hairy

EVOLUTIONARY HOMOLOGS

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).

Hairy-related proteins - protein interactions

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).

Hairy-related proteins interact with Runt-related proteins

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 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 ICís 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).

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).

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).

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 transcription