h Interactive Fly, Drosophila

cubitus interruptus


Cubitus interruptus homologs: Protein structure

Gli family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling in vertebrates. The question remains unanswered, however, as to how these Gli proteins participate in the Shh signaling pathway. Regulatory activities associated with the Gli2 protein were investigated in relation to the Shh signaling. Although Gli2 acts as a weak transcriptional activator, it is in fact a composite of positive and negative regulatory domains. In cultured cells, truncation of the activation domain in the C-terminal half results in a protein with repressor activity, while removal of the repression domain at the N terminus converts Gli2 into a strong activator. In transgenic mouse embryos, N-terminally truncated Gli2, unlike the full length protein, activates a Shh target gene, HNF3beta, in the dorsal neural tube, thus mimicking the effect of Shh signal. This suggests that unmasking of the strong activation potential of Gli2 through modulation of the N-terminal repression domain is one of the key mechanisms of the Shh signaling. A similar regulatory mechanism involving the N-terminal region was also found for Gli3, but not for Gli1. When the Shh signal derived from the notochord is received by the neural plate, the widely expressed Gli2 and Gli3 proteins are presumably converted to their active forms in the ventral cells, leading to activation of transcription of their target genes, including Gli1. The possible roles of Gli proteins in Shh-dependent gene repression have not yet been elucidated and need to be studied in the future (Sasaki, 1999).

Cubitus interruptus homologs: Conservation of the Hedgehog signaling pathway

The three mouse Gli genes are putative transcription factors that are the homologs of cubitus interruptus in Drosophila. Along with the gene patched, ci has been implicated in the Hedgehog (Hh) signal transduction pathway. To assess the role of Gli in embryogenesis, its expression was compared with that of Ptc and Hh family members in mouse. Gli and Ptc are expressed in similar domains in diverse regions of the developing mouse embryo and these regions are adjacent to Hh signals. Gli and different Hh isoforms show reciprocal relationships in the limb, digits, brain, gut, and whisker follicles. Gli is expressed ectopically along with Ptc and Shh in Strong's luxoid mutant mice. It is likely that Shh is expressed ectopically in the dominant Strong's Luxoid mutation. These results are consistent with conservation of the Hh signal transduction pathway in mice with Gli potentially mediating Hh signaling in multiple regions of the developing embryo (Platt, 1997).

Drosophila Suppressor of fused [Su(fu)] encodes a novel 468-amino-acid cytoplasmic protein that, by genetic analysis, functions as a negative regulator of the Hedgehog segment polarity pathway. The primary structure, tissue distribution, biochemical and functional analyses of a human Su(fu) [hSu(fu)] is described. Two alternatively spliced isoforms of hSu(fu) were identified, predicting proteins of 433 and 484 amino acids, with a calculated molecular mass of 48 and 54 kDa, respectively. The two proteins differ only by the inclusion or exclusion of a 52 amino-acid extension at the carboxy terminus. Both isoforms are expressed in multiple embryonic and adult tissues, and exhibit a developmental profile consistent with a role in Hedgehog signaling. The hSu(fu) contains a high-scoring PEST-domain, and exhibits an overall 37% sequence identity (63% similarity) with the Drosophila protein and 97% sequence identity with the mouse Su(fu). The hSu(fu) locus maps to chromosome 10q24-q25, a region that is deleted in glioblastomas, prostate cancer, malignant melanoma and endometrial cancer. HSu(fu) represses activity of the zinc-finger transcription factor Gli, which mediates Hedgehog signaling in vertebrates, and physically interacts with Gli, Gli2 and Gli3 as well as with supernumerary limbs (Slimb), an F-box containing protein that, in the fly, suppresses the Hedgehog response, in part by stimulating the degradation of the fly Gli homolog. Coexpression of Slimb with Su(fu) potentiates the Su(fu)-mediated repression of Gli. Taken together, these data provide biochemical and functional evidence for the hypothesis that Su(fu) is a key negative regulator in the vertebrate Hedgehog signaling pathway. The data further suggest that Su(fu) can act by binding to Gli and inhibiting Gli-mediated transactivation as well as by serving as an adaptor protein, which links Gli to the Slimb-dependent proteasomal degradation pathway (Stone, 1999).

Ci/Gli zinc finger proteins mediate the transcriptional effects of Hedgehog protein signals. In Drosophila, Ci action as a transcriptional repressor or activator is contingent upon Hedgehog-regulated, PKA-dependent proteolytic processing. Processing of Drosophila Ci155 protein depends upon activity of the cyclic AMP-dependent protein kinase (PKA). In addition, responses to Shh signaling in vertebrates are blocked either by pharmacological manipulations that increase cAMP levels or by coexpression with the constitutively active form of the PKA catalytic subunit. These findings are consistent with a possible role for PKA phosphorylation activity in the formation of a Gli repressor. Transfected cells were treated either with forskolin (FSK), a membrane-permeable activator of adenylate cyclase, and its inactive analog dideoxy forskolin (ddFSK) as a control, or 3-isobutyl-1-methyl-xanthine (IBMX), a membrane-permeable inhibitor of cAMP phosphodiesterase. Treatment of both COS1 cells and primary limb bud cultures with FSK or IBMX but not with ddFSK or the DMSO vehicle causes the appearance of a novel species detectable by antiGli3 antiserum. This novel species has been designated Gli3-83 based upon its electrophoretic mobility. If Gli3-83 is formed by a single cleavage of the Gli3-190 precursor, as appears to occur in the processing of Ci155, this cleavage occurs between residues 700 and 740 within the Gli3-190 precursor. The Gli3-83 protein would thus contain amino-terminal sequences extending just C-terminal to the zinc finger region, corresponding to the Drosophila Ci75 repressor in its structure. The cAMP-stimulated appearance of Gli3-83 in COS1 cells and in primary limb bud cultures suggests that Gli3 processing may be dependent on PKA activity (Wang, 2000).

PKA-dependent processing of vertebrate Gli3 in the developing limb similarly generates a potent repressor in a manner antagonized by apparent long-range signaling from posteriorly localized Sonic hedgehog protein. The resulting anterior/posterior Gli3 repressor gradient can be perturbed by mutations of Gli3 in human genetic syndromes or by misregulation of Gli3 processing in the chicken mutant talpid2, producing a range of limb patterning malformations. The high relative abundance and potency of Gli3 repressor suggest specialization of Gli3 and its products for negative Hedgehog pathway regulation (Wang, 2000).

The large proportion of total Gli3 protein that exists as the Gli3-83 species suggests that Gli3-190 is readily processed. The extent, if any, of endogenous Gli1 and Gli2 processing cannot be examined until high-affinity antibodies are available. It is interesting to note, however, that neither Gli1 nor Gli2 appear to be processed in transfection experiments with established cell lines, even though Gli2 is phosphorylated upon stimulation of PKA. Gli proteins display biologically distinct properties upon high-level expression in Xenopus embryos, such that ectopic development of Shh-inducible cell fates appears to be promoted by Gli1, by Gli2 to a lesser extent, and not at all by Gli3, which actually seems to suppress the positive activities of Gli1 and Gli2. Differences in propensity to be processed may help account for these differences, with Gli3 representing a potent repressing activity for Shh-inducible fates simply because it is readily processed to form Gli3-83 repressor, particularly at sites distant from endogenous Shh activity (Wang, 2000).

The pattern of Gli-dependent transcription of a developing structure would depend upon the combined activities of all Gli protein species present. In the case of Gli3, Shh signaling appears to reduce transcription, and the protein has a high propensity for repressor formation; these properties of the Gli3 gene and of the Gli3 protein help insure that highest repressor levels are found in cells experiencing the lowest levels of Shh pathway activation. Conversely, transcription of Gli1 and Gli2 is respectively stimulated or unaffected by Shh pathway activation, at least outside the immediate zone of Shh gene expression: this neutral or positive effect is coupled to the more positive effects of Gli1 and Gli2 proteins on Gli-dependent transcription of target genes. These more positive effects of Gli1 and Gli2 proteins may be in part due to a reduced propensity for processing and repressor formation. As compared to Drosophila, the vertebrate innovation of distinctly regulated Gli genes encoding proteins with distinctly regulated propensities for processing may provide the opportunity to establish a more robust and finer gradation of Gli-dependent transcription within a developing field of cells (Wang, 2000).

In both vertebrates and Drosophila, limb development is organized by a posteriorly located source of the signaling protein Hedgehog (Hh). In Drosophila, the expression of Hh target genes is controlled by two opposing activities of the transcriptional regulator Cubitus interruptus (Ci), which activates target genes in response to Hh signaling but is converted into a repressor form in the absence of Hh. Three homologs of Ci (Gli1, Gli2, and Gli3) have been implicated in mediating responses to Sonic hedgehog (Shh) in vertebrates. Much attention has been devoted to the expression pattern of GLI genes; GLI1 is induced by Shh, whereas GLI3 transcription appears to be repressed by Shh signaling. The regulation of GLI gene expression is therefore one important mechanism by which GLI genes organize pattern. It is not well understood, however, whether Shh signaling also controls the activities of Gli proteins post-translationally and whether these activities have activating or repressing effects on target genes in vivo. Here, the human proteins Gli1 and Gli3 have been subjected to the precise and well-defined Hh signaling assay of Drosophila wing development and it has been established that Gli1 functions as an activator and Gli3 as a repressor of Hh target genes; that the activating transcriptional activity of Gli1 and the repressing activity of Gli3 are both subject to Hh regulation in vivo; and that the combined activities of Gli1 and Gli3 can substitute for Ci in controlling Hh target gene expression during embryonic and larval development (von Mering, 1999).

One key finding of this analysis of Gli protein activities is that Drosophila Hh signaling can not only tightly control the formation of an activator form (Gli1act) but can also negatively regulate the activity of a repressor form (Gli3rep). In Drosophila, these two modes of regulation operate on the product of a single gene, ci. It has been shown that both mechanisms are essential for Hh-mediated patterning. The repressor function seems to be dispensable in early development, however, because a regulated 'activator-only' form of Ci, CiU, is sufficient to substitute for wild-type Ci during embryogenesis and early larval development. Indeed, Gli1 can also provide this function, since it rescues ci null mutant animals to late larval stages when expressed from a tubulin alpha1-GLI1 transgene. Thus, Gli1 is sufficient to mediate essential aspects of Hh signaling in embryos. During later stages of limb patterning, however, the repressor function of Ci is required to repress dpp and hh expression and neither CiU nor Gli1 can provide this function. Instead, Gli3 may have taken over the important role of providing regulated repressor activity. A prediction of this assumption is that the combination of Gli1 and Gli3 activities should be able to substitute for Ci in limb patterning. Indeed, proper regulation of Hh target gene expression is restored if ci null mutant animals are rescued by the concurrent introduction of transgenes encoding Gli1 and Gli3 (von Mering, 1999).

These findings indicate that, even without transcriptional regulation of GLI expression, the superimposition of two Gli protein activities can result in a Gli activity profile that mediates a precise Shh signaling output. The multiplication of an ancestral GLI gene might have enabled a more complex regulation of target genes and an increased flexibility in mediating the response to Hh. The fact that neither Gli1 nor Gli3 protein seems to have retained the entire complement of essential functions compared with its ancestor might have balanced the coexistence of their genes by rendering them functionally interdependent (von Mering, 1999).

The Cubitus interruptus (Ci) and Gli proteins are transcription factors that mediate responses to Hedgehog proteins (Hh) in flies and vertebrates, respectively. During development of the Drosophila wing, Ci transduces the Hh signal and regulates transcription of different target genes at different locations. In vertebrates, the three Gli proteins are expressed in overlapping domains and are partially redundant. To assess how the vertebrate Glis correlate with Drosophila Ci, each was expressed in Drosophila and their behaviors and activities were monitored. Each Gli has distinct activities that are equivalent to portions of the regulatory arsenal of Ci. Gli2 and Gli1 have activator functions that depend on Hh. Gli2 and Gli3 are proteolyzed to produce a repressor form able to inhibit hh expression. However, while Gli3 repressor activity is regulated by Hh, Gli2 repressor activity is not. These observations suggest that the separate activator and repressor functions of Ci are unevenly partitioned among the three Glis, yielding proteins with related yet distinct properties (Aza-Blanc, 2000).

Gli1 and Gli3 proteins can transduce Hh signals in Drosophila and both proteins are regulated by Hh -- Gli1, as an inducible activator, and Gli3 as a regulated repressor. Gli2, the Gli protein whose role and properties are least well understood, has also been characterized. Gli2 shares functional properties with Gli1 and Gli3. Gli2 contains a repressor activity able to inhibit hh expression in vivo, as well as an activator activity that is Hh sensitive. Recent studies have shown that Gli1 function is dispensable in mice if both copies of the Gli2 gene are present, suggesting that Gli2 can compensate for the absence of Gli1 if it is present in sufficient amounts. An explanation for this interaction is suggested, since Gli2 can mimic Gli1 as a regulated activator. Both Gli2 and Gli3 can be proteolyzed in Drosophila in a manner similar to Ci. In addition, these experiments have allowed for the detection of subtle, yet significant differences between Gli2 and Gli3 that might explain their distinct properties in vivo. (1) Gli2 accumulates less proteolytic product than Gli3, and this correlates with its lower repressor activity. This difference is thought to not be an artifact of the Drosophila system, since transfection of Gli2 and Gli3 constructs into 10T1/2 cells generates a similar profile of proteolysis (more Gli3 proteolytic product than Gli2). (2) Hh affects Gli2 and Gli3 differently. No change in the proportion of cleaved and uncleaved Gli2 is observed in these studies, indicating that the production of Gli2 repressor is not regulated by Hh. In contrast, Hh inhibits the accumulation of cleaved Gli3, reversing the proportion of full-length and processed forms. Consistent with this behavior in the fly system, production of Gli3 repressor is also regulated in the vertebrate limb bud, where it forms an anteroposterior gradient in response to Sonic Hh. Further studies on Gli2 in vertebrate systems will be required to validate these observations (Aza-Blanc, 2000).

There are numerous examples of conserved function and regulation between vertebrate proteins and their Drosophila counterparts. Nevertheless, the conservation of the many aspects of Ci function and regulation seems remarkable. Ci is thought to interact with a number of different proteins, both as a resident in a protein complex tethered in the cytoplasm and as a nuclear transcription factor, and it is assumed that these interactions are critical to the ability of Ci to regulate its activities. Domains responsible for these presumed interactions that provide activities for transcriptional activation and repression, cytoplasmic and nuclear localization, DNA binding, regulated proteolysis and association with the tethered complex, map to multiple regions of the protein. Gli1 can be co-immunoprecipitated with Fu and regulated by it, providing direct evidence for its ability to associate physically with a Drosophila partner. Since all three Gli proteins appear to function in a regulated manner in Drosophila, it is presumed that they retain many or most functional contacts, and it must be that the many regions responsible for these contacts are conserved. However, it seems likely that differences imposed by their particular structure delimit how they interact with other components of the pathway, since each of them retains some but not all aspects of Hh regulation (Aza-Blanc, 2000).

Although in aggregate the Gli proteins appear to embody the many different attributes of Ci, only some of the Ci activities are in each. Most intriguing, perhaps, is the differential activation of ptc and dpp expression by Gli1 and Gli2, respectively. The basis for the selectivity of Gli1 for ptc and Gli2 for dpp is not understood, but it has many conceivable causes. One is that Gli2 interacts with proteins known to associate with Ci, such as CBP, but that Gli1 does not. Alternatively, the ability of Gli2 to activate dpp more strongly could be related to the conversion of Gli2 to a repressor form. It is formally possible that the activator and repressor forms can cooperate in some manner to enhance dpp transcription, or that the repressor form competes with the activator for binding sites at the ptc promoter. Consistent with this latter proposal, the level of ptc induction in wing discs is inversely related to the level of Gli2 expression: higher levels of expression produce lower levels of ptc. Since Ci75 is abundant in A cells that express high levels of dpp, but it is not in cells closer to the compartment border where ptc is expressed, this model may be relevant to Ci. Perhaps the most interesting possibility to consider is that the reason for the differential activation of dpp and ptc may be that Gli1 and Gli2 represent different forms of Ci Act, one with a preference for ptc and the other for dpp (Aza-Blanc, 2000).

The finding that the individual Gli proteins contain a subset of the activities retained by Ci suggests that evolution has dispersed these functions in the course of gene duplication and diversification. It also suggests that proteins like Ci can be considered to represent composites whose multiple functions are compressed into a single polypeptide. If Ci is only one example of many such proteins, then such composite proteins might contribute significantly to the complexity of functions encoded by the Drosophila genome. The Drosophila genome is thought to be especially compact, with fewer genes even than C. elegans. It has been assumed that splicing variants and alternative promoters account for additional complexity. The Ci paradigm suggests that composite proteins may contribute as well (Aza-Blanc, 2000).

Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved

A central question in Hedgehog (Hh) signaling is how evolutionarily conserved components of the pathway might use the primary cilium in mammals but not fly. This study focussed on Suppressor of fused (Sufu), a major Hh regulator in mammals, and reveals that Sufu controls protein levels of full-length Gli transcription factors, thus affecting the production of Gli activators and repressors essential for graded Hh responses. Surprisingly, despite ciliary localization of most Hh pathway components, regulation of Gli protein levels by Sufu is cilium-independent. It is proposed that Sufu-dependent processes in Hh signaling are evolutionarily conserved. Consistent with this, Sufu regulates Gli protein levels by antagonizing the activity of Spop (speckle-type POZ protein), a conserved Gli-degrading factor [a homolog of the Drosophila MATH and BTB domain-containing protein Hib (Roadkill or Rdx). Furthermore, addition of zebrafish or fly Sufu restores Gli protein function in Sufu-deficient mammalian cells. In contrast, fly Smo is unable to translocate to the primary cilium and activate the mammalian Hh pathway. A novel positive role of Sufu in regulating Hh signaling was uncovered, resulting from its control of both Gli activator and repressor function. Taken together, these studies delineate important aspects of cilium-dependent and cilium-independent Hh signal transduction and provide significant mechanistic insight into Hh signaling in diverse species (Chen, 2009).

