotypes, it is also possible that ref-1 affects cell fate in a cell-type-specific manner (Alper, 2001).

Sexual dimorphism in the nervous system is required for sexual behavior and reproduction in many metazoan species. However, little is known of how sex determination pathways impose sex specificity on nervous system development. In C. elegans, the conserved sexual regulator MAB-3 controls several aspects of male development, including formation of V rays, male-specific sense organs required for mating. MAB-3 promotes expression of the proneural Atonal homolog LIN-32 in V ray precursors by transcriptional repression of ref-1, a member of the Hes family of neurogenic factors. Mutations in ref-1 restore lin-32::gfp expression and normal V ray development to mab-3 mutants, suggesting that ref-1 is the primary target of MAB-3 in the V ray lineage. Proteins related to MAB-3 (DM domain proteins: see Drosophila Doublesex) control sexual differentiation in diverse metazoans. It is therefore suggested that regulation of Hes genes by DM domain proteins may be a general mechanism for specifying sex-specific neurons (Ross, 2005).

MAB-3 promotes development of male-specific sensory neurons by regulating two bHLH factors. In V ray precursors, MAB-3 indirectly promotes expression of the proneural protein LIN-32 by preventing expression of REF-1, a distant homolog of the Hes family of neurogenic proteins. REF-1 is a negative regulator of lin-32; lin-32::gfp expression is dramatically reduced in the mab-3 V ray lineage, but is restored by the introduction of a ref-1 mutation. This REF-1-mediated repression of lin-32 is necessary to prevent V ray formation in mab-3 mutants, as evidenced by the observation that ref-1 mutations restore V ray development in mab-3 mutants. Furthermore, ectopic ref-1 expression is sufficient to cause V ray defects in wild-type males. These results indicate that MAB-3 acts in parallel to Hox proteins to promote activation of lin-32 by preventing expression of ref-1, a gene with antineural activity (Ross, 2005).

Two conserved ref-1 regulatory elements (A and B) have been identified and putative MAB-3 binding sites have been identified within these elements. Wild-type MAB-3, is required to prevent ref-1::gfp expression during V ray development. Based on these observations, the following model is proposed. In wild-type V ray precursors, MAB-3 promotes LIN-32-mediated V ray development by binding within one or both of the conserved elements to prevent activation of ref-1 by an unknown factor, X. In the mab-3 mutant V ray lineage, ref-1 is inappropriately activated by X and disrupts V ray formation by preventing Hox-mediated activation of lin-32. ref-1-inactivating mutations relieve this repression of lin-32, restoring normal V ray formation in mab-3 ref-1 double mutants (Ross, 2005).

ref-1 is initially expressed in the posterior hypodermal seam cells in young males and is downregulated when mab-3 is first expressed. Although the identities of ref-1 activators are unknown, a binding site for one such factor may overlap the MAB-3 binding site in element B; disruption of this MAB-3 site eliminates ref-1 expression in the seam. Thus, MAB-3 may repress ref-1 by physically interfering with binding or function of activators bound to nearby sites. Similarly, overlapping binding sites for DSX and a bZIP transcription factor coordinate regulation of yolk expression in Drosophila. The structure of DM domain proteins may be particularly suited for interaction with transcription factors that bind overlapping DNA sites. DSX binds in the minor groove of DNA, which might allow close apposition with major groove binding transcription factors (Ross, 2005).

It is possible that the weak mab-3-suppressing mutation ref-1(ez6) reduces ref-1 expression by disrupting a second positive regulatory site in element A. However, ref-1 transgenes containing the ez6 lesion are expressed in the seam and rescue the ref-1(ez11) V ray phenotype. The rescuing activity and expression of ref-1 transgenes driven by ez6 mutant regulatory sequences may be a consequence of high copy number of the reporter or may indicate a minor role for this element (Ross, 2005).

All sex-specific development in the C. elegans soma occurs downstream of the zinc finger transcription factor TRA-1, the terminal global regulator in the sex determination cascade. However, the connection between TRA-1 and male-specific effectors that drive V ray development remains obscure. MAB-3 represses ref-1 expression in males to allow specification of V rays by LIN-32. While it might follow that REF-1 normally prevents V ray formation in hermaphrodites, this does not appear to be the case. Although ref-1 is expressed in hermaphrodite seam cells, ref-1 mutant hermaphrodites do not produce ectopic V rays or express ectopic lin-32::gfp. TRA-1 must somehow prevent V ray formation in hermaphrodites. TRA-1 might regulate lin-32 expression directly or might prevent lin-32 activation indirectly by regulating Hox gene activity. EGL-5 expression in the V6 lineage is sex specific and could be regulated by TRA-1. MAB-5 is expressed in the V6 lineage in both sexes, but TRA-1 might modulate MAB-5 activity, for example by controlling factors that modify MAB-5 posttranslationally (Ross, 2005).

Male-specific regulation of ref-1 by MAB-3 also must require additional regulators. Although mab-3 is expressed in hermaphrodites, it only represses ref-1 in males. It is possible that mab-3 requires a male-specific coregulator. Alternatively, MAB-3 may be posttranslationally modified such that it is active only in males (Ross, 2005).

Proteins of the Hes family of neurogenic regulators typically share a characteristic bHLH domain, an Orange domain that may confer functional specificity, and a C-terminal WRPW sequence required for interaction with the corepressor Groucho (Gro). Although the bHLH domains of REF-1 are most similar to those of the Hes family, the overall resemblance of REF-1 to Hes proteins is weak. The six C. elegans REF-1-like proteins are unusual in that they each possess two bHLH domains. Furthermore, the REF-1 bHLH domains are only 28% and 22% identical to the bHLH domain of Hairy and lack a basic domain proline that is conserved in other Hes proteins. By contrast, the bHLH domain of LIN-22, a second C. elegans Hairy homolog, is 51% identical to that of Hairy. Additionally, REF-1 lacks an Orange domain and contains a C-terminal FRPWE, rather than WRPW, sequence (Ross, 2005).

Despite sequence and structural differences, REF-1 bears striking functional homology to other Hes proteins. In flies, Hairy and E(spl) proteins progressively limit domains of neurogenesis in the peripheral nervous system by interfering with the activity of proneural factors like Achaete (Ac), Scute (Sc), and Atonal. During sensory bristle formation, Hairy binds directly to an ac-sc enhancer to restrict spatial expression of ac. E(spl) proteins act later, in response to Notch signaling, to downregulate proneural gene expression in presumptive epidermal cells by interfering with an autostimulatory feedback loop. E(spl) proteins also antagonize proneural proteins by interfering with activation of proneural target genes. In both cases, E(spl)-mediated repression can occur by direct DNA binding or by protein-protein interactions with proneural activators that are bound to their own sites. Repression by either mechanism requires recruitment of Gro. Vertebrate Hes proteins can act as transcriptional repressors and are also thought to prevent activation by sequestering the MASH or MATH proneural proteins in inactive heterodimer complexes (Ross, 2005).

Like Hes proteins, REF-1 prevents neurogenesis by negatively regulating a proneural protein, LIN-32. ref-1-dependent reduction of lin-32::gfp expression in mab-3 mutant males suggests that REF-1 is likely to repress lin-32 transcription. Consistent with this, REF-1 proteins with substitutions in the first basic domain (mu220 and ez11) fail to repress neurogenesis and lin-32::gfp expression, suggesting that DNA binding is required. In addition, the lin-32 promoter contains many potential REF-1 binding sites (E boxes and N boxes). It is unclear whether REF-1 interacts with the Gro homolog UNC-37 to negatively regulate lin-32. ref-1 transgenes lacking the FRPWE domain weakly rescue the ref-1 phenotype, suggesting that this sequence is partially dispensable for ref-1 function. It is possible that another sequence mediates REF-1/UNC-37 interactions or that REF-1 interacts with a different corepressor. The possibility cannot be excluded that REF-1 regulates lin-32 posttranscriptionally, perhaps by forming an unstable heterodimer with LIN-32 or by interfering with a positive feedback mechanism that would normally increase lin-32 expression (Ross, 2005).

Both ref-1 and lin-32 are required for normal development of two neuronal structures derived from seam cells. REF-1 negatively regulates development of V5- and V6-derived sensory rays and production of the postdeirid, a neuroblast normally derived from V5. In contrast, LIN-32 promotes sensory ray and postdeirid formation (Ross, 2005)

The Hes protein LIN-22 prevents neurogenesis in anterior seam cells V1-V4 . In lin-22 mutants, V1-V4 undergo a V5-like lineage to produce a postdeirid and two sensory rays. The ectopic postdeirid depends on lin-32, suggesting that LIN-22 negatively regulates lin-32 in V1-V4. REF-1 and LIN-22 appear to affect postdeirid production regionally, with LIN-22 acting in the anterior and REF-1 in the posterior seam (Ross, 2005).

Ectopic sensory ray production in lin-22 mutants requires Hox, lin-32, and mab-3 activity, suggesting that LIN-22 acts upstream of the network of V ray regulators. ref-1 and lin-22 interact to inhibit V ray formation in V1-V4. ref-1 mutations cause ectopic ray formation in mab-3; lin-22 double mutants, which normally do not produce V1-V4-derived rays. This suggests that, at least in mab-3 mutants, LIN-22 acts upstream of REF-1 in a hierarchy of bHLH proteins controlling V ray neurogenesis (Ross, 2005).

During Drosophila peripheral neurogenesis, Hairy acts early to establish a prepattern of cells competent to become neurons. E(spl) proteins subsequently define the subgroup of these cells that will form sensory organs. The inappropriate neurogenesis in V1-V4 in lin-22 mutants suggests that LIN-22, like Hairy, acts globally to define which seam cells are competent to produce neuronal lineages. The experiments suggest that REF-1, like E(spl), may then act downstream within these lineages to refine which cells will become neurons (Ross, 2005).

One mechanism by which DM domain proteins regulate sexual dimorphism is the sex-specific modulation of developmental programs. For example, DSX^F inhibits Wingless and FGF pathway activity and DSX^M sex specifically inhibits Dpp signaling. Although direct targets are not known, this inhibition is likely to occur by transcriptional regulation of key pathway components. The DM domain protein MAB-3 represses the Hes family bHLH protein REF-1 in males to modulate sex-specific nervous system development (Ross, 2005).

Hes proteins regulate both the extent of neurogenesis and the specification of neuronal subtypes. In Drosophila, E(spl) mutations lead to ectopic neurogenesis, while overexpression prevents neurogenesis. In the mouse brain, Hes proteins control timing of cell differentiation to regulate brain size, shape, and cell arrangement, possibly via interactions with cell-cycle regulators. Thus, it is clear that sex-specific regulation of Hes activity in the developing nervous system could achieve sexual dimorphism in organ shape, size, cell fate, or timing of differentiation. MAB-3/REF-1 interactions provide an example of such regulation (Ross, 2005).

mab-3 mutant males produce epidermal cells at the expense of neuronal cells, a phenotype like that caused by Hes overexpression in other organisms. In addition, V ray precursor cell divisions of mab-3 mutants are often delayed relative to wild-type. This delay may reflect an interaction between ref-1 and cell-cycle regulators, similar to that seen for mouse Hes proteins. While no interactions between bHLH proteins and DSX have been described, these seem likely based on functional homology between MAB-3 and DSX. Male sex combs are a likely candidate for this mode of regulation, since bristle formation in flies is regulated by Hes proteins. DSX also regulates sexual dimorphism in abdominal neuroblasts, which undergo more cell divisions in males than in females. It is possible that this sex-specific proliferation is controlled by DSX/bHLH interactions (Ross, 2005).

This work establishes that sex-specific regulation of REF-1 and LIN-32 by MAB-3 can regulate development of male-specific neurons in C. elegans. Future studies will reveal whether sex-specific regulation of bHLH proteins by DM domain transcription factors is a conserved mechanism for generating sexual dimorphism in the nervous system (Ross, 2005).

Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider: Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen)

The Wnt genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration. In bilaterians, there are at least 12 subfamilies of Wnt genes. Wnt3 and Wnt8 are required for somitogenesis in vertebrates and are thought to be involved in posterior specification in deuterostomes in general. Although TCF and β-catenin have been implicated in the posterior patterning of some short-germ insects, the specific Wnt ligands required for posterior specification in insects and other protostomes remained unknown. This study investigated the function of Wnt8 in a chelicerate, the common house spider Achaearanea tepidariorum. Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. These results suggest that in the spider, Wnt8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, it is proposed that Wnt8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes (McGregor, 2008).

The posterior truncation phenotypes resulting from pRNAi against Wnt8 in the spider are at least superficially similar to those observed when Wnt8 and/or Wnt3 are perturbed in vertebrate embryos. Removal or blocking Wnt8 and/or Wnt3 in Xenopus, zebrafish, and mouse results in truncated embryos with only a few anterior somites and no tail bud. Although analysis of TCF and β-catenin in Oncopeltus and Gryllus, respectively, indicated that Wnt signaling might be involved in the development of the growth zone and posterior segments in arthropods, the current data show that in fact the same ligand, Wnt8, is employed in posterior development in both vertebrates and arthropods (McGregor, 2008).

In class II and III At-Wnt8^pRNAi embryos exhibiting fused L4 limb buds, it also appeared that the most ventral part of this segment is missing. This phenotype shows similarities to the phenotype when short-gastrulation is knocked down in this spider. It suggests that, in addition to A-P patterning, At-Wnt8 is involved in D-V patterning in the spider, a role Wnt8 genes also perform in vertebrates (McGregor, 2008).

There is evidence that Wnt signaling acts upstream of Delta/Notch in vertebrate somitogenesis. Although the expression of Wnt3a and Wnt8 is not cyclical during somitogenesis in vertebrates, some downstream components of Wnt signaling, such as Axin2, are cyclically expressed in mice and possibly are integral to the Delta/Notch-dependent segmentation clock. However, recent experiments have shown that Axin2 and components of the Delta/Notch pathway continue to oscillate in the presence of stabilized β-catenin, which suggests that in mice, Wnt signaling may be permissive for the segmentation clock rather than instructive. Similarly, in zebrafish it is thought that Wnt8 may act to maintain a precursor population of stem cells in the PSM and tailbud rather than directly regulate the segmentation clock. It is proposed that the same ligand, Wnt8, could play a similar permissive role for segmentation in the growth zone of the spider by establishing and possibly maintaining a pool of cells that develop into the opisthosomal segments. When At-Wnt8 activity is reduced, cells are ectopically used in L3/L4 or internalized, depleting the putative growth-zone pool. This depletion manifests as a smaller opisthosoma, separated clusters of cells that give rise to separate irregular germbands, or even no opisthosoma (McGregor, 2008).

It was previously shown that Delta/Notch signaling is also involved in posterior development in the spiders Cupiennius. These new results reveal that in the spider, Wnt8 is required for the clearing of Dl and h expression in the posterior and that this is necessary for repression of twi, activation of cad, and establishment of the growth zone (McGregor, 2008).

The involvement of Wnt8, Delta/Notch signaling, and cad in the posterior development of other arthropods has also been directly demonstrated by functional analysis or inferred from expression patterns, and in vertebrates, Wnt3a and Wnt8 probably act upstream of Delta/Notch and cad during somitogenesis. Taken together, this suggests that a regulatory genetic network for posterior specification including Wnt8, Delta/Notch signaling, and cad could have been present in the last common ancestor of protostomes and deuterostomes, but has subsequently been modified in some lineages. For example, in Drosophila, Delta/Notch signaling is not involved in segmentation, and although the Drosophila Wnt8 ortholog, WntD, is required for D-V patterning, it is not involved in posterior development. Segments arise almost simultaneously in Drosophila, rather than sequentially from a growth zone, so this may suggest that the role of Wnt8 in posterior development was not required for this mode of development and therefore was lost during the evolution of these insects (McGregor, 2008).

These results suggest that Wnt8 regulates formation of the posterior growth zone and then maintains a pool of undifferentiated cells in this tissue required for development of the opisthosoma. Wnt signaling thus regulates the establishment and maintenance of an undifferentiated pool of posterior cells in both vertebrates and spiders and in fact the same Wnt ligand, Wnt8, is used in both phyla. Therefore, Wnt8 could be part of an ancient genetic regulatory network, also including Dl, Notch, h, and cad, that was used for posterior specification in the last common ancestor of deuterostomes and protostomes (McGregor, 2008).

Divergent Hairy-related proteins

Members of the Hairy/Enhancer of Split family of basic helix-loop-helix (bHLH) transcription factors are regulated by the Notch signaling pathway in vertebrate and Drosophila embryos and control cell fates and establishment of sharp boundaries of gene expression. A new subclass of bHLH proteins [HRT1 (Hairy-related transcription factor 1), HRT2, and HRT3], is described that shares high homology with the Hairy family of proteins yet has characteristics that are distinct from those of Hairy and other bHLH proteins. Sequence comparison of HRT1-3 reveals that each encodes a protein containing a bHLH domain toward the amino-terminus. HRT2 and HRT3 share 57% and 45% homology with HRT1 over the entire protein, but display regions of increased homology at the amino- and carboxy-termini. The highest region of homology is observed in the bHLH domain, where HRT2 and HRT3 shared 96% and 91% homology with HRT1, respectively. Comparison of the bHLH domain of HRT1-3 with the bHLH domain of other known bHLH proteins reveals the presence of critical residues typical of bHLH proteins. Only two amino acid mismatches, in identical residues for each HRT protein, are present compared to a predicted model of bHLH factors. The basic regions, or DNA-binding domains, of HRT1-3 are nearly identical, suggesting that HRT1-3 may interact with common promoter/enhancer DNA sequences. The HRT proteins have closest homology to a family of transcriptional repressors, including Hairy and HES-1. They typically contain a conserved region adjacent to the bHLH region, known as an orange domain, that confers functional specificity among Hairy family proteins. The three HRT proteins are highly conserved in a region just C-terminal to the bHLH region and share significant similarity in this stretch with the orange domain of Hairy-related proteins. Each HRT gene is expressed in distinct cell types within numerous organs, particularly in those patterned along the anterior-posterior axis. HRT1 and HRT2 are expressed in atrial and ventricular precursors, respectively, and are also expressed in the cardiac outflow tract and aortic arch arteries. HRT1 and HRT2 transcripts are also detected in precursors of the pharyngeal arches and subsequently in the pharyngeal clefts. Within somitic precursors, HRT1 and HRT3 exhibit dynamic expression in the presomitic mesoderm, mirroring the expression of other components of Notch-Delta signaling pathways. The HRT genes are expressed in other sites of epithelial-mesenchymal interactions, including the developing kidneys, brain, limb buds, and vasculature. The unique and complementary expression patterns of this novel subfamily of bHLH proteins suggest a previously unrecognized role for Hairy-related pathways in segmental patterning of the heart and pharyngeal arches, among other organs (Nakagawa, 1999).

The amphioxus Hairy family: differential fate after duplication

Vertebrate Hairy genes are highly pleiotropic and have been implicated in numerous functions, such as somitogenesis, neurogenesis and endocrine tissue development. In order to gain insight into the timing of acquisition of these roles by the Hairy subfamily, the Hairy genes in amphioxus have been cloned and the expression patterns have been studied. The cephalochordate amphioxus is widely believed to be the living invertebrate more closely related to vertebrates, the genome of which has not undergone the massive gene duplications that took place early during vertebrate evolution. Surprisingly, eight Hairy genes were isolated from the 'pre-duplicative' amphioxus genome. In situ hybridization on amphioxus embryos showed that Hairy genes had experienced a process of subfunctionalization that is predicted in the DDC model (for duplication-degeneration-complementation). Only the summation of four out of the eight Amphi-Hairy genes expression resembles the expression pattern of vertebrate Hairy genes, i.e., in the central nervous system, presomitic mesoderm, somites, notochord and gut. In addition, Amphi-Hairy genes expression suggest that amphioxus early somites are molecularly prefigured in an anteroposterior sequence in the dorsolateral wall of the archenteron, and the presence of a midbrain/hindbrain boundary. The expansion of the amphioxus Hairy subfamily suggests caution when deducing the evolutionary history of a gene family in chordates based in the singularity of the amphioxus genome. Amphioxus may resemble the ancestor of the vertebrates, but it is not the ancestor, only its closest living relative, a privileged position that should not assume the freezing of its genome (Minguillón, 2003).

Hairy-related proteins, patterning, and differentation

During embryonic development in vertebrates, the endoderm becomes patterned along the anteroposterior axis to produce distinct derivatives. How this regulation is controlled is not well understood. The zebrafish hairy/enhancer of split [E(spl)]-related gene her5 plays a critical role in this process. At gastrulation, following endoderm induction and further cell interaction processes, including a local release of Notch/Delta signaling, her5 expression is progressively excluded from the presumptive anterior- and posterior-most mesendodermal territories to become restricted to an adjacent subpopulation of dorsal endodermal precursors. Ectopic misexpressions of wild-type and mutant forms of her5 reveal that her5 functions primarily within the endodermal/endmost mesendodermal germ layer to inhibit cell participation to the endmost-fated mesendoderm. In this process, her5 acts as an active transcriptional repressor. These features are strikingly reminiscent of the function of Drosophila Hairy/E(spl) factors in cell fate decisions. These results provide the first model for vertebrate endoderm patterning where an early regulatory step at gastrulation, mediated by her5, controls cell contribution jointly to the anterior- and posterior-most mesendodermal regions. These results identify the first regionalized, endoderm-specific factor in vertebrates, and illustrate its pivotal role in patterning the deep embryonic layers in the zebrafish gastrula (Bally-Cuif, 2000).