Studies on Sufu provide important mechanistic insight into how Sufu regulates Hh signaling. Largely based on physical interactions between Sufu and Gli proteins, the traditional model proposed that Sufu tethers Gli protein in the cytoplasm, preventing nuclear translocation and subsequent activation of target genes. This study showed that Sufu antagonizes Spop, preventing degradation of full-length Gli2 and Gli3. The process of Sufu-Spop antagonism is evolutionarily conserved since Drosophila Sufu protects Ci from Hib-mediated degradation through competitive binding to Ci (Zhang, 2006). As a result, loss of Sufu affects production of Gli2/Gli3 activator and repressor forms, which are both derived from full-length proteins. This is achieved by Hib/Spop forming a complex with Ci/Gli2/Gli3 and Cul3, thus promoting Ci/Gli ubiquitination through the Cul3-based E3 ubiquitin ligase and resulting in complete degradation by the 26S proteasome (Chen, 2009).

Drosophila Sufu is able to partially restore the defects in Gli2/Gli3 protein levels, ciliary localization, and Hh pathway activation in Sufu-/- MEFs, supporting conservation of this process. Interestingly, overexpression of Drosophila Sufu in imaginal discs inhibits Hh target gene expression in anterior cells that receive the Hh signal, but activates Hh target gene expression in the most anterior region that does not receive the Hh signal. This is consistent with a dual role of fly Sufu and whether a conserved mechanism underlies these effects needs to be further investigated (Chen, 2009).

Nevertheless, important differences in the Sufu-Spop-Gli circuit exist between flies and mammals. Gli1, unlike Gli2 and Gli3, does not appear to be subject to Spop regulation. Furthermore, while sufu mutant flies are viable, Sufu-/- mice die during early embryogenesis. Therefore, the gain-of-function phenotype in Sufu-null mice may result from increased levels of Gli1, triggered by Spop-mediated degradation of full-length Gli2/Gli3. Gli1 may have lost a requisite Spop-interacting domain, allowing it to escape regulation by Spop. Identification of domains in Gli2 and Gli3 that interact with Spop will further clarify this issue. Notably, full-length Ci and Ci repressor levels appear to be proportionately reduced in sufu mutant flies, implying that sufu affects Ci protein stability. Duplication of the ancestral Ci/Gli gene, coupled with subfunctionalization (including the distribution of activator and repressor function) and evolution of negative and positive transcriptional regulatory loops, may account for the vastly different effects of loss of Sufu in insects and vertebrates (Chen, 2009).

Regulation of Gli protein stability is a key step in controlling Hh pathway activity, and multiple, distinct degradation signals have been identified in the three Gli proteins. For instance, two degradation signals are present in Gli1, one of which contains recognition sequences for the β-TrCP adapter protein, and two β-TrCP-binding motifs also exist in Gli2. This allows utilization of the β-TrCP adapter protein for Gli1/2 proteolysis via the Cul1-based E3 ligase, distinct from Spop-mediated Gli2/3 degradation through the Cul3-based E3 ligase.β-TrCP is also required for limited proteolysis of Gli3 into a truncated repressor form. A critical unresolved issue is to understand how multiple degradation signals in Gli proteins are used to regulate full-length protein stability as well as generation of repressor forms. Further investigation is required to determine if the role of Sufu is specific in antagonizing Spop-mediated degradation, or if it is capable of opposing additional degradative pathways. It is also formally possible that Sufu has a direct effect on Gli repressor stability (Chen, 2009).

Sufu was postulated to function in both the nucleus and the cytoplasm, as overexpressed Sufu protein in cultured cells could be detected in both compartments. Furthermore, Sufu can be coimmunoprecipitated with all three Gli proteins and was shown to cooperate with SAP18-Sin3 corepressor complex in repressing transcription from a multimerized Gli-binding site luciferase reporter. Thus, it was proposed that Sufu may have a direct role in repressing Gli-mediated transcription in the nucleus in addition to sequestering Gli proteins in the cytoplasm. Recent work has challenged Sufu's cytoplasmic function by demonstrating that an overexpressed Gli1-eGFP fusion protein has a similar cytoplasmic distribution in wild-type or Sufu-deficient MEFs; in both cell types, Gli1 is largely cytoplasmic and becomes predominantly nuclear when nuclear export is blocked. However, the distributions of overexpressed Gli proteins may fail to reflect those of endogenous Gli proteins. Importantly, conclusions based on Gli1 studies may not be applicable to Gli2 and Gli3 given their distinct properties. This study observed that knockdown of Spop in Sufu-/- MEFs partially restored levels of cytoplasmic Gli2 and Gli3, resembling the wild-type nuclear-cytoplasmic distribution. While the data suggest that Sufu may have minimal effect on shuttling Gli1, Gli2, and Gli3, potential alternations in kinetics of Gli trafficking or possible post-transcription degradation events cannot be ruled out at this time. Contrary to previous reports, this stidu failed to observe any discernable effects of SAP18 either singly or in conjunction with other Hh pathway components on Hh pathway activity in MEFs. Nevertheless, although these studies highlight a major function of Sufu in regulating cytoplasmic Gli protein levels, potential minor roles in the nucleus cannot be conclusively excluded (Chen, 2009).

Protein kinase A acts at the basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube

Protein kinase A (PKA) is an evolutionarily conserved negative regulator of the hedgehog (Hh) signal transduction pathway. PKA is known to be required for the proteolytic processing event that generates the repressor forms of the Ci and Gli transcription factors that keep target genes off in the absence of Hh. This study shows that complete loss of PKA activity in the mouse leads to midgestation lethality and a completely ventralized neural tube, demonstrating that PKA is as strong a negative regulator of the sonic hedgehog (Shh) pathway as patched 1 (Ptch1) or suppressor of fused (Sufu). Genetic analysis shows that although PKA is important for production of the repressor form of Gli3, the principal function of PKA in the Shh pathway in neural development is to restrain activation of Gli2. Activation of the Hh pathway in PKA mutants depends on cilia, and the catalytic and regulatory subunits of PKA are localized to a compartment at the base of the primary cilia, just proximal to the basal body. The data show that PKA does not affect cilia length or trafficking of smoothened (Smo) in the cilium. Instead, there is a significant increase in the lev el of Gli2 at the tips of cilia of PKA-null cells. The data suggest a model in which PKA acts at the base of the cilium after Gli proteins have transited the primary cilium; in this model the sequential movement of Gli proteins between compartments in the cilium and at its base controls accessibility of Gli proteins to PKA, which determines the fates of Gli proteins and the activity of the Shh pathway (Tuson, 2011).

Positive and negative regulation of gli activity by kif7 in the zebrafish embryo

Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, this study shows that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. Kif7 interacts with both Gli1 and Gli2a, suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. Zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu) protein and mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles (Maurya, 2013).

Gli protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling

Gli proteins are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. This paper describes a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA), a master negative regulator of the Hh pathway. Activation of signaling leads to a global remodeling of the Gli phosphorylation landscape: the PKA target sites become dephosphorylated, while a second cluster of sites undergoes phosphorylation. The pattern of Gli phosphorylation can regulate Gli transcriptional activity in a graded fashion, suggesting a phosphorylation-based mechanism for how a gradient of Hh signaling in a morphogenetic field can be converted into a gradient of transcriptional activity (Niewiadomski, 2014).

The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins

The transcriptional program orchestrated by Hedgehog signaling depends on the Gli family of transcription factors. Gli proteins can be converted to either transcriptional activators or truncated transcriptional repressors. The interaction between Gli3 and Suppressor of Fused (Sufu) regulates the formation of either repressor or activator forms of Gli3. In the absence of signaling, Sufu restrains Gli3 in the cytoplasm, promoting its processing into a repressor. Initiation of signaling triggers the dissociation of Sufu from Gli3. This event prevents formation of the repressor and instead allows Gli3 to enter the nucleus, where it is converted into a labile, differentially phosphorylated transcriptional activator. This key dissociation event depends on Kif3a, a kinesin motor required for the function of primary cilia. It is proposed that the Sufu-Gli3 interaction is a major control point in the Hedgehog pathway, a pathway that plays important roles in both development and cancer (Humke, 2010).

Suppressor of fused and Spop regulate the stability, processing and function of Gli2 and Gli3 full-length activators but not their repressors

Gli2 and Gli3 are primary transcriptional regulators that mediate hedgehog (Hh) signaling. Mechanisms that stabilize and destabilize Gli2 and Gli3 are essential for the proteins to promptly respond to Hh signaling or to be inactivated following the activation. This study show that loss of suppressor of fused (Sufu; an inhibitory effector for Gli proteins) results in destabilization of Gli2 and Gli3 full-length activators but not of their C-terminally processed repressors, whereas overexpression of Sufu stabilizes them. By contrast, RNAi knockdown of Spop (a substrate-binding adaptor for the cullin3-based ubiquitin E3 ligase) in Sufu mutant mouse embryonic fibroblasts (MEFs) can restore the levels of Gli2 and Gli3 full-length proteins, but not those of their repressors, whereas introducing Sufu into the MEFs stabilizes Gli2 and Gli3 full-length proteins and rescues Gli3 processing. Consistent with these findings, forced Spop expression promotes Gli2 and Gli3 degradation and Gli3 processing. The functions of Sufu and Spop oppose each other through their competitive binding to the N- and C-terminal regions of Gli3 or the C-terminal region of Gli2. More importantly, the Gli3 repressor expressed by a Gli3 mutant allele (Gli3Delta699) can mostly rescue the ventralized neural tube phenotypes of Sufu mutant embryos, indicating that the Gli3 repressor can function independently of Sufu. This study provides a new insight into the regulation of Gli2 and Gli3 stability and processing by Sufu and Spop, and reveals the unexpected Sufu-independent Gli3 repressor function (Wang, 2010).

Kif7 promotes hedgehog signaling in growth plate chondrocytes by restricting the inhibitory function of Sufu

Proper regulation of Indian hedgehog (Ihh) signaling is vital for chondrocyte proliferation and differentiation in the growth plate. Its dysregulation causes skeletal dysplasia, osteoarthritis or cartilaginous neoplasia. This study shows that Suppressor of fused (Sufu) and Kif7 are essential regulators of Ihh signaling. While Sufu acts as a negative regulator of Gli transcription factors, Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in the turnover of Sufu and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving the dose of Sufu restores normal hedgehog pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive role of Kif7 is to restrict the inhibitory activity of Sufu. Furthermore, Kif7 also inhibits Gli transcriptional activity in the chondrocytes when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and -independent mechanisms (Hsu, 2011).

moothened transduces Hedgehog signal by forming a complex with Evc/Evc2

Hedgehog (Hh) signaling plays pivotal roles in embryonic development and adult tissue homeostasis in species ranging from Drosophila to mammals. The Hh signal is transduced by Smoothened (Smo), a seven-transmembrane protein related to G protein coupled receptors. Despite a conserved mechanism by which Hh activates Smo in Drosophila and mammals, how mammalian Hh signal is transduced from Smo to the Gli transcription factors is poorly understood. This study provides evidence that two ciliary proteins, Evc and Evc2, the products of human disease genes responsible for the Ellis-van Creveld syndrome, act downstream of Smo to transduce the Hh signal. Loss of ciliary protein complex Evc/Evc2 does not affect Sonic Hedgehog-induced Smo phosphorylation and ciliary localization but impedes Hh pathway activation mediated by constitutively active forms of Smo. Evc/Evc2 are dispensable for the constitutive Gli activity in Sufu(-/-) cells, suggesting that Evc/Evc2 act upstream of Sufu to promote Gli activation. Furthermore, it was demonstrated that Hh stimulates binding of Evc/Evc2 to Smo depending on phosphorylation of the Smo C-terminal intracellular tail and that the binding is abolished in Kif3a(-/-) cilium-deficient cells. It is propose that Hh activates Smo by inducing its phosphorylation, which recruits Evc/Evc2 to activate Gli proteins by antagonizing Sufu in the primary cilia (Yang, 2012).

This study has obtained evidence that Hh stimulates the binding of Kif7 to Smo, which is facilitated by Evc/Evc2. Increased binding of Cos2 to activated Smo has also been observed in Drosophila. It has been shown that binding of Cos2 to activated Smo promotes Fu dimerization, phosphorylation and activation, and activated Fu then regulates Ci by inhibiting its repressor form and stimulating its activator form. The mammalian Fu homolog is dispensable for Hh signaling; however, it is possible that another kinase(s) may substitute for the Fu function in the mammalian Hh pathway. It has been shown that Hh stimulates Gli3 phosphorylation, which correlates with formation of Gli3. Therefore, it would be interesting to determine whether Hh-induced Gli3 phosphorylation is affected by loss of Evc/Evc2. It is tempting to speculate that the activated Smo/Evc/Evc2 complex may recruit one or more kinases to phosphorylate Gli proteins and promote their activation (Yang, 2012).

Cubitus interruptus homologs: Interaction with DNA

GLI3 represents an important control gene for development and differentiation of several body structures. Reduction in gene dosage already leads to severe perturbation, especially of limb morphogenesis. The gene encodes a zinc finger protein that likely functions as a transcriptional modulator. The five zinc fingers should be capable of recognizing an extended stretch of genomic DNA. The sequence bound by the GLI3 zinc fingers consists of 16 nucleotides and shows a high degree of similarity to sequences bound by the GLI and tra-1 proteins. Comparison with protein-DNA interactions in the known crystal structure of the GLI-DNA complex suggests relevant interactions of additional amino acids of GLI3 with its target site (Vortkamp, 1995).

Cubitus interruptus homologs: Protein Interactions

The hedgehog (Hh) signaling pathway is crucial for pattern formation during metazoan development. Although originially characterized in Drosophila, vertebrate homologs have been identified for several, but not all, genes in the pathway. Analysis of mutants in Drosophila demonstrates that Suppressor of fused [Su(fu)] interacts genetically with genes encoding proteins in the Hh signal transduction pathway, and its protein product physically interacts with two of the proteins in the Hh pathway. The molecular cloning and characterization of chicken and mouse homologs of Su(fu) is reported here. The chick and mouse proteins are 27% identical and 53% similar at the amino acid level to the Drosophila melanogaster and Drosophila virilis proteins. Vertebrate Su(fu) is widely expressed in the developing embryo with higher levels in tissues that are known to be patterned by Hh signaling. The chick Su(fu) protein can physically interact with factors known to function in Hh signal transduction including the Drosophila serine/threonine kinase, Fused, and the vertebrate transcriptional regulators Gli1 and Gli3. This interaction may be significant for transcriptional regulation, as recombinant Su(fu) enhances the ability of Gli proteins to bind DNA in electrophoretic mobility shift assays (Pearse, 1999).

The human Suppressor-of-Fused (SUFUH) complementary DNA has been identified and the gene product has been shown to interact physically with the transcriptional effector GLI-1. SUFUH can sequester GLI-1 in the cytoplasm, but can also interact with GLI-1 on DNA. Functionally, SUFUH inhibits transcriptional activation by GLI-1, as well as osteogenic differentiation in response to signaling from Sonic hedgehog. Localization of GLI-1 is influenced by the presence of a GLI-1 nuclear-export signal, and GLI-1 becomes constitutively nuclear when this signal is mutated or nuclear export is inhibited. These results show that SUFUH is a conserved negative regulator of GLI-1 signaling that may affect nuclear-cytoplasmic shuttling of GLI-1 or the activity of GLI-1 in the nucleus and thereby modulate cellular responses (Kogerman, 1999).

To test whether vertebrate Sufu is expressed in a pattern consistent with a potential role in mediating Shh signaling during embryogenesis, whole-mount in situ hybridization was used to analyse Sufu expression in mouse embryos at days 8.5 to 15.5 of development. Throughout the entire period signals were observed in the neural tube and, at the later stages, in the neural tube derivatives -- the brain and spinal cord. The somites express Sufu at all stages; the vibrissae field stain positively for Sufu from day 12.5 and onwards, with the vibrissae themselves being spared. The Sufu expression pattern during limb-bud development appears to be separated into two distinct phases, with strong homogeneous staining all over the limb buds being observed from their emergence at 9.5 days, whereas at 12.5 days only the interdigital mesenchyme of the limbs stain positively. This expression pattern partially overlaps with the expression of Ptch and the Ci homologs Gli 1-3, and is compatible with a conserved role for Sufu in Shh signaling (Kogerman, 1999).

To substantiate this observation in more detail and in the human system, the expression of SUFUH and PTCH1 was analyzed in the developing limb of a 12-week-old human embryo by radioactive in situ hybridization. The results show marked SUFUH expression in the osteoblasts of the perichondrium, where PTCH1 is also highly expressed. These findings are consistent with earlier observations in the avian and murine systems, in which Ptch1 and Gli1 are highly expressed in the same type of cells in response to Ihh secretion by prehypertrophic chondrocytes. Taken together, these results show that SUFUH is preferentially expressed in cells that receive a Hedgehog signal, and indicate that, during embryogenesis, SUFUH may be co-regulated with PTCH1 and GLI1 (Kogerman, 1999).

The retention of GLI-1 in the cytoplasm by SUFUH when nuclear export is compromised, and the similar SUFUH-mediated retention in the cytoplasm of an otherwise constitutively nuclear GLI-1 variant (truncated so that it lacks the NES) indicates that SUFUH could block nuclear entry of GLI-1, possibly by masking a nuclear-localization signal, and thereby inhibit transcriptional activation of target genes. Consistent with this idea, a truncated SUFUH variant unable to repress GLI1-induced transcriptional activation is also unable to modify the subcellular localization of GLI-1. What remains an interesting question for future studies is whether or not binding of SUFUH to GLI-1 on DNA, or elsewhere in the nuclear compartment, actually acts to repress or block activation of transcription, alone or in combination with cytoplasmic retention of GLI-1. The expression of Sufu in cells next to Shh- or Ihh-producing cells during mouse and human embryogenesis, coupled with the ability of Sufu to inhibit Gli-mediated transcriptional activation, indicates that an important function of Sufu may be to act in an intracellular negative feedback mechanism and to impose thresholds on the responsiveness of cells to Shh and Ihh. A similar role for D-Axin has been proposed as regards Wingless signaling in Drosophila (Kogerman, 1999).

Hedgehog (Hh) proteins are secreted factors that control cell proliferation and cell-fate specification. Hh signaling is mediated in vertebrates by the Gli zinc-finger transcription factors (Gli1, Gli2 and Gli3) and in Drosophila by the Gli homolog Cubitus interruptus (Ci). However, the mechanisms that regulate Gli/Ci activity are not fully understood. Genetic studies in Drosophila have identified a putative serine-threonine kinase, Fused (Fu), and a new protein, Suppressor of Fused [Su(fu)], as modulators of Ci activity. A human homologue of Drosophila Fu, hFu, regulates the activity of Gli1 and Gli2 on several levels. hFu converts Gli2 from a weak to a strong transcriptional activator, antagonizes the repressive effect of the human Su(fu) homolog, [hSu(fu)], on Gli1 and Gli2, and promotes nuclear localization of Gli1 and Gli2 (Murone, 2000).

To identify possible regulators of Gli proteins, complementary DNAs were isolated encoding hFu, which shares a significant level of homology with Drosophila Fu in the kinase domain (55%), but only a limited amount of homology over the remaining 1,052 amino acids. The gene encoding hFu was mapped to chromosome 2q35, close to the PAX3 gene, which is implicated in the Klein-Waardenburg syndrome. PAX3 is a target of Sonic hedgehog (Shh) and it has been suggested that additional loci in the 2q35 region may regulate the PAX3 locus, thereby influencing the Klein-Waardenburg phenotype. Northern-blot analysis has showen that a single 5-kb hFu transcript is expressed at low levels in most fetal tissues and adult ovaries, and at high levels in adult testes, where it is localized in germ cells with other components of the Hh pathway. Examination of a mouse embryo at day 13.5 of development by in situ hybridization shows that mouse Fu (mFu) mRNA is widely distributed in Shh-responsive tissues, including the forebrain, midbrain, hindbrain, spinal cord, somites, developing limb buds and skin (Murone, 2000).

To determine whether hFu can regulate Gli activity, hFu was cotransfected with a Gli-binding-site (Gli-BS) luciferase reporter in the Hh-responsive cell line C3H10T1/2. hFu alone is capable of weakly inducing transcription of the Gli-BS reporter, indicating that it may be a positive regulator of the Hh pathway. Although hFu contains a putative kinase domain, no substantial kinase activity for hFu was detected; a similar lack of kinase activity has been reported for Drosophila Fused (Murone, 2000).

To determine the function of the kinase domain of hFu, a putative catalytically dead version of hFu [hFu(K33R)] was constructed by mutating a conserved lysine residue in the ATP-binding site at position 33. This residue is crucial to the catalytic activity of all kinases, and the corresponding mutation in Drosophila leads to a fu phenotype. hFu(K33R) is able to activate the Gli-BS reporter as efficiently as wild-type hFu, indicating that the putative kinase activity of hFu may not contribute significantly to Gli activation under these conditions. A similar result has been obtained for a hFu construct [hFu(270-1,315)] lacking the entire kinase domain (amino acids 1-269). The activity of hFu was tested in combination with various Gli-family members. Whereas human Gli1 alone strongly induces the luciferase reporter, mouse Gli2 exhibits only weak activity and human Gli3 shows no activity at all. hFu does not affect the activity of Gli1 and Gli3, but strongly synergizes with Gli2. Moreover, activation of Gli2 by hFu is antagonized by hSu(fu). In contrast, Gli1 is constitutively active and its ability to activate the Gli-BS reporter is inhibited by hSu(fu) and restored in the presence of hFu (Murone, 2000).

To investigate further the mechanisms by which hFu regulates Gli activity, whether hFu forms a physical complex with hSu(fu) or the various Gli proteins was determined. Cultured cells were cotransfected with epitope-tagged versions of hFu, hSu(fu), Gli1, Gli2 and Gli3 and the resulting interactions were observed. hFu co-immunoprecipitates with hSu(fu) and with Gli1, Gli2 and Gli3. In vertebrates, Su(fu) represses Gli1 function in part by tethering it in the cytoplasm. In contrast, hFu and hFu(K 33R) promote nuclear localization of Gli1. An assessment was made of whether hFu could influence the subcellular localization of Gli1 when co-expressed with hSu(fu). In the presence of hSu(fu), roughly 3% of cells exhibit nuclear staining of Gli1. In contrast, when both hSu(fu) and hFu are present, 20% of cells possess nuclear Gli1. Identical results are obtained for Gli2. Overall, these results indicate that hFu controls the activity of Gli1 and Gli2 by opposing the effect of hSu(fu). Whereas hSu(fu) constrains Gli1 and Gli2 in the cytoplasm, hFu promotes their nuclear localization. Gli2 also requires an additional function of hFu to become transcriptionally active, as Gli2 transfected in the absence of hSu(fu) is unable to activate transcription unless hFu is present, despite the fact that it enters the nucleus. The mechanisms by which hFu activates Gli2 remain to be elucidated but may include a hFu-mediated modification of Gli2 to mask the inhibitory Gli2 amino-terminal domain (Murone, 2000).

The activity of hFu described here does not seem to require a functional kinase domain, since overexpression of kinase-mutant forms of Fu are as active as wild-type forms. Catalytically dead versions of other serine-threonine kinases, such as the RIPs8 and IRAKs14, show comparable activity to their wild-type counterparts in inducing apoptosis or activating NFkappaB respectively. Although some Drosophila kinase-domain fu mutants suffer a complete lack of induction of Hh target genes in the embryo, they show only a partial fu phenotype in the wing discs, indicating that there may be different requirements for the kinase activity of Fu in different cellular contexts (Murone, 2000).

The Suppressor of fused [Su(fu)] gene of Drosophila encodes a protein containing a PEST sequence [a sequence enriched in proline (P), glutamic acid (E), serine (S) and threonine (T)] that acts as an antagonist to the serine-threonine kinase Fused in Hedgehog (Hh) signal transduction during embryogenesis. The Su(fu) gene isolated from a distantly related Drosophila species, D. virilis, shows significantly high homology throughout its protein sequence with its D. melanogaster counterpart. These two Drosophila homologs of Su(fu) are functionally interchangeable in enhancing the fused phenotype. Mammalian homologs of Su(fu) have been isolated. The absence of the PEST sequence in the mammalian Su(fu) protein suggests a different regulation for this product between fly and vertebrates. Using the yeast two-hybrid method, the murine Su(fu) protein is shown to interact directly with the Fused and Cubitus interruptus proteins, known partners of Su(fu) in Drosophila. Su(fu) could be regulated posttranslationally in the fly and at another level in vertebrates. A similar divergence is observed for the regulation of the ci gene and its homologs, the Gli genes: in Drosophila, there is only one ci gene whose product is regulated posttranslationally; in vertebrates, there are three ci-related genes Gli, Gli2 and Gli3 that are regulated at a transcriptional level (Delattre, 1999).

The Suppressor of Fused [Su(fu)] protein plays a conserved role in the regulation of Gli transcription factors of the hedgehog (Hh) signaling pathway that controls cell fate and tissue patterning during development. In both Drosophila and mammals, Su(fu) represses Gli-mediated transcription, but the mode of its action is not completely understood. Recent evidence suggests that Su(fu) physically interacts with the Gli proteins and, when overexpressed, sequesters Gli in the cytoplasm. However, Su(fu) also traverses into the nucleus under the influence of a serine-threonine kinase, Fused (Fu), and has the ability to form a DNA-binding complex with Gli, suggesting that it has a nuclear function. This study reports that the mouse homolog of Su(fu) [mSu(fu)] specifically interacts with SAP18, a component of the mSin3 and histone deacetylase complex. In addition, mSu(fu) functionally cooperates with SAP18 to repress transcription by recruiting the SAP18-mSin3 complex to promoters containing the Gli-binding element. These results provide biochemical evidence that Su(fu) directly participates in modulating the transcriptional activity of Gli (Cheng, 2002).

Sonic hedgehog signaling plays a critical role during development and carcinogenesis. While Gli family members govern the transcriptional output of Shh signaling, little is known how Gli-mediated transcriptional activity is regulated. The actin-binding protein Missing in Metastasis (MIM) has been identified as a new Shh-responsive gene. MIM is a member of the Wiskott-Aldrich Syndrome family of proteins and contains a conserved coiled-coil protein interaction domain and a C-terminal WH2 domain. Previous independent studies have shown that MIM binds monomeric actin through its WH2 domain and bundles F-actin using its N-terminal coiled-coil domain. Together, Gli1 and MIM recapitulate Shh-mediated epidermal proliferation and invasion in regenerated human skin. MIM is part of a Gli/Suppressor of Fused complex and potentiates Gli-dependent transcription using domains distinct from those used for monomeric actin binding. These data define MIM as both a Shh-responsive gene and a new member of the pathway that modulates Gli responses during growth and tumorigenesis (Callahan, 2004).

Hedgehog-regulated processing of the transcription factor Cubitus interruptus (Ci) in Drosophila depends on phosphorylation of the C-terminal region of Ci by cAMP-dependent protein kinase and subsequently by Casein kinase 1 and Glycogen synthase kinase 3. Ci processing also requires Slimb, an F-box protein of SCF (Skp1/Cullin/F-box proteins) complex, and the proteasome, but the interplay between phosphorylation and the activity of Slimb and the proteasome remains unclear. This study shows that processing of the Gli3 protein, a homolog of Ci, also depends on phosphorylation of a set of four cAMP-dependent protein kinase sites that primes subsequent phosphorylation of adjacent casein kinase 1 and glycogen synthase kinase 3. Gain- and loss-of-function analyses in cultured cells further reveal that ßTrCP, the vertebrate homolog of Slimb, is required for Gli3 processing, and ßTrCP can bind phosphorylated Gli3 both in vitro and in vivo. Gli3 protein is polyubiquitinated in the cell, and its processing depends on proteasome activity. These findings provide evidence for a direct link between phosphorylation of Gli3/Ci proteins and ßTrCP/Slimb action, thus supporting the hypothesis that the processing of Gli3/Ci is affected by the proteasome (Wang, 2006).

The Hedgehog (Hh) and Wingless (Wnt) families of secreted signaling molecules have key roles in embryonic development and adult tissue homeostasis. In the developing neural tube, Wnt and Shh, emanating from dorsal and ventral regions, respectively, have been proposed to govern the proliferation and survival of neural progenitors. Surprisingly, Shh is required for the growth and survival of cells in both ventral and dorsal neural tube. This study demonstrates that inhibition of Shh signaling causes a reduction in Wnt-mediated transcriptional activation. This reduction requires Gli3. Assays in embryos and cell lines indicate that repressor forms of the Hh-regulated transcription factor, Gli3 (Gli3R), which are generated in the absence of Hh signaling, inhibit canonical Wnt signaling. Gli3R acts by antagonizing active forms of the Wnt transcriptional effector, β-catenin. Consistent with this, Gli3R appears to physically interact with the carboxy-terminal domain of β-catenin, a region that includes the transactivation domain. These data offer an explanation for the proliferative defects in Shh null embryos and suggest a novel mechanism for crosstalk between the Hh and Wnt pathways (Ulloa, 2007).

Zic genes encode a conserved family of zinc finger proteins with essential functions in neural development and axial skeletal patterning in the vertebrate embryo. Zic proteins also function as Gli co-factors in Hedgehog signaling. This study reports that Zic genes have a role in Myf5 regulation for epaxial somite myogenesis in the mouse embryo. In situ hybridization studies show that Zic1, 2, and 3 transcripts are expressed in Myf5-expressing epaxial myogenic progenitors in the dorsal medial dermomyotome of newly forming somites, and immunohistological studies show that Zic2 protein is co-localized with Myf5 and Pax3 in the dorsal medial lip of the dermomyotome, but is not expressed in the forming myotome. In functional reporter assays, Zic1 and Zic2, but not Zic3, potentiate the transactivation of Gli-dependent Myf5 epaxial somite-specific (ES) enhancer activity in 3T3 cells, and Zic1 activates endogenous Myf5 expression in 10T1/2 cells and in presomitic mesoderm explants. Zic2 also co-immunoprecipitates with Gli2, indicating that Zic2 forms complexes with Gli2 to promote Myf5 expression. Genetic studies show that, although Zic2 and Zic1 are activated normally in sonic hedgehog−/− mutant embryos, Myf5 expression in newly forming somites is deficient in both sonic hedgehog−/− and in Zic2kd/kd mutant mouse embryos, providing further evidence that these Zic genes are upstream regulators of Hedgehog-mediated Myf5 activation. Myf5 activation in newly forming somites is delayed in Zic2 mutant embryos until the time of Zic1 activation, and both Zic2 and Myf5 require noggin for their activation (Pan, 2011).

Structural insight into the mutual recognition and regulation between Suppressor of Fused and Gli/Ci

Hedgehog (Hh) signalling regulates embryonic development and adult tissue homoeostasis. Mutations of its pathway components including Suppressor of Fused (Sufu) and Gli/Ci predispose to cancers and congenital anomalies. The Sufu-Gli protein complex occupies a central position in the vertebrate Hh signalling pathway, especially in mammals. Structures of full-length human and Drosophila Sufu, the human Sufu-Gli complex, along with normal mode analysis and FRET measurement results, reveal that Sufu alternates between 'open' and 'closed' conformations. The 'closed' form of Sufu is stabilized by Gli binding and inhibited by Hh treatment, whereas the 'open' state of Sufu is promoted by Gli-dissociation and Hh signalling. Mutations of critical interface residues disrupt the Sufu-Gli complex and prevent Sufu from repressing Gli-mediated transcription, tethering Gli in the cytoplasm and protecting Gli from the 26S proteasome-mediated degradation. This study thus provides mechanistic insight into the mutual recognition and regulation between Sufu and Gli/Ci (Zhang, 2013).

Sufu is a crucial regulator of the Gli/Ci family of transcription factors in both vertebrates and invertebrates, and the Sufu-Gli protein complex forms the core of the vertebrate Hh signalling pathway downstream of Smoothened. Crystal structures of FL hSufu, FL dSufu and the hSufuΔ60-hGli1 (112-128) complex, as well as the normal mode analysis, reveal that the Sufu protein possesses an intrinsic conformational flexibility, with the arrangement of its NTD and CTD domains alternating between 'open' and 'closed' states. Binding to Gli stabilizes Sufu in the 'closed' state, with the β-strand of Gli acting as a 'glue' to bring strands β5 of Sufu-NTD and β9 of Sufu-CTD together. In response to Hh, the conformation of Sufu relaxes to the 'open' state and it is dissociated from Gli. Therefore, the 'closed' state of Sufu is favored when it is associated with Gli and when the Hh signal is absent, and the 'open' conformation of Sufu prevails when it dissociates from Gli and is promoted by Hh signalling (Zhang, 2013).

The Sufu-binding fragment of Gli/Ci, the 'SYGHLS' motif, is completely surrounded by conserved residues from Sufu. It was reported that two of the major HIB-binding sites on Ci are at residues 216-227 and 368-376, which are nearby its 'SYGHIS' motif, residues 255-260. In the case of Gli3, it is only known that its N-terminal region (residues 242-477 of mouse Gli3) contains a Spop-binding site (Spop/HIB is a negative regulator of the HH pathway), but its exact location has not been pinpointed. It is likely that the complex formation between Sufu and Gli/Ci creates a steric hindrance for Spop/HIB, thus prohibiting the Spop/HIB-mediated ubiquitination and degradation of Gli/Ci. Moreover, Spop/HIB has been reported to form a dimer and make multivalent interactions with Gli/Ci, therefore the complex formation between Sufu and Gli/Ci would indeed leave barely enough space for a Spop/HIB dimer to occupy two adjacent binding sites on Gli/Ci at the same time. It would be worthwhile to investigate further the underlying mechanism(s) of how Sufu inhibits Gli/Ci degradation, which could be Spop/HIB-dependent or -independent (Zhang, 2013).

Previous investigations on the molecular mechanism of interaction between Sufu and Gli/Ci have obtained somewhat contradictory results. Part of the reason might be that Sufu uses both its NTD and CTD to clamp Gli/Ci in the middle, hence any attempt using the deletion mapping approach might be hampered by the pitfall of damaging the structural integrity of FL Sufu and thus yield misleading results. Studies conducted by mutation-based approaches also need to be carefully evaluated so as not to be over-interpreted. For example, the highly conserved 'HGRHFTYK' motif (residues 391-398 in hSufu) is reported to be required for stable interaction with Gli/Ci, and mutation of this motif disrupted the binding and inhibition of Gli by Sufu. However, structure of the hSufu-hGli1 complex shows that these residues are not in direct contact with Gli. This fragment forms strand β13 of hSufu-CTD and juxtaposes strand β9, which engages hGli1 by β-sheet interaction. Mutation of the 'HGRHFTYK' motif to alanines would potentially affect the structural integrity of β13, which would in turn obstruct the formation of β9 and even the entire Sufu-CTD. Without the participation of Sufu-CTD, especially that of β9, a stable complex between Sufu and the 'SYGHLS' motif of Gli would not be achieved (Zhang, 2013).