Dickkopf1 (Dkk1) is a secreted protein that acts as a Wnt inhibitor and, together with BMP inhibitors, is able to induce the formation of ectopic heads in Xenopus. Dkk1 null mutant embryos lack head structures anterior of the midbrain. Analysis of chimeric embryos implicates the requirement of Dkk1 in anterior axial mesendoderm but not in anterior visceral endoderm for head induction. In addition, mutant embryos show duplications and fusions of limb digits. Characterization of the limb phenotype strongly suggests a role for Dkk1 both in cell proliferation and in programmed cell death. These data provide direct genetic evidence for the requirement of secreted Wnt antagonists during embryonic patterning and implicate Dkk1 as an essential inducer during anterior specification as well as a regulator during distal limb patterning (Mukhopadhyay, 2001).

In contrast to Xenopus, the role of Dkk1 in mammals is unknown. In the mouse, Dkk1 is first expressed in the anterior domain of the gastrulating embryo. In this domain, head induction is thought to be mediated by the anterior visceral endoderm (AVE), an extraembryonic tissue essential for initiating head formation in mammalian embryos, and the anterior mesendoderm (AME), a node-derived embryonic tissue involved in anterior specification. Murine Dkk1 inhibits the axis-inducing ability of XWnt8 in Xenopus embryos, indicating that the mouse gene functions as a Wnt inhibitor comparable to its Xenopus homolog. There has been no genetic evidence that Wnt inhibitors play a role in anteroposterior patterning in the mouse. For example, no axis defects have been noted in mice that lack the function of the Cerberus homolog Cer1. Also, there has been no genetic evidence implicating Wnt signaling in antagonizing head induction during gastrulation, although a requirement has been established for Wnt/ß-catenin signaling during early axis and node induction. Mice with a Dkk1 null mutation were generated in an effort to establish the function of this gene in the early mouse embryo. Dkk1 knockout mice have two major phenotypes: they lack anterior head structures and they display forelimb malformations. These results reveal a requirement for the inhibition of Wnt signaling during mouse axis formation and limb morphogenesis (Mukhopadhyay, 2001 and references therein).

No morphological defect can be detected in the Dkk1^-/- embryos prior to the headfold stage. At the molecular level, the absence of Hesx1 gene expression in the prospective anterior neuroectodermal (ANE) cells of late streak mutant embryos is the earliest defect detected in this study. The complete absence of Hesx1 at E7.5 suggests that Dkk1 function is required for the proper expression of this gene in the AVE as well. Hesx1 expression in the ANE is required for forebrain development. Expression of Six3, another gene activated in the ANE at late streak stages, is also undetectable in Dkk1^-/- mutants. Hesx1 and Six3 are the earliest known ANE markers in the mouse. Expression of these genes in the ANE of a wild-type embryo starts at E7.5 and continues through early somite stages. In the Hesx1^-/- mutant, Six3 expression in the ANE appears normal at late streak stages but is reduced at the early somite stage. Therefore, the absence of Six3 expression at the early somite stage of Dkk1^-/- mutants may be a consequence of the loss of Hesx1 expression (Mukhopadhyay, 2001).

Proper anterior positioning of the early Dkk1-expressing AVE cells appears to be controlled by Otx2, a marker which seems unaffected in these Dkk1^-/- mutants. The severe anterior phenotype of Otx2^-/- embryos suggests that Otx2 is a key factor in the head developmental process. Since Dkk1 acts downstream of Otx2, it most likely mediates forebrain induction pathways activated by Otx2 (Mukhopadhyay, 2001).

Forebrain patterning in the mouse is initiated by the inductive activity of the AVE and subsequently requires the function of the node-derived AME. Chimeric embryos, largely composed of Dkk1^+/+ epiblast cells developing within the confines of a Dkk1^-/- visceral endoderm, display a seemingly normal anterior morphology at E9.5. This shows that Dkk1 gene expression in the AVE is not required for head induction. Notably, a recent study has demonstrated that the function of Hesx1, the gene that is essential for forebrain development and acts downstream of Dkk1, is dispensable in the AVE. Thus, cells of the elongating axial mesendoderm are the likely source of the Dkk1 signal that mediates the rescue of head organization in the chimera. This conclusion is based on studies in other vertebrates showing that Dkk1 function in the AME is required for head development. In Xenopus, injection of Dkk1 mRNA, together with a BMP inhibitor, is able to induce the complete duplication of head structures. It is believed that the head duplication resulted from an ectopic expression of prechordal plate (AME) markers, such as Xhex and Xgsc. This work identifies Dkk1, a secreted factor associated with the role of AME, in early rostral specification of the mouse embryo (Mukhopadhyay, 2001).

It is proposed that Hesx1 function in the ANE is mediated through Dkk1, which is secreted by adjacent AME. Dkk1 fits the role of a ligand that interacts with a receptor to protect the prospective anterior neuroectoderm via Wnt inhibition from caudalizing effects of the node, thereby exerting an early and indispensable function in head induction. Interestingly, Dkk1 has recently been shown to bind the Wnt coreceptor LRP6 (LDL receptor-related protein 6), thus most likely repressing type I Wnt signaling. It is not yet known which type I Wnt protein is mediating negative control of head formation in the mouse. However, in Xenopus, Wnt8 has been implicated in this process. This study clearly shows that the ablation of Dkk1 function affects not only brain development but also that of surrounding head structures. Various degrees of head truncation have also been observed in mice carrying null mutations in other factors expressed during early stages of head induction, including Hesx1, Otx, and Lim1. This suggests that there may be a coordinated morphogenetic response of neural and nonneural precursor cells to head-inducing signals (Mukhopadhyay, 2001).

The most obvious defect in mice mutant for the bHLH gene Hes1, is their lack of thymus; this gene is known to keep cells in a proliferative state. Transfer of Hes1-null fetal liver cells into RAG2-null host mice normally reconstitutes B cells but fails to generate mature T cells in the thymus. In the reconstituted thymus, T cell differentiation is arrested at the CD4(-)CD8(-) double negative (DN) stage. Both the initial T cell receptor (TCR)-independent and the subsequent TCR-dependent selective expansion during the DN stage are severely affected. Thus, Hes1 is essential for the earliest thymocyte expansion in a cell-autonomous manner (Tomita, 1999).

It has been shown that the selective proliferation of DN cells that express functional TCRs involves various transcription factors such as HMG-box factors LEF-1 and TCF-1 and zinc finger transcription factor GATA-3. However, none of these factors appears to be involved in cell expansion before TCR gene rearrangement. Thus, Hes1 is unique in regulating TCR-independent expansion of thymocytes at the earliest stage. Interestingly, in the absence of IL-7 receptor, thymocyte expansion is severely disturbed before TCR gene rearrangement, suggesting that IL-7 is one of the major growth factors involved in initial TCR-independent thymocyte proliferation. It is therefore tempting to speculate that Hes1 functions downstream of the IL-7 signaling pathway. In addition to TCR-independent expansion, Hes1 seems to be involved in expansion at later stages, because Hes1-null thymocytes are still confined exclusively to the DN fraction with negligible increase in cell number. In the developing nervous system, Hes1 is known to keep cells in a proliferative state and down regulation of Hes1 expression leads to transition to a nonproliferative differentiation stage. Present results have indicated that, as in the thymus, Hes1 promotes the initial expansion of immigrant progenitor cells, which is essential for the extensive clonal diversification and selection to generate mature T cells (Tomita, 1999 and references).

Hes1 is also known as a target gene of the Notch signaling pathway. It has been shown that both Notch and Hes1 regulate CD4 single positive versus CD8 single positive fate choice of T cell development. Although this function could not be examined here, as T cell development is arrested much earlier by Hes1 mutation, collectively these data suggest that Hes1 might function at multiple steps of T cell development (Tomita, 1999 and references).

HES6 is a novel member of the family of basic helix-loop-helix mammalian homologs of Drosophila Hairy and Enhancer of split. The biochemical and functional roles of HES6 in myoblasts has been analyzed. HES6 interacts with the corepressor transducin-like Enhancer of split 1 in yeast and mammalian cells through its WRPW COOH-terminal motif. HES6 represses transcription from an N box-containing template and also when tethered to DNA through the GAL4 DNA binding domain. On N box-containing promoters, HES6 cooperates with HES1 to achieve maximal repression. An HES6-VP16 activation domain fusion protein activates the N box-containing reporter, confirming that HES6 binds the N box in muscle cells. The expression of HES6 is induced when myoblasts fuse to become differentiated myotubes. Constitutive expression of HES6 in myoblasts inhibits expression of MyoR, a repressor of myogenesis, and induces differentiation, as evidenced by fusion into myotubes and expression of the muscle marker myosin heavy chain. Reciprocally, blocking endogenous HES6 function by using a WRPW-deleted dominant negative HES6 mutant leads to increased expression of MyoR and completely blocks the muscle development program. These results show that HES6 is an important regulator of myogenesis and suggest that MyoR is a target for HES6-dependent transcriptional repression (Gao, 2001).

Limb growth in higher vertebrate embryos is initially due to the outgrowth of limb buds and later continues as a result of elongation of the skeletal elements. The distal limb mesenchyme is crucial for limb bud outgrowth. Members of the Hairy/Enhancer of Split family of DNA binding transcriptional repressors can be effectors of Notch signaling and often act to maintain cell populations in an undifferentiated, proliferating state, properties predicted for the distal limb mesenchyme. A member of this family, c-hairy1, is expressed in this region and two alternatively spliced isoforms, c-hairy1A and c-hairy1B, of this gene are produced, predicting proteins that differ in their basic, DNA binding, domains. Viral misexpression of c-hairy1A causes a reduction in size of the limb and shortened skeletal elements, without affecting the chondrocyte differentiation program. Misexpression of c-hairy1B leads to a significantly lesser shortening of the bones, implying functional differences between the two isoforms. It is concluded that c-hairy1 regulates the size of the limb, suggesting a role for Notch signaling in the distal mesenchyme (Vasiliauskas, 2003).

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 (a bHLH transcription factor) in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, it has been found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, it was further demonstrated that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation (Esni, 2004).

In teleosts and amphibians, the proneuronal domains, which give rise to primary-motor, primary-inter and Rohon-Beard (RB) neurons, are established at the beginning of neurogenesis as three longitudinal stripes along the anteroposterior axis in the dorsal ectoderm. The proneuronal domains are prefigured by the expression of basic helix-loop-helix (bHLH) proneural genes, and separated by domains (inter-proneuronal domains) that do not express the proneural genes. Little is known about how the formation of these domains is spatially regulated. The zebrafish hairy- and enhancer of split-related (Her) genes her3 and her9 are expressed in the inter-proneuronal domains, and are required for their formation. her3 and her9 expression is not regulated by Notch signaling, but rather controlled by positional cues, in which Bmp signaling is involved. Inhibition of Her3 or Her9 by antisense morpholino oligonucleotides leads to ectopic expression of the proneural genes in part of the inter-proneuronal domains. Combined inhibition of Her3 and Her9 induces ubiquitous expression of proneural and neuronal genes in the neural plate, and abolishes the formation of the inter-proneuronal domains. Furthermore, inhibition of Her3/Her9 and Notch signaling leads to ubiquitous and homogeneous expression of proneural and neuronal genes in the neural plate, revealing that Her3/Her9 and Notch signaling have distinct roles in neurogenesis. These data indicate that her3 and her9 function as prepattern genes that link the positional dorsoventral polarity information in the posterior neuroectoderm to the spatial regulation of neurogenesis (Bae, 2005).

It is proposed that Her3 and Her9 function as prepattern genes and control the position of the proneuronal and inter-proneuronal domains through a conserved mechanism, by which Hairy also controls the position of sensory organs in the Drosophila leg. Among the Her genes in vertebrates, only a few are reported to function in a similar way. Mouse Hes1 is expressed in the olfactory placodal domains independent of Mash1 activity, thus Hes1 expression is suggested to be Notch signal independent. Combined disruption of Hes1 and the Hes5 gene, whose expression is dependent on Mash1 and thus possibly controlled by Notch signaling, leads to a strong upregulation of neurog1 expression in the olfactory epithelium. The situation is similar to the effect of the combined inhibition of Her3/Her9 and Notch signaling in the zebrafish posterior neuroectoderm. Zebrafish her5 and another hairy family member him, both of which are expressed in the MHB, function to repress neurogenesis in the MHB. Although the mechanism that induces the expression of her5 and him is not clear, the maintenance of her5 and him1 in the MHB involves Pax2.1, Eng2/3 and Fgf8, which provide positional information in the MHB. In this sense, the function of her3 and her9 is similar to that of her5 and him. Mouse Hes1 and zebrafish her5 function downstream of positional information related to the anteroposterior (AP) axis and control neurogenesis in the specific position of the AP axis, whereas her3 and her9 function as prepattern genes that control neurogenesis in the context of the dorsoventral (DV) axis in the neuroectoderm. Intriguingly, the inhibition of Her5 and Him function leads to ectopic neurog1 expression in the MHB, while leaving a neurog1-negative domain in the lateral region of MHB. her3 is expressed in the lateral region of the MHB . It is possible that her3 contributes to the repression of proneural genes in the lateral region of the MHB. her3, her5 and him might redundantly function in this region; the inhibition of Her3 function does not lead to ectopic neurog1 expression in the MHB. All of these data support the role of a subset of Her genes in the prepatterning that functions downstream of the positional information linked to the DV and AP axes (Bae, 2005).

The intervening zone (IZ) is a pool of progenitor cells located at the midbrain-hindbrain boundary (MHB) and important for MHB maintenance, midbrain-hindbrain growth and the generation of midbrain-hindbrain neurons. The Hairy/E(spl) transcription factor Her5 has been implicated in the formation of the medial (most basal) part of the IZ (MIZ) in zebrafish; the molecular bases for lateral IZ (LIZ) formation, however, remain unknown. her5 is physically linked to a new family member, him, displaying an identical MHB expression pattern. Using single and double knockdowns of him and her5, as well as a him+her5 deletion mutant background (b404), it has been demonstrated that Him and Her5 are equally necessary for MIZ formation, and that they act redundantly in LIZ formation in vivo. These processes do not involve cross-regulation between Him and Her5 expression or activities, although Him and Her5 can heterodimerize with high affinity. Increasing the function of one factor when the other is depleted further shows that Him and Her5 are functionally interchangeable. Together, these results demonstrate that patterning and neurogenesis are integrated by the her5-him gene pair to maintain a progenitor pool at the embryonic MHB. A molecular mechanism for this process is proposed where the global `Him+Her5' activity inhibits ngn1 expression in a dose-dependent manner and through different sensitivity thresholds along the medio-lateral axis of the neural plate (Ninkovic, 2005).

The absence of Her5 leads to disappearance of the entire MIZ and its replacement by ngn1-expressing cells, which later differentiate into Hu-, HNK1- and acetylated-tubulin-positive neurons. Surprisingly, Him plays an equally important role in MIZ formation, since an exactly identical phenotype is triggered by lack of Him activity. An interdependent regulation of him and her5 expression is ruled out. Thus, another important implication of this work is that MIZ formation relies on prepatterning by both Him and Her5 (Ninkovic 2005).

A priori, the finding that loss of Him or Her5 function result in identical phenotypes can have three different molecular interpretations: first, Him and Her5 might act in distinct pathways that converge on and are both necessary for neurogenesis control at the MIZ; second, the activities of Him and Her5 might be interdependent; third, Him and Her5 might have equivalent functions, a minimal dose of 'Him + Her5' activity being required for MIZ formation. The first two mechanisms are unlikely, given the observation that increased levels of Him alone to three doses (as in her5PAC::egfp/+ heterozygote transgenic embryos injected with her5MO) can compensate for the lack of Her5 function within the MIZ. Genetic means of assessing whether a high dose of Her5 alone would also suffice for MIZ formation are unavailable. However, the findings that Him and Her5 are equally potent to prevent lateral neurogenesis strongly suggest that this is the case. Thus, it is proposed that the crucial determinant of MIZ formation, is a total level of 'Him + Her5' inhibitory activity. Hence, above a threshold of Him + Her5, ngn1 expression is prevented medially and the MIZ is formed, while ngn1 expression is induced below this threshold. The results indicate that three doses of one factor alone is the minimum level of inhibitory activity required for MIZ formation. Interestingly, however, two doses are sufficient when both Him and Her5 are present, as in b404/+ heterozygote embryos. This result might be related to the higher propensity of Him and Her5 to hetero- than homodimerize, or to an increased activity of heterodimers versus homodimers or oligomers. Because the same factors Him and Her5 account for LIZ formation, and can functionally replace each other in this domain as well, a parsimonious interpretation of these findings is to implicate the same dose-dependent mechanism within the LIZ, albeit with a lower threshold level. The LIZ minimal level of inhibition would be achieved with one dose of Him or Her5 alone. Together, these results thus lead to a unified model where the maintenance of a pool of progenitor cells at the MHB is orchestrated by a variable dose-dependency to the Him/Her5 pair (Ninkovic 2005). <>Even in the absence of Him and Her5, ngn1 expression was not induced within a small intermediate field located between the MIZ and LIZ. In this domain, an additional (as yet unknown) factor might increase the total inhibitory activity and/or prevent neurogenesis in addition to Him and Her5. No additional IZ-expressed E(spl) genes were recoved following a search through the zebrafish genome and expression studies. Because the intermediate field is aligned with the longitudinal domains of non-differentiation in the hindbrain and spinal cord, it is perhaps more likely that this factor is expressed along the AP axis of the neural plate, like other known neurogenesis inhibitors. An interesting open question remains to identify the cues controlling the differential sensitivity of the MIZ versus LIZ to Him + Her5, and their functional significance. The MIZ and LIZ differ in their proliferation rates: the MIZ exhibits more cells in M phase than the LIZ at late gastrulation, based on anti-phosphoH3 immunostaining. It will be crucial to investigate the possible relationship between MIZ and LIZ cell cycle properties and their response to Him + Her5. Also, several morphogens acting in this region are expressed following a mediolateral gradient. For instance, wnt1 is expressed in a spatio-temporal pattern similar to her5 and him at late gastrulation, thus with initially higher levels laterally than medially, and might enhance cell sensitivity to neurogenesis inhibitors. This might be related to the delay of dorsal differentiation proposed to result from the gradient of Wnt signaling from the spinal cord roof plate. Conversely Shh signaling from the ventral midline and specifically active at the MHB could increase 'neurogenic competence'. These hypotheses will be important to test experimentally to gain insight into the prepatterning of IZ formation (Ninkovic 2005).

The Wnt/β-catenin pathway plays an essential role during regionalisation of the vertebrate neural plate and its inhibition in the most anterior neural ectoderm is required for normal forebrain development. Hesx1 is a conserved vertebrate-specific transcription factor that is required for forebrain development in Xenopus, mice and humans. Mouse embryos deficient for Hesx1 exhibit a variable degree of forebrain defects, but the molecular mechanisms underlying these defects are not fully understood. This study shows that injection of a hesx1 morpholino into a 'sensitised' zygotic headless (tcf3) mutant background leads to severe forebrain and eye defects, suggesting an interaction between Hesx1 and the Wnt pathway during zebrafish forebrain development. Consistent with a requirement for Wnt signalling repression, a synergistic gene dosage-dependent interaction occurs between Hesx1 and Tcf3, a transcriptional repressor of Wnt target genes, to maintain anterior forebrain identity during mouse embryogenesis. In addition, it is revealed that Tcf3 is essential within the neural ectoderm to maintain anterior character and that its interaction with Hesx1 ensures the repression of Wnt targets in the developing forebrain. By employing a conditional loss-of-function approach in mouse, it was demonstrated that deletion of β-catenin, and concomitant reduction of Wnt signalling in the developing anterior forebrain of Hesx1-deficient embryos, lead to a significant rescue of the forebrain defects. Finally, transcriptional profiling of anterior forebrain precursors from mouse embryos expressing eGFP from the Hesx1 locus provides molecular evidence supporting a novel function of Hesx1 in mediating repression of Wnt/β-catenin target activation in the developing forebrain (Andoniadou, 2011).

Multiple roles of Notch signaling in the regulation of epidermal development

Recent studies have shown that Notch signaling plays an important role in epidermal development, but the underlying molecular mechanisms remain unclear. By integrating loss- and gain-of-function studies of Notch receptors and Hes1, molecular information is described about the role of Notch signaling in epidermal development. Notch signaling is shown to determine spinous cell fate and induces terminal differentiation by a mechanism independent of Hes1, but Hes1 is required for maintenance of the immature state of spinous cells. Notch signaling induces Ascl2 expression to promote terminal differentiation, while simultaneously repressing Ascl2 through Hes1 to inhibit premature terminal differentiation. Despite the critical role of Hes1 in epidermal development, Hes1 null epidermis transplanted to adult mice showed no obvious defects, suggesting that this role of Hes1 may be restricted to developmental stages. Overall, it is concluded that Notch signaling orchestrates the balance between differentiation and immature programs in suprabasal cells during epidermal development (Moriyama, 2008).

Conditional ablation of Notch signaling in epidermal development results in loss of the spinous and granular layers due to hypoproliferation of the epidermis, indicating that Notch signaling is required for commitment of basal keratinocytes to spinous cell differentiation at early stages of epidermal development. By contrast, postnatal ablation of Notch1 causes hyperproliferation of basal keratinocytes, suggesting that signaling from Notch1 is required for cell cycle withdrawal of the basal keratinocytes to promote terminal differentiation in the postnatal epidermis. These apparently contradictory functions of Notch signaling in the regulation of keratinocyte proliferation and differentiation may reflect differences in the cell-context-specific functions of Notch signaling between embryonic and postnatal keratinocytes or may be due to differential use of either canonical or noncanonical pathways in the regulation of epidermal keratinocytes. In both cases, however, the molecular mechanisms underlying the regulation of epidermal development by Notch signaling remain largely unknown (Moriyama, 2008).

The current study demonstrates multiple roles of Notch signaling in epidermal development. By combining both loss- and gain-of-function studies, it was confirmed that Notch signaling promotes spinous cell commitment from basal cells and induces their terminal differentiation into granular cells. Moreover, a crucial role is revealed for the Hes1 transcriptional repressor in determining the outcome of Notch signaling via coordination of the balance between maintenance of the spinous cell fate and the induction of granular cell differentiation. The present data thus provide new insights into how Notch signaling accomplishes apparently contradictory tasks simultaneously, i.e., activating cell fate determination and terminal differentiation programs while also preventing a terminal differentiation (Moriyama, 2008).

Different types of oscillations in Notch and Fgf signaling regulate the spatiotemporal periodicity of somitogenesis

Somitogenesis is controlled by cyclic genes such as Notch effectors and by the wave front established by morphogens such as Fgf8, but the precise mechanism of how these factors are coordinated remains to be determined. This study shows that effectors of Notch and Fgf pathways oscillate in different dynamics and that oscillations in Notch signaling generate alternating phase shift, thereby periodically segregating a group of synchronized cells, whereas oscillations in Fgf signaling released these synchronized cells for somitogenesis at the same time. These results suggest that Notch oscillators define the prospective somite region, while Fgf oscillators regulate the pace of segmentation (Niwa, 2011).

Somite formation occurs periodically by segmentation and maturation of a block of cells in the anterior presomitic mesoderm (PSM). It is thought that the pace of segmentation depends on the clock controlled by cyclic genes such as Notch signaling molecules, while the timing of maturation depends on the wave front established by morphogens such as Fgf8. However, Notch signaling oscillations become slower than the pace of segmentation as the oscillations are propagated anteriorly, raising the question of whether such a slowing oscillator regulates the segmentation pace. Furthermore, Fgf signaling seems to sweep back at a steady speed as the PSM grows, raising another question of whether the release from Fgf signaling occurs at different times between the anterior and posterior cells even in the same prospective somites (Niwa, 2011).

In the mouse PSM, Hes7 is expressed in an oscillatory manner and induces oscillatory expression of Lunatic fringe (Lfng), a modulator of Notch signaling. Lfng oscillations in turn lead to cyclic formation of the Notch intracellular domain (NICD), an active form of Notch, which then periodically induces expression of Mesp2, an essential gene for the segmentation and rostro-caudal patterning of each somite. Mesp2 expression depends on NICD and Tbx6 and occurs after the release from Fgf and Wnt signaling in the whole S-1 region, a group of cells that forms a prospective somite. High-resolution in situ hybridization demonstrated that S-1 cells synchronously exhibit nuclear dots of Mesp2 signals, indicating synchronous initiation of Mesp2 transcription in the whole S-1 region. In Lfng-null mice, which have segmentation defects, Mesp2 expression becomes randomized in S-1 cells, displaying a salt-and-pepper pattern. These results suggest that synchronous Mesp2 expression in S-1 cells is important for somite formation. However, how slowing Notch signaling oscillators and steadily regressing Fgf and Wnt signaling regulate periodic and synchronous Mesp2 expression in S-1 cells remains to be determined (Niwa, 2011).

This study found that Notch and Fgf signaling effectors oscillate with different dynamics and that oscillations in Notch signaling periodically segregate a group of synchronized cells, whereas oscillations in Fgf signaling release these synchronized cells for somitogenesis at the same time. These results suggest that Notch oscillators define the prospective somite region, while Fgf oscillators regulate the pace of segmentation, thereby linking the clock and the wave front (Niwa, 2011).

Modifying transcript lengths of cycling mouse segmentation genes

Regular production of somites, precursors of the axial skeleton and attached muscles is controlled by a molecular oscillator, the segmentation clock, which drives cyclic transcription of target genes in the unsegmented presomitic mesoderm (PSM). The clock is based on a negative feedback loop which generates pulses of transcription that oscillate with the same periodicity as somite formation. Mutants in several oscillating genes including the Notch pathway gene Lunatic fringe (Lfng) and the Notch target Hes7, result in defective somitogenesis and disorganised axial skeletons. Both genes encode negative regulators of Notch signalling output, but it is not yet clear if they are just secondary clock targets or if they encode components of a primary, pacemaker oscillator. This study tries to identify components in the primary oscillator by manipulating delays in the feedback circuitry. Recombinant mice were characterized in which Lfng and Hes7 introns are lengthened in order to delay mRNA production. Lengthening the third Hes7 intron by 10 or 20 kb disrupts accurate RNA splicing and inactivates the gene. Lfng expression and activity is normal in mice whose Lfng is lengthened by 10 kb, but no effects on segmentation are evident. These results are discussed in terms of the relative contributions of transcriptional and post-transcriptional delays towards defining the pace of segmentation, and of alternative strategies for manipulating the period of the clock (Stauber, 2012).

Hairy and heart development

Notch signaling is required for multiple aspects of cardiovascular development, including arterial-venous differentiation, septation and cushion formation. Despite recognition of the importance of the Notch pathway in normal cardiovascular development, the proximate downstream effectors are not yet known. Likely candidate effectors are members of the hairy and enhancer of split related (hesr) family of bHLH transcription factors. However, mutational analysis of individual hesr genes has so far failed to elucidate their role in all Notch-mediated cardiovascular signaling events. An example of this is evident for mutants of gridlock, the zebrafish counterpart of mouse hesr2; they have vascular defects, whereas mouse hesr2 mutants have only cardiac defects. One possible explanation for these differences could be functional redundancy between hesr family members. Mice lacking the hesr1 gene are shown to be viable and fertile, whereas a knockout mouse of both hesr1 and hesr2 is embryonic lethal at 11.5 days of development and recapitulates most of the known cardiovascular phenotypes of disrupted Notch pathway mutants including defects in arterial-venous specification, septation and cushion formation. Taken together, these results demonstrate a requirement for hesr1 and hesr2 in mediating Notch signaling in the developing cardiac and vascular systems (Kokubo, 2005).

The establishment of chamber specificity is an essential requirement for cardiac morphogenesis and function. Hesr1 (Hey1) and Hesr2 (Hey2) are specifically expressed in the atrium and ventricle, respectively, implicating these genes in chamber specification. Forced expression of Hesr1 or Hesr2 in the entire cardiac lineage of the mouse results in the reduction or loss of the atrioventricular (AV) canal. In the Hesr1-misexpressing heart, the boundaries of the AV canal are poorly defined, and the expression levels of specific markers of the AV myocardium, Bmp2 and Tbx2, are either very weak or undetectable. More potent effects were observed in Hesr2-misexpressing embryos, in which the AV canal appears to be absent entirely. These data suggest that Hesr1 and Hesr2 may prevent cells from expressing the AV canal-specific genes that lead to the precise formation of the AV boundary. These findings suggest that Tbx2 expression might be directly suppressed by Hesr1 and Hesr2. Furthermore, the expression of Hesr1 and Hesr2 is independent of Notch2 signaling. Taken together, these data demonstrate that Hesr1 and Hesr2 play crucial roles in AV boundary formation through the suppression of Tbx2 (Kokubo, 2007).

Hairy/Enhancer-of-split and testis development

During testis development, fetal Leydig cells increase their population from a pool of progenitor cells rather than from proliferation of a differentiated cell population. However, the mechanism that regulates Leydig stem cell self-renewal and differentiation is unknown. This study shows that blocking Notch signaling, by inhibiting gamma-secretase activity or deleting the downstream target gene Hairy/Enhancer-of-split 1, results in an increase in Leydig cells in the testis. By contrast, constitutively active Notch signaling in gonadal somatic progenitor cells causes a dramatic Leydig cell loss, associated with an increase in undifferentiated mesenchymal cells. These results indicate that active Notch signaling restricts fetal Leydig cell differentiation by promoting a progenitor cell fate. Germ cell loss and abnormal testis cord formation were observed in both gain- and loss-of-function gonads, suggesting that regulation of the Leydig/interstitial cell population is important for male germ cell survival and testis cord formation (Tang, 2008).

Hairy-related proteins and neurogenesis

A rat gene structurally homologous to hairy (RHL) behaves as an immediate-early gene in its response to growth factors. Like its Drosophila homolog, it can suppress neuronal differentiation events. The human hairy gene homolog (HRY) is contained within four exons. The predicted amino acid sequence reveals only four amino acid differences between the human and rat genes. Analysis of the DNA sequence 5' to the coding region reveals a putative untranslated exon (Feder, 1994).

The transcriptional repressor HES-1, a basic helix-loop-helix (bHLH) factor structurally related to the Drosophila hairy gene, is expressed at high levels throughout the ventricular zone, but the level decreases as neural differentiation proceeds. Continuous expression of HES-1 inhibits neural differentiation. Cells overexpressing HES-1 remain in the ventricular/subventricular zone, decreased to approximately 10% in number as compared with that of the newborn during the postnatal 4-5 weeks. When they survive they are present exclusively in the ependymal layer. Cells overexpressing HES-1 fail to became immunoreactive for neuronal and glial markers. Thus persistent expression of HES-1 severely perturbs neuronal and glial differentiation (Ishibashi, 1994).

Mammalian hairy and Enhancer of split homolog-1 (HES-1) encodes a helix-loop-helix (HLH) factor that is thought to act as a negative regulator of neurogenesis. Knockout mice homozygous for HES-1 mutation exhibite severe neurulation defects and die during gestation or just after birth. In the developing brain of HES-1-null embryos, expression of the neural differentiation factor Mash-1 and other neural HLH factors are up-regulated and postmitotic neurons appeare prematurely. These results suggest that HES-1 normally controls the proper timing of neurogenesis and regulates neural tube morphogenesis (Ishibashi, 1995).

In the mammalian central nervous system, a diverse group of basic helix-loop-helix (bHLH) proteins is involved in the determination of progenitor cells and, subsequently, in regulating neuronal differentiation. A novel subfamily of bHLH proteins, defined by two mammalian enhancer-of-split- and hairy-related proteins, termed SHARP-1 and SHARP-2, has been identified. In contrast to known bHLH genes, detectable transcription of SHARP genes begins at the end of embryonic development marking differentiated neurons that have reached a final position, and increases as postnatal development proceeds. In the adult, SHARP genes are expressed in subregions of the CNS that have been associated with adult plasticity. In PC12 cells, a model system to study neurite outgrowth, SHARP genes can be induced by NGF with the kinetics of an immediate-early gene. Similarly, within 1 h after the administration of kainic acid in vivo, SHARP-2 is induced in neurons throughout the rat cerebral cortex. This suggests that neuronal bHLH proteins are also involved in the "adaptive" changes of mature CNS neurons which are coupled to glutamatergic stimulation (Rossner, 1998).

During the development of the vertebrate nervous system, neurogenesis is promoted by proneural bHLH proteins such as the neurogenins, which act as potent transcriptional activators of neuronal differentiation genes. The pattern by which these proteins promote neuronal differentiation is thought to be governed by inhibitors, including a class of transcriptional repressors called the WRPW-bHLH proteins, which are similar to Drosophila proteins encoded by hairy and genes in the enhancer of split complex [E-(SPL)-C]. Hes6, which encodes a novel WRPW-bHLH protein expressed during neurogenesis in mouse and Xenopus embryos has been isolated and characterized. Hes6 expression follows that of neurogenins but precedes that of the neuronal differentiation genes. Several lines of evidence show that Hes6 expression occurs in developing neurons and is induced by the proneural bHLH proteins but not by the Notch pathway. When ectopically expressed in Xenopus embryos, Hes6 promotes neurogenesis. The properties of Hes6 distinguish it from other members of the WRPW-bHLH family in vertebrates, and suggest that it acts in a positive-feedback loop with the proneural bHLH proteins to promote neuronal differentiation (Koyano-Nakagawa, 2000).

The mechanism by which Hes6 promotes the differentiation of neurogenin-expressing progenitor cells remains an unanswered question. Based on its structural features as a transcriptional repressor, one likely model is that Hes6 binds target sites and represses the expression of genes that normally act to inhibit neuronal differentiation. The main argument against this model is that Hes6 still promotes neuronal differentiation even when its DNA-binding domain is mutant, or the WRPW motif is deleted. However, this argument is inconclusive as there are several published examples in which WRPW-bHLH proteins act as wild-type molecules, even when their DNA-binding or WRPW domains are removed. As further support of this model, the expression of Xngn1 and neuronal differentiation are repressed by a form of Hes6 that is converted from a transcriptional repressor into a transcription activator by substituting the WRPW domain with the activation domain of VP16. The simplest interpretation of this result is that this form of Hes6 induces the expression of repressors that inhibit neuronal differentiation, while, by extension, Hes6 would normally repress these repressors. If Hes6 normally acts as a transcriptional repressor, it does not promote neuronal differentiation by repressing the expression of genes in the lateral inhibitory pathway such as Xdelta1 and the genes coding for E(SPL)-related, WRPW-bHLH (Esr) proteins, or other proposed repressors of neuronal differentiation such as the hairy-like genes or Zic2. This implies that Hes6 represses the expression of a novel class of repressors that negatively regulate the expression and activity of the proneural bHLH proteins (Koyano-Nakagawa, 2000).

An alternative, but not mutually exclusive, model is based on the striking induction of Hairy gene expression by Hes6. Because the expression of the Hairy genes is repressed by their own products, the simplest interpretation of this result is that Hes6 inhibits the activity of these WRPW-bHLH proteins post-transcriptionally. In support of this possibility, Hes6 induces the expression of Xhairy1 in an animal cap assay. Moreover in the same assay, Xhairy2A represses Xhairy1 expression, and this effect can be reversed by co-injection of Hes6 RNA. Finally, Hes6 can bind to the hairy proteins both in vitro and in vivo. These observations raise the possibility that Hes6 inhibits hairy protein activity by forming, for example, nonfunctional heterodimers with the hairy-like proteins, in much the same way that the Ids heterodimerize with and inhibit the positive acting bHLHs. Hes6 could also compete for accessory molecules that are required for repression by the hairy proteins, although this is not likely to be the Groucho co-repressors, since the WRPW-deletion mutant of Hes6 retains wild-type activity. Conversely, an Esr7 mutant containing the WRPW but lacking the DNA-binding domain is apparently inactive when overexpressed, indicating that overexpression of the WRPW domain is not sufficient to promote neuronal differentiation. Regardless of the exact mechanistic details, the ability of Hes6 to interfere with the activity of these proteins raises the possibility that the hairy class of proteins is one target disabled by Hes6 when it promotes neuronal differentiation (Koyano-Nakagawa, 2000).

An intracellular timer in oligodendrocyte precursor cells is thought to help control the timing of their differentiation. The expression of the Hes5 and Mash1 genes, which encode neural-specific bHLH proteins, decrease and increase, respectively, in these cells with a time course expected if the proteins are part of the timer. Enforced expression of Hes5 in purified precursor cells strongly inhibits the normal increase in the thyroid hormone receptor protein TRbeta1, which is thought to be part of the timing mechanism; it also strongly inhibits the differentiation induced by either mitogen withdrawal or thyroid hormone treatment. Enforced expression of Mash1, by contrast, somewhat accelerates the increase in TRbeta1 protein. These findings suggest that Hes5 and Mash1 may be part of the cell-intrinsic timer in the precursor cells (Kondo, 2000).

While the transmembrane protein Notch plays an important role in various aspects of development, and in diseases (including tumors and neurological disorders), the intracellular pathway of mammalian Notch remains very elusive. To understand the intracellular pathway of mammalian Notch, the role of the bHLH genes Hes1 and Hes5 (mammalian hairy and the Enhancer-of-split homolog, respectively) was examined by retrovirally misexpressing the constitutively active form of Notch (caNotch) in neural precursor cells prepared from wild-type, Hes1-null, Hes5-null and Hes1-Hes5 double-null mouse embryos. caNotch, which induces the endogenous Hes1 and Hes5 expression, inhibits neuronal differentiation in the wild-type, Hes1-null and Hes5-null background, but not in the Hes1-Hes5 double-null background. These results demonstrate that Hes1 and Hes5 are essential Notch effectors in the regulation of mammalian neuronal differentiation (Ohtsuka, 1999).

The isthmic organizer, which is located at the midbrain-hindbrain boundary, plays an essential role in development of the midbrain and anterior hindbrain. Homeobox genes regulate establishment of the isthmic organizer, but the mechanism by which the organizer is maintained is not well understood. In mice doubly mutant for the basic helix-loop-helix genes Hes1 and Hes3, the midbrain and anterior hindbrain structures are missing without any significant cell death. In these mutants, the isthmic organizer cells prematurely differentiate into neurons and terminate expression of secreting molecules such as Fgf8 and Wnt1 and the paired box genes Pax2/5, all of which are essential for the isthmic organizer function. These results indicate that Hes1 and Hes3 prevent premature differentiation and maintain the organizer activity of the isthmic cells, thereby regulating the development of the midbrain and anterior hindbrain (Hirata, 2001).

In the developing cerebellar cortex, granule neuron precursors (GNPs) proliferate and commence differentiation in a superficial zone, the external granule layer (EGL). The molecular basis of the transition from proliferating precursors to immature differentiating neurons remains unknown. Notch signaling is an evolutionarily conserved pathway regulating the differentiation of precursor cells of many lineages. Notch2 is specifically expressed in proliferating GNPs in the EGL. Treatment of GNPs with soluble Notch ligand Jagged1, or overexpression of activated Notch2 or its downstream target HES1, maintains precursor proliferation. The addition of GNP mitogens Jagged1 or Sonic Hedgehog (Shh) upregulates the expression of HES1, suggesting a role for HES1 in maintaining precursor proliferation (Solecki, 2001).

The molecular mechanisms that govern early patterning of anterior neuroectoderm (ANE) for the prospective brain region in vertebrates are largely unknown. Screening a cDNA library of Xenopus ANE led to the isolation of a Hairy and Enhancer of split- (HES)-related transcriptional repressor gene, Xenopus HES-related 1 (XHR1). XHR1 is specifically expressed in the midbrain-hindbrain boundary (MHB) region at the tailbud stage. The localized expression of XHR1 is detected as early as the early gastrula stage in the presumptive MHB region, an area just anterior to the involuting dorsal mesoderm, demarcated by the expression of the gene Xbra. Expression of XHR1 is detected much earlier than that of other known MHB genes (XPax-2 and En-2) and also before the formation of the expression boundary between Xotx2 and Xgbx-2, suggesting that the early patterning of the presumptive MHB is independent of Xotx2 and Xgbx-2. Instead, the location of XHR1 expression appears to be determined in relation to the Xbra expression domain, since reduced or ectopic expression of Xbra alters the XHR1 expression domain according to the location of Xbra expression. In functional assays using mRNA injection, overexpression of dominant-negative forms of XHR1 in the MHB region led to marked reduction of XPax-2 and En-2 expression, and this phenotype was rescued by coexpression of wild-type XHR1. Furthermore, ectopically expressed wild-type XHR1 near the MHB region enhances En-2 expression only in the MHB region but not in the region outside the MHB. These data suggest that XHR1 is required, but not sufficient by itself, to initiate MHB marker gene expression. Based on these data, it is proposed that XHR1 demarcates the prospective MHB region in the neuroectoderm in Xenopus early gastrulae (Shinga, 2001).

The iroquois (iro) homeobox genes participate in many developmental processes both in vertebrates and invertebrates -- among them are neural plate formation and neural patterning. The Xenopus Iro (Xiro) function in primary neurogenesis has been studied in detail. Misexpression of Xiro genes promotes the activation of the proneural gene Xngnr1 but suppresses neuronal differentiation. This is probably due to upregulation of at least two neuronal-fate repressors: XHairy2A and XZic2. Accordingly, primary neurons arise at the border of the Xiro expression domains. In addition, XGadd45-gamma has been identified as a new gene repressed by Xiro. XGadd45-gamma encodes a cell-cycle inhibitor and is expressed in territories where cells will exit mitosis, such as those where primary neurons arise. Indeed, XGadd45-gamma misexpression causes cell cycle arrest. It is concluded that during Xenopus primary neuron formation, in Xiro expressing territories neuronal differentiation is impaired, while in adjacent cells, XGadd45-gamma may help cells stop dividing and differentiate as neurons (de la Calle-Mustienes, 2002).