ITC and Ni2+ column pull-down results suggested that the NTD or CTD of Sufu alone was not sufficient for stable complex formation with the 'SYGHLS' motif of Gli/Ci). However, it was also reported that Sufu-NTD and -CTD had reduced but detectable interactions with FL Gli1 and Sufu-NTD was sufficient for the inhibition of Gli1- and Gli2-dependent transcriptional activation. Gli/Ci proteins contain a second Sufu-binding site in their C-terminal halves, but its precise location has not been pinpointed. Determination of the exact position of this C-terminal Sufu-binding site on Gli/Ci and elucidation of its role in the regulation of Gli/Ci by Sufu deserve further attention (Zhang, 2013).

In vertebrates, especially in mammals, primary cilium has an important role in Hh signalling. Many key Hh signalling components such as Patched and Smoothened are localized to primary cilia and their ciliary localizations are regulated by Hh. Sufu translocates to cilia coordinately with Gli and its ciliary localization depends on Gli. The regulation of Gli by Sufu is independent of cilia, but the Hh/Smo-induced release of Sufu from Gli requires cilia. It is conceivable that in response to Hh, some kind of cilia-specific post-translational modification event such as phosphorylation happens on Gli or Sufu, resulting in the dissociation of Gli from Sufu in cilia and the subsequent translocation of Gli to the nucleus. Intriguingly, there are many potential phosphorylation sites within and surrounding the Sufu-binding 'SYGHLS' motif in Gli/Ci. It might be warranted to investigate whether any Hh-induced and cilia-dependent phosphorylation occurs on Gli or Sufu and whether there exists any cilia-localized kinase specifically mediating these phosphorylation events (Zhang, 2013).

Mutations of the human Sufu gene predispose individuals to cancers. The tumour suppressor function of Sufu is considered mainly as working through restraining the activities of the Gli transcription factors, whose mutations are also correlated with tumours and congenital malformations. Understanding the complex interplay between Sufu and Gli at the structural level would deepen understanding of the molecular mechanism of how pathogenic mutations of Sufu and Gli work. For example, a frameshift mutation IVS8+1G->A was found in both medulloblastoma and Gorlin syndrome patients. This mutation resulted in a truncated Sufu-Δex8 protein (residues 1-322, instead of WT hSufu residues 1-484), in which the majority of the CTD domain of hSufu was removed. This result is fully consistent with the finding that Sufu-CTD is indispensible in the recognition and regulation of Gli proteins. The Gli transcription factor represents a potential therapeutic target, and understanding of the Sufu-Gli protein complex might also inspire the development of pharmaceutical approaches to rein in aberrant activities of Gli in cancers (Zhang, 2013).

Cilia and hedgehog signal transduction

Sonic Hedgehog (Shh) signals are transduced into nuclear ratios of Gli transcriptional activator versus repressor. The initial part of this process is accomplished by Shh acting through Patched (Ptc) to regulate Smoothened (Smo) activity. The mechanisms by which Ptc regulates Smo, and Smo activity is transduced to processing of Gli proteins remain unclear. Recently, a forward genetic approach in mice identified a role for intraflagellar transport (IFT) genes in Shh signal transduction, downstream of Patched (Ptc) and Rab23. This study shows that the retrograde motor for IFT is required in the mouse for the phenotypic expression of both Gli activator and repressor function and for effective proteolytic processing of Gli3. Furthermore, the localization of Smo to primary cilia is disrupted in mutants. These data indicate that primary cilia act as specialized signal transduction organelles required for coupling Smo activity to the biochemical processing of Gli3 protein (May, 2005).

Several studies have linked cilia and Hedgehog signaling, but the precise roles of ciliary proteins in signal transduction remain enigmatic. A mouse mutation, hennin (hnn), causes coupled defects in cilia structure and Sonic hedgehog (Shh) signaling. The hnn mutant cilia are short with a specific defect in the structure of the ciliary axoneme, and the hnn neural tube shows a Shh-independent expansion of the domain of motor neuron progenitors. The hnn mutation is a null allele of Arl13b, a small GTPase of the Arf/Arl family, and the Arl13b protein is localized to cilia. Double mutant analysis indicates that Gli3 repressor activity is normal in hnn embryos, but Gli activators are constitutively active at low levels. Thus, normal structure of the ciliary axoneme is required for the cell to translate different levels of Shh ligand into differential regulation of the Gli transcription factors that implement Hedgehog signals (Caspary, 2007).

In mutants that lack cilia altogether, such as Ift172 and Ift88 mutants, no Gli activator is produced. In contrast, in hnn mutants, it appears there is a constitutive low level of Gli activator in all cells in the neural tube. It is therefore proposed that there are two cilia-dependent steps in the formation of Gli activators. It is proposed that full-length Gli proteins are modified within the cilium in a Hh-independent process to have a low level of activator function, and the low-level activator is normally tethered in the cilium in the absence of ligand. The idea that there are ligand-independent steps in Hh signaling that depend on cilia is not novel: processing of Gli3 to make Gli3 repressor is also cilia dependent and occurs in the absence of ligand. In response to Hh, full-length Gli proteins can be further modified to create high-level activator, which is then released from the cilium to the nucleus. In the abnormal cilia that lack Arl13b, the modification that produces full-length Gli protein with low-level activity takes place, but this low-level activator is not effectively tethered in the cilium and is released inappropriately to the nucleus in the absence of Hh ligand. This step retains a small amount of sensitivity to upstream signals in hnn mutant, as the phenotype of Ptch1 hnn double mutants is not identical to the hnn phenotype (Caspary, 2007).

Two alternative views of the relationship between cilia and Hh signaling have been proposed. In the simpler view, cilia represent a site where Hh pathway components are enriched, and the high local concentration of the proteins allows efficient signaling transduction. Alternatively, dynamic trafficking within the cilium may allow a sequence of protein interactions that promote the activity of the pathway. The hnn phenotype supports the latter model, as it reveals a complexity of events that occur within the cilium (Caspary, 2007).

Cilia have been implicated in Hedgehog (Hh) and Wnt signaling in mouse but not in Drosophila. To determine whether the role of cilia is conserved in zebrafish, maternal-zygotic (MZ) oval (ovl; ift88) mutants were generated that lack all cilia. MZovl mutants display normal canonical and non-canonical Wnt signaling but show defects in Hh signaling. As in mouse, zebrafish cilia are required to mediate the activities of Hh, Ptc, Smo and PKA. However, in contrast to mouse Ift88 mutants, which show a dramatic reduction in Hh signaling, zebrafish MZovl mutants display dampened, but expanded, Hh pathway activity. This activity is largely due to gli1, the expression of which is fully dependent on Hh signaling in mouse but not in zebrafish. These results reveal a conserved requirement for cilia in transducing the activity of upstream regulators of Hh signaling but distinct phenotypic effects due to differential regulation and differing roles of transcriptional mediators (Huang, 2009).

Primary cilia have essential functions in vertebrate development and signaling. However, little is known about cilia function in brain morphogenesis, a process that is severely affected in human ciliopathies. Telencephalic morphogenesis was studied in a mouse mutant for the ciliopathy gene Ftm (Rpgrip1l). The olfactory bulbs are present in an ectopic location in the telencephalon of Ftm-/- fetuses and do not display morphological outgrowth at the end of gestation. Investigating the developmental origin of this defect, it was established that E12.5 Ftm-/- telencephalic neuroepithelial cells lack primary cilia. Moreover, in the anterior telencephalon, the subpallium is expanded at the expense of the pallium, a phenotype reminiscent of Gli3 mutants. This phenotype indeed correlates with a decreased production of the short form of the Gli3 protein. Introduction of a Gli3 mutant allele encoding the short form of Gli3 into Ftm mutants rescues both telencephalic patterning and olfactory bulb morphogenesis, despite the persistence of cilia defects. Together, these results show that olfactory bulb morphogenesis depends on primary cilia and that the essential role of cilia in this process is to produce processed Gli3R required for developmental patterning. This analysis thus provides the first in vivo demonstration that primary cilia control a developmental process via production of the short, repressor form of Gli3. Moreover, these findings shed light on the developmental origin of olfactory bulb agenesis and of other brain morphogenetic defects found in human diseases affecting the primary cilium (Besse, 2011).

Broad-minded links cell cycle-related kinase to cilia assembly and hedgehog signal transduction

Recent findings indicate that mammalian Sonic hedgehog (Shh) signal transduction occurs within primary cilia, although the cell biological mechanisms underlying both Shh signaling and ciliogenesis have not been fully elucidated. This study shows that an uncharacterized TBC domain-containing protein, Broad-minded (Bromi), is required for high-level Shh responses in the mouse neural tube. Bromi controls ciliary morphology and proper Gli2 localization within the cilium. By use of a zebrafish model, it was further shown that Bromi is required for proper association between the ciliary membrane and axoneme. Bromi physically interacts with cell cycle-related kinase (CCRK), whose Chlamydomonas homolog regulates flagellar length. Biochemical and genetic interaction data indicate that Bromi promotes CCRK stability and function. It is proposed that Bromi and CCRK control the structure of the primary cilium by coordinating assembly of the axoneme and ciliary membrane, allowing Gli proteins to be properly activated in response to Shh signaling (Ko, 2010).

Bromi controls ciliogenesis, yet its mutant ciliary phenotype appears distinct from that of other mutants previously described. While Bromi regulates the manner in which cilia are assembled, it is not required for their generation in the embryo. Rather, Bromi appears to control axonemal shape and the close apposition of axonemes with ciliary membranes. IFT proteins and electron-dense material accumulate between the membranes and axonemes of mutants with defective retrograde IFT leading to mild, symmetric expansion of ciliary/flagellar membranes around axonemes without affecting axonemal shape. In contrast, disruption of bromi in zebrafish results in a more dramatic expansion of the ciliary membrane. This expansion is limited to one side of the cilium, and the space between the membrane and axoneme is devoid of ectopic material. Moreover, IFT88 does not accumulate at the distal ends of bromi mutant cilia as it does in mouse, C. elegans, and Chlamydomonas mutants with retrograde IFT defects. Thus, the expansion of the ciliary membrane away from the axoneme that was observed more likely results from defective axonemal/membrane growth control rather than membrane displacement resulting from accumulation of IFT or structural proteins in the cilium (Ko, 2010).

The ciliary membrane does not arise from evagination of the plasma membrane, but, rather, it is a specialized membrane with unique lipid and protein composition produced from specific Golgi-derived vesicles. With the exception of specialized cilia (e.g., photoreceptors), flagellar and ciliary membranes remain tightly associated with axonemes during their growth. This suggests that the deposition and growth of ciliary membranes are coordinated with growth of the axoneme. However, work on trypanosome flagella indicate that flagellar membranes can form without axonemes, and vice versa, when IFT proteins or TbLRTP (a protein important for faithful basal body duplication and flagellum biogenesis) are disrupted, respectively. Thus, at least in trypanosomes, biogenesis of flagellar membranes and axonemes are separable under experimental conditions (Ko, 2010).

Bromi may control cilia assembly through several mechanisms. Its primary role could be in proper assembly of the axonemal structure, which, in turn, controls the growth of the ciliary membrane. However, because the axonemal structure of zebrafish bromi morphant cilia appeared intact, whereas ciliary membranes showed a dramatic defect, it is suggested that the curling of their axonemes results from stress placed on the axoneme by the asymmetric growth of the ciliary membrane along one side. Alternatively, Bromi may directly control the addition of newly synthesized ciliary membranes from within the cell. Although many of the details of ciliary membrane growth remain obscure, Rab8a, IFT20, and BBS proteins have been shown to function in transporting Golgi-derived membranes to the cilium. Bromi may function to control the delivery of membrane to the cilium or to coordinate this process with axonemal growth through membrane trafficking. The data suggest that this function of Bromi is due to its regulation of CCRK function, rather than through Rab-GAP activity of its TBC domain (Ko, 2010).

One clue as to the cellular function of Bromi is its physical association with CCRK. Like Bromi, CCRK localizes to the cytoplasm, and, in zebrafish, its disruption results in curling of ciliary axonemes. CCRK is particularly interesting, because it is the closest vertebrate homolog to the LF2p of Chlamydomonas. Together with two other proteins, LF1p and LF3p, LF2p is a component of the length regulatory complex (LRC). Although the biochemical function of the LRC remains elusive, mutations in any one of these components cause flagella to be unusually long or short, and of unequal length within the same cell. Homologs of LF1p and LF3p have not been identified in other species, and a role for LF2p homologs outside of Chlamydomonas in ciliogenesis has not been described. It is unclear whether LF1p and LF3p regulate or potentiate LF2p activity and, if so, whether vertebrate Bromi performs an analogous function with CCRK (Ko, 2010).

This work suggests that the Bromi/CCRK complex helps to stabilize CCRK in the cell, but Bromi may provide additional functions, such as recruiting CCRK substrates. One of the few CCRK substrates identified thus far is MRK (also known as ICK), a member of the MAK/MOK/MRK subfamily of MAP kinases. Interestingly, this subfamily shares considerable homology with Chlamydomonas LF4p, C. elegans DYF-5, and Leishmania mexicana LmxMPK9, which negatively regulate flagellar and ciliary length. The Chlamydomonas lf4 mutant phenotype, like that of lf2, perturbs flagellar length. However, null alleles of these genes show distinct phenotypic features and genetic interactions, suggesting that LF2p and LF4p have related, yet separable, cell biological functions. Identification of additional substrates for these kinases should clarify their functions with respect to the assembly of cilia and flagella (Ko, 2010).

In mice, the primary cilium appears to have diverse and complex functions in regulating the Shh signaling pathway. In mouse mutants with ciliary defects, the specific manner in which the output of the pathway is disrupted depends on the way in which ciliogenesis is affected. Mutations that disrupt ciliogenesis altogether (e.g., Ift172, Ift88, and Kif3a null mutants) have two effects on Shh signaling: while Gli activators lose their abilities to activate Shh target genes, diminished processing of Gli3 to form the Gli3 repressor leads to weak derepression of Shh target genes. Disruption of Arl13b results in constitutive, intermediate-level activity of Shh pathway throughout the neural tube. These mutants generate cilia, but they are short and exhibit open axonemal B tubules. Disruption of THM1/aln results in ball-shaped cilia with defects in retrograde IFT and constitutive, high-level pathway activity in the neural tube. In contrast to these mutants, the Shh pathway in bromi mutants retains appropriate responses to all but the highest levels of Shh signaling in the neural tube. This phenotype is most similar to mouse mutants with hypomorphic C2cd3 or IFT88 function. The phenotype of bromi,Ptch1 double mutants indicates that loss of bromi places an upper boundary on the extent to which the pathway may be activated, but, at lower levels of Shh signaling, responses to graded Shh signals are intact. The suppression of the Rab23 phenotype in bromi,Rab23 double mutants indicates that Bromi acts downstream in the Shh pathway, and that strong, constitutive activation of Gli2 in the absence of Rab23 relies on Bromi function (Ko, 2010).

The notion that cilia merely provide a permissive environment where Hh signaling occurs has been challenged with the characterization of mouse ciliogenesis mutants with complex Shh responses. The localization of Gli proteins to cilia is regulated, at least in part, by activity of the Hh pathway (Chen, 2009; Endoh-Yamagami, 2009), and it appears that the precise level of Gli activity in response to a given level of stimulus depends on the way in which ciliary transport and structure are affected in various mutants. In bromi neuroepithelial cells, Gli2 fails to colocalize with Arl13b and Ift88 at cilia tips. It is proposed that Ift88, Arl13b, and acetylated α-tubulin label the axonemes within the mutant cilia, and that Gli2 localizes interior to, and distinct from, the curled axonemes. This dissociation may attenuate the efficacy of the Glis, possibly by affecting their flux into and out of cilia (Kim, 2009), or by preventing efficient interactions between Gli proteins and their upstream regulators (Ko, 2010).

Regulation of Gli1 localization by the cAMP/protein kinase A signaling axis through a site near the nuclear localization signal

The hedgehog (Hh) pathway plays a critical role during embryo development and in cancer. Although the molecular basis by which protein kinase A (PKA) regulates the stability of Hedgehog's downstream transcription factor Cubitus interruptus (the Drosophila homologue of vertebrate Gli molecules) is well documented, the mechanism by which PKA inhibits the functions of Gli molecules in vertebrates remains elusive. This study reports that activation of PKA retains Gli1 in the cytoplasm. Conversely, inhibition of PKA activity promotes nuclear accumulation of Gli1. Mutation analysis identifies Thr374 as a major PKA site determining Gli1 protein localization. In the three-dimensional structure, Thr374 resides adjacent to the basic residue cluster of the nuclear localization signal (NLS). Phosphorylation of this Thr residue is predicted to alter the local charge and consequently the NLS function. Indeed, mutation of this residue to Asp (Gli1/T374D) results in more cytoplasmic Gli1 whereas a mutation to Lys (Gli1/T374K) leads to more nuclear Gli1. Disruption of the NLS causes Gli1/T374K to be more cytoplasmic. The change of Gli1 localization is correlated with the change of its transcriptional activity. These data provide evidence to support a model that PKA regulates Gli1 localization and its transcriptional activity, in part, through modulating the NLS function (Sheng, 2006).