This may be due to redundancy between different Gadd45 proteins. The spatial and temporal patterns of expression of Gadd45-gamma and the Notch ligand XDl1 largely coincide. Moreover, both XGadd45-gamma and XDl1 are positively regulated by proneural genes and negatively controlled by Notch signaling. According to the lateral inhibition model, activation of the Notch pathway within a cell, by signaling from neighboring cells, maintains the cell's mitotic potential and prevents its differentiation. In contrast, a cell that expresses high levels of Notch ligands and signals strongly, escapes lateral inhibition, exits the cell cycle and differentiates. XGadd45-gamma may provide a link between Notch signaling, cell-cycle arrest and differentiation. Thus, in the neural plate, cells with high levels of proneural genes have also high levels of XDl1 and XGadd45-gamma. The first allows them to escape lateral inhibition, and the second to exit the cell cycle. These cells can then differentiate. Mitotic arrest mediated by XGadd45-gamma probably occurs through interaction with cyclin and inhibitors of cyclin-dependent kinases. In neighboring cells, the Notch pathway is activated, proneural genes and XGadd45-gamma are downregulated, and cell-cycle arrest and differentiation cannot occur. It is of interest that induction of Gadd45 genes in cell culture stops the cell cycle in G1 phase. This phase is compatible with exiting the cell cycle, a requirement for terminal neuronal differentiation. Cells that differentiate outside the neural plate may resort to genes different from the proneural ones to accumulate Notch ligands and XGadd45-gamma (de la Calle-Mustienes, 2002).

This study compares the effects of overexpressing either Xiro1, -2 or -3 in neural development. To make comparisons more meaningful, equivalent constructs were prepared in the pCS2MT plasmid. The overexpression of each Xiro gene causes similar effects, although Xiro3 was approximately five to ten times more potent. Paradoxically, the overexpressions activated Xngnr1 and repressed neuronal differentiation. This may be explained at least in part by the finding that Xiro upregulates the neuronal repressors XHairy2A and XZic2. Indeed, it has been shown that XZic2 antagonizes development of Xngnr1-promoted ectopic neurons. XZic2 antagonizes Xngnr1-promoted XGadd45-gamma and XDl1 expression. Consistently with these findings, in wild type embryos the intermediate stripes of expression of XHairy2A and XZic2 are within the Xiro1 domains. Also in accordance with these results, in the prospective spinal chord, the Xiro1 domain is contained within the broader Xngnr1 domain and neurons arise at the border of the Xiro1 domain. Taken together, these observations suggest that Xiro proteins simultaneously participate in the activation of Xngnr1 and of genes that antagonize primary neuron formation (de la Calle-Mustienes, 2002).

Overexpressions of Xiro genes represses both XGadd45-gamma and XDl1 in territories where primary neurons arise. Consistently, in wild type embryos, XGadd45-gamma and XDl1 are expressed at the borders of Xiro domains. Moreover, XDl1 is activated in embryos expressing a Xiro1 chimera that converts the Xiro1 repressor into an activator (HD-GR-E1A). This activation occurs even in the absence of protein synthesis. Thus, XDl1 is probably directly repressed by Xiro. However, XGadd45-gamma is repressed by HD-GR-E1A, probably because Xngnr1 is also downregulated. Indeed, coinjection of HD-GR-E1A and Xngnr1 mRNAs rescues the expression of XGadd45-gamma. Thus, Xiro-mediated repression of XGadd45-gamma is probably indirect and may take place, at least in part, by Xiro-upregulated neuronal repressors. In this case, interference with Xiro function would suppress neuronal repressors, but would also downregulate Xngnr1, which is needed for XGadd45-gamma expression (de la Calle-Mustienes, 2002).

A model is proposed for the function of Xiro in neural patterning that integrates the above data. Xiro proteins, as well as other factors, participate in the activation of Xngnr1. Within the Xiro domains, Xngnr1 does not activate XDl1 or XGadd45-gamma, and cannot promote differentiation of primary neurons due to the upregulation by Xiro of neuronal repressors, such as XHairy2A and XZic2. In addition, Xiro probably represses XDl1 directly. Outside the Xiro domains, other factors, such as the Gli proteins, activate Xngnr1, which in turn promotes the expression of XDl1 and XGadd45-gamma in those cells that will become primary neurons. XDl1 switches on the lateral inhibition mechanism by which the Notch signaling pathway is activated in neighboring cells. This pathway downregulates proneural genes, XDl1 and XGadd45-gamma. As a consequence, these cells keep dividing and do not differentiate. In contrast, cells with high levels of Xngnr1, XDl1 and XGadd45-gamma escape lateral inhibition, exit the cell cycle (in part due to the presence of XGadd45-gamma) and differentiate as primary neurons. This differentiation is triggered by a genetic program activated by Xngnr1. Thus, Xiro proteins may help coordinate cell cycle and differentiation (de la Calle-Mustienes, 2002).

The midbrain-hindbrain (MH) domain of the vertebrate embryonic neural plate displays a stereotypical profile of neuronal differentiation, organized around a neuron-free zone ('intervening zone', IZ) at the midbrain-hindbrain boundary (MHB). The mechanisms establishing this early pattern of neurogenesis are unknown. The MHB is globally refractory to neurogenesis, and forced neurogenesis in this area interferes with the continued expression of genes defining MHB identity. Expression of the zebrafish bHLH Hairy/E(spl)-related factor Her5 prefigures and then precisely delineates the IZ throughout embryonic development. Using morpholino knock-down and conditional gain-of-function assays, it has been demonstrated that Her5 is essential to prevent neuronal differentiation and promote cell proliferation in a medial compartment of the IZ. One probable target of this activity has been identified; the zebrafish Cdk inhibitor p27Xic1. Although the her5 expression domain is determined by anteroposterior patterning cues, Her5 does not retroactively influence MH patterning. Together, these results highlight the existence of a mechanism that actively inhibits neurogenesis at the MHB, a process that shapes MH neurogenesis into a pattern of separate neuronal clusters and might ultimately be necessary to maintain MHB integrity. Her5 appears as a partially redundant component of this inhibitory process that helps translate early axial patterning information into a distinct spatiotemporal pattern of neurogenesis and cell proliferation within the MH domain (Geling, 2003).

Neurogenesis in both vertebrates and invertebrates is tightly controlled in time and space involving both positive and negative regulators. The bHLH factor Her5 acts as a prepattern gene to prevent neurogenesis in the anlage of the midbrain/hindbrain boundary in the zebrafish neural plate. This involves selective suppression of both neurogenin1 (ngn1) and coe2 mRNA expression in a process that is independent of Notch signalling, and where inhibition of either ngn1 or coe2 expression is sufficient to prevent neuronal differentiation across the midbrain-hindbrain boundary. A ngn1 transgene faithfully responds to Her5 and deletion analysis of the transgene identifies an E-box in a ngn1 upstream enhancer to be required for repression by Her5. Together these data demonstrate a role for Her5 as a prepattern factor in the spatial definition of proneural domains in the zebrafish neural plate, in a manner similar to its Drosophila homolog Hairy (Geling, 2004).

Both Ngn1 and Coe2 functions are necessary for the progression of neurogenesis and for the early events of neuronal differentiation in the midbrain-hindbrain domain. Blocking Coe2 activity downregulates ngn1 expression throughout the neural plate, suggesting a requirement for Coe2 in all primary neurons. The absence of ngn1 function prevents deltaB expression in the anterior proneural clusters, including the presumptive motorneurons of rhombomeres 2 and 4, and the first anterior neuronal cluster (ventrocaudal cluster, vcc), and is also necessary for neuronal differentiation of vcc derivatives, which comprise at least the first differentiating populations of the reticulospinal nMLF neurons. This, together with previous reports, indicates a strict requirement for Ngn1 in spinal sensory neurons and the MH area of the embryonic zebrafish CNS. By contrast, Ngn1 is not essential for motorneuron and interneuron development in the trunk and spinal cord, and for epiphysial neurons. Differential requirements for Ngn in CNS neuronal differentiation were also observed in other vertebrates, a typical example being the complementary requirements for Ngn2 and Mash1 in the mouse embryonic neural tube. Other bHLH factors, such as Achaete-scute or Olig, may play redundant or prominent roles in neurogenic areas that differentiate normally in ngn1-deficient embryos (Geling, 2004 and references therein).

These results point to synergistic roles of Ngn1 and Coe2 in MH neurogenesis, possibly reflecting the positive cross-regulation of their expression, and a parallel activity of these factors rather than their action in a linear cascade. It is possible that the crossregulation of ngn1 and coe2 expression helps stabilize the committed state of neuronal progenitors, as described for Xenopus Xcoe2. Together, these results led to a model for the spatial control of MH neurogenesis. In this process, ngn1 and coe2 expression are crucial elements that permit neurogenesis throughout the MH, which is initially identified as a single territory competent to form neurons. At the MHB, ngn1 and coe2 expression are the targets of Her5 inhibition. This inhibition prevents the specification of a proneural cluster in this location and permits the generation of the 'intervening zone' (Geling, 2004).

The midbrain-hindbrain domain (MH) of the vertebrate embryonic neural tube develops in response to the isthmic organizer (IsO), located at the midbrain-hindbrain boundary (MHB). MH derivatives are largely missing in mutants affected in IsO activity; however, the potentialities and fate of MH precursors in these conditions have not been directly determined. To follow the dynamics of MH maintenance in vivo, artificial chromosome transgenesis was used in zebrafish to construct lines where egfp transcription is driven by the complete set of regulatory elements of her5, the first known gene expressed in the MH area. In these lines, egfp transcription faithfully recapitulates her5 expression from its induction phase onwards. Using the stability of GFP protein as lineage tracer, her5, first demonstrated at gastrulation, is a selective marker of MH precursor fate. By comparing GFP protein and her5 transcription, the spatiotemporal dynamics of her5 expression that conditions neurogenesis progression towards the MHB over time was further revealed. The molecular identity of GFP-positive cells was traced in the acerebellar (ace) and no-isthmus (noi) mutant backgrounds to analyze directly fgf8 and pax2.1 mutant gene activities for their ultimate effect on cell fate. Most MH precursors are maintained in both mutants but express abnormal identities, in a manner that strikingly differs between the ace and noi contexts. These observations directly support a role for Fgf8 in protecting anterior tectal and metencephalic precursors from acquiring anterior identities, while Pax2.1 controls the choice of MH identity as a whole. Together, these results suggest a model where an ordered MH pro-domain is identified at gastrulation, and where cell identity choices within this domain are subsequently differentially controlled by Fgf8 and Pax2.1 functions (Tallafuß, 2003).

These results correlatively demonstrate that primary neurogenesis converges from anterior and posterior towards the MHB over time, and suggest that neurogenesis progression is permitted by the dynamic downregulation of her5 expression. Along the DV axis of the neural tube, the combinatorial differentiation-promoting and differentiation-inhibiting activities of Shh and Wnt signaling, respectively, has been proposed to account for the global ventral-to-dorsal progression of neuronal maturation. Her5 might be regarded as a counterpart to Shh and Wnt along DV; Her5 controls the spatial order of neurogenesis progression along AP within the MH domain. Within the MH basal plate, neuronal identity varies according to, and has been postulated to depend upon, the position of the population considered relative to the MHB. For example, nMLF reticulospinal neurons lie at the anterior border of the mesencephalon, while motoneurons (of cranial nerves III and IV) are found adjacent to the MHB. The results on her5 and neurogenesis dynamics also imply that these neurons are generated at different times, the former being an early and the latter a late neuronal type. Along this line, the combined action of the two E(spl)-like factors Hes1 and Hes3 is required for IZ maintenance in the E10.5 mouse embryo, and premature neurogenesis at the MHB in Hes1-/-;Hes3-/- embryos is correlated with the loss of some but not all neuronal identities that normally develop around the MHB after E10.5. Whether the primary determinant of neuronal identity is the AP location of the different populations, or rather is the timing of their engagement into the differentiation process, primarily controlled by her5 restriction, becomes an important aspect of MH development to address in future studies (Tallafuß, 2003).

A wide variety of in vivo manipulations influence neurogenesis in the adult hippocampus. It is not known, however, if adult neural stem/progenitor cells (NPCs) can intrinsically sense excitatory neural activity and thereby implement a direct coupling between excitation and neurogenesis. Moreover, the theoretical significance of activity-dependent neurogenesis in hippocampal-type memory processing networks has not been explored. This study demonstrates that excitatory stimuli act directly on adult hippocampal NPCs to favor neuron production. The excitation is sensed via Ca_v1.2/1.3 (L-type) Ca²⁺ channels and NMDA receptors on the proliferating precursors. Excitation through this pathway acts to inhibit expression of the glial fate genes Hes1 and Id2 and increase expression of NeuroD, a positive regulator of neuronal differentiation. These activity-sensing properties of the adult NPCs, when applied as an 'excitation-neurogenesis coupling rule' within a Hebbian neural network, predict significant advantages for both the temporary storage and the clearance of memories (Deisseroth, 2004).

Using an array of approaches, the coupling of excitation to neurogenesis in proliferating adult-derived NPCs was studied both in vitro and in vivo. Adult neurogenesis is potently enhanced by excitatory stimuli and involves Ca_v1.2/1.3 channels and NMDA receptors. These Ca²⁺ influx pathways are located on the proliferating NPCs, allowing them to directly sense and process excitatory stimuli. No effect of excitation was found on the extent of differentiation in individual cells (measured by extent of MAP2ab expression in the NPC-derived neurons) nor were effects observed on proliferative rate or fraction, survival, or apoptosis. Instead, excitation increased the fraction of NPC progeny that were neurons, both in vitro and in vivo, and total neuron number was increased as well. The Ca²⁺ signal in NPCs leads to rapid induction of a proneural gene expression pattern involving the bHLH genes HES1, Id2, and NeuroD, and the resulting cells become fully functional neurons defined by neuronal morphology, expression of neuronal structural proteins (MAP2ab and Doublecortin), expression of neuronal TTX-sensitive voltage-gated Na⁺ channels, and synaptic incorporation into active neural circuits. A monotonically increasing function characterizes excitation-neurogenesis coupling, and incorporation of this relationship into a layered Hebbian neural network suggests surprising advantages for both the clearance of old memories and the storage of new memories. Taken together, these results provide a new experimental and theoretical framework for further investigation of adult excitation-neurogenesis coupling (Deisseroth, 2004).

In the hippocampal formation, neural stem cells exist either within the adjacent ventricular zone or within the subgranular zone proper at the margin between the granule cell layer and the hilus, where proliferative activity is most robust. These cells do not express neuronal markers but proliferate and produce dividing progeny that incrementally commit to differentiated fates (such as the neuronal lineage) over successive cell divisions. Native NPC populations in vivo are therefore heterogenous with regard to lineage potential, and markers are not available that distinguish between the multipotent stem cell and the subtly committed yet proliferative progenitor cell. Excitation may therefore act on either or both types of proliferating precursor, in vitro and in vivo. The functional consequences of coupling excitation to insertion of new neurons for the neural network, however, is independent of which precursor cell types respond to excitation (Deisseroth, 2004).

The enhancement of hippocampal neurogenesis by behavioral stimuli such as environmental enrichment and running may, at least in part, be implemented at the molecular level by excitation-neurogenesis coupling. Notably, running and environmental enrichment increase adult neurogenesis in the hippocampus but not in the subventricular zone. Of course, not every neurogenic region in the brain need follow the excitation-neurogenesis coupling rule outlined here. An activity rule appropriate for the unique information processing or storage function of that brain region might be expected to operate. In this context, it is interesting to note that, while subventricular zone/olfactory bulb precursor neurogenesis is not enhanced by behavioral activity, proliferation and survival in this system can be influenced by olfactory sensory stimuli. This suggests that a different form of activity rule, appropriate for that local circuit, may govern olfactory bulb neurogenesis (Deisseroth, 2004).

Radial glial cells derive from neuroepithelial cells, and both cell types are identified as neural stem cells. Neural stem cells are known to change their competency over time during development: they initially undergo self-renewal only and then give rise to neurons first and glial cells later. Maintenance of neural stem cells until late stages is thus believed to be essential for generation of cells in correct numbers and diverse types, but little is known about how the timing of cell differentiation is regulated and how its deregulation influences brain organogenesis. Inactivation of Hes1 and Hes5, known Notch effectors, and additional inactivation of Hes3 extensively accelerates cell differentiation and causes a wide range of defects in brain formation. In Hes-deficient embryos, initially formed neuroepithelial cells are not properly maintained, and radial glial cells are prematurely differentiated into neurons and depleted without generation of late-born cells. Furthermore, loss of radial glia disrupts the inner and outer barriers of the neural tube, disorganizing the histogenesis. In addition, the forebrain lacks the optic vesicles and the ganglionic eminences. Thus, Hes genes are essential for generation of brain structures of appropriate size, shape and cell arrangement by controlling the timing of cell differentiation. The data also indicate that embryonic neural stem cells change their characters over time in the following order: Hes-independent neuroepithelial cells, transitory Hes-dependent neuroepithelial cells and Hes-dependent radial glial cells (Hatakeyama, 2004).

Proper development of the hypothalamic-pituitary axis requires precise neuronal signaling to establish a network that regulates homeostasis. The developing hypothalamus and pituitary utilize similar signaling pathways for differentiation in embryonic development. The Notch signaling effector gene Hes1 is present in the developing hypothalamus and pituitary and is required for proper formation of the pituitary, which contains axons of arginine vasopressin (AVP) neurons from the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). It was hypothesized that Hes1 is necessary for the generation, placement and projection of AVP neurons. It was found that Hes1 null mice show no significant difference in cell proliferation or death in the developing diencephalon at embryonic day 10.5 (e10.5) or e11.5. By e16.5, AVP cell bodies are formed in the SON (supraoptic nucleus) and PVN, but are abnormally placed, suggesting that Hes1 may be necessary for the migration of AVP neurons. GAD67 immunoreactivity is ectopically expressed in Hes1 null mice, which may contribute to cell body misplacement. Additionally, at e18.5 Hes1 null mice show continued misplacement of AVP cell bodies in the PVN and SON and additionally exhibit abnormal axonal projection. Using mass spectrometry to characterize peptide content, it was found that Hes1 null pituitaries have aberrant somatostatin (SS) peptide, which correlates with abnormal SS cells in the pituitary and misplaced SS axon tracts at e18.5. These results indicate that Notch signaling facilitates the migration and guidance of hypothalamic neurons, as well as neuropeptide content (Aujla, 2011).

Combinatorial actions of patterning and HLH transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing spinal cord

During development, the three major neural cell lineages, neurons, oligodendrocytes and astrocytes, differentiate in specific temporal orders at topologically defined positions. How the timing and position of their generation are coordinately regulated remains poorly understood. Evidence is presented that the transcription factors Pax6, Olig2 and Nkx2.2 (Nkx2-2), which define the positional identity of multipotent progenitors early in development, also play crucial roles in controlling the timing of neurogenesis and gliogenesis in the developing ventral spinal cord. Each of these factors has a unique ability to either enhance or inhibit the activities of the proneural helix-loop-helix (HLH) factors Ngn1 (Neurog1), Ngn2 (Neurog2), Ngn3 (Neurog3) and Mash1 (Ascl1), and the inhibitory HLH factors Id1 and Hes1, thereby regulating both the timing of differentiation of multipotent progenitors and their fate. Consistent with this, dynamic changes in their co-expression pattern in vivo are closely correlated to stage- and domain-specific generation of three neural cell lineages. Genetic manipulations of their temporal expression patterns in mice alter the timing of differentiation of neurons and glia. A molecular code model is proposed whereby the combinatorial actions of two classes of transcription factors coordinately regulate the domain-specific temporal sequence of neurogenesis and gliogenesis in the developing spinal cord (Sugimori, 2007).

This study demonstrates the combinatorial actions of two classes of transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing ventral spinal cord. In vitro data have shown that the proneural HLH factors Ngns and Mash1 intrinsically possess the activity to induce neurons and oligodendrocytes, respectively, whereas the inhibitory HLH factors Id1 and Hes1 stimulate astrogenesis. Yet, the timing of differentiation of neurons and glia in vivo is not determined a priori by the expression of these HLH factors. The data have shown that they do so in collaboration with Pax6, Olig2 and Nkx2.2, the primary function of which has been thought to be to specify the positional identity of progenitors (Sugimori, 2007).

These patterning factors participate in controlling both the timing of differentiation and cell fate by two mechanisms. First, they act to maintain progenitors undifferentiated by suppressing otherwise strong neurogenic and astrogenic activities of Ngns and Id1 and/or Hes1. The suppression of the neurogenic activity of Ngn2 by Olig2 is in accordance with the fact that the Olig2+ domain markedly expands while producing a large number of motoneurons. Such an activity, however, is not limited to Olig2, but common among three patterning factors. Second, three patterning factors differentially modulate the activity of Mash1. Mash1 itself promotes differentiation of both neurons and oligodendrocytes. Pax6, however, converts Mash1 to become selectively neurogenic, whereas Olig2 selectively enhances Mash1-dependent oligodendrogenesis. Thus, it is proposed that these two classes of transcription factors comprise a molecular code for the coordinated spatiotemporal control of neuro/gliogenesis. According to this model, the relative expression levels of patterning and HLH factors at the single cell level are crucial to determine the fate of multipotent progenitors. How the timing and expression level of individual factors are precisely controlled remains to be further investigated. How these two classes of transcription factors coordinately regulate genetic programs for differentiation of neurons and glia also needs to be examined in the future studies (Sugimori, 2007).

Floor plate (FP) cells, the ventral midline cells of the developing neural tube, have long been thought to be non-neurogenic organizer cells that control neuronal patterning and axonal guidance. Recent studies have revealed that mesencephalic FP (mesFP) cells have neurogenic activity and generate dopaminergic neurons. However, the mechanisms underlying the control of neurogenic potential in FP cells are not yet fully understood. This study identified the bHLH factor Nato3 (Drosophila homolog: 48 related 3) as an FP-specific transcription factor. In Nato3-null mutant mice, FP cells in the spinal cord are correctly specified, but cannot properly mature. By contrast, in the developing mesencephalon, loss of Nato3 does not affect FP differentiation, but leads to loss of neurogenic activity in the medial subpopulation of mesFP cells by suppressing proneural gene expression and inducing cell cycle arrest. As a consequence, the number of midbrain dopaminergic neurons generated is decreased in mutants. It was also found that Hes1, which is known to be required for non-dividing organizer cell development in the neural tube, is aberrantly upregulated in the mesFP cells of Nato3 mutants. Consistently, forced expression of Nato3 repressed Hes1 expression and consequently induces premature neurogenesis. Finally, it was shown that forced expression of Hes1 in mesFP cells induces cell cycle arrest and downregulation of proneural factors. Taken together, these results suggest that Nato3 confers neurogenic potential on mesFP cells by suppressing classical non-neurogenic FP cell differentiation, at least in part, through repressing Hes1 (Ono, 2010).

The level of the transcription factor Pax6 is essential for controlling the balance between neural stem cell self-renewal and neurogenesis

Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. Pax6 was used as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. The genomic binding locations were identified of Pax6 in neocortical stem cells during normal development, and the functional significance of genes were ascertained that were found to be regulated by Pax6. Pax6 was found to positively and directly regulate cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, a core network regulating neocortical stem cell decision-making was identified in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. It was found that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal (Sansom, 2009).

Loss of Hes5 suppresses neurogenesis defects in Fezf1- and Fezf2-deficient telencephalon

Precise control of neuronal differentiation is necessary for generation of a variety of neurons in the forebrain. However, little is known about transcriptional cascades, which initiate forebrain neurogenesis. This study shows that zinc finger genes Fezf1 and Fezf2, homologs of Drosophila Earmuff, that encode transcriptional repressors, are expressed in the early neural stem (progenitor) cells and control neurogenesis in mouse dorsal telencephalon. Fezf1- and Fezf2-deficient forebrains display upregulation of Hes5 and downregulation of neurogenin 2, which is known to be negatively regulated by Hes5. FEZF1 and FEZF2 bind to and directly repress the promoter activity of Hes5. In Fezf1- and Fezf2-deficient telencephalon, the differentiation of neural stem cells into early-born cortical neurons and intermediate progenitors is impaired. Loss of Hes5 suppresses neurogenesis defects in Fezf1- and Fezf2-deficient telencephalon. These findings reveal that Fezf1 and Fezf2 control differentiation of neural stem cells by repressing Hes5 and, in turn, by derepressing neurogenin 2 in the forebrain (Shimizu, 2010).

An important question about neural development is how the differentiation of neural stem cells is precisely controlled in the forebrain. Asymmetric cell division of neural stem cells is thought to contribute to the differentiation of neural stem cells (radial glial cells) into either neurons or intermediate progenitors. Recent reports suggest that the orientation of stem cell division in the VZ might not directly control which of the two asymmetrically divided cells becomes a stem cell and which of the two becomes a differentiated cell. Although asymmetric centrosome inheritance during the asymmetric cell divisions was reported to play a role in the maintenance of the neural stem cells, it is not clear what factors determine cell fate. It is known that oscillation of Hes1 and neurogenin 2 expression in the telencephalic VZ plays an important role in maintenance of the neural stem cells and that stabilization of neurogenin 2 expression supports differentiation of the neural stem cells. However, it is still not understood what factor(s) control stabilization of neurogenin 2 expression and what factor(s) induce their differentiation. These reports imply that, besides asymmetric distribution of cell-fate determinants, extrinsic and intrinsic factors might bias the neural stem cells toward differentiation. Notch signaling plays an essential role in maintenance of the neural stem cells. Thus, regulators of Notch signaling and its downstream effectors might be involved in the decision as to whether to be a stem cell or a differentiated cell. This report demonstrates that Fezf1 and Fezf2, which are expressed in the neural stem cells at the beginning of mouse cortical development, inhibit the expression of the Notch effector Hes5 and promote differentiation of the neural stem cells. The findings suggest that Fezf1 and Fezf2 function as intrinsic factors to bias the neural stem cells toward differentiation (Shimizu, 2010).

Expression of fezf2 takes place in the radial glial cells of the telencephalic VZ of adult zebrafish (Berberoglu, 2009). fezf2 is also expressed in the neural progenitors and neurons in the pre-optic region and hypothalamus of the adult zebrafish brains (Berberoglu, 2009). In zebrafish, neurogenesis continuously takes place in adult brains. It is possible that fezf2 might control differentiation of the neural stem cells in the adult zebrafish forebrain as Fezf1 and Fezf2 do during early mouse cortical development (Shimizu, 2010).

Expression of Fezf1 or Fezf2 repressed both NOTCH1-dependent and NOTCH1-independent Hes5 promoter activity, but did not repress the Hes1 promoter or the artificial CBS-dependent promoter. Hes1 expression was not upregulated in the telencephalon of Fezf1^-/-Fezf2^-/- mice. Furthermore, FEZF1 and FEZF2 bound to the Hes5 promoter in vivo in the mouse forebrain. All of these data indicate that FEZF1 and FEZF2, rather than inhibit Notch cytoplasmic signaling, specifically bind to and directly repress the Hes5 promoter. FEZF1 and FEZF2 have an EH1 repressor motif. The data support the assertion that FEZF1 and FEZF2 function as transcriptional repressors and repress the Hes5 promoter at least during early cortical development. Hes5 deficiency suppressed neurogenesis defects in Fezf1^-/-Fezf2^-/- telencephalon, supporting the hypothesis that Fezf1 and Fezf2 suppress the expression of Hes5 and thereby control differentiation of the neural stem cells (Shimizu, 2010).

FEZF1 and FEZF2 repress only Hes5. Hes1 and Hes5 function redundantly in the maintenance of neural stem cells in the mouse central nervous system, whereas only Hes1 is reported to exhibit oscillatory expression in the neural stem cells, suggesting that Hes1 and Hes5 might have distinct roles in neurogenesis. Previous research has revealed that oscillation of Hes1 is involved in the maintenance of neural stem cells and, in the current study, it is speculated that Hes5 plays a different role in neurogenesis; specifically, it is proposed that Hes5, in contrast to Hes1, sets up the overall expression levels of Hes genes and neurogenin 2 in the forebrain. Once Fezf1 and Fezf2 expression exceeds a threshold, FEZF1 and FEZF2 might repress Hes5 expression, stabilize neurogenin 2 expression and thereby bias the neural stem cells toward differentiation (Shimizu, 2010).

The Drosophila homolog of Fezf1/2 (dFezf or Earmuff) has been shown to restrict the developmental potential of intermediate progenitors by negatively regulating Notch signaling. Although the mechanism by which dFezf represses Notch signaling is unknown, Fezf family genes function to negatively regulate Notch signaling in both vertebrates and invertebrates (Shimizu, 2010).

Fezf1 and Fezf2 function to repress the caudal diencephalon fate and their function is involved in proper rostro-caudal patterning of the forebrain (see Jeong, 2007). The prospective telencephalon domain is already smaller in Fezf1^-/-Fezf2^-/- mouse embryos than in the wild type at E9.5, before neurogenesis is initiated in the telencephalon. Therefore, the defect in rostro-caudal patterning is attributable to reduction of the telencephalon domain. In addition, Fezf2^-/- or Fezf1^-/-Fezf2^-/- telencephalon lacks layer-V subcerebral projection neurons. Hes5 deficiency did not suppress the defects in rostro-caudal patterning of the forebrain or specification of layer-V neurons in Fezf1^-/-Fezf2^-/- forebrains. Therefore, Fezf1/2-mediated downregulation of Hes5 is not involved in the rostro-caudal patterning of the forebrain and the specification of layer-V neurons. Fezf1 and/or Fezf2 probably control genes other than Hes5 to elicit these functions (Shimizu, 2010).

Fezf1^-/-Fezf2^-/- telencephalon exhibited reduced formation of early-born neurons such as SP neurons and CR cells. A birthdate analysis revealed that the reduction of SP neurons and CR cells was not due to mis-specification of these neurons to other types of neurons. The data suggest that generation of the neural stem cells into SP neurons and CR cells is impaired in Fezf1^-/-Fezf2^-/- telencephalon. This finding is consistent with a reduction of differentiated (anti-neuron-specific βIII tubulin antibody TUJ1⁺) neurons in the Fezf1^-/-Fezf2^-/- telencephalon at E10.5, when subplate (SP) neurons and Cajal-Retzius (CR) cells were born in the VZ. Hes5 deficiency rescued neurogenin 2 expression at E10.5 and the generation of SP neurons and CR cells in Fezf1^-/-Fezf2^-/- telencephalon, indicating that Fezf1- and/or Fezf2-mediated repression of Hes5 plays an important role in the generation of these early-born cortical neurons. It is reported that formation of CR cells in the choroid plexus region, near the cortical hem, is controlled by a Hes-neurogenin cascade but that the Notch signal-mediated lateral inhibition is not involved in regulation of the Hes-neurogenin cascade in the CR cell development. Fezf1 and Fezf2 are expressed in the dorsomedial telencephalon. The current data suggest that Fezf1 and Fezf2 might control the development of CR cells by regulating Hes5 and neurogenin 2 expression in the choroid plexus domain (Shimizu, 2010). Fezf1^-/-Fezf2^-/- telencephalon had normal upper-layer (layer II, III) neurons but displayed a reduction of layer-IV neurons. There are two plausible explanations for this finding: Fezf1 and Fezf2 regulate the specification of layer-IV neurons or Fezf1 and Fezf2 control the generation of layer-IV neurons. Neither Fezf1 nor Fezf2 is expressed in differentiated layer-IV neurons, but both are expressed in their progenitors (neural stem cells or intermediate progenitors). Layer-IV neurons are normally born (differentiated) from E13.5 through E15.5. Birthdate analysis indicated that Fezf1^-/-Fezf2^-/- telencephalon contained a reduced number of Rorβ-positive neurons that were born at E13.5, suggesting that Fezf1 and Fezf2 control the generation of layer-IV neurons either from the neural stem cells or the intermediate progenitors. In Fezf1^-/-Fezf2^-/- telencephalon, differentiation of the neural stem cells into the TBR2⁺ intermediate progenitors was impaired. Tbr2 is an essential regulator of the intermediate progenitors and is directly regulated by neurogenin 2. These data suggest that the gene cascade Fezf1/Fezf2 -> Hes5 -> neurogenin 2 regulates the expression of Tbr2 and controls differentiation of the neural stem cells into the intermediate progenitors. The reduction of the TBR2⁺ intermediate progenitors in the Fezf1^-/-Fezf2^-/- telencephalon might contribute to a reduction of layer-IV neurons. Consistent with this idea, Hes5 deficiency rescued the development of TBR2⁺ intermediate progenitors as well as layer-IV neurons in Fezf1^-/-Fezf2^-/- telencephalon. It is reported that TBR1⁺ layer-VI neurons are increased in Fezf2^-/- telencephalon, suggesting the transfate of layer-V to layer-VI neurons. However, they were not increased in Fezf1^-/-Fezf2^-/- telencephalon, implying that the gene cascade Fezf1/Fezf2 -> Hes5 ->neurogenin 2 controls the generation of layer-VI neurons. Future studies will clarify these issues (Shimizu, 2010).

In summary, FEZF1 and FEZF2 are transcriptional repressors that repress Hes5 expression and subsequently activate neurogenin expression. The Fezf1/Fezf2 -> Hes5 -> neurogenin 2 gene cascade controls differentiation of the neural stem cells into neurons or intermediate progenitors and contributes to the generation of a variety of neurons in the forebrain (Shimizu, 2010).

Hairy-related proteins and retinal development

Mammalian hairy and Enhancer of split homolog 1 (HES1), a basic helix-loop-helix factor gene, is expressed in retinal progenitor cells, and its expression decreases as differentiation proceeds. Retinal progenitor cells infected with HES1-transducing retrovirus do not differentiate into mature retinal cells, suggesting that persistent expression of HES1 blocks retinal development. In contrast, in the retina of HES1-null mutant mice, differentiation is accelerated, and rod and horizontal cells appear prematurely and form abnormal rosette-like structures. Lens and cornea development is also severely disturbed. There is extensive bipolar cell death in the mutant retina, to the point of complete disappearance of such cells. These studies provide evidence that HES1 regulates differentiation of retinal neurons and is essential for eye morphogenesis (Tomita, 1996).

Genes that can direct the formation of glia in the retina have been identified. rax, a homeobox gene (Drosophila homolog Rx); Hes1, a basic helix-loop-helix gene, and notch1, a transmembrane receptor gene, are all expressed in retinal progenitor cells, downregulated in differentiated neurons, and expressed in Müller glia. Retroviral transduction of any of these genes results in expression of glial markers. In contrast, misexpression of a dominant-negative Hes1 gene reduces the number of glia. Cotransfection of rax with reporter constructs containing the Hes1 or notch1 regulatory regions leads to the upregulation of reporter transcription. These data suggest a regulatory heirarchy that controls the formation of glia at the expense of neurons (Furukawa, 2000).

Thus, rax, Hes1, and notch1 are expressed by retinal progenitor cells and by differentiating Müller glia. In addition, when individually transduced, all three genes are capable of promoting the formation of cells that express markers of Müller glia. Since all three of these genes are presumably transcription factors, these observations raise the possibility that they either directly or indirectly regulate each other. There is evidence that activated notch1 directly upregulates Hes1. Evidence is provided that rax leads to upregulation of Hes1 and notch1. Following infection with rax-GFP virus, both notch1 and Hes1 RNA are detected using a RT/PCR assay. The upregulation of Hes1 and notch1 by rax may be direct. Reporter constructs with either the notch1 or Hes1 regulatory regions showed a 5-fold induction in RNA levels when rax is cotransfected. The Hes1 upstream region encodes two putative sites for a paired-type homeobox gene, such as rax. The sequence of the 11 kb notch1 regulatory regions is not yet known, but the data predict that such a site is present in notch1 as well. Since rax is expressed prior to notch1 or Hes1 in the retinal anlagen, it is likely that at least the initial period of rax transcription is independent of Hes1 and notch, while the subsequent expression of Hes1 and notch1 may be dependent upon rax (Furukawa, 2000).

Neurons and glial cells differentiate from common precursors. Whereas the gene glial cells missing determines the glial fate in Drosophila, current data about the expression patterns suggest that, in mammals, gcm homologs are unlikely to regulate gliogenesis. In mouse retina, the bHLH gene Hes5 is specifically expressed by differentiating Müller glial cells and misexpression of Hes5 with recombinant retrovirus significantly increases the population of glial cells at the expense of neurons. Conversely, Hes5-deficient retina show 30%-40% decrease of Müller glial cell number without affecting cell survival. These results indicate that Hes5 modulates glial cell fate specification in mouse retina (Hojo, 2000).

It remains to be determined how Hes5 specifies the glial fate in the retina. One possible mechanism is that Hes5, a DNA-binding repressor, may repress expression of neuronal bHLH genes such as Mash1, NeuroD and Math3 and lead to the glial fate. Supporting this idea, in the retina of Mash1- or NeuroD-deficient mice, Müller glial cells increase in number. The notion that Hes5 may repress Mash1 expression is also supported by the finding that Mash1 expression is prematurely upregulated in the regions where Hes5 expression disappears in RBP-J or Notch1 mutant mice. Thus, the antagonistic regulation between Hes5 and the neuronal bHLH genes such as Mash1 may determine the ratio of neuronal to glial cell numbers. However, in the retina of Hes5-deficient mice, many Müller glial cells still are differentiated, suggesting that Hes5 may be redundant in gliogenesis (Hojo, 2000 are references therein).

Members of a subclass of hairy/Enhancer of split [E(spl)] homologs, called hesr genes, are structurally related to another subclass of hairy/E(spl) homologs, Hes genes, which play an important role in neural development. To characterize the roles of hesr genes in neural development, the retina was used as a model system. In situ hybridization analysis indicates that all hesr genes are expressed in the developing retina, but only hesr2 expression is associated spatially with gliogenesis. Each member was misexpressed with retrovirus in the retinal explant cultures prepared from mouse embryos or neonates; this mimics well the in vivo retinal development. Interestingly, hesr2 but not hesr1 or hesr3 promotes gliogenesis while inhibiting rod genesis without affecting cell proliferation or death, suggesting that the cells that normally differentiate into rods adopt the glial fate by misexpression of hesr2. The gliogenic activity of hesr2 is more profound when it is misexpressed postnatally than prenatally. In addition, double mutation of the neuronal determination genes Mash1 and Math3, which increases Müller glia at the expense of bipolar cells, upregulates hesr2 expression. These results indicate that, among structurally related hesr genes, only hesr2 promotes glial versus neuronal cell fate specification in the retina and that antagonistic regulation between hesr2 and Mash1-Math3 may determine the ratios of neurons and glia (Satow, 2001).

In the developing retina, the production of ganglion cells is dependent on the proneural proteins NGN2 and ATH5, whose activities define stages along the pathway converting progenitors into newborn neurons. Crossregulatory interactions between NGN2, ATH5 and HES1 maintain the uncommitted status of ATH5-expressing cells during progenitor patterning, and later on regulate the transition from competence to cell fate commitment. Prior to exiting the cell cycle, a subset of progenitors is selected from the pool of ATH5-expressing cells to go through a crucial step in the acquisition of a definitive retinal ganglion cell (RGC) fate. The selected cells are those in which the upregulation of NGN2, the downregulation of HES1 and the autostimulation of ATH5 are coordinated with the progression of progenitors through the last cell cycle. This coordinated pattern initiates the transcription of ganglion cell-specific traits and determines the size of the ganglion cell population (Matter-Sadzinski, 2005).

Spatial cell patterning and RGC commitment correlate with the two main phases of ATH5 expression. During the period of patterning, crossregulatory interactions between HES1, NGN2 and ATH5 keep ATH5 expression low, thereby maintaining the uncommitted status of ATH5-expressing cells and enabling the expansion and intermingling of pools of progenitors initially partitioned in distinct domains. Once progenitors are properly distributed throughout the retina, about one-third of ATH5-expressing cells become committed to acquire a definitive RGC fate immediately before exiting the cell cycle. This requires a tight coordination between downregulation of HES1, upregulation of NGN2, cell progression through the last S-phase and the upregulation of ATH5. Cells that upregulate ATH5 expression initiate transcription of early RGC-specific traits, then exit the cell cycle and express Neuro M and other post-mitotic RGC-specific genes. This study highlights how changes in the transcriptional patterns correlate with the progression of progenitors through the last cell cycle and with their commitment to the RGC fate, underlining the role of HES1 as a key prompt of the molecular events leading to RGC genesis (Matter-Sadzinski, 2005).

A specific feature of retinogenesis is that it proceeds from the centre to the periphery such that all seven retinal cell types are distributed at the proper ratio throughout the retina. At early stages of development, the retinal neuroepithelium is subdivided into two developmentally distinct territories. Low levels of HES1 transcripts outline a broad region of the posterior retina where ATH5, NGN2 and ASH1 are expressed, whereas a robust accumulation of HES1 transcripts throughout the anterior retina prevents the onset of proneural gene expression. HES1 functions similarly at the onset of neurogenesis in the olfactory placode, where it circumscribes a domain of Mash1 expression. It thus appears that HES1 is acting, much like hairy in Drosophila, as a prepattern gene. Neurogenesis starts within a rather broad central region defined by expression of ATH5, NGN2 and Neuro M. Cells expressing ATH5 at a high level and Neuro M-positive cells are evenly distributed throughout the neurogenic domain, indicating that the first newborn RGCs are produced with similar frequency throughout the central retina. In the posterior retina, cells that initiated expression of proneural genes are initially organized in two separate domains corresponding to two retinal lineages: cells that express NGN2/ATH5 constitute the progenitor pools from which early-born retinal neurons will emerge, whereas ASH1-expressing cells form a pool for late-born neurons. The opposite effects of NGN2 on ATH5 and ASH1 expression combined with the inhibitory activity of ASH1 on ATH5 transcription account for the distribution of ASH1 and ATH5/NGN2 cells in two distinct progenitor domains, the more peripheral expression of ASH1 perhaps reflecting its lower sensitivity towards HES1. The initial patterning of the posterior retina resembles the neuroepithelial partitioning detected in other areas of the developing CNS. However, whereas in other CNS regions the refining of borders is essential for the precise spatial generation of different classes of neurons along the dorsoventral axis, the blurring of borders and intermingling of initially distinct progenitor pools are necessary for a proper spatial distribution of neurons and glia throughout the retina. Although ATH5/NGN2 and ASH1 expressions are mutually exclusive, a small fraction of ATH5-expressing cells co-express ASH1, indicating that they are in a transient state prior to acquiring a definite progenitor status. Because the ATH5, NGN2 and ASH1 genes crossregulate and display different sensitivities towards HES1, it is supposed that various balances between these four factors may mediate alternate fate choices. Such dynamic regulatory interactions are, in part, responsible for the progressive loss of patterning in the posterior retina. The ATH5/NGN2 domain remains restricted to the posterior retina until E4 and expands to keep pace with growth of the whole retina at a rate similar to that reported for the differentiation of RGCs. Despite significant changes in the expression pattern of ATH5, similar proportions of retinal cells express this gene at stages 18 and 29-30, suggesting that ATH5-expressing cells propagate at a rate comparable with that of the other progenitors during the period of patterning (Matter-Sadzinski, 2005).