FKBP8 acts as a negative regulator of mouse sonic hedgehog signaling in neural tissues: FKBP8 activities ultimately converge on the GLI transcription factors

Sonic hedgehog (SHH) is a secreted morphogen that regulates the patterning and growth of many tissues in the developing mouse embryo, including the central nervous system (CNS). A member of the FK506-binding protein family, FKBP8, is an essential antagonist of SHH signaling in CNS development. Loss of FKBP8 causes ectopic and ligand-independent activation of the Shh pathway, leading to expansion of ventral cell fates in the posterior neural tube and suppression of eye development. Although it is expressed broadly, FKBP8 is required to antagonize SHH signaling primarily in neural tissues, suggesting that hedgehog signal transduction is subject to cell-type specific modulation during mammalian development. FKBP8 shares sequence similarity with an uncharacterized Drosophila gene product, CG5482. The predicted CG5482 protein has the same overall domain structure, as well as a membrane insertion site at the extreme C terminus. There are no known mutations in the CG5482 gene, nor are there mutations in other Drosophila FKBP genes that suggest their involvement in hedgehog signaling (Bulgakov, 2004).

It is not yet understood how FKBP8 acts in the hedgehog pathway at the molecular level. Some endogenous FKBP8 protein is present in a complex with the RII subunit of protein kinase A in vivo, raising the possibility that FKBP8 has a role in PKA-dependent phosphorylation of the GLI proteins. This possibility is supported by the similarity in phenotype between mutants deficient in FKBP8 and PKA. Total PKA activity in Fkbp8 mutant embryos, measured in vitro, is not diminished, suggesting that FKBP8 is not required for general PKA activity. FKBP8 has been found to associate with and inhibits the phosphatase calcineurin. This raises the possibility that activated calcineurin can promote hedgehog signaling. Regardless of the biochemical functions of FKBP8 in hedgehog signaling, it seems likely that its activities ultimately converge on the GLI transcription factors. Given that Gli2-null mutants have a considerably more severe neural patterning phenotype than Gli3 or Gli1 null mutants, GLI2 might be the principal target of FKBP8 function. This hypothesis is currently being tested though genetic epistasis (Bulgakov, 2004).

Molecular properties of Zic proteins as transcriptional regulators and their relationship to GLI proteins

Zic family genes encode zinc finger proteins, which play important roles in vertebrate development. The zinc finger domains are highly conserved between Zic proteins and show a notable homology to those of Gli family proteins. In this study, the functional properties of Zic proteins and their relationship to the GLI proteins has been investigated. An optimal binding sequence for Zic1, Zic2, and Zic3 proteins was establised by electrophoretic mobility shift assay-based target selection and mutational analysis. The selected sequence is almost identical to the GLI binding sequence. However, the binding affinity is lower than that of GLI. Consistent results were obtained in reporter assays, in which transcriptional activation by Zic proteins is less dependent on the GLI binding sequence than GLI1. Moreover, Zic proteins activate a wide range of promoters irrespective of the presence of a GLI binding sequence. When Zic and GLI proteins are cotransfected into cultured cells, Zic proteins enhance or suppress sequence-dependent, GLI-mediated transactivation depending on cell type. Taken together, these results suggest that Zic proteins may act as transcriptional coactivators and that their function may be modulated by the GLI proteins and possibly by other cell type-specific cofactors (Mizugishi, 2001).

In the present study, a consensus binding sequence for Zics was established by EMSA-based target selection and mutational analysis. The Zic binding sequence is essentially identical to the GLI-BS, 5'-TGGGTGGTC -3', and has a minimum consensus sequence of 5'-GGGTGGTC-3'. The binding affinities for this sequence are very similar among the three Zic proteins examined. However, the Zics-ZF bind the GLI-BS much more weakly than GLI3-ZF, as shown by competition experiments and the calculated binding constant. The Kd values of Zics are much higher than those of other transcription factors that function in a sequence-specific manner. Therefore, it is unlikely that Zic proteins compete with GLI for the GLI-BS (Mizugishi, 2001).

The binding properties are consistent with the results of the reporter assay, in which the dependence of Zic proteins on the GLI-BS for transcriptional activation is much less than that of GLI1. Instead, Zics activated transcription even in the absence of the GLI-BS via various promoters (TK promoter, adenovirus major late promoter, SV40 early promoter, and Zic1 promoter). On the basis of these facts, rather than being the transcription factors that regulate transcription by direct binding to DNA, Zic family proteins may function as transcriptional coactivators, which potentiate the activity of other transcription regulatory factors. It is possible that Zics interact with the transcription machinery or other factors that regulate transcriptional efficiency (Mizugishi, 2001).

The relationship between Zic and GLI proteins was examined. In C3H10T1/2 cells, Zic-GLI1 or Zic-GLI3 coactivates reporter gene expression, whereas in 293T cells, coexistence of the Zic and GLI proteins has a reverse effect. These results suggest a significant regulatory relationship between Zic and GLI proteins; however, the nature of this interaction remains unclear (Mizugishi, 2001).

The interaction between Zic and GLI proteins may be entirely independent of DNA binding. This direct or indirect interaction between Zic and GLI proteins may be modulated by cell type-specific cofactors. One well characterized cell type-specific cofactor is Oct-binding factor 1 (OBF-1); this is expressed in B-lymphocyte lineages and interacts with the POU-homeodomain proteins Oct-1 and Oct-2 to enhance transcriptional activation in the B-cell lineage. Similar cell type-specific cofactors might modify the GLI-Zic interactions. It is also possible that GLI proteins may be differentially modified post-translationally depending on the presence of Zics in different cell types. Recently, it was shown that Gli3 was processed depending on cAMP-activated protein kinase to generate a phosphorylated repressor form. Zic proteins might be involved in this pathway (Mizugishi, 2001).

Alternatively, the differential binding affinities of the Zic and GLI proteins for the target sequence may underlie the regulatory relationship between these two protein families. Although Zics-ZF have much lower binding affinities to the GLI-BS, there is a DNA binding transcription factor that has a Kd value similar to those of Zics. Moreover, the different human homeodomain proteins, despite having similar homeodomains, bind their target sequence with different affinities and thereby generate a complex regulatory network in the developmental process. In that case, less conserved domains other than the zinc finger may modulate binding in vivo to determine final binding specificity, because the recombinant proteins used in these experiments only included zinc finger domains. It is necessary to examine the downstream target genes in the developmental context to understand the Zic-DNA interaction in detail (Mizugishi, 2001).

Zic1/Gli3 double mutant mice showed severe abnormalities of vertebral arches not found in single mutants, strongly suggesting that these two proteins act synergistically in the development of the vertebral arches. However, in Xenopus laevis it was shown that Zic2 antagonizes the Gli proteins in the patterning of the neural plate. These findings suggest that Zic and GLI proteins may interact to variously repress or activate gene expression in vivo (Mizugishi, 2001).

In conclusion, Zic1, Zic2, and Zic3-ZF specifically recognize and bind the GLI-BS but with a much lower binding affinity than that of the GLI3-ZF. Zic proteins activated a wide range of promoters. These results suggest that Zic proteins may function as transcriptional coactivators or as factors generally involved in the gene expression process. How can such general factors regulate specific developmental processes, including the patterning of forebrain, cerebellum, axial skeleton, vasculature, and visceral organs? A clue to solving this problem may be the relationship with Gli family proteins as shown in this study. To clarify the regulatory networks under a broad range of developmental processes, the relationships between Zic proteins and other molecules in the hedgehog signaling pathway and transforming growth factor beta superfamily, which are closely related to each other, should also be examined in both in vitro and in vivo studies (Mizugishi, 2001).

Zic and Gli family proteins are transcription factors that share similar zinc finger domains. Recent studies indicate that Zic and Gli collaborate in neural and skeletal development. Evidence suggests that the Zic and Gli proteins physically and functionally interact through their zinc finger domains. Moreover, Gli proteins were translocated to cell nuclei by coexpressed Zic proteins, and both proteins regulated each other's transcriptional activity. These result suggests that the physical interaction between Zic and Gli is the molecular basis of their antagonistic or synergistic features in developmental contexts and that Zic proteins are potential modulators of the hedgehog-mediated signaling pathway (Koyabu, 2001).

A CUL-2 ubiquitin ligase containing three FEM proteins degrades TRA-1 to regulate C. elegans sex determination

In C. elegans, the Gli-family transcription factor TRA-1 is the terminal effector of the sex-determination pathway. TRA-1 activity inhibits male development and allows female fates. Genetic studies have indicated that TRA-1 is negatively regulated by the fem-1, fem-2, and fem-3 genes. However, the mechanism of this regulation has not been understood. This study shows that TRA-1 is regulated by degradation mediated by a CUL-2-based ubiquitin ligase complex that contains FEM-1 as the substrate-recognition subunit, and FEM-2 and FEM-3 as cofactors. CUL-2 physically associates with both FEM-1 and TRA-1 in vivo, and cul-2 mutant males share feminization phenotypes with fem mutants. CUL-2 and the FEM proteins negatively regulate TRA-1 protein levels in C. elegans. When expressed in human cells, the FEM proteins interact with human CUL2 and induce the proteasome-dependent degradation of TRA-1. This work demonstrates that the terminal step in C. elegans sex determination is controlled by ubiquitin-mediated proteolysis (Starostina, 2007).

Cubitus interruptus and butterfly eyespot evolution

The origin of new morphological characters is a long-standing problem in evolutionary biology. Novelties arise through changes in development, but the nature of these changes is largely unknown. In butterflies, eyespots have evolved as new pattern elements that develop from special organizers called foci. Formation of these foci is associated with novel expression patterns of the Hedgehog signaling protein, its receptor Patched, the transcription factor Cubitus interruptus, and the engrailed target gene, all of which break the conserved compartmental restrictions on this regulatory circuit in insect wings. Redeployment of preexisting regulatory circuits may be a general mechanism underlying the evolution of novelties. hh is expressed in all cells of the posterior compartment of the butterfly wing disc, as it is in Drosophila, but hh transcript levels are increased in a striking pattern in cells just outside of the subdivision midlines at specific positions along the proximodistal axis of the wing. These domains of increased hh transcription flank cells that have the potential to form foci. Higher levels of hh transcripts accumulate specifically in cells that flank the developing foci. In the presence of high levels of Hh, Patched function is inhibited, resulting in the accumulation of the activator form of Ci. Because ptc is a direct target of Ci, cells that receive and transduce the Hh signal have increased levels of ptc transcription. Activation of ptc transcription, accompanied by the accumulation of Ci protein occurs in cells that are flanked by the domains of highest hh transcription and are destined to become eyespot foci. these results indicate that the Hh signal is received and transduced by cells that will differentiate as foci. These expression patterns break the A/P compartmental restrictions on gene expression known in Drosophila. During the course of eyespot evolution, there is a relaxation of the strict En-mediated repression of ci that occurs in the posterior compartment of Drosophila. During focal establishment, en and invected are targets, rather than inducers of Hh signaling. In most species of butterflies, eyespots are found only in the posterior compartment of the wing. But in those species in which eyespots are found in the anterior compartment, both En/Inv and Ci are coexpressed in eyespot foci, including the one in the anterior compartment. Thus the expression of the Hh signaling pathway and en/inv is associated with the development of all eyespot foci and has become independent of A/P compartmental restrictions. It is suggested that during eyespot evolution, the Hh-dependent regulatory circuit that establishes foci is recruited from the circuit that acts along the A/P boundary of the wing. This recruitment of an entire regulatory circuit through changes in the regulation of a subset of components increases the facility with which new developmental functions can evolve and may be a general theme in the evolution of novelties within extant structures (Keys, 1999).

Cubitus interruptus homologs in fish

Zebrafish you-too (yot) mutations interfere with Hedgehog (Hh) signaling during embryogenesis. Using a comparative synteny approach, yot was isolated as a zinc finger transcription factor homologous to the Hh target gli2. Two alleles of yot contain nonsense mutations resulting in carboxy-terminally truncated proteins. In addition to causing defects in midline development, muscle differentiation, and retinal axon guidance. yot mutations disrupt anterior pituitary and ventral forebrain differentiation. yot mutations also cause ectopic lens formation in the ventral diencephalon. These findings reveal that truncated zebrafish Gli2 proteins interfere with Hh signaling necessary for differentiation and axon guidance in the ventral forebrain (Karlstrom, 1999).

The specification of different muscle cell types in the zebrafish embryo requires signals that emanate from the axial mesoderm. Overexpression of different members of the Hedgehog protein family can induce the differentiation of two types of slow-twitch muscles: the superficially located slow-twitch fibers and the medially located muscle pioneer (MP) cells. The requirement for Hedgehog signaling in the specification of these distinct muscle cell types has been investigated in two ways: (1) by characterizing the effects on target gene expression and muscle cell differentiation of the u-type (you; you-too; sonic you; chameleon; u-boot). mutants, members of a phenotypic group previously implicated in Hedgehog signaling, and (2) by analyzing the effects of overexpression of the Patched1 protein, a negative regulator of Hedgehog signaling. Embryos mutant for u-type genes all have normal notochords, leading to the suggestion that they may directly disrupt the signaling pathway required for MP induction. Two members of this class map to genes encoding components of the Hh signaling pathway. The syu mutations map to the shh gene itself, while mutations in the gene encoding the transcription factor Gli2, a homolog of the Drosophila Ci protein, are responsible for the yot mutant phenotype. The results support the idea that most u-type genes are required for Hedgehog signaling. The analysis of ptc1 expression has confirmed a role for two other members of the u-type class, con and you, in the propagation or transduction of the Hh signals between the notochord and the paraxial mesoderm. It is striking that the effects of both these mutants are like those of syu, initially weak and increasing in severity with developmental time. Whether this reflects a hypomorphism of the you and con alleles or a specificity in the function of the you and con gene products remains to be elucidated. While hedgehog signaling is essential for slow myocyte differentiation, the loss of activity of one signal, Sonic hedgehog, can be partially compensated for by other Hedgehog family proteins (Lewis, 1999b).

Gli proteins regulate the transcription of Hedgehog (Hh) target genes. Genetic studies in mouse have shown that Gli1 is not essential for embryogenesis, whereas Gli2 acts as an activator of Hh target genes. In contrast, misexpression studies in Xenopus and cultured cells have suggested that Gli1 can act as an activator of Hh-regulated genes, whereas Gli2 might function as a repressor of a subset of Hh targets. To clarify the roles of gli genes during vertebrate development, the requirements were analyzed for gli1 and gli2 during zebrafish embryogenesis. detour (dtr) mutations encode loss-of-function alleles of gli1. In contrast to mouse Gli1 mutants, dtr mutants and embryos injected with gli1 antisense morpholino oligonucleotides display defects in the activation of Hh target genes in the ventral neuroectoderm. Mutations in you-too (yot) encode C-terminally truncated Gli2. These truncated proteins act as dominant repressors of Hh signaling, in part by blocking Gli1 function. In contrast, blocking Gli2 function by eliminating full-length Gli2 results in minor Hh signaling defects and uncovers a repressor function of Gli2 in the telencephalon. In addition, Gli1 and Gli2 have activator functions during somite and neural development. These results reveal divergent requirements for Gli1 and Gli2 in mouse and zebrafish and indicate that zebrafish Gli1 is an activator of Hh-regulated genes, while zebrafish Gli2 has minor roles as a repressor or activator of Hh targets (Karlstrom, 2003).

Hedgehog signaling regulates cell differentiation and patterning in a wide variety of embryonic tissues. In vertebrates, at least three Gli transcription factors (Gli1, Gli2, and Gli3) are involved in Hh signal transduction. Comparative studies have revealed divergent requirements for Gli1 and Gli2 in zebrafish and mouse. This study addresses the question of whether Gli3 function has also diverged in zebrafish and the regulatory interactions between Hh signaling and Gli activity has been analyzed. Zebrafish Gli3 has an early function as an activator of Hh target genes that overlaps with Gli1 activator function in the ventral neural tube. In vitro reporter analysis shows that Gli3 cooperates with Gli1 to activate transcription in the presence of high concentrations of Hh. During late somitogenesis stages, Gli3 is required as a repressor of the Hh response. Gli3 shares this repressor activity with Gli2 in the dorsal spinal cord, hindbrain, and midbrain, but not in the forebrain. Consistently, zebrafish Gli3 blocks Gli1-mediated activation of a reporter gene in the absence of Hh in vitro. In the eye, Gli3 is also required for proper ath5 expression and the differentiation of retinal ganglion cells (RGCs). These results reveal a conserved role for Gli3 in vertebrate development and uncover novel regional functions and regulatory interactions among gli genes (Tyurina, 2005).

Three major axon pathways cross the midline of the vertebrate forebrain early in embryonic development: the postoptic commissure (POC), the anterior commissure (AC) and the optic nerve. A small population of Gfap+ astroglia spans the midline of the zebrafish forebrain in the position of, and prior to, commissural and retinal axon crossing. These glial 'bridges' form in regions devoid of the guidance molecules slit2 and slit3, although a subset of these glial cells express slit1a. Hh signaling is required for commissure formation, glial bridge formation, and the restricted expression of the guidance molecules slit1a, slit2, slit3 and sema3d, but Hh does not appear to play a direct role in commissural and retinal axon guidance. Reducing Slit2 and/or Slit3 function expands the glial bridges and causes defasciculation of the POC, consistent with a 'channeling' role for these repellent molecules. By contrast, reducing Slit1a function leads to reduced midline axon crossing, suggesting a distinct role for Slit1a in midline axon guidance. Blocking Slit2 and Slit3, but not Slit1a, function in the Hh pathway mutant yot (gli2DR) dramatically rescues POC axon crossing and glial bridge formation at the midline, indicating that expanded Slit2 and Slit3 repellent function is largely responsible for the lack of midline crossing in these mutants. Hh signaling appears to affect axon guidance indirectly through its role in patterning of the midline, including the formation of the glial bridge and the regulation of axon guidance-molecule expression. This analysis shows that Hh signaling helps to pattern the expression of Slit guidance molecules that then help to regulate glial cell position and axon guidance across the midline of the forebrain (Barresi, 2005).

iguana encodes a novel zinc-finger protein with coiled-coil domains essential for Hedgehog signal transduction in the zebrafish embryo

Signaling by lipid-modified secreted glycoproteins of the Hedgehog family play fundamental roles during pattern formation in animal development and in humans; dysfunction of Hedgehog pathway components is frequently associated with a variety of congenital abnormalities and cancer. Transcriptional regulation of Hedgehog target genes is mediated by members of the Gli zinc-finger transcription factors. The relative nuclear concentrations of Gli activator (Gliact) and repressor (Glirep) forms, together with their nucleocytoplasmic trafficking, appear to be critical determinants for target gene expression. Whereas such stringent controls of Gli activity are critical in ensuring appropriate levels of pathway activation, the mechanisms by which these processes are regulated remain inadequately understood. Genetic analysis has been used to show that the zebrafish iguana gene product acts downstream of the Smoothened protein to modulate Gli activity in the somites of the developing embryo. Positional cloning reveals that iguana encodes the zebrafish ortholog of Dzip1, a novel zinc-finger/coiled-coil domain protein that can shuttle between the cytoplasm and nucleus in a manner correlated with Hedgehog pathway activity (Wolff, 2004).