Even though the population of ATH5-expressing cells is established at E2.5, only a small fraction of these will differentiate into RGCs until E4. Retinogenesis is controlled by components of the Notch pathway, which may employ two strategies to keep the majority of cells in the central retina from differentiating during the patterning period. Cells that express proneural genes may promote the upregulation of HES1 in neighbouring cells, thereby preventing them from expressing proneural genes. The proximity in central retina of individual cells that highly express HES1 or ATH5 is indeed indicative of ongoing lateral inhibition. However, cells strongly expressing Notch effectors are rare in the posterior retina, whereas a high proportion of ATH5-expressing progenitors co-express HES1. Thus, it appears that the low level of HES1 in cells that have already initiated NGN2 and ATH5 expression suffices to prevent the upregulation of these genes. The proliferative state is thereby maintained in most ATH5-expressing cells, as required to ensure the proper ratio of RGC progenitors in the posterior retina and as expected of HES genes, which function to keep neuroepithelial cells undifferentiated, thereby regulating the size and cell architecture of brain structures and retina. In anterior retina, progenitor cell patterning becomes evident by E4 and the expansion of proneural gene expression proceeds, much as in zebrafish, in a wave-like fashion as HES1 expression recedes to the retinal margin. The ASH1 and NGN2 expression domains expand to the periphery at similar rates, whereas the progression of the ATH5 domain is slightly delayed. The full patterning of the retina accomplished around E6 coincides with the upregulation of proneural gene expression throughout the retina and with the peak of RGC production (Matter-Sadzinski, 2005).

To analyse how ATH5 is regulated along the course of RGC specification, a promoter region extending 775 bp upstream of the initiation codon was used. The cloned sequence accurately reproduces the activity and the mode of regulation of the endogenous promoter. It contains essential regulatory elements that are well conserved across distant vertebrate species, but it is unclear whether the different species use similar strategies to regulate ATH5 expression. Whereas a proximal cis-regulatory region of the Xenopus Xath5 gene suffices, much as in the chick retina, to drive retina specific reporter gene expression in a bHLH-dependent manner, the mouse ATH5 promoter appears to be regulated differently. It is tempting to speculate that the different modes regulating ATH5 across species may account for differences in the spatiotemporal progenitor patterning of the retinal neuroepithelium. Differences in the developments of the anterior and posterior retinas may have permitted the evolution of a specialized structure such as the macula (Matter-Sadzinski, 2005).

This study reveals that NGN2 acts at different regulatory levels during RGC specification. In early retina, NGN2 is a principal regulator of ATH5 expression and exerts this function through direct activation of ATH5 transcription and through crossregulatory interactions with HES1. In addition, NGN2 drives ATH5-expressing cells out of S phase. Whereas the capacity of NGN2 to promote cell cycle arrest is part of its panneuronal activities and is in evidence in other compartments of the developing CNS, its capacity to activate ATH5 expression is largely retina specific. The quasi-simultaneous onset of NGN2 and ATH5 expression in the central retina shortly after formation of the eye cup, the capacity of NGN2 to activate ATH5 transcription and to bind the ATH5 promoter at the early stages of development suggest that NGN2 may be directly involved in the activation of ATH5 expression. The finding that the expansion of the NGN2 domain towards the anterior edge of the retina precedes that of ATH5 argues in favour of this interpretation. In the retina of the Ngn2^–/– mouse, the much increased expression of ASH1 and the downregulation of ATH5 when compared with the wild type, may result from an increase in the population of ASH1-expressing cells at the expense of the ATH5/NGN2 progenitors, thus underlining the importance of NGN2 in establishing and maintaining a pool of ATH5-expressing cells. Both the NGN2 and ATH5 genes fail to be activated in the retinal precursors of the Pax6^–/– mouse and Pax6 has been proposed to regulate NGN2 directly in the mouse retina. There are multiple E-boxes but no consensus Pax6 binding site in the chicken ATH5 promoter, and therefore the idea is favored that Pax6 regulates ATH5 via NGN2. The expression of NGN2 in many regions of the nervous system anlage where ATH5 is not detected and the demonstration that recruitment of NGN2 on the ATH5 promoter is retina specific provide evidence that a retina-specific context accounts for the capacity of NGN2 to activate ATH5 expression. The ability of bHLH factors to regulate the development of distinct neurons has been proposed to depend upon the cellular contexts in which they function. In retina, this context may be determined, among other possibilities, by the balance between NGN2 and HES1: as show, HES1 inhibits the NGN2-mediated activation of ATH5 in a dose-dependent manner. Likewise, the upregulation of NGN2 correlates with the dowregulation of HES1. Moreover, single cell transcriptional analysis reveals that overexpressing NGN2 diminishes the pool of cells that co-express ATH5 and HES1, an indication that NGN2 may contribute to the downregulation of HES1 in early neural progenitors, thereby providing a cellular environment permissive for ATH5 autostimulation (Matter-Sadzinski, 2005).

The upregulation of both NGN2 and ATH5 occurs later in development, around E6, but by then ATH5 has become the main regulator of its own transcription. NGN2 occupies the ATH5 promoter similarly at E3 and at E6, suggesting that it still directly participates in the control of ATH5 transcription. However, its main contribution to ATH5 expression may occur through other, indirect regulatory pathways. As ATH5-expressing progenitors exit the cell cycle, NGN2 promotes the expression first of Neuro M and then of Neuro D, both stimulators of ATH5 promoter activity. These distinct functions of NGN2 in the ontogenesis of RGCs illustrate how, depending on specific combinations of transcription factors and of other cellular components, neurogenic proteins may contribute to neuronal identity (Matter-Sadzinski, 2005).

Hairy1 acts as a node downstream of Wnt signaling to maintain retinal stem cell-like progenitor cells in the chick ciliary marginal zone

In the vertebrate retina, stem cell-like progenitor cells are maintained in a distinct region called the ciliary marginal zone (CMZ). Canonical Wnt signaling regulates the maintenance of the progenitor cells in the CMZ. However, its downstream molecular mechanisms have remained largely unclear. This study shows that chick Hairy1, an established Notch signaling effector, mediates the Wnt-dependent maintenance of CMZ progenitor cells in chicken. Interestingly, unlike other developmental contexts in which Hes gene expression is regulated by Notch signaling, Hairy1 expression in the CMZ is regulated by Wnt signaling. Hairy1 is necessary and sufficient for the expression of a set of molecular markers characteristic of the CMZ, and Wnt2b fails to induce CMZ markers when Hairy1 activity is inhibited. Furthermore, microarray analysis identifies multiple Wnt-responsive transcription factors that activate Hairy1 expression. It is proposed that Hairy1 functions as a node downstream of Wnt signaling to maintain progenitor cells in the chick CMZ (Kubo, 2009).

Hairy-related proteins and segmentation

her1, the zebrafish homolog of hairy is transcribed in a pattern similar to both that of hairy and to that of its Tribolium orthologue, suggesting that her1 is functionally related to hairy. During late gastrulation and somitogenesis, her1 is transcribed in the advancing epibolic margin and in metameric stripes of paraxial mesodermal cells, which alternate with transcriptionally silent zones. The distribution of her1 transcripts argues for a role of her1 in formation and/or differentiation of somites (Müller, 1996).

There is an ongoing discussion as to whether segmentation in different phyla has a common origin sharing a common genetic program. However, before comparing segmentation between phyla, it is necessary to identify the ancestral condition within each phylum. Even within the arthropods it is not clear which parts of the genetic network leading to segmentation are conserved in all groups. In this paper, the expression of three segmentation genes of the pair-rule class is examined in the spider Cupiennius salei. Spiders are representatives of the Chelicerata, a monophyletic basic arthropod group. During spider embryogenesis, segments are sequentially added at the posterior end of the embryo, which resembles the formation of the abdominal segments in short-germ insect embryos. In spider embryos, the orthologues for the Drosophila primary pair-rule genes hairy, even-skipped, and runt are expressed in stripes in the growth zone, where the segments are forming, suggesting a role for these genes in chelicerate segmentation. These data imply that the involvement of hairy, even-skipped, and runt in arthropod segmentation is an ancestral character for arthropods and is not restricted to a particular group of insects (Damen, 2000).

Previous expression data had suggested a conserved role for the Hes genes in the Notch signaling pathway, but not in segmentation. Here, Hes3 expression during mouse embryogenesis is described. During early development of the central nervous system, Hes3 is expressed specifically in the region of the midbrain/hindbrain boundary, and in rhombomeres 2, 4, 6 and 7. This pattern occurs at approximately the same time that Krox20 expression appears in r3 and r5 and precedes the morphological appearance of rhombomeres. The regulatory interactions between Krox20 and Hes3 are currently unknown. The segmental pattern of Hes3 suggests that it may have a conserved role as a segmentation gene. Later in development, Hes3 is co-expressed with other neurogenic gene homologs in the developing central nervous system and epithelial cells undergoing mesenchyme induction (Lobe, 1997).

The cyclic gene Hes1 contributes to diverse differentiation responses of embryonic stem cells

Stem cells do not all respond the same way, but the mechanisms underlying this heterogeneity are not well understood. This study found that expression of Hes1 and its downstream genes oscillate in mouse embryonic stem (ES) cells. Those expressing low and high levels of Hes1 tended to differentiate into neural and mesodermal cells, respectively. Furthermore, inactivation of Hes1 facilitated neural differentiation more uniformly at an earlier time. Thus, Hes1-null ES cells display less heterogeneity in both the differentiation timing and fate choice, suggesting that the cyclic gene Hes1 contributes to heterogeneous responses of ES cells even under the same environmental conditions (Kobayashi, 2009).

The oscillations continued throughout the cell cycle, but Hes1 tended to be expressed at higher levels during S-G2 phases compared with G1 phase. The oscillations seemed to be synchronized between neighboring daughter cells after cell division, although they easily became asynchronous in large colonies. The period of Hes1 oscillations was variable from cycle to cycle and from cell to cell. The power spectrum of Hes1 oscillations after Fourier transformation showed that the periodicity was ~3-5 h, although it included many noises of short periodicity. The period of 3-5 h in ES cells was longer than in other cell types (2-3 h). The half-life of Hes1 protein was ~16 min in ES cells, which was similar to fibroblasts, whereas that of Hes1 mRNA was about two to four times longer in ES cells (~46 min) than in fibroblasts, implying that the stabilization of Hes1 mRNA contributes to a longer periodicity in ES cells (Kobayashi, 2009).

Because Hes1 expression oscillated, some downstream genes might be also expressed in an oscillatory manner. To determine the relationship of Hes1 expression with the downstream gene expression, 32 single ES cells were randomly picked up and cDNAs were made from each cell to perform quantitative real-time PCR (single-cell Q-PCR). These ES cells expressed variable levels of Hes1 and were classified into three groups according to the Hes1 expression level. When Hes1 expression is high, both Dll1 and Gadd45g expression are also high and vice versa, whereas the other downstream genes p57, Lef1, Jag1, and Crabp2 display different patterns. Thus, expression levels of Hes1, Dll1, and Gadd45g changed in a similar manner in individual ES cells, suggesting that these genes oscillate in phase. When the Hes1 protein level is high, transcription of all Hes1, Dll1, and Gadd45g genes may be repressed, but they may be activated when the Hes1 protein level is low. Due to this delayed negative regulation by Hes1, expression of all these genes probably oscillates in phase. To obtain direct evidence that both Dll1 and Gadd45g expression also oscillate in ES cells, their expression dynamics were further analyzed by a real-time imaging method using a ubiquitin-fused luciferase reporter under the control of Dll1 or Gadd45g promoter. The expression of both Dll1 and Gadd45g dynamically changed in many individual ES cells. These results suggest that the differentiation competency of ES cells can be changed rapidly by oscillations of Dll1, a ligand of Notch signaling that induces neural differentiation, and Gadd45g, which inhibits cell cycle progression. Interestingly, while Nanog was mostly expressed by picked-up ES cells, its expression level seemed to be variable and became higher when Hes1 was highly expressed, whereas there was no such relationship with Oct3/4 and Sox2, suggesting that Hes1 oscillations may have some correlation with Nanog fluctuation but not with Oct3/4 or Sox2 (Kobayashi, 2009).

Recent reports show that reversible changes in gene expression occur slowly, over several days, in ES cells and hematopoietic progenitor cells, resulting in different potentials for differentiation, although the mechanism for such slow changes remains to be elucidated. This study has shown that Hes1 and its downstream gene expression dynamically change much faster, over several hours, in ES cells. Hes1-high cells tend to adopt the mesodermal fate whereas Hes1-low cells tend to differentiate into neural cells. Furthermore, virtually all Hes1-null ES cells differentiated into the neural cells within 6 d under the neural differentiation condition, whereas only subsets of wild-type ES cells did so. Thus, in the absence of Hes1, ES cells display less heterogeneity in both the differentiation timing and fate choice, suggesting that the cyclic gene Hes1 contributes to heterogeneous responses of ES cells. Such rapid cycling of gene expression might be suitable to make multiple cell types even under a single differentiation condition. Hes1 expression also oscillates in neural progenitor cells, but this oscillation seems to contribute to maintenance of the undifferentiated state rather than the diversity in responses. Thus, it is likely that Hes1 oscillations have different functions in different cell types (Kobayashi, 2009).

Hairy homologs axial segmentation and mesoderm

c-hairy1, an avian homolog of the Drosophila segmentation gene hairy, is identified as a molecular clock linked to vertebrate segmentation and somitogenesis. c-hairy1 is strongly expressed in the presomitic mesoderm, where its mRNA exhibits cyclic waves of expression whose temporal periodicity corresponds to the formation time of one somite (90 min). The apparent movement of these waves is due to coordinated pulses of c-hairy1 expression, not to cell displacement along the anteroposterior axis, nor to propagation of an activating signal. c-hairy1 was studied in explant cultures of presomitic mesoderm isolated from all the surrounding tissue that might provide extrinsic signals. The presomitic mesoderm from half of the 15- to 25-somite embryos was separated from ectoderm, endoderm, neural tube, notochord, lateral plate, and tail bud, while the other half remained intact. The two halves were cultured separately for up to 180 minutes. c-hairy1 expression patterns are similar in both types of explant, suggesting that the kinetics of c-hairy1 are independent of surrounding tissues. The rhythmic c-hairy mRNA expression appears to be an autonomous property of the paraxial mesoderm. Progression of the c-hairy1 wavefront and operation of the c-hairy1 clock are insensitive to blocking protein synthesis by cycloheximide. This tends to exclude c-hairy1from a role in the clock mechanism itself. Instead, it is likely that c-hairy1 expression acts posttranslationally, using protein modifications such as phosphorylation. These results provide molecular evidence for a developmental clock linked to segmentation and somitogenesis of the paraxial mesoderm, and support the possibility that segmentation mechanisms used by invertebrates and vertebrates have been conserved (Palmeirim, 1997).

Somitogenesis has been linked both to a molecular clock that controls the oscillation of gene expression in the presomitic mesoderm (PSM) and to Notch pathway signaling. The oscillator, or clock, is thought to create a prepattern of stripes of gene expression that regulates the activity of the Notch pathway that subsequently directs somite border formation. The zebrafish gene after eight (aei) that is required for both somitogenesis and neurogenesis encodes the Notch ligand DeltaD. Additional analysis has revealed that stripes of hairy relate her1 expression oscillate within the PSM and that aei/DeltaD signaling is required for this oscillation. aei/DeltaD expression does not oscillate, indicating that the activity of the Notch pathway upstream of her1 may function within the oscillator itself. Moreover, her1 stripes are expressed in the anlage of consecutive somites, indicating that her1 expression pattern is not pair-rule. Analysis of her1 expression in aei/DeltaD, fused somites (fss), and aei;fss embryos has uncovered a wave-front activity that is capable of continually inducing her1 expression de novo in the anterior PSM in the absence of the oscillation of her1. The wave-front activity, in reference to the clock and wave-front model, is defined as such because it interacts with the oscillator-derived pattern in the anterior PSM and is required for somite morphogenesis. This wave-front activity is blocked in embryos mutant for fss but not aei/DeltaD. Thus, this analysis indicates that the smooth sequence of formation, refinement, and fading of her1 stripes in the PSM is governed by two separate activities (Holley, 2000).

Somite formation is thought to be regulated by an unknown oscillator mechanism that causes the cells of the presomitic mesoderm to activate and then repress the transcription of specific genes in a cyclical fashion. These oscillations create stripes/waves of gene expression that repeatedly pass through the presomitic mesoderm in a posterior-to-anterior direction. In both the mouse and the zebrafish, it has been shown that the notch pathway is required to create the stripes/waves of gene expression. However, it is not clear if the notch pathway comprises part of the oscillator mechanism or if the notch pathway simply coordinates the activity of the oscillator among neighboring cells. In the zebrafish, oscillations in the expression of a hairy-related transcription factor, her1 and the notch ligand deltaC precede somite formation. This study focuses on how the oscillations in the expression of these two genes areaffected in the mutants aei/deltaD and des/notch1, in 'morpholino knockdowns' of deltaC and her1 and in double 'mutant' combinations. This analysis indicates that these oscillations in gene expression are created by a genetic circuit comprised of the notch pathway and the notch target gene her1. A later function of the notch pathway can create a segmental pattern even in the absence of prior oscillations in her1 and deltaC expression (Holley, 2002).

Both aei/deltaD and des/Notch1 are necessary to promote the expression of the oscillating genes her1 and deltaC. Meanwhile, her1 regulates deltaC expression and functions, directly or indirectly, in a negative feedback loop to repress its own transcription. Thus, the notch pathway functions upstream of her1 to promote the transcription of her1 mRNA, and her1 functions upstream of the Notch pathway to create the oscillating pattern of deltaC transcription. This identifies a rudimentary genetic loop (notch pathway > her1 > notch pathway) that functions within the PSM. Further, fused somites (fss) functions downstream of the notch pathway but upstream of her1 in the anterior PSM, and the notch pathway and fss function downstream of her1 slightly later in the anteriormost PSM. Therefore, the regulatory circuit consisting of her1 and the notch pathway exists throughout the PSM. Because this genetic circuit comprises genes that are required to create the oscillations in gene expression, these findings suggest that her1 and the notch pathway have cyclical functions at the center of the somitogenesis oscillator (Holley, 2002).

The genetic analysis of her1 and the notch pathway suggest a model in which these genes somehow generate the oscillations in gene expression. The initiation of the oscillations may be coupled to the commitment to become paraxial mesoderm. The expression of each of these genes (her1, deltaC, aei/deltaD and des/notch1) is initiated at the tip of the tailbud as cells subduct to form the paraxial mesoderm. The subsequent activities of these proteins could then initiate the interactions that create the oscillations in gene expression. deltaC, aei/deltaD and des/notch1 signaling would activate the transcription of her1 and deltaC. The subsequent increase in Her1 protein would then act to block the transcription of her1. Since the hairy proteins typically function as transcriptional repressors, an increase in Her1 should result in an increase in repressive activity, and the gradual degradation of this protein would produce a gradual decrease in this repressive activity. Therefore, the anterior progression/activation of a stripe of gene expression could be driven by the gradual loss of a repressive activity generated during the previous somite cycle. The positive regulation via notch could also display a cyclical variation, but ultimately the re-initiation of her1 and deltaC transcription would not occur until the level of Her1 drops below a specific threshold. In essence, this model suggests that the anterior progression of a stripe of gene expression is, at least in part, driven by the degradation of an existing, repressive activity (Her1), as opposed to the de novo synthesis of an activating component (Holley, 2002).

The analysis of deltaC expression in her1^mo embryos uncovers an additional Notch-dependent patterning activity in the anterior PSM. This activity can create a segmental pattern of gene expression in the absence of any evidence of oscillations in her1 and deltaC expression: a smooth domain of deltaC expression is refined anteriorly to create stripes of expression that persist in the somitic mesoderm. This refinement requires the activity of fss, aei/deltaD, des/notch1, deltaC and beamter (bea), indicating that each of these genes has an additional function in the anterior-most PSM, downstream of her1. This is consistent with the fact that aei/deltaD, deltaC and des/notch1 are each transcribed within the PSM and later in the somitic mesoderm. In fact, this refining pattern is likely to be revealed only within the her1^mo embryos because her1 is the only one of these cloned genes whose expression is restricted to the PSM. Ultimately, this indicates that the phenotypes observed in aei/deltaD and des/notch1 embryos are composites of defects that occur both upstream and downstream of her1 (oscillator) function. It has been shown that notch pathway signaling is involved in establishing the anteroposterior pattern within each somite. The late activity of the notch pathway described here probably represents this same anteroposterior patterning function. What is remarkable is that this late function can create a segmental pattern in the absence of prior oscillations in her1 and deltaC expression (Holley, 2002).

Somitic segmentation provides the framework on which is established the segmental pattern of the vertebrae, some muscles and the peripheral nervous system. Recent evidence indicates that a molecular oscillator, the 'segmentation clock', operates in the presomitic mesoderm (PSM) to direct periodic expression of c-hairy1 and lunatic fringe (l-fng). The identification and characterization of a second avian hairy-related gene, c-hairy2, is reported, that also cycles in the PSM and whose sequence is closely related to the mammalian HES1 gene, a downstream target of Notch signaling in vertebrates. HES1 mRNA is also expressed in a cyclic fashion in the mouse PSM, similar to that observed for c-hairy1 and c-hairy2 in the chick. In HES1 mutant mouse embryos, the periodic expression of l-fng is maintained, suggesting that HES1 is not a critical component of the oscillator mechanism. In contrast, dynamic HES1 expression is lost in mice mutant for Delta1 that are defective for Notch signaling. In order to investigate the relationship between the dynamic HES1 expression in the PSM and the Notch signaling pathway, HES1 expression was examined in Dll1 homozygous mutant mice in which Notch activation is impaired in the PSM. Homozygous null mutants for the Dll1 gene exhibit strong segmentation defects and a severe down-regulation of l-fng expression. The expression of HES1 at E10.5 was compared in wild type, heterozygous and homozygous null mutants by in situ hybridization. The dynamic expression of HES1 in the PSM is maintained in Dll1+/- embryos as shown by the different expression patterns observed in the PSM. In contrast, all Dll1-/- embryos show the same global downregulation of HES1 expression in the PSM (n=7). This observation suggests that HES1 expression in the PSM is dependent on the Notch signaling pathway and suggests that Notch signaling is required for hairy-like genes cyclic expression in the PSM (Jouve, 2000).

Thus HES1 and l-fng dynamic expression are lost in the PSM of Dll1 mutants, in which Notch signaling is disrupted. Various clock outputs appear, therefore, to be severely downregulated when Notch signaling is disrupted. These observations raise the possibility that in addition to being an output of the segmentation clock as previously proposed, the Notch signaling pathway might also be an important component of the oscillator. Notch activation upon ligand binding involves a proteolytic cleavage liberating the intracytoplasmic domain (NICD), which translocates into the nucleus where together with Su(H)/RBPjk it activates the transcription of genes such as HES1 in vertebrates. The observations in the Dll1-/- mice indicate that HES1 is downstream of the Notch pathway in the PSM. Since c-hairy1 and c-hairy2 share a high similarity in their sequence and in their expression patterns to HES1, they are also likely targets of Notch signaling in the chick PSM. A direct regulation of c-hairy1 and c-hairy2 expression by oscillating Notch activation would explain why c-hairy1 expression is insensitive to cycloheximide, since protein synthesis is not required for transduction of the Notch signal. To achieve oscillations of Notch signaling, the activity of the pathway would need to be modulated by a feedback mechanism. However, known output events resulting from Notch signaling are transcriptional regulation of target genes and the clock is partly independent of protein synthesis. Notch1 and Delta1 are present along the whole presomitic mesoderm and could generate constitutive activation of the pathway in the tissue. The rhythmic modification of this activation could, in principle, be achieved by the periodic expression of l-fng (Jouve, 2000).

Vertebrate hairy genes are expressed in patterns thought to be readouts of a 'segmentation clock' in the presomitic mesoderm (PSM). Transgenic Xenopus embryos were used to show that two types of regulatory elements are required to reconstitute the segmental pattern of Xenopus hairy2. The first is a promoter element containing two binding sites for Xenopus Su(H), a transcriptional activator of Notch target genes. The second is a short sequence in the hairy2 3' untranslated region (UTR), which most likely functions posttranscriptionally to modulate hairy2 RNA levels. 3' UTRs of other hairy-related, segmentally expressed genes can substitute for that of hairy2. These results demonstrate a novel mechanism regulating the segmental patterns of Notch target genes and suggest that vertebrate segmentation requires the intersection of two regulatory pathways (Davis, 2001).

To function in PSM expression, the UTR must be in its normal position and orientation in the transcript of the transgene, a requirement consistent with a modulator of RNA levels after transcription initiation, possibly entirely at the posttranscriptional level. That 3' UTR function can be reduced to a discrete 25 bp motif suggests this motif is the target for trans-acting factors controlling PSM expression. The role for this sequence could in principle affect a nuclear or a cytoplasmic event required for hairy2a RNA levels to accumulate in anterior PSM cells. The hairy2a 3' UTR also increases the rate of RNA turnover. Based on these findings, one plausible mechanism for UTR function is to both destabilize RNA in general and to provide a specific sequence for factors that transiently stabilize the RNA in the anterior PSM. To produce a transient stripe, these factors must themselves be rapidly inactivated soon after hairy2a expression and before somite formation. The results do not rule out a role for the UTR in transcriptional control of hairy2a, although such a role would likely occur after transcription initiation. Because neither the promoter, nor the 3' UTR, alone yield an obvious subpattern of the PSM stripe, a more speculative interpretation of these results would posit a molecular interaction between the promoter and 3' UTR through bound trans-acting factors (Davis, 2001).

The substitution of the hairy2a 3' UTR with UTRs from other segmentally expressed genes, even from other species, suggests a common mechanism controlling the segmental expression of these other genes. The c-hairy1 and mouse HES1 3' UTRs generate a somewhat broader, more diffuse PSM stripe than the hairy2a or other Xenopus 3' UTRs. This suggests that while there is some functional recognition of these UTRs, they do not show the more focused spatial specificity of the natural 3' UTR. Possibly, Xenopus 3' UTR binding factors may have higher affinity, or slower turnover, on the c-hairy1 and mouse HES1 UTRs, and thus widen the domain of cells where the transgene RNA accumulates. Consistent with this broader PSM expression, the cardiac actin promoter transgene with the hairy2a 3' UTR is expressed in a much wider PSM domain, as is the endogenous cardiac actin gene, but is still appreciably downregulated in newly formed somites and remains at low levels in more anterior somites. This suggests that factors that recognize the hairy2a 3' UTR are, in fact, distributed over a much wider domain of the PSM than is hairy2a RNA, whose expression is further restricted to the anterior PSM by the hairy2a promoter. Taken together, these results support a role for the 3' UTR in controlling the segmental pattern of Notch target genes (Davis, 2001).

A similar situation may exist in Drosophila, where it has been shown that the 3' UTRs of E(spl) genes and the bearded class genes (which coincidentally negatively regulate Notch activity) can negatively regulate their own respective expression levels and patterns. This negative regulation affects neural tissue patterning controlled by these genes, while no 3' UTR-dependent regulation of hairy has been described for segmentation in Drosophila. It is possible that the 3' UTR function has been coopted in evolution for regulating vertebrate segmentation genes (based on the conservation of Notch signaling), or perhaps lost in the evolution of segmentation in Drosophila and other long germ-band insects (Davis, 2001).

The hairy2a PSM pattern shows two types of temporal asynchrony. (1) Expression is consistently activated first in the dorsal half of the PSM stripe. A similar asynchrony exists in the anterior PSM stripes of esr5, esr4, and 8C9, as well as the stripe patterns of Delta1 and Delta2. It seems reasonable to suggest that this aspect of hairy2a expression, as well as that of the esr genes, may be driven, in part, by the similar pattern of Delta1 or Delta2 (i.e., timing of Notch activation). (2) The hairy2a pattern is consistently phase-advanced on the right side of the embryo (as are the esr, Delta1, and Delta2 stripes). Such bilateral asynchrony has not been described in other vertebrates, though its observation in the frog suggests that there is no obligate bilateral synchronization in vertebrate segmentation. It may be that genes involved in left-right asymmetry, such as Xenopus nodal-related 1, cause a phase shift in segmentation on the two sides of the embryo (Davis, 2001).

Rather than oscillating over the whole PSM, the hairy2a pattern behaves as a single wavefront translocating through the anterior PSM and prefiguring new somite formation. In fact, none of the genes whose orthologs are reported to oscillate in the fish, chick, or mouse PSM do so in Xenopus, including hairy2a/b, hairy1 (orthologous to mouse and human HES1 and c-hairy2, lunatic fringe, or esr5 [related to her-1 in zebrafish]). This suggests that cycling in the PSM, exemplified by c-hairy1, is not an invariant property of vertebrate segmentation. Though there are some differences in the exact temporal pattern of hairy and E(spl)-related genes in the PSM of different vertebrates, it is likely that the basic timing mechanisms and the readout of these timing mechanisms are very similar. The pattern differences are probably a function of upstream events, such as activation of Su(H) by regulation of Notch signaling. It is predicted that the c-hairy1 promoter will be very similar to the Xenopus hairy2a and human HES4 promoters, while the c-hairy2 promoter will be very similar to that of the mouse and human HES1 promoters (Davis, 2001).

A schematic is presented of the molecular targets that control the hairy2a pattern, and, by implication, the targets of the segmentation clock. There are at least three types of cis regulatory inputs. The first one is represented by the two Su(H) binding sites, which are presumably bound by a Su(H) containing complex that represses the hairy2a promoter, until Notch signaling switches Su(H) to an activator. The second one is represented by the conserved hexamer between the Su(H) binding sites. Although mutation of the hexamer lowers expression levels in tissues other than the PSM, the possibility that the hexamer has a specific function in modulating the paired Su(H) motif function in the PSM cannot be ruled out. The central role of the paired Su(H) motif for transcriptional activation of hairy2a suggests that Notch signaling must be a fundamental component of the segmentation clock. The third input occurs through the 3' UTR, in particular through the 25 bp motif. The 3' UTR confers global instability on the hairy2a RNA, but apparently local stability in the anterior PSM. How inputs through these three types of small sequences are integrated to control the dynamic hairy2a PSM pattern is a major question for understanding the molecular mechanisms of segmentation (Davis, 2001).

The basic helix-loop-helix (bHLH) gene Hes7, a putative Notch effector, encodes a transcriptional repressor. Hes7 expression oscillates in 2-h cycles in the presomitic mesoderm (PSM). In Hes7-null mice, somites are not properly segmented and their anterior-posterior polarity is disrupted. As a result, the somite derivatives such as vertebrae and ribs are severely disorganized. Although expression of Notch and its ligands is not affected significantly, the oscillator and Notch modulator lunatic fringe is expressed continuously throughout the mutant PSM. These results indicate that Hes7 controls the cyclic expression of lunatic fringe and is essential for coordinated somite segmentation (Bessho, 2001).

The expression of a Hairy/E(spl)-related (Her) gene, her7, has been studied in the zebrafish; its expression in the presomitic mesoderm cycles similarly to her1 and deltaC. A decrease in her7 function generated by antisense oligonucleotides disrupts somite formation in the posterior trunk and tail, and disrupts the dynamic expression domains of her1 and deltaC, suggesting that her7 plays a role in coordinating the oscillations of neighboring cells in the presomitic mesoderm. This phenotype is reminiscent of zebrafish segmentation mutants with lesions in genes of the Delta/Notch signaling pathway, which also show a disruption of cyclic her7 expression. The interaction of HER genes with the Delta/Notch signaling system was investigated by introducing a loss of her7 function into mutant backgrounds. This leads to segmental defects more anterior than in either condition alone. Combining a decrease of her7 function with reduction of her1 function results in an enhanced phenotype that affects all the anterior segments, indicating that Her functions in the anterior segments are also partially redundant. In these animals, gene expression does not cycle at any time, suggesting that a complete loss of oscillator function had been achieved. Consistent with this, combining a reduction of her7 and her1 function with a Delta/Notch mutant genotype does not worsen the phenotype further. Thus, these results identify members of the Her family of transcription factors that together behave as a central component of the oscillator, and not as an output. This indicates, therefore, that the function of the segmentation oscillator is restricted to the positioning of segmental boundaries. Furthermore, these data suggest that redundancy between Her genes and genes of the Delta/Notch pathway is in part responsible for the robust formation of anterior somites in vertebrates (Oates, 2002).

The formation of somites, reiterated structures that will give rise to vertebrae and muscles, is thought to be dependent upon a molecular oscillator that may involve the Notch pathway. hairy/Enhancer of split related [E(spl)]-related (her or hes) genes, potential targets of Notch signaling, have been implicated as an output of the molecular oscillator. A zebrafish deficiency, b567, has been isolated that deletes two linked her genes, her1 and her7. Homozygous b567 mutants have defective somites along the entire embryonic axis. Injection of a combination of her1 and her7 (her1+7) morpholino modified antisense oligonucleotides (MOs) phenocopies the b567 mutant somitic phenotype, indicating that her1 and her7 are necessary for normal somite formation and that defective somitogenesis in b567 mutant embryos is due to deletion of her1 and her7. Analysis at the cellular level indicates that somites in her1+7-deficient embryos are enlarged in the anterior-posterior dimension. Weak somite boundaries are often found within these enlarged somites that are delineated by stronger, but imperfect, boundaries. In addition, the anterior-posterior polarity of these enlarged somites is disorganized. Analysis of her1 MO-injected embryos and her7 MO-injected embryos indicates that although these genes have partially redundant functions in most of the trunk region, her1 is necessary for proper formation of the anteriormost somites and her7 is necessary for proper formation of somites posterior to somite 11. By following somite development over time, it has been demonstrated that her genes are necessary for the formation of alternating strong somite boundaries. Thus, even though two potential downstream components of Notch signaling are lacking in her1+7-deficient embryos, somite boundaries form, but do so with a one and a half to two segment periodicity (Henry, 2002).

Somite formation in vertebrates depends on a molecular oscillator in the presomitic mesoderm (PSM). In order to get a better insight into how oscillatory expression is achieved in the zebrafish Danio rerio, the regulation of her1 and her7, two bHLH genes that are co-expressed in the PSM, has been analyzed. Using specific morpholino oligonucleotide mediated inhibition and intron probe in situ hybridization, her7 was found to be required for initiating the expression in the posterior PSM, while her1 is required to propagate the cyclic expression in the intermediate and anterior PSM. Reporter gene constructs with the her1 upstream sequence driving green fluorescent protein (GFP) expression show that separable regulatory regions can be identified that mediate expression in the posterior versus intermediate and anterior PSM. These results indicate that the cyclic expression is generated at the transcriptional level and that the resulting mRNAs have a very short half-life. A specific degradation signal for her1 mRNA must be located in the 5'-UTR, since this region also destabilizes the GFP mRNA such that it mimics the dynamic pattern of the endogenous her1 mRNA. In contrast to the mRNA, GFP protein is stable and all somitic cells are found to express the protein, proving that her1 mRNA is transiently expressed in all cells of the PSM (Gajewski, 2003).

Hes7, a bHLH gene essential for somitogenesis, displays cyclic expression of mRNA in the presomitic mesoderm (PSM). Hes7 protein is also expressed in a dynamic manner, which depends on proteasome-mediated degradation. Spatial comparison reveals that Hes7 and Lunatic fringe (Lfng) transcription occurs in the Hes7 protein-negative domains. Furthermore, Hes7 and Lfng transcription is constitutively up-regulated in the absence of Hes7 protein and down-regulated by stabilization of Hes7 protein. Thus, periodic repression by Hes7 protein is critical for the cyclic transcription of Hes7 and Lfng, and this negative feedback represents a molecular basis for the segmentation clock (Bessho, 2003).

The temporal expression profiles are thought to occur as follows: Hes7 transcription leads to accumulation of Hes7 mRNA and Hes7 protein but is turned off as soon as Hes7 protein is accumulated, but Hes7 mRNA persists for a while. Recent studies have revealed that oscillatory expression of Lfng is controlled at the transcriptional level. Among several elements in the Lfng promoter, the region 2 or the region A contains two E-boxes, which are critical for the cyclic expression. The data indicate that Hes7, which represses transcription via E-boxes, is likely to regulate the cyclic expression of Lfng through the E-boxes in the region 2/A (Bessho, 2003).

Lfng represses its own expression through modulation of the Notch pathway in chick; the negative feedback loop of Lfng is a molecular basis for the segmentation clock. It is likely that Lfng also constitutes a negative feedback loop in mouse, because the expression domains of the lacZ gene, which is knocked into the Lfng allele, become wider in the Lfng-null mutant mouse. However, although Hes7 is required for the dynamic expression of Lfng, Lfng is not required for the dynamic expression of Hes7. Thus, Hes7 is an upstream regulator of Lfng oscillation, and the negative feedback loop of Lfng could be involved in refinement of cyclic expression or keeping accuracy of the 2-h cycle. It was recently reported that Wnt signaling is also involved in the segmentation clock. Interestingly, in hypomorphic mutants for Wnt3a, Lfng oscillation is lost, suggesting the cross-talk between Wnt and Notch signaling. However, the relationship between Hes7 oscillation and Wnt signaling remains to be determined (Bessho, 2003).

Suppressor of Hairless [Su(H)] codes for a protein that interacts with the intracellular domain of Notch to activate the target genes of the Delta-Notch signalling pathway. The zebrafish homolog of Su(H) has been cloned and characterized and its function has been analyzed by morpholino mediated knockdown. While there are at least four notch and four delta homologs in zebrafish, there appears to be only one complete Su(H) homolog. The function of Su(H) in the somitogenesis process was analyzed and its influence on the expression of notch pathway genes was examined, in particular her1, her7, deltaC and deltaD. The cyclic expression of her1, her7 and deltaC in the presomitic mesoderm is disrupted by the Su(H) knockdown mimicking the expression of these genes in the notch1a mutant deadly seven. deltaD expression is similarly affected by Su(H) knockdown like deltaC but shows in addition an ectopic expression in the developing neural tube. The inactivation of Su(H) in a fss/tbx24 mutant background leads furthermore to a clear breakdown of cyclic her1 and her7 expression, indicating that the Delta-Notch pathway is required for the creation of oscillation and not only for the synchronization between neighboring cells. The strongest phenotypes in the Su(H) knockdown embryos show a loss of all somites posterior to the first five to seven. This phenotype is stronger than the known amorphic phenotypes for notch1 (des) or deltaD (aei) in zebrafish, but mimicks the knockout phenotype of RBP-Jkappa gene in the mouse that is the homolog of Su(H). This suggests that there is some functional redundancy among the Notch and Delta genes. This fact that the first five to seven somites are only weakly affected by Su(H) knockdown indicates that additional genetic pathways may be active in the specification of the most anterior somites (Sieger, 2003).

Alterations of the Delta/Notch signalling pathway cause multiple morphogenetic abnormalities in somitogenesis, including defects in intersomitic boundary formation and failure in maintenance of somite regularity. Notch signalling has been implicated in establishing the anteroposterior polarity within maturing somites and in regulating the activity of a molecular segmentation clock operating in the presomitic mesoderm. The pleiotropy of Notch signalling obscures the roles of this pathway in different steps of somitogenesis. One possibility is that distinct Notch effectors mediate different aspects of Notch signalling. In this study, focus was placed on two zebrafish Notch-dependent hairy/Enhancer-of-split-related transcription factors, Her6 and Her4, which are expressed at the transition zone between presomitic mesoderm and the segmented somites. The results of overexpression/gain-of-function and of morpholino-mediated loss-of-function experiments show that Her6 and Her4 are Notch signalling effectors that feedback on the clock and take part in the maintenance of cyclic gene expression coordination among adjacent cells in the presomitic mesoderm. Her6 and Her4 are necessary for normal paraxial mesoderm segmentation and the activities of their protein products are required to maintain synchronization of the cyclical expression of both deltaC and her1 (Pasini, 2004).

During most of somitogenesis, expression of her6 is confined to two stripes in the anterior PSM and to the posterior compartment of the mature somites. Expression in the tailbud is only observed early during somitogenesis and no expression is detected in the intermediate PSM at any stage. Although the two her6 stripes in the anterior PSM show some variability in their strength, their distances from one another, from the myod stripes in the PSM or from the last formed intersomitic cleft do not vary among embryos with the same number of somites. In addition to this, and in contrast to the cycling zebrafish hairy/E(Spl)-related genes her1 and her7, the formation and maintenance of the her6 stripes do not depend on the activity of Her6 protein. Thus, her6 does not show an oscillatory behavior and, despite its high degree of homology to the cycling genes mouse Hes1 and chicken hairy2, its expression pattern in the PSM resembles that of the non-cycling frog gene x-hairy2 (Pasini, 2004).

Differences in the PSM expression pattern of Notch pathway components between zebrafish in one case and mouse and chicken in the other have already been noted. Zebrafish lfng, in contrast to its mouse and chicken homologs, does not cycle in the PSM, whereas Delta genes cycle in zebrafish but not in mouse or chicken. One possible explanation for these discrepancies is that different vertebrate classes exploit different cycling components of Notch pathway to fulfil the same functions during somitogenesis. Alternatively, it is possible that Hes1 and chick hairy2 exert different functions in the PSM and in the segmented somites and that in zebrafish such functions have been shared among distinct hairy/E(Spl)-related genes, of which some – her1 and her7 – cycle within the PSM, while others, such as her6, are expressed in a static fashion within the anterior PSM and the somites (Pasini, 2004).

The data show that the pattern of expression of her6 is dependent on the integrity of the Notch signalling pathway and on its spatially restricted activation: a block of the Notch signal by dominant-negative Su(H) results in a loss of her6 expression in somites and the anterior PSM, while ubiquitous and sustained activation of Notch signalling by constitutively active Su(H) leads to ectopic expression of her6 throughout the PSM. Four zebrafish Notch genes with spatially restricted expression patterns have been identified to date. Ubiquitous expression of constitutively active Notch1a, NIC, which leads to increased and ectopic expression of her1 and her4, fails to induce an ectopic expression of her6 in the posterior PSM. However, the expression pattern of her6 in the anterior PSM and the segmented somites is remarkably similar to that of notch5. Thus, it is possible that Her6 is a specific effector of the Notch5-mediated signal (Pasini, 2004).