The phenotypic similarity and synergistic interaction between igu mutants and Su(fu) depletion and overexpression could imply a mechanistic similarity between igu and Su(fu) function. Moreover, like Su(fu), the Igu protein localizes to the cytoplasm, but translocates to the nucleus in response to the same signal that activates Gli activity. This translocation could be driven by an interaction between Igu and the Gli proteins, as seems to be the case for Su(fu); according to this scenario, the nuclear-localized Igu might function by modulating nuclear Gli activity. Alternatively, Igu might itself regulate the nuclear-cytoplasmic shuttling of the Glis. Consistent with this notion, it was found that like Su(fu) morphants, igu enhances the dominant effect of yot heterozygotes, suggesting an increased nuclear accumulation of the constitutively active Gli2rep encoded by the yot mutant allele. In this regard, it is striking that the C-terminally truncated form of Igu is constitutively localized to the nucleus. Although this could imply a possible mechanism for the increased activity of both Gli1 as well as the Gli2rep in igu mutants, the fact that the igu mutant alleles are completely recessive, and that misexpression of a truncated form of Igu has no effect on muscle specification argues against this. Furthermore, the MO-mediated inhibition of Igu expression has identical phenotypic consequences to the mutant alleles that encode truncated forms of the protein, implying that the increased activities of Gli1 and Gli2rep are independent of the aberrant nuclear localization of the mutant proteins (Wolff, 2004).

As with Su(fu), the cytoplasmic retention of Igu may reflect its physical interaction with one or more Gli proteins. Such an interaction could be direct or via an intermediary, perhaps Su(fu) itself. The molecular structure of the Igu protein is certainly consistent with these possibilities, the single zinc-finger and the coiled-coil domains both capable of mediating protein-protein interactions; however, given the properties of the truncated protein, it is predicted that any such Gli interaction would be mediated by the coiled-coil domain. Alternatively, Igu distribution in uninduced cells may be controlled primarily by the NES, which is deleted in the C-terminally truncated form of the protein. Unraveling the details of interactions between Igu and its potential partners, and the requirements for the NLS and NES, will be essential in elucidating the basis of its contrasting effects on Gli1 and Gli2 activity (Wolff, 2004).

Fine-tuning of Hh signaling by the RNA-binding protein Quaking to control muscle development

The development of the different muscles within the somite is a complex process that involves the Hedgehog (Hh) signaling pathway. To specify the proper number of muscle cells and organize them spatially and temporally, the Hh signaling pathway needs to be precisely regulated at different levels, but only a few factors external to the pathway have been described. This study reports the role of the STAR family RNA-binding protein Quaking A (QkA) in somite muscle development. The loss of QkA function in zebrafish affects fast muscle fiber maturation as well as Hh-induced muscle derivative specification and/or morphogenesis. Mosaic analysis reveals that fast fiber maturation depends on the activity of QkA in the environment of fast fiber progenitors. It was further shown that Hh signaling requires QkA activity for muscle development. By an in silico approach, 3'UTRs of known Hh signaling component mRNAs were screened for the Quaking response element, and the transcription factor Gli2a, a known regulator of muscle fate development, was identified. Using destabilized GFP as a reporter, it was shown that the gli2a mRNA 3'UTR is a functional QkA target. Consistent with this notion, the loss of QkA function rescued slow muscle fibers in yot mutant embryos, which express a dominant-negative Gli2a isoform. Thus, these results reveal a new mechanism to ensure muscle cell fate diversity by fine-tuning of the Hh signaling pathway via RNA-binding proteins (Lobbardi, 2011).

Cubitus interruptus homologs in amphibians

In frog embryos, Gli1 is expressed transiently in the prospective floor plate during gastrulation and in cells lateral to the midline during late gastrula and neurula stages. In contrast, Gli2 and Gli3 are absent from the neural plate midline with Gli2 expressed widely and Gli3 in a graded fashion, with highest levels in lateral regions. In mouse embryos, the three Gli genes show a similar pattern of expression in the neural tube but are coexpressed throughout the early neural plate. Gli1 is the only Gli gene expressed in the prospective floor plate cells of frog embryos: it therefore seemed likely that this gene would be involved in ventral neural tube development. Sonic hedgehog (Shh) signaling activates Gli1 transcription and widespread expression of endogenous frog Gli1, but not Gli3, in developing frog embryos. This results in the ectopic differentiation of floor plate cells and ventral neurons within the neural tube. Floor-plate-inducing ability is retained when cytoplasmic Gli1 proteins are either forced into the nucleus or are fused to the VP16 transactivating domain. Gli1 induces HNF-3ß, a ventral marker, in the neural tube as well as in the epidermal ectoderm. In addition, ectopic expression of Gli1 induces the ectopic expression of Sonic hedgehog and the floor-plate-specific marker F-spondin. Embryos injected with Gli1 display ectopic cells in the midbrain resembling putative neurons. These ectopic cells can be identified as 5HT (serotonin) producing neurons These results identify Gli1 as a midline target of Shh and suggest that it mediates the induction of floor plate cells and ventral neurons when Shh acts as a transcriptional regulator (Lee, 1997).

In Xenopus, the Gli-type proteins XGli-3 and XGli-4 are first expressed in earliest stages of mesoderm and neural development. Transient transfection assays reveal that XGli-3 and XGli-4 can function as transcription repressors. Counteracting the Gli-protein repressor activity by ectopic expression of a fusion protein that contains the Gli-zinc finger cluster connected to the E1A activator domain in Xenopus embryos results in specific morphological alterations in the developing somites and in the central nervous system. Altered expression characteristics for a broad set of molecular markers highlighting specific aspects of mesodermal and neural differentiation demonstrate an important role for Gli-type zinc finger proteins in the early mesodermal and neural patterning of Xenopus embryos. Muscle development is severly disturbed by expression of the fusion protein activator, as can be seen by the failure to form ordered somatic segments and from effects on the expression of mesoderm/muscle-specific markers such as MyoD and cardiac actin Gene expression in the neural tube is grossly disturbed. For example, there is an increase in Delta expression; eye vesicles are absent, and Pax-6 transcription is strongly reduced. Neural crest precursors fail to express the twist gene, a molecular marker for cephalic neural crest (Marine, 1997).

The Drosophila homeoproteins Ara and Caup are members of a combination of factors (prepattern) that control the highly localized expression of the proneural genes achaete and scute. Two Xenopus homologs of ara and caup (Xiro1 and Xiro2) have been identified. Like their Drosophila counterparts, they control the expression of proneural genes and, probably as a consequence, the size of the neural plate. In Xenopus, ectopic expression of these genes expands the neural plate, similar to the effect of overexpressing XASH-3 and ATH-3. Xiro expression precedes expression of the proneural genes, and partially overlaps the domains of expression of XASH-3 and ATH-3 and those of X-ngnr-1, another proneural gene. When overexpressed, X-ngnr-1 causes the differentiation of ectopic neurons. Xiro1 and Xiro2 are themselves controlled by noggin and retinoic acid. Like ara and caup, they are overexpressed in Xenopus embryos as a result of the expression of Drosophila cubitus interruptus gene, suggesting that neurogenesis is induced by the hedgehog family of proteins. These and other findings suggest the conservation of at least part of the genetic cascade that regulates proneural genes, and the existence in vertebrates of a prepattern of factors important to control the differentiation of the neural plate (Gómez-Skarmeta, 1998).

Patterning along the anteroposterior (A/P) axis involves the interplay of secreted and transcription factors that specify cell fates in the mesoderm and neuroectoderm. While FGF and homeodomain proteins have been shown to play different roles in posterior specification, the network coordinating their effects remains elusive. The function of Gli zinc-finger proteins in mesodermal A/P patterning has been examined. Gli2 is sufficient to induce ventroposterior development, functioning in the FGF-brachyury regulatory loop. Gli2 directly induces brachyury, a gene required and sufficient for mesodermal development, and Gli2 is in turn induced by FGF signaling. Moreover, the homeobox gene Xhox3, a critical determinant of posterior development, is also directly regulated by Gli2. Gli3, but not Gli1, has an activity similar to that of Gli2 and is expressed in ventroposterior mesoderm after Gli2. These findings uncover a novel function of Gli proteins, previously only known to mediate hedgehog signals, in the maintenance and patterning of the embryonic mesoderm. More generally, these results suggest a molecular basis for an integration of FGF and hedgehog inputs in Gli-expressing cells that respond to these signals (Brewster, 2000).

Previous work has shown that Gli2 can be induced by SHH signaling in frog embryos, and that it can mediate some of the effects of SHH. FGF also induces Gli2, although it remains unclear which factors directly initiate its expression in mesoderm, as this is difficult to separate from the general induction of mesoderm by FGF or TGFbeta family signals. While Gli2/3 function in mesoderm may have nothing to do with HH signaling, HH genes have been reported to be expressed at low levels throughout the gastrula marginal zone, raising the possibility that Gli2 and Gli3 activity in mesoderm could be responsive to HH signals by analogy with some of its later roles in neural development. For example, a low tonic HH signal throughout the marginal zone could attenuate the formation of putative Gli3 repressors, a process regulated by the SHH signaling pathway, thus allowing Gli2 and Gli3 activator forms to function in ventroposterior development. The fact that misexpression of HHs at early stages has no obvious consequence on mesodermal development could be consistent with this possibility if repressor forms were not required in mesoderm. In mice, loss of SHH, Gli2 or Gli3 function does not appear to affect the early embryonic mesoderm, possibly indicating that Gli proteins could have partially divergent roles in different organisms (Brewster, 2000).

The role of Gli2 in FGF signaling, the ability of FGF and SHH to induce its expression and its partial mediation of SHH functions suggest a mechanism for a possible integration of FGF and HH signaling in tissues in which these signals act on the same Gli-expressing cells. SHH can act through Gli1, and Gli3 has an antagonistic relationship with SHH/Gli1. In contrast, SHH can also act through Gli2 in some contexts, but in others, Gli2 can instead antagonize the actions of SHH and Gli1. A context-dependent function of Gli2 could therefore underlie the sometimes synergistic and sometimes antagonistic effects of FGFs and HHs. Similarly, antagonism between HH and FGF signaling could result from their use of Gli1 and Gli3, respectively. This model may be particularly relevant for Gli-expressing precursor cells. For example, SHH is a known mitogen for cerebellar granule precursors and FGF can partially inhibit this effect. Because Wnt signaling has been recently suggested to affect Gli2 and Gli3 expression in chick somites, a challenge of ongoing studies is to elucidate how different signaling inputs regulate Gli function in vertebrate development and disease (Brewster, 2000).

Sonic hedgehog is involved in eye field separation along the proximodistal axis. As the optic vesicle and optic cup mature, Hh signalling continues to be important in defining aspects of the proximodistal axis. Two other Hedgehog proteins, Banded hedgehog and Cephalic hedgehog, related to the mouse Indian hedgehog and Desert hedgehog, respectively, are strongly expressed in the central retinal pigment epithelium but excluded from the peripheral pigment epithelium surrounding the ciliary marginal zone. By contrast, downstream components of the Hedgehog signalling pathway, Gli2, Gli3 and X-Smoothened, are expressed in this narrow peripheral epithelium. This zone contains cells that are in the proliferative state. This equivalent region in the adult mammalian eye, the pigmented ciliary epithelium, has been identified as a zone in which retinal stem cells reside. These data, combined with double labelling and the use of other retinal pigment epithelium markers, show that the retinal pigment epithelium of tadpole embryos has a molecularly distinct peripheral to central axis. In addition, Gli2, Gli3 and X-Smoothened are also expressed in the neural retina, in the most peripheral region of the ciliary marginal zone, where retinal stem cells are found in Xenopus, suggesting that they are good markers for retinal stem cells. To test the role of the Hedgehog pathway at different stages of retinogenesis, the pathway was activated by injecting a dominant-negative form of PKA or blocking it by treating embryos with cyclopamine. Embryos injected or treated at early stages display clear proximodistal defects in the retina. Interestingly, the main phenotype of embryos treated with cyclopamine at late stages is a severe defect in RPE differentiation. This study thus provides new insights into the role of Hedgehog signalling in the formation of the proximodistal axis of the eye and the differentiation of retinal pigment epithelium (Perron, 2003).

Cubitus interruptus homologs in birds and mammals

Three proteins identified in mammals, GLI, GLI2, and GLI3, all share a highly conserved zinc finger domain with Drosophila Cubitus interruptus and are believed to function as transcription factors in the vertebrate Sonic hedgehog-Patched signaling pathway. The transcriptional regulatory properties of GLI and its contribution of specific domains to transcriptional regulation have been characterized in order to better understand the role GLI plays in the Sonic hedgehog-Patched pathway and mechanisms of GLI-induced transcriptional regulation. GLI activates expression of reporter constructs in HeLa cells in a concentration-dependent manner through the GLI consensus binding motif; a GAL4 binding domain-GLI fusion protein activates reporter expression through the GAL4 DNA binding site. GLI-induced transcriptional activation requires the carboxyl-terminal amino acids 1020-1091, which include an 18-amino acid region highly similar to the alpha-helical herpes simplex viral protein 16 activation domain, including the consensus recognition element for the human TFIID TATA box-binding protein-associated factor TAFII31 and conservation of all three amino acid residues believed to directly contact chemically complementary residues in TAFII31. The presence of this 18-amino acid region in the GLI activation domain provides a mechanism for GLI-induced transcriptional regulation (Yoon, 1998).

Two members of the GLI family have been isolated from the chick, GLI and GLI3. Their expression patterns in a variety of tissues during embryogenesis suggest that these genes may be targets of Sonic hedgehog signals. The two GLI genes are differentially regulated by Sonic hedgehog during limb development. Sonic hedgehog up-regulates GLI transcription, while down-regulating GLI3 expression in the mesenchymal cells of the developing limb bud. An activated form of GLI can induce expression of Patched a known target of Sonic hedgehog, thus inplicating GLI as a key transcription factor in the vertebrate hedgehog signaling pathway. In conjunction with evidence from a mouse Gli3 mutant, these data suggest that GLI and GLI3 may have taken two different functions of their Drosophila homolog CI. These two functions are the mediation of hedgehog signaling and the repression of hedgehog transcription (Domínguez, 1996). In Drosophila the same transcription factor can be utilized for both purposes because the cells expressing hedgehog and the cells reponsive to it are mutually exclusive populations. In the vertebrate limb, where the responsive cells overlap the cells producing Sonic hedgehog, the same factor cannot be used (Marigo, 1996).

The regulation of the Gli genes during somite formation has been investigated in quail embryos. The Gli genes are a family encoding three related zinc finger transcription factors, Gli1, Gli2 and Gli3, which are effectors of Shh signaling in responding cells. A quail Gli3 cDNA has been cloned and its expression compared with Gli1 and Gli2. These studies show that Gli1, Gli2 and Gli3 are co-activated at the time of somite formation, thus providing a mechanism for regulating the initiation of Shh signaling in somites. Embryo surgery and paraxial mesoderm explant experiments show that each of the Gli genes is regulated by distinct signaling mechanisms. Gli1 is activated in response to Shh produced by the notochord, which also controls the dorsalization of Gli2 and Gli3 following their activation by Wnt signaling from the surface ectoderm and neural tube. This surface ectoderm/neural tube Wnt signaling has both negative and positive functions in Gli2 and Gli3 regulation: these signals repress Gli3 in segmental plate mesoderm prior to somite formation and then promote somite formation and the somite-specific activation of Gli2 and Gli3. These studies, therefore, establish a role for Wnt signaling in the control of Shh signal transduction through the regulation of Gli2 and Gli3, and provide a mechanistic basis for the known synergistic actions of surface ectoderm/neural tube and notochord signaling in somite cell specification (Borycki, 2000).

A model is presented for Wnt and Shh signaling in the control of Gli gene activation during somite formation. In this model, in the segmental plate mesoderm, Gli3 is maintained in a repressed state by Wnt signaling through beta-catenin. When anteriormost segmental plate mesoderm initiates somite formation, Wnt/beta-catenin signaling undergoes a negative to positive switch, leading to derepression of Gli3, to the initiation of somite formation, and to activation of the somite-specific expression of Gli2 and Gli3. It is suggested that this switch in Wnt/beta-catenin function might be mediated by transcription cofactors such as Groucho, NLK and CtBP, factors that are known to control the transcription activities of beta-catenin/LEF1/tcf complexes in segmental plate mesoderm. The process of somite formation and the regulated expression of beta-catenin cofactors might be be under the control of the segmentation genes. Quantitative changes in Wnt signaling at the time of somite formation, resulting from the activation of Wnt expression in the neural tube and loss of Wnt inhibitors in newly forming somites, would then mediate increased levels of beta-catenin. This high level of beta-catenin would participate in both the cytoplasmic cell adhesion processes to initiate somite formation as well as in new beta-catenin/LEF1/tcf transcription complexes for Gli2 and Gli3 activation. The Gli2 and Gli3 proteins produced in newly formed somites would then become activated as nuclear transcription factors in response to the Shh that is produced by the notochord, leading to their participation in the activation of Shh response genes, including Gli1 and Ptc1 (Borycki, 2000).

Three mouse genes, Gli, Gli-2, and Gli-3, which share a similar zinc finger domain with the products of Cubitus interruptus and the Caenorhabditis elegans sex-determining gene tra-1 have been cloned and characterized. Expression is first detected during gastrulation in both the ectoderm and mesoderm. Later in development, their expression becomes more restricted in various ectoderm- and mesoderm-derived tissues and is not detectable after completion of organogenesis. Interestingly, in the developing neural tube, Gli shows a narrow ventral domain of expression, whereas Gli-2 and Gli-3 show a broad and dorsally restricted domain. Expression of these three Gli genes in various ectoderm- and mesoderm-derived tissues suggests that they play multiple roles during postimplantation development. Consistent with this hypothesis, a naturally occurring Gli-3 mutation, the mouse extra-toes mutant; shows defects in both mesoderm- and ectoderm-derived tissues (Hui, 1994).

The secreted factor Sonic hedgehog (SHH) is both necessary and sufficient to induce multiple developmental processes, including ventralization of the CNS, branching morphogenesis of the lungs and anteroposterior patterning of the limbs. Based on analogy to the Drosophila Hh pathway, the multiple GLI transcription factors in vertebrates are likely to both transduce SHH signaling and repress Shh transcription. In order to discriminate between overlapping versus unique requirements for the three Gli genes in mice, a Gli1 mutant was produced and the phenotypes of Gli1/Gli2 and Gli1/3 double mutants were analyzed. Gli3xt mutants have polydactyly and dorsal CNS defects associated with ectopic Shh expression, indicating GLI3 plays a role in repressing Shh. In contrast, Gli2 mutants have five digits, but lack a floorplate, indicating that it is required to transduce SHH signaling in some tissues. Remarkably, mice homozygous for a Gli1zfd mutation that deletes the exons encoding the DNA-binding domain are viable and appear normal. Transgenic mice expressing a GLI1 protein lacking the zinc fingers can not induce SHH targets in the dorsal brain, indicating that the Gli1zfd allele contains a hypomorphic or null mutation. Interestingly, Gli1zfd/zfd;Gli2zfd/+, but not Gli1zfd/zfd;Gli3zfd/+ double mutants have a severe phenotype; most Gli1zfd/zfd;Gli2zfd/+ mice die soon after birth and all have multiple defects including a variable loss of ventral spinal cord cells and smaller lungs that are similar to, but less extreme than, Gli2zfd/zfd mutants. Gli1/Gli2 double homozygous mutants have more extreme CNS and lung defects than Gli1zfd/zfd;Gli2zfd/+ mutants, however, in contrast to Shh mutants, ventrolateral neurons develop in the CNS and the limbs have 5 digits with an extra postaxial nubbin. These studies demonstrate that the zinc-finger DNA-binding domain of GLI1 protein is not required for SHH signaling in mouse. Furthermore, Gli1 and Gli2, but not Gli1 and Gli3, have extensive overlapping functions that are likely downstream of SHH signaling (Park, 2000).

Drosophila transcription factor cubitus interruptus (Ci) and its co-activator CRE (cAMP response element)-binding protein (CBP) activate a group of target genes on the anterior-posterior border in response to Hedgehog protein (Hh) signaling. In contrast, in the anterior region, the carboxyl-truncated form of Ci generated by protein processing represses Hh expression. In vertebrates, three Ci-related transcription factors (glioblastoma gene products [GLIs] 1, 2, and 3) have been identified, but their functional difference in Hh signal transduction is unknown. Distinct roles are reported for GLI1 and GLI3 in Sonic hedgehog (Shh) signaling. GLI3, which contains both repression and activation domains, acts both as an activator and a repressor, as does Ci, whereas GLI1 contains only the activation domain. Consistent with this, GLI3, but not GLI1, is processed to generate the repressor form. Transcriptional co-activator CBP binds to GLI3, but not to GLI1. The trans-activating capacity of GLI3 is positively and negatively regulated by Shh and cAMP-dependent protein kinase, respectively, through a specific region of GLI3, which contains the CBP-binding domain and the phosphorylation sites of cAMP-dependent protein kinase. GLI3 directly binds to the Gli1 promoter and induces Gli1 transcription in response to Shh. Thus, GLI3 may act as a mediator of Shh signaling in the activation of the target gene Gli1 (Dai, 1999).

The hedgehog signal transduction network performs critical roles in mediating cell-cell interactions during embryogenesis and organogenesis. Loss-of-function or misexpression mutation of hedgehog network components can cause birth defects, skin cancer, and other tumors. The Gli gene family (Gli1, Gli2, and Gli3) encodes zinc finger transcription factors that act as mediators of hedgehog signal transduction. The role of Gli2 in mammary gland development has been investigated. Mammary expression of Gli2 is developmentally regulated in a tissue compartment-specific manner. Expression is exclusively stromal during virgin stages of development but becomes both epithelial and stromal during pregnancy and lactation. The null phenotype with respect to both ductal and alveolar development was examined by transplantation rescue of embryonic mammary glands into physiologically normal host females. Glands derived from both wild type and null embryo donors show ductal outgrowths that develop to equivalent extents in virgin hosts. However, in null transplants, ducts are frequently distended or irregularly shaped and show a range of histological alterations similar to micropapillary ductal hyperplasias in the human breast. Alveolar development during pregnancy is not overtly affected by loss of Gli2 function. Ductal defects are not observed when homozygous null epithelium is transplanted into a wild type stromal background, indicating that Gli2 function is required primarily in the stroma for proper ductal development. Gli2 heterozygotes also demonstrate an elevated frequency and severity of focal ductal dysplasia relative to that of wild type littermate- and age-matched control animals (Lewis, 2001).

In mice, three Gli genes are thought to collectively mediate sonic hedgehog (Shh) signaling. Mis-expression studies and analysis of null mutants for each gene have indicated that the Gli proteins have different functions. In particular, Gli1 appears to be a constitutive activator, and Gli2 and Gli3 have repressor functions. To determine the precise functional differences between Gli1 and Gli2, Gli1 has been expressed in place of Gli2 from the endogenous Gli2 locus in mice. Strikingly, a low level of Gli1 can rescue all the Shh signaling defects in Gli2 mutants; however, this is the case only in the presence of a wild-type Shh gene. These studies demonstrate that only the activator function of Gli2 is actually required, and indicate that in specific situations, Shh can modulate the ability of Gli1 to activate target genes. Furthermore, expression of both copies of Gli1 in place of Gli2 does not disrupt spinal cord patterning, but does result in new gain-of-function defects that lead to lethality. The defects are enhanced when Gli3 function is reduced, demonstrating that an important difference between Gli1 and Gli2 is the ability of Gli1 to antagonize Gli3 function (Bai, 2001).

Cubitus interruptus: transcriptional targets

There is growing evidence that Gli proteins participate in the mediation of Hedgehog and FGF signaling in neural and mesodermal development. However, little is known about which genes act downstream of Gli proteins. The regulation of members of the Wnt family by Gli proteins in different contexts is shown in this study. These findings indicate that Gli2 regulates Wnt8 expression in the ventral marginal zone of the early frog embryo: activating Gli2 constructs induce ectopic Wnt8 expression in animal cap explants, whereas repressor forms inhibit its endogenous expression in the marginal zone. Using truncated Frizzled and dominant-negative Wnt constructs, the requirement of at least two Wnt proteins, Wnt8 and Wnt11, for Gli2/3-induced posterior mesodermal development is shown. Blocking Wnt signals, however, inhibits Gli2/3-induced morphogenesis, but not mesodermal specification. Gli2/3 may therefore normally coordinate the action of these two Wnt proteins, which regulate distinct downstream pathways. In addition, the finding that Gli1 consistently induces a distinct set of Wnt genes in animal cap explants and in skin tumors suggests that Wnt regulation by Gli proteins is general. Such a mechanism may link signals that induce Gli activity, such as FGFs and Hedgehogs, with Wnt function (Mullor, 2001).

The zinc finger transcription factor GLI3 is considered a repressor of vertebrate Hedgehog (Hh) signaling. In humans, the absence of GLI3 function causes Greig cephalopolysyndactyly syndrome, affecting the development of the brain, eye, face, and limb. Because the etiology of these malformations is not well understood, the phenotype of mouse Gli-/- mutants was examined as a model to investigate this. An up-regulation of Fgf8 is observed in the anterior neural ridge, isthmus, eye, facial primordia, and limb buds of mutant embryos, sites coinciding with the human disease. Intriguingly, endogenous apoptosis is reduced in Fgf8-positive areas in Gli-/- mutants. Since SHH is thought to be involved in Fgf8 regulation, Fgf8 expression was compared in Shh-/- and Gli-/-;Shh-/- mutant embryos. Whereas Fgf8 expression is almost absent in Shh-/- mutants, it is up-regulated in Gli-/-;Shh-/- double mutants, suggesting that SHH is not required for Fgf8 induction, and that GLI3 normally represses Fgf8 independently of SHH. In the limb bud, evidence is provided that ectopic expression of Gremlin in Gli-/- mutants might contribute to a decrease in apoptosis. Together, these data reveal that GLI3 limits Fgf8-expression domains in multiple tissues, through a mechanism that may include the induction or maintenance of apoptosis. It is concluded that Fgf8 may not be a direct target of GLI3 but that the size of the Fgf domain may be regulated by GLI3 indirectly; when GLI3 is present, it activates the expression of Bmps, which regulates cell death to alter the size of FGF8 domains (Aoto, 2002).

Bone morphogenetic protein 2 (BMP-2) plays a critical role in osteoblast function. In Drosophila, Cubitus interruptus (Ci), which mediates hedgehog signaling, regulates gene expression of dpp, the ortholog of mammalian BMP-2. Null mutation of the transcription factor Gli2, a mammalian homolog of Ci, results in severe skeletal abnormalities in mice. It was hypothesized that Gli2 regulates BMP-2 gene transcription and thus osteoblast differentiation. The present study shows that overexpression of Gli2 enhances BMP-2 promoter activity and mRNA expression in osteoblast precursor cells. In contrast, knocking down Gli2 expression by Gli2 small interfering RNA or genetic ablation of the Gli2 gene results in significant inhibition of BMP-2 gene expression in osteoblasts. Promoter analyses, including chromatin immunoprecipitation and electrophoretic mobility shift assays, provided direct evidence that Gli2 physically interacts with the BMP-2 promoter. Functional studies showed that Gli2 is required for osteoblast maturation in a BMP-2-dependent manner. Finally, Sonic hedgehog (Shh) stimulates BMP-2 promoter activity and osteoblast differentiation, and the effects of Shh are mediated by Gli2. Taken together, these results indicate that Gli2 mediates hedgehog signaling in osteoblasts and is a powerful activator of BMP-2 gene expression, which is required in turn for normal osteoblast differentiation (Zhao, 2006; full text of article).

Sonic hedgehog (Shh) acts as a morphogen to mediate the specification of distinct cell identities in the ventral neural tube through a Gli-mediated (Gli1-3) transcriptional network. Identifying Gli targets in a systematic fashion is central to the understanding of the action of Shh. This issue was examined in differentiating neural progenitors in mouse. An epitope-tagged Gli-activator protein was used to directly isolate cis-regulatory sequences by chromatin immunoprecipitation (ChIP). ChIP products were then used to screen custom genomic tiling arrays of putative Hedgehog (Hh) targets predicted from transcriptional profiling studies, surveying 50-150 kb of non-transcribed sequence for each candidate. In addition to identifying expected Gli-target sites, the data predicted a number of unreported direct targets of Shh action. Transgenic analysis of binding regions in Nkx2.2, Nkx2.1 (Titf1) and Rab34 established these as direct Hh targets. These data also facilitated the generation of an algorithm that improved in silico predictions of Hh target genes. Together, these approaches provide significant new insights into both tissue-specific and general transcriptional targets in a crucial Shh-mediated patterning process (Volkes, 2007).

Sonic hedgehog (Shh) signals via Gli transcription factors to direct digit number and identity in the vertebrate limb. This study characterized the Gli-dependent cis-regulatory network through a combination of whole-genome chromatin immunoprecipitation (ChIP)-on-chip and transcriptional profiling of the developing mouse limb. These analyses identified approximately 5000 high-quality Gli3-binding sites, including all known Gli-dependent enhancers. Discrete binding regions exhibit a higher-order clustering, highlighting the complexity of cis-regulatory interactions. Further, Gli3 binds inertly to previously identified neural-specific Gli enhancers, demonstrating the accessibility of their cis-regulatory elements. Intersection of DNA binding data with gene expression profiles predicted 205 putative limb target genes. A subset of putative cis-regulatory regions were analyzed in transgenic embryos, establishing Blimp1 (see Drosophila Blimp-1) as a direct Gli target and identifying Gli activator signaling in a direct, long-range regulation of the BMP antagonist Gremlin. In contrast, a long-range silencer cassette downstream from Hand2 likely mediates Gli3 repression in the anterior limb. These studies provide the first comprehensive characterization of the transcriptional output of a Shh-patterning process in the mammalian embryo and a framework for elaborating regulatory networks in the developing limb (Vokes, 2008).

The transcriptional response to the Hedgehog (Hh) pathway is mediated by Gli proteins, which function as context-dependent transcriptional activators or repressors. However, the mechanism by which Gli proteins regulate their target genes is poorly understood. This is the first genetic characterization of a Gli-dependent cis-regulatory module (CRM), focusing on its regulation of Grem1 in the mouse limb bud. The CRM, termed GRE1 (Gli responsive element 1), can act as both an enhancer and a silencer. The enhancer activity requires sustained Hh signaling. As a Gli-dependent silencer, GRE1 prevents ectopic transcription of Grem1 driven through additional CRMs. In doing so, GRE1 works with additional GREs to robustly regulate Grem1. It is suggested that multiple Gli CRMs may be a general mechanism for mediating a robust transcriptional response to the Hh pathway (Li, 2014).

Cubitus interruptus homologs: Brain development

In the developing vertebrate CNS, members of the Wnt gene family are characteristically expressed at signaling centers that pattern adjacent parts of the neural tube. To identify candidate signaling centers in the telencephalon, Wnt gene fragments were isolated from cDNA derived from embryonic mouse telencephalon. In situ hybridization experiments demonstrate that one of the isolated Wnt genes, Wnt7a, is broadly expressed in the embryonic telencephalon. By contrast, three others, Wnt3a, 5a and a novel mouse Wnt gene, Wnt2b, are expressed only at the medial edge of the telencephalon, defining the hem of the cerebral cortex. The Wnt-rich cortical hem is a transient, neuron-containing, neuroepithelial structure that forms a boundary between the hippocampus and the telencephalic choroid plexus epithelium (CPe) throughout their embryonic development. Indicating a close developmental relationship between the cortical hem and the CPe, Wnt gene expression is upregulated in the cortical hem both before and just as the CPe begins to form, and persists until birth. In addition, although the cortical hem does not show features of differentiated CPe, such as expression of transthyretin mRNA, the CPe and cortical hem are linked by shared expression of members of the Bmp and Msx gene families. In the extra-toesJ (XtJ) mouse mutant, telencephalic CPe fails to develop. Wnt gene expression is shown to be deficient at the cortical hem in XtJ/XtJ mice, but the expression of other telencephalic developmental control genes, including Wnt7a, is maintained. The XtJ mutant carries a deletion in Gli3, a vertebrate homolog of the Drosophila gene cubitus interruptus (ci), which encodes a transcriptional regulator of the Drosophila Wnt gene, wingless. These observations indicate that Gli3 participates in Wnt gene regulation in the vertebrate telencephalon, and suggest that the loss of telencephalic choroid plexus in XtJ mice is due to defects in the cortical hem that include Wnt gene misregulation (Grove, 1998).

D/V patterning of the anterior neural plate is controlled by several signaling centers. Signals from the anterior neural ridge (ANR) regulate expression of the Forkhead related factor Brain factor1 (Bf1), which is required for growth and patterning of the telencephalon. Fgf8 represents an important component of this signal as Fgf8 applied to the prosencephalic neural plate mimics the effects of the ANR. In addition, the anterior non-neural ectoderm, the ANR and later the roof of the forebrain produce several secreted factors of the bone morphogenetic protein (Bmp) family. Bmps have been shown to induce the expression of Msx1 in the dorsal midline of the forebrain and to repress the expression of Bf1. Also, noggin, encoding a secreted protein that binds to Bmps and prevents the latter from interacting with its receptor, is expressed in the telencephalic roof plate, suggesting that Bmp activity is under stringent control during dorsal forebrain development (Theil, 1999 and references).

The prechordal mesendoderm represents a key determinant in the specification of the ventral forebrain and produces Sonic hedgehog protein. Shh is expressed throughout the axial mesendoderm and has been implicated in ventral patterning throughout the neuraxis. Mice mutant for Shh are cyclopic and exhibit disruptions of ventral forebrain formation. Mutations of the human SHH gene have also been identified in patients with holoprosencephaly. These studies therefore implicate Shh as an essential mediator of the inductive effects of the prechordal mesendoderm (Theil, 1999 and references).