To explore whether the progressive disruption of somitogenesis correlates with a gradual breakdown of the segmentation clock as in Notch mutants or embryos depleted of Her7 or Her7 and Her1, deltaC and her1 expression were analysed at different time points on batches of her6MO- and her6MO+her4MO-injected embryos. Regardless of the dose and type of injected MO, embryos at early stages of somitogenesis show normal sharp stripes of deltaC and her1 expression. However, in embryos analyzed at progressively later time points, this periodicity of expression is gradually lost. The time at which abnormalities of deltaC and her1 expression pattern appear depends on the dose and type of injected MO and correlates with the onset of somitic defects. In embryos injected with her6MO+her4MO, the homogeneous deltaC stripes in the anterior PSM are progressively replaced by a broad and irregular band within which cells expressing deltaC at strong and weak levels are intermixed. A similar mixture of cells expressing different levels of deltaC is observed in the posterior PSM. It is concluded that Her6 and Her4 are necessary for normal paraxial mesoderm segmentation and the activities of their protein products are required to maintain synchronization of the cyclical expression of both deltaC and her1 (Pasini, 2004).

During vertebrate embryogenesis, the formation of reiterated structures along the body axis is dependent upon the generation of the somite by segmentation of the presomitic mesoderm (PSM). Notch signaling plays a crucial role in both the generation and regulation of the molecular clock that provides the spatial information for PSM cells to form somites. In a screen for novel genes involved in somitogenesis, a gene was identified encoding a Wnt antagonist, Nkd1, which is transcribed in an oscillatory manner, and may represent a new member of the molecular clock constituents. The transcription of nkd1 is extremely downregulated in the PSM of vestigial tail (vt/vt), a hypomorphic mutant of Wnt3a, whereas nkd1 oscillations have a similar phase to lunatic fringe (L-fng) transcription and they are arrested in Hes7 (a negative regulator of Notch signaling) deficient embryos. These results suggest that the transcription of nkd1 requires Wnt3a, and that its oscillation patterns depend upon the function of Hes7. Wnt signaling has been postulated to be upstream of Notch signaling but it is demonstrated in this study that a Wnt-signal-related gene may also be regulated by Notch signaling. Collectively, these data suggest that the reciprocal interaction of Notch and Wnt signals, and of their respective negative feedback loops, function to organize the segmentation clock required for somitogenesis (Ishikawa, 2004).

Notch and fibroblast growth factor (FGF) signaling pathways have been implicated in the establishment of proper periodicity of vertebrate somites. Evidence is provided that a Hes6-related hairy/Enhancer of split-related gene, her13.2, links FGF signaling to the Notch-regulated oscillation machinery in zebrafish. Expression of her13.2 is induced by FGF-soaked beads and decreased by an FGF signaling inhibitor. her13.2 is required for periodic repression of the Notch-regulated genes her1 and her7, and for proper somite segmentation. Furthermore, Her13.2 augments autorepression of her1 in association with Her1 protein. Therefore, FGF signaling appears to maintain the oscillation machinery by supplying a binding partner, Her13.2, for Her1 (Kawamura, 2005).

Periodic formation of somites is controlled by the segmentation clock, where the oscillator Hes7 regulates cyclic expression of the Notch modulator Lunatic fringe. This study shows that Hes7 also regulates cyclic expression of the Fgf signaling inhibitor Dusp4/MKP2 (MAP kinase phosphatase 2) and links Notch and Fgf oscillations in phase. Strikingly, inactivation of Notch signaling abolishes the propagation but allows the initiation of Hes7 oscillation. By contrast, transient inactivation of Fgf signaling abolishes the initiation, whereas sustained inactivation abolishes both the initiation and propagation of Hes7 oscillation. It is thus proposed that Hes7 oscillation is initiated by Fgf signaling and propagated/maintained anteriorly by Notch signaling (Niwa, 2007).

The Notch pathway has been implicated in mesenchymal progenitor cell (MPC) differentiation from bone marrow-derived progenitors. However, whether Notch regulates MPC differentiation in an RBPjkappa-dependent manner, specifies a particular MPC cell fate, regulates MPC proliferation and differentiation during early skeletal development or controls specific Notch target genes to regulate these processes remains unclear. To determine the exact role and mode of action for the Notch pathway in MPCs during skeletal development, tissue-specific loss-of-function [Prx1Cre; Rbpjk(f/f)], gain-of-function [Prx1Cre; Rosa-NICD(f/+)] and RBPjkappa-independent Notch gain-of-function [Prx1Cre; Rosa-NICD(f/+); Rbpjk(f/f)] mice were analyzed for defects in MPC proliferation and differentiation. These data demonstrate for the first time that the RBPjkappa-dependent Notch signaling pathway is a crucial regulator of MPC proliferation and differentiation during skeletal development. This study also implicates the Notch pathway as a general suppressor of MPC differentiation that does not bias lineage allocation. Finally, Hes1 was identified as an RBPjkappa-dependent Notch target gene important for MPC maintenance and the suppression of in vitro chondrogenesis (Dong, 2010).

The spatial and temporal periodicity of somite formation is controlled by the segmentation clock, in which numerous cells cyclically express hairy-related transcriptional repressors with a posterior-to-anterior phase delay, creating 'traveling waves' of her1 expression. In zebrafish, the first traveling wave buds off from the synchronous oscillation zone in the blastoderm margin. This study shows that the emergence of a traveling wave coincides with the anterior expansion of Fgf signaling; transplanted Fgf8b-soaked beads induce ectopic traveling waves. It is thus proposed that as development proceeds, the activity of Fgf signaling gradually expands anteriorly, starting from the margin, so that cells initiate her1 oscillation with a posterior-to-anterior phase delay. Furthermore, it is suggested that Fgf has an essential role in establishing the period gradient that is required for the her1 spatial oscillation pattern at the emergence of the traveling wave (Ishimatsu, 2010).

Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers

Satellite cells (skeletal muscle stem cells) divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. This study shows that Hesr1 (Hey1) and Hesr3 (Hey3), which are known Notch target genes, are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7⁺ MyoD^- undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis (Fukada, 2011).

Antagonistic regulation of p57kip2 by Hes/Hey downstream of Notch signaling and muscle regulatory factors regulates skeletal muscle growth arrest

A central question in development is to define how the equilibrium between cell proliferation and differentiation is temporally and spatially regulated during tissue formation. This study addresses how interactions between cyclin-dependent kinase inhibitors essential for myogenic growth arrest (p21^cip1 and p57^kip2), the Notch pathway and myogenic regulatory factors (MRFs) orchestrate the proliferation, specification and differentiation of muscle progenitor cells. It was first shown that cell cycle exit and myogenic differentiation can be uncoupled. In addition, it was establish that skeletal muscle progenitor cells require Notch signaling to maintain their cycling status. Using several mouse models combined with ex vivo studies, it was demonstrated that Notch signaling is required to repress p21^cip1 and p57^kip2 expression in muscle progenitor cells. Finally, a muscle-specific regulatory element of p57^kip2 directly activated by MRFs was identified in myoblasts but was found to be repressed by the Notch targets Hes1/Hey1 in progenitor cells. A molecular mechanism is proposed whereby information provided by Hes/Hey downstream of Notch as well as MRF activities are integrated at the level of the p57^kip2 enhancer to regulate the decision between progenitor cell maintenance and muscle differentiation (Zalc, 2014).

Hairy-related proteins and olfactory development

The functions of the bHLH transcriptional repressors HES1 and HES5 in neurogenesis have been characterized, using the development of the olfactory placodes in mouse embryos as a model. Hes1 and Hes5 are expressed with distinct patterns in the olfactory placodes and are subject to different regulatory mechanisms. Hes1 is expressed in a broad placodal domain, which is maintained in the absence of the neural determination gene Mash1. In contrast, expression of Hes5 is restricted to clusters of neural progenitor cells and requires Mash1 function. Mutations in Hes1 and Hes5 also have distinct consequences on olfactory placode neurogenesis. Loss of Hes1 function leads both to expression of Mash1 outside of the normal domain of neurogenesis and to increased density of MASH1-positive progenitors within this domain, and results in an excess of neurons after a delay. A mutation in Hes5 does not produce any apparent defect. However, olfactory placodes that are double mutant for Hes1 and Hes5 upregulate Ngn1, a neural bHLH gene activated downstream of Mash1, and show a strong and rapid increase in neuronal density. Together, these results suggest that Hes1 regulates Mash1 transcription in the olfactory placode in two different contexts, initially as a prepattern gene defining the placodal domain undergoing neurogenesis and, subsequently, as a neurogenic gene controlling the density of neural progenitors in this domain. Hes5 synergises with Hes1 and regulates neurogenesis at the level of Ngn11 expression. Therefore, the olfactory sensory neuron lineage is regulated at several steps by negative signals acting through different Hes genes and targeting the expression of different proneural gene homologs (Cau, 2000).

The genes of the Hes family encode bHLH transcription factors that are most closely related to two groups of negative regulators of neurogenesis in Drosophila: hairy and the products of the Enhancer of Split complex (Espl). Both hairy and Espl products have been shown to directly repress transcription of the proneural gene achaete, but their activity is required in different contexts. hairy is a prepattern gene. It is required in large areas of the wing and leg imaginal discs to prevent ectopic expression of the proneural gene achaete and the formation of ectopic bristles. The genes of the Espl complex are neurogenic genes that are activated by Notch signaling in a process of lateral inhibition during embryonic and adult neurogenesis. Activation of the Espl genes blocks the accumulation of high amounts of proneural protein in most cells of the proneural clusters, thereby preventing them from adopting a neural fate. Hes1 regulation and mutant phenotype in the developping OE suggest that Hes1 has a dual role, acting as a prepattern (hairy-like) gene at the onset of neurogenesis in the olfactory placode and subsequently as a neurogenic (Espl-like) gene regulating Mash1 expression in olfactory sensory epithelium (OE) progenitors. In addition, the regulation of Hes5 expression in the olfactory placode and the placodal phenotype of Hes1;Hes5 double mutants support a role for Hes5 as a neurogenic gene acting at a later step in the olfactory sensory neuron (OSN) lineage (Cau, 2000).

Distinct stages of OSN progenitor maturation have been defined by the sequential expression of the three neural bHLH genes Mash1, Ngn1 and NeuroD. Mash1 is involved in the generation of basal OSN progenitors, whereas Ngn1 is required for their differentiation, and the function of NeuroD in this lineage has not yet been characterized. Analysis of the Hes1 mutant phenotype demonstrates that Hes1 regulates OSN development at the level of Mash1 expression. Despite the enlargement of the Mash1-positive cell population in Hes1 mutant olfactory placodes, there is only a relatively small increase in expression of Ngn1, suggesting that the step of Ngn1 expression is also subject to negative regulation. Indeed, the phenotype of Hes1;Hes5 double mutant placodes shows a further increase in the number of Ngn1-positive cells and of SCG10-positive neurons (SCG10 is a panneural marker that functions to depolymerize microtubules: there is no Drosophila homolog, but Drosophila CG5981 contains several domains that are partially homologous to SCG10) without change in the expression of Mash1, indicating that Hes5 is likely to regulate OSN development at the level of Ngn1 expression. Altogether, these data suggest that Notch signaling, acting through different Hes genes, regulates the production of OSNs by targeting the expression of two bHLH genes that control the development of the lineage at two distinct steps at least. This complex negative regulation of the OSN lineage at two levels, the determination and the differentiation of basal progenitors, allows for a fine tuning of the rate of neuronal production in the OE (Cau, 2000).

Hairy-related proteins and ear development

Hair cell fate determination in the inner ear has been shown to be controlled by specific genes. Recent loss-of-function and gain-of-function experiments have demonstrated that Math1, a mouse homolog of the Drosophila gene atonal, is essential for the production of hair cells. To identify genes that may interact with Math1 and inhibit hair cell differentiation, a focus was placed on Hes1, a mammalian hairy and enhancer of split homolog, which is a negative regulator of neurogenesis. Targeted deletion of Hes1 leads to formation of supernumerary hair cells in the cochlea and utricle of the inner ear. RT-PCR analysis shows that Hes1 is expressed in the inner ear during hair cell differentiation and its expression is maintained in adulthood. In situ hybridization with late embryonic inner ear tissue reveals that Hes1 is expressed in supporting cells, but not hair cells, of the vestibular sensory epithelium. In the cochlea, Hes1 is selectively expressed in the greater epithelial ridge and lesser epithelial ridge regions that are adjacent to inner and outer hair cells. Co-transfection experiments in postnatal rat explant cultures show that overexpression of Hes1 prevents hair cell differentiation induced by Math1. Therefore Hes1 can negatively regulate hair cell differentiation by antagonizing Math1. These results suggest that a balance between Math1 and negative regulators such as Hes1 is crucial for the production of an appropriate number of inner ear hair cells (Zheng, 2000).

The expression of mouse HES-6, a new member of the Hairy/Enhancer of split family of basic helix-loop-helix transcription factors, was studied. HES-6 is expressed in all neurogenic placodes and their derivatives and in the brain, where it is patterned along both the anteroposterior and dorsoventral axes. HES-6 is also expressed in the trunk, in the dorsal root ganglia and in the myotomes. In the limb buds HES-6 is expressed in skeletal muscle and presumptive tendons (Vasiliauskas, 2000).

Hey2 regulation by FGF provides a Notch-independent mechanism for maintaining pillar cell fate in the organ of Corti

The organ of Corti, the auditory organ of the inner ear, contains two types of sensory hair cells and at least seven types of supporting cells. Most of these supporting cell types rely on Notch-dependent expression of Hes/Hey transcription factors to maintain the supporting cell fate. Notch signaling is not necessary for the differentiation and maintenance of pillar cell fate, that pillar cells are distinguished by Hey2 expression, and, unlike other Hes/Hey factors, Hey2 expression is Notch independent. Hey2 is activated by FGF and blocks hair cell differentiation, whereas mutation of Hey2 leaves pillar cells sensitive to the loss of Notch signaling and allows them to differentiate as hair cells. It is speculated that co-option of FGF signaling to render Hey2 Notch independent also liberated pillar cells from the need for direct contact with surrounding hair cells, and enabled evolutionary remodeling of the complex cellular mosaic of the inner ear (Doetzlhofer, 2009).

The postnatal organ of Corti can be divided into four regions based on the expression of different combinations of Hes and Hey genes. Hes1 and HeyL define the neural region of the organ of Corti, being expressed in Kölliker's organ and inner phalangeal cells, whereas the abneural region is defined by the expression of Hes1 and Hey1 in Hensen's cells. Hes5, in combination with Hey1 and HeyL, defines the Deiters' cells that lie beneath outer hair cells, whereas Hey2 defines the pillar cell region. This combinatorial expression may have functional consequences, as Hes and Hey genes can form heterodimers that are often more stable than homodimers of each family member. The data also suggest a basis for the relatively mild cochlear phenotypes seen in single or double mutants of Hes1 and Hes5, since both Hes1 and Hes5 are expressed in supporting cells with an accompanying Hey gene family member (HeyL and Hey1, respectively), which might act redundantly with Hes1 or Hes5. Similarly, no hair cell phenotypes were observed in Hey1 or HeyL mutant mice and only very minor changes in hair cell density in Hey2 mutants. Future studies will address whether, at embryonic stages, signals initiating hair cell differentiation are responsible for the upregulation of Hes1, Hes5, and HeyL and/or for the restriction of Hey1 and Hey2 to specific cell types (Doetzlhofer, 2009).

The data reveal the existence of regulatory hierarchies between different Hes and Hey gene family members. In the absence of Hey2, the domain of Hes5 expression expanded laterally into the pillar cell region, suggesting that Hey2 can repress Hes5 expression. Such crossregulation may help to establish asymmetry in the organ of Corti, whereby inner hair cells are separated from outer hair cells by a hair cell-free region of Hey2-expressing pillar cells (Doetzlhofer, 2009).

It is interesting to note that, in contrast to the more recently derived cochlea, the mammalian vestibular system lacks pillar-like supporting cells, does not express Hey2, and contains no supporting cells that are resistant to DAPT (γ-secretase inhibitor IX). Based on the observation that extant basal monotreme mammals, such as the duck-billed platypus and echidna, have three to four rows of pillar cells separating inner from outer hair cells, it is speculated that co-option of Hey2 and its regulation by FGF signaling rather than the Notch pathway resulted in a lack of lateral inhibition between the multiple rows of pillar cells and their hair cell counterparts. In this evolutionary context, it would be interesting to determine whether Hey2 is expressed in the expanded pillar cell domain of monotremes and whether it plays a similar Notch-independent role in pattern formation in the monotreme inner ear (Doetzlhofer, 2009).

Ligand-dependent Notch signaling strength orchestrates lateral induction and lateral inhibition in the developing inner ear

During inner ear development, Notch exhibits two modes of operation: lateral induction, which is associated with prosensory specification, and lateral inhibition, which is involved in hair cell determination. These mechanisms depend respectively on two different ligands, jagged 1 (Jag1) and delta 1 (Dl1), that rely on a common signaling cascade initiated after Notch activation. In the chicken otocyst, expression of Jag1 and the Notch target Hey1 correlates well with lateral induction, whereas both Jag1 and Dl1 are expressed during lateral inhibition, as are Notch targets Hey1 and Hes5. This study shows that Jag1 drives lower levels of Notch activity than Dl1, which results in the differential expression of Hey1 and Hes5. In addition, Jag1 interferes with the ability of Dl1 to elicit high levels of Notch activity. Modeling the sensory epithelium when the two ligands are expressed together shows that ligand regulation, differential signaling strength and ligand competition are crucial to allow the two modes of operation and for establishing the alternate pattern of hair cells and supporting cells. Jag1, while driving lateral induction on its own, facilitates patterning by lateral inhibition in the presence of Dl1. This novel behavior emerges from Jag1 acting as a competitive inhibitor of Dl1 for Notch signaling. Both modeling and experiments show that hair cell patterning is very robust. The model suggests that autoactivation of proneural factor Atoh1, upstream of Dl1, is a fundamental component for robustness. The results stress the importance of the levels of Notch signaling and ligand competition for Notch function (Petrovic, 2014).

Hairy-related proteins and neural crest development

The neural crest is a population of cells that originates at the interface between the neural plate and non-neural ectoderm. The role that Notch and the homeoprotein Xiro1 play in the specification of the neural crest has been anayzed. Xiro1, Notch and the Notch target gene Hairy2A are all expressed in the neural crest territory, whereas the Notch ligands Delta1 and Serrate are expressed in the cells that surround the prospective crest cells. Inducible dominant-negative and activator constructs of both Notch signaling components and Xiro1 were used to analyze the role of these factors in neural crest specification without interfering with mesodermal or neural plate development (Galvic, 2004).

Activation of Xiro1 or Notch signaling leads to an enlargement of the neural crest territory, whereas blocking their activity inhibits the expression of neural crest markers. It is known that BMPs are involved in the induction of the neural crest and, thus, whether these two elements might influence the expression of Bmp4 was assessed. Activation of Xiro1 and of Notch signaling upregulates Hairy2A and inhibits Bmp4 transcription during neural crest specification. These results, in conjunction with data from rescue experiments, allow a model to be proposed wherein Xiro1 lies upstream of the cascade regulating Delta1 transcription. At the early gastrula stage, the coordinated action of Xiro1, as a positive regulator, and Snail, as a repressor, restricts the expression of Delta1 at the border of the neural crest territory. At the late gastrula stage, Delta1 interacts with Notch to activate Hairy2A in the region of the neural fold. Subsequently, Hairy2A acts as a repressor of Bmp4 transcription, ensuring that levels of Bmp4 optimal for the specification of the neural plate border are attained in this region. Finally, the activity of additional signals (WNTs, FGF and retinoic acid) in this newly defined domain induces the production of neural crest cells. These data also highlight the different roles played by BMP in neural crest specification in chick and Xenopus or zebrafish embryos (Galvic, 2004).

In conclusion, Notch signaling activates the expression of Hairy2A in the region of the neural folds, and thereby represses Bmp4 transcription. This effect of Notch signaling is dependent on Xmsx1 activity, since the inhibition of Notch by Su(H)DBMGR can be reversed by Xmsx1, and the effects produced by activating Notch can be blocked by a dominant-negative Xmsx1 construct. These results also provide a possible explanation for the apparent discrepancy in the role played by BMP in chick and Xenopus or zebrafish neural crest induction. At the time of neural crest induction, the levels of BMP at the neural plate border are high in both Xenopus and zebrafish, and low in the chick. If it is assumed that an intermediate level is required to induce neural crest in all these vertebrates, then an increase in BMP levels in the chick would establish similar levels to those generated by a decrease in Xenopus and zebrafish. Thus, because of the initial differences in the levels of BMP in these two groups of organisms, the molecular machinery that induces neural crest formation (e.g. Notch/Delta, Xiro1) must adjust the specific levels of BMP by producing opposing effects on BMP expression. Thus, Notch/Delta signaling induces the neural crest by increasing BMP expression in the chick, and decreasing it in Xenopus (Galvic, 2004).

Hairy-related proteins and tumorogenesis

The achaete-scute genes encode essential transcription factors in normal Drosophila and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble the neoplastic cells. Mechanisms regulating achaete-scute homolog expression outside of Drosophila are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. The protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its Drosophila homolog, Hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines that lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in Drosophila is conserved in human lung cancer cells. Modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer (Chen, 1997).

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