The dentate gyrus and hippocampus as centers for spatial learning, memory and emotional behaviour have been the focus of much interest in recent years. The molecular information on their development, however, has been relatively poor. To date, only Emx genes are known to be required for dorsal telencephalon development. Forebrain development in the extra toes (XtJ) mouse mutant, which carries a null mutation of the Gli3 gene, is described. Gli3 is a mediator of Shh signaling. The XtJ defect leads to a failure to establish the dorsal di-telencephalic junction and finally results in a severe size reduction of the neocortex. In addition, XtJ/XtJ mice show absence of the hippocampus (Ammon's horn plus dentate gyrus) and the choroid plexus in the lateral ventricle. The medial wall of the telencephalon, which gives rise to these structures, fails to invaginate during embryonic development. On a molecular level, disruption of dorsal telencephalon development in XtJ/XtJ embryos correlates with a loss of Emx1 and Emx2 expression. Furthermore, the expression of Fgf8 and Bmp4 in the dorsal midline of the telencephalon is altered. However, expression of Shh, which is negatively regulated by Gli3 in the spinal cord, is not affected in the XtJ/XtJ forebrain. This study therefore implicates Gli3 as a key regulator for the development of the dorsal telencephalon and implies that Gli3 is upstream of Emx genes in a genetic cascade controlling dorsal telencephalic development (Theil, 1999).

Based on their expression pattern, Emx genes are candidates for playing a role in subdividing the prosencephalon. Furthermore, their Drosophila homolog, empty spiracles, functions as a gap gene as well as a segment identity gene during head segmentation suggesting that Emx genes might also be involved in specifying dorsal telencephalon identity. However, the phenotypes of mice in which Emx genes have been inactivated have not provided clues on these potential roles. Analysis of the forebrain phenotype of XtJ/XtJ mice therefore provides the first evidence for a gene controlling formation of the di-telencephalic boundary and specification of dorsal telencephalon identity (Theil, 1999).

The Gli3 mutation also affects development of the telencephalic roof and the juxtaposed medium pallium. Moreover, formation of the choroid plexus is disrupted in the lateral ventricle while its development occurs normally in the 4th ventricle. The expression patterns of several regulatory genes were found to be altered in the telencephalic dorsal midline of XtJ/XtJ embryos. While Fgf8 is ectopically activated in the trp, Bmp signaling is negatively affected by the Gli3 mutation as judged by the loss of Bmp4 and Msx1 expression and by the maintenance of noggin expression. Interestingly, Fgf8 and Bmp2/Bmp4 have been shown to act antagonistically on cell proliferation and differentiation in the dorsal forebrain. Ectopic Fgf8 expression and loss of Bmp signaling in the roof plate as observed in XtJ/XtJ embryos might therefore disrupt the balance between these two processes (Theil, 1999).

Little is known about the mechanisms that control the development of regional identity in the mammalian telencephalon. The Gli family of transcription factor genes is involved in the regulation of pattern at many sites in the embryo and is expressed in the embryonic mouse telencephalon. Telencephalic patterning has been analyzed in the extra-toesJ (XtJ) mouse mutant, which carries a deletion in the Gli family member Gli3. Dorsoventral patterning of the telencephalon is dramatically disrupted in the XtJ mutant. Specific dorsal telencephalic cell types and gene expression patterns are lost in homozygous XtJ mutants, and features of ventral telencephalic identity develop ectopically in the dorsal telencephalon. This partial ventralization of the dorsal telencephalon does not appear to be induced by an expansion of Sonic hedgehog expression in the telencephalon, but may be due to a loss of Bmp and Wnt gene expression in a putative dorsal telencephalic signaling center, the cortical hem. These findings suggest that in dorsal telencephalon, Gli3 is needed to repress ventral telencephalic identity (Tole, 2000).

At least two alternative explanations are possible of the development of a partially 'ventralized' dorsal telencephalon in the XtJ mouse. (1) Rather than causing a direct expansion of a ventralizing signal, the Gli3 mutation may result in a failure to supply a dorsalizing signal. In the spinal cord and hindbrain, a balance between signals mediated by Shh, and by Bmp proteins and other TGFbeta family members, appears to regulate basic dorsoventral patterning of the neural tube and the development of specific regional cell types. The XtJ mutant lacks Wnt and Bmp signals supplied by the cortical hem. Thus signals may be lost that are normally required to specify and expand dorsal telencephalic cell populations and to antagonize signals that ventralize the telencephalon. The loss of cortical hem expression of Wnt3a, for example, could alone account for the loss of the hippocampus in XtJ homozygous mutants. A striking loss of the hippocampus has been observed in mice deficient in expression of Wnt3a, indicating that a local Wnt3a signal, derived from the cortical hem, is required for hippocampal development. (2) Another possible explanation is that the Gli3 mutation in XtJ mutants results in the loss of a mechanism that normally suppresses a Shh-activated ventralizing pathway (Tole, 2000).

The mechanisms that regulate the growth of the brain remain unclear. Sonic hedgehog (Shh) is expressed in a layer-specific manner in the perinatal mouse neocortex and tectum, whereas the Gli genes, which are targets and mediators of SHH signaling, are expressed in proliferative zones. In vitro and in vivo assays show that SHH is a mitogen for neocortical and tectal precursors and that it modulates cell proliferation in the dorsal brain. Together with its role in the cerebellum, these findings indicate that SHH signaling unexpectedly controls the development of the three major dorsal brain structures. A variety of primary human brain tumors and tumor lines consistently express the GLI genes and cyclopamine, a SHH signaling inhibitor, inhibits the proliferation of tumor cells. Using the in vivo tadpole assay system, it has been further shown that misexpression of GLI1 induces CNS hyperproliferation that depends on the activation of endogenous Gli1 function. SHH-GLI signaling thus modulates normal dorsal brain growth by controlling precursor proliferation, an evolutionarily important and plastic process that is deregulated in brain tumors (Dahmane, 2001).

Considerable data suggest that sonic hedgehog (Shh) is both necessary and sufficient for the specification of ventral pattern throughout the nervous system, including the telencephalon. The regional markers induced by Shh in the E9.0 telencephalon are dependent on the dorsoventral and anteroposterior position of ectopic Shh expression. This suggests that by this point in development regional character in the telencephalon is established. To determine whether this prepattern is dependent on earlier Shh signaling, the telencephalon was examined in mice carrying either Shh- or Gli3-null mutant alleles. This analysis revealed that the expression of a subset of ventral telencephalic markers, including Dlx2 and Gsh2, although greatly diminished, persists in Shh-/- mutants, and that these same markers are expanded in Gli3-/- mutants. To understand further the genetic interaction between Shh and Gli3, Shh/Gli3 and Smoothened/Gli3 double homozygous mutants were examined. Notably, in animals carrying either of these genetic backgrounds, genes such as Gsh2 and Dlx2, which are expressed pan-ventrally, as well as Nkx2.1, which demarcates the ventral most aspect of the telencephalon, appear to be largely restored to their wild-type patterns of expression. These results suggest that normal patterning in the telencephalon depends on the ventral repression of Gli3 function by Shh and, conversely, on the dorsal repression of Shh signaling by Gli3. In addition, these results support the idea that, in addition to hedgehog signaling, a Shh-independent pathways must act during development to pattern the telencephalon (Rallu, 2002).

Regionalization of the neural plate and the early neural tube is controlled by several signaling centers that direct the generation of molecularly distinct domains. In the developing telencephalon, the anterior neural ridge (ANR) and the roof and floor plate act as such organizing centers via the production of Fgfs, Bmps/Wnts, and Shh, respectively. It remains largely unknown, however, how the combination of these different signals is used to coordinate the generation of different telencephalic territories. Telencephalic development has been examined in Pdn mutant mice, which carry an integration of a retrotransposon in the Gli3 locus. Homozygous mutant animals are characterized by a partial dorsal-to-ventral transformation of the telencephalon and by an increased size of the septum. On a molecular level, these alterations correlate with a reduction and/or loss of Bmp/Wnt expression and a concomitant expansion of Fgf8 transcription. Evidence that the ectopic activation of Fgf signaling in the dorsal telencephalon provides an explanation for the ventralization of the Gli3 mutant telencephalon since application of Fgf8-soaked beads to dorsal telencephalic explants leads to the specific induction and repression of ventral marker and dorsal marker genes, respectively. In summary, Gli3 regulates the generation and specification of distinct dorsal and ventral telencephalic domains not only by restricting the dorsal extent of Shh signaling but also by setting up the antagonizing Fgf and Bmp/Wnt signaling centers. This dual mechanism ensures the coordinated development of distinct dorsal and ventral telencephalic progenitor domains (Kuschel, 2003).

Spatial pattern of sonic hedgehog signaling through Gli genes during cerebellum development

The cerebellum consists of a highly organized set of folia that are largely generated postnatally during expansion of the granule cell precursor (GCP) pool. Since the secreted factor sonic hedgehog (Shh) is expressed in Purkinje cells and functions as a GCP mitogen in vitro, it is possible that Shh influences foliation during cerebellum development by regulating the position and/or size of lobes. How Shh and its transcriptional mediators, the Gli proteins, regulate GCP proliferation in vivo was studied, and whether they influence foliation was tested. Shh expression correlates spatially and temporally with foliation. Expression of the Shh target gene Gli1 is also highest in the anterior medial cerebellum, but is restricted to proliferating GCPs and Bergmann glia. By contrast, Gli2 is expressed uniformly in all cells in the developing cerebellum except Purkinje cells and Gli3 is broadly expressed along the anteroposterior axis. Whereas Gli mutants have a normal cerebellum, Gli2 mutants have greatly reduced foliation at birth and a decrease in GCPs. In a complementary study using transgenic mice, it was shown that overexpressing Shh in the normal domain does not grossly alter the basic foliation pattern, but does lead to prolonged proliferation of GCPs and an increase in the overall size of the cerebellum. Taken together, these studies demonstrate that positive Shh signaling through Gli2 is required to generate a sufficient number of GCPs for proper lobe growth (Corrales, 2004).

Cerebellar development is a carefully orchestrated process that produces an exquisitely foliated structure with a simple layered cytoarchitecture. In mammals, the cerebellum is divided into three regions with distinct anteroposterior (AP) foliation patterns: a central vermis and two bilaterally symmetric hemispheres. The most abundant neurons in the cerebellum, as well as the entire brain, are the granule cells. Whereas Purkinje cells and cerebellar interneurons originate in the ventricular neuroepithelium, cerebellar granular cell precursors (GCPs) arise from a germinal zone in the rhombic lip situated in dorsal posterior rhombomere 1. The GCPs begin to leave the rhombic lip at approximately embryonic day (E) 13 and migrate over the cerebellar anlage to form the external granule layer (EGL). Although the EGL is formed by E15, GCPs in the EGL remain mitotically active until 2 weeks postnatal. Granule cells start to exit the cell cycle after birth and as part of their differentiation program migrate internally past the Purkinje cells to form the inner granule layer (IGL). Over the course of the first two postnatal weeks, cerebellar folia form, suggesting the increase in granule cells is largely responsible for foliation. The process of foliation begins with the formation of four principal fissures, which divide the cerebellum into five cardinal lobes. As GCP proliferation continues, these lobes expand and are further subdivided to give rise to the species-specific foliation pattern observed in the mature cerebellum. The fissures that divide the central cardinal lobe into lobes VI-VIII are among the last to form in the vermis (Corrales, 2004).

It has been shown that an interaction between Purkinje cells and GCPs is important for granule cell proliferation and foliation. For example, when Purkinje cells are ablated or as in mouse mutants that lack Purkinje cells, such as Lurcher and Staggerer, the GCP population is diminished and foliation is arrested. One key GCP mitogen expressed in Purkinje cells is sonic hedgehog (Shh), since it can induce proliferation of GCPs in culture, and injection of Shh antibodies into the cerebellum reduces granule cell proliferation. Shh signaling is mediated by the Gli family of transcription factors. In the spinal cord Gli2 is the primary activator of Shh signaling, whereas Gli3 functions mainly as a repressor but is also a weak activator. By contrast, in the limb only Gli3 is required for digit patterning and to regulate a normal level of proliferation. An important question, therefore, is whether Shh functions in the cerebellum primarily by inhibiting the Gli3 repressor as in the limb, and/or by inducing the activator Gli2. Due to the embryonic lethality of Gli2 and Gli3 mutants, the in vivo requirements for these two genes during postnatal cerebellum development have not been addressed. Gli1 (Gli: Mouse Genome Informatics) however, is not required for mouse development, although it plays a redundant activator function with Gli2, which is revealed only in Gli2 heterozygotes. Furthermore, unlike that of Gli2 and Gli3, Gli1 transcription is regulated by Shh signaling. In particular, all transcription of Gli1 is absolutely dependent on induction of Gli2 and Gli3 activators by Hh signaling. Since Gli1 is a transcriptional target of Shh signaling, lacZ expression in Gli-lacZ knock-in mice (Gli1lz/+) is a readout of positive Shh signaling (Corrales, 2004).

Gli1-lacZ mice were utilized to characterize the precise spatial and temporal pattern of positive Shh signaling in the developing cerebellum. Strikingly, Shh expression and signaling (Gli-lacZ expression) in the developing vermis is spatially patterned from E18 to P10 with highest levels in anterior lobes (III-VIa) and the most posterior lobe (X). Both Gli1 and Gli2 are primarily excluded from Purkinje cells, and Gli expression is strongest in Bergmann glia and in the GCPs in the outer layer of the EGL. Gli3 is expressed in most cell types along the AP axis. In the absence of Gli2, normal expansion of GCPs in the EGL is impaired, and foliation is reduced at birth. Gli1-lacZ expression is undetectable in Gli2 mutants, demonstrating that Gli2 is the major activator required to transduce Shh-positive signaling in the developing cerebellum. In support of this, the thickness of the EGL appears normal in Gli3 mutants. In transgenic mice overexpressing Shh in a normal pattern in the cerebellum, the basic pattern of cerebellum foliation is maintained, although the entire cerebellum is enlarged and the lobes that normally express higher levels of Shh have an irregular IGL. In addition, the EGL persists longer than normal in transgenics. This study utilizes in vivo experiments to establish a role for positive Shh signaling in regulating expansion of the cerebellar lobes by regulating GCP proliferation, and demonstrates that Gli2 is a required mediator for this signaling (Corrales, 2004).

Cubitus interruptus homologs: Effects of mutation

X-linked heterotaxy (HTX1) is a rare developmental disorder characterized by disturbances in embryonic laterality and other midline developmental field defects. HTX1 results from mutations in ZIC3, a member of the GLI transcription factor superfamily. A targeted deletion of the murine Zic3 locus has been created to investigate its function and interactions with other molecular components of the left-right axis pathway. Embryonic lethality is seen in approximately 50% of null mice with an additional 30% lethality in the perinatal period. Null embryos have defects in turning, cardiac development and neural tube closure. Malformations in live born null mice include complex congenital heart defects, pulmonary reversal or isomerism, CNS defects and vertebral/rib anomalies. Investigation of nodal expression in Zic3-deficient mice indicates that, although nodal is initially expressed symmetrically in the node, there is failure to maintain expression and to shift to asymmetric expression. Subsequent nodal and Pitx2 expression in the lateral plate mesoderm in these mice is randomized, indicating that Zic3 acts upstream of these genes in the determination of left-right asymmetry. The phenotype of these mice correctly models the defects found in human HTX1 and indicates an important role for Zic3 in both left-right and axial patterning (Purandare, 2002).

The Shh signaling pathway is required in many mammalian tissues for embryonic patterning, cell proliferation and differentiation. In addition, inappropriate activation of the pathway has been implicated in many human tumors. Based on transfection assays and gain-of-function studies in frog and mouse, the transcription factor Gli1 has been proposed to be a major mediator of Shh signaling. To address whether this is the case in mouse, a Gli1 null allele expressing lacZ was generated. Strikingly, Gli1 is not required for mouse development or viability. Of relevance, it has been shown that all transcription of Gli1 in the nervous system and limbs is dependent on Shh and, consequently, Gli1 protein is normally not present to transduce initial Shh signaling. To determine whether Gli1 contributes to the defects seen when the Shh pathway is inappropriately activated and Gli1 transcription is induced, Gli1;Ptc double mutants were generated. It has been shown that Gli1 is not required for the ectopic activation of the Shh signaling pathway or to the early embryonic lethal phenotype in Ptc null mutants. Instead, it has been found that Gli2 is required for mediating some of the inappropriate Shh signaling in Ptc mutants. These studies demonstrate that, in mammals, Gli1 is not required for Shh signaling and that Gli2 mediates inappropriate activation of the pathway due to loss of the negative regulator Ptc (C. B. Bai, 2002).

A concentration gradient of Shh is thought to pattern the ventral neural tube, and these ventral cell types are absent in shh-/- mice. Based on in vitro and genetic studies, the zinc finger-containing transcription factors Gli 1, 2, and 3 are mediators of the Shh intracellular response. The floorplate and adjacent cell types are absent in gli1-/-;gli2-/- mice, but part of the Shh-/- phenotype in the neural tube is alleviated in the Shh-/-;gli3-/- double mutant. This is consistent with the predicted role of Gli3 as a repressor of the Shh response. Gli3 repressor activity is blocked by Shh. In order to test the role of the repressor form of Gli3 in the neural tube, a truncated version of Gli3 (Gli3R*) was modeled after an allele found in Pallister Hall Syndrome, an autosomal dominant disorder that includes hypothalamic hamartomas, a bifid epiglottis, polysydactyly, and abnormal craniofacial features. Gli3R* acts as a constitutive repressor independent of Shh signaling. Misexpression of Gli3R* in the chick neural tube caused a ventral expansion of class-I, dorsal progenitor proteins and a loss of class-II, ventral progenitor proteins consistent with expected activity as a repressor of the Shh response. Activation of the BMP response is sufficient to maintain gli3 expression in neural plate explants, which might be a mechanism by which BMPs antagonize the Shh response (Meyer, 2003).

Cubitus interruptus homologs: Neural tube midline

Continued: Cubitus interruptus Evolutionary homologs part 2/3 | part 3/3

cubitus interruptus continued:

Biological Overview | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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