pangolin

DEVELOPMENTAL BIOLOGY

Embryonic

pangolin is already found in embryos of 0-2 hours, indicative of maternal expression (van de Wetering, 1997). pangolin is expressed ubiquitously throughout embryogenesis. By the end of germ band retraction, pan transcriptions are arrayed in a segmentally modulated pattern, with high accumulation in the visceral mesoderm and endoderm of the gut, as well as in part of the procephalon (Brunner, 1997).

Pangolin function during wing development: Nemo antagonizes Wingless signaling

The cellular events that govern patterning during animal development must be precisely regulated. This is achieved by extrinsic factors and through the action of both positive and negative feedback loops. Wnt/Wg signals are crucial across species in many developmental patterning events. Drosophila nemo (nmo) acts as an intracellular feedback inhibitor of Wingless (Wg) and it is a novel Wg target gene. Nemo antagonizes the activity of the Wg signal, as evidenced by the finding that reduction of nmo rescues the phenotypic defects induced by misexpression of various Wg pathway components. In addition, the activation of Wg-dependent gene expression is suppressed in wing discs ectopically expressing nmo and enhanced cell autonomously in nmo mutant clones. nmo itself is a target of Wg signaling in the imaginal wing disc. nmo expression is induced upon high levels of Wg signaling and can be inhibited by interfering with Wg signaling. Finally, alterations are observed in Arm stabilization upon modulation of Nemo. These observations suggest that the patterning mechanism governed by Wg involves a negative feedback circuit in which Wg induces expression of its own antagonist Nemo (Zeng, 2004).

In Drosophila, several examples of Wg feedback inhibition have been identified. (1) It has been shown that Wg downregulates its own transcription in the wing pouch to narrow the RNA expression domain at the DV boundary. (2) Wg signaling can repress the expression of its receptor Dfz2 in the wg-expressing cells of the wing disc. Wg regulation of Dfz2 creates a negative feedback loop in which newly secreted Wg is stabilized only once it moves away from the DV boundary to cells expressing higher levels of Drosophila Fz2. (3) The Wg target gene naked cuticle (nkd) acts through Dsh to limit Wg activity. (4) Wingful (Wf), an extracellular inhibitor of Wg, is itself induced by Wg signaling (Zeng, 2004).

This research adds Nemo to this list of inducible antagonists participating in Wg signaling. Nemo antagonizes the Wg signal in wing development, as evidenced by phenotypic rescue, suppression of Wg-dependent gene expression in discs ectopically expressing nmo, and ectopic expression of a Wg-dependent gene in nmo mutant clones (Zeng, 2004).

In support that Wg signaling regulates the transcription of nmo, several dTCF consensus binding sites have been found in the 5' region of the nmo gene that may represent enhancer elements. Indeed, two sites match 9 out of 11 bp (GCCTTTGAT) of the T1 site (GCCTTTGATCT) in the dpp BE enhancer that has been shown both in vitro and in vivo to bind and respond to dTCF. The presence of these sites suggests that the observed transcriptional regulation of nmo by Wg may involve direct binding to the nmo DNA sequence by dTCF (Zeng, 2004).

As a result of comparing the endogenous expression pattern of nmo with stabilized Arm, it was noticed that the highest levels of Nemo exclude Arm stabilization, while high levels of Arm are present in cells in which nmo levels are lower. Since Arm protein stabilization is a direct consequence of Wg pathway activation, attempts were made to examine whether Nemo may function to inhibit Wg by promoting Arm destabilization and subsequent breakdown. Indeed, ectopic expression of Nemo can lead to cell-autonomous reduction in Arm protein levels. This preliminary result suggests a mechanism in which Nemo may contribute to the destabilization of Arm that involves the Axin/APC/GSK3 complex. One explanation to account for such a finding would concern the interaction with TCF in the nucleus and the role of dTCF as an anchor for Arm. Given what is known about NLKs, it is likely that Nemo may act on the ability of the dTCF/Arm complex to bind DNA and activate transcription. It has been proposed that dTCF acts as an anchor for Arm in the nucleus. It remains to be determined how efficient this anchor is and whether there are conditions in which the interaction may become compromised, such as is seen with elevated Nemo. NLKs have been shown to affect the DNA-binding ability of TCF/ß-catenin. Perhaps in the absence of DNA binding, this complex is less stable and Arm could be free to shuttle to the cytoplasm where it could associate with Axin or APC and become degraded. It is proposed that the ectopic nmo leads to destabilization of the dTCF/Arm/DNA complex, thus causing Arm to exit the nucleus and be degraded through interaction with Axin, APC and GSK3. The observation that ectopic expression of full-length Arm cannot induce any activated Wg phenotypes has been explained by the hypothesis that even these high levels of protein are not sufficient to overcome the degradation machinery. Thus, the finding that there is no elevated Arm in nmo clones is consistent with an inability to overcome the endogenous degradation machinery; even though less Nemo could lead to more stabilized DNA interactions, this would not lead to higher levels of stabilized Arm than are normally found (Zeng, 2004).

Developmental roles of the Mi-2/NURD-associated protein p66 in Drosophila: Overexpression of p66 can repress activation of a TCF reporter by ß-catenin

The NURD and Sin3 histone deacetylase complexes are involved in transcriptional repression through global deacetylation of chromatin. Both complexes contain many different components that may control how histone deacetylase complexes are regulated and interact with other transcription factors. In a genetic screen for modifiers of wingless signaling in the Drosophila eye, mutations were isolated in the Drosophila homolog of p66, a protein previously purified as part of the Xenopus NURD/Mi-2 complex. p66 encodes a highly conserved nuclear zinc-finger protein that is required for development and it is proposed that the p66 protein acts as a regulatory component of the NURD complex. Animals homozygous mutant for p66 display defects during metamorphosis possibly caused by misregulation of ecdysone-regulated expression. Although heterozygosity for p66 enhances a wingless phenotype in the eye, loss-of-function clones in the wing and the eye discs do not have any detectable phenotype, possibly due to redundancy with the Sin3 complex. Overexpression of p66, in contrast, can repress wingless-dependent phenotypes. Furthermore, p66 expression can repress multiple reporters in a cell culture assay, including a Wnt-responsive TCF reporter construct, implicating the NURD complex in repression of Wnt target genes. By co-immunoprecipitation, p66 associates with dMi-2, a known NURD complex member (Kon, 2005).

Thus, loss of p66 enhances the sev-wg^ts phenotype in the eye; i.e., the resulting phenotype resembles that of increased wingless signaling. Since loss of p66 does not affect other transgenes expressed using the same promoter as sev-wg^ts, it is believed that removal of p66 does not affect wingless expression through the sevenless promoter, but instead represses wingless target genes involved in bristle formation. The results are in agreement with experiments that have implicated the NURD complex in Wnt signaling. It has been reported that Lef-1 repression involves HDAC-1 function. This interaction takes place in the absence of mSin3A, leading to the hypothesis that Lef-1 repression involves recruitment of the NURD complex. Three pieces of evidence are presented to further support this hypothesis: (1) loss of p66 enhances a wg overexpression phenotype; (2) overexpression of p66 can repress activation of a TCF reporter by ß-catenin in tissue culture cells; (3) overexpression of p66 in vivo represses the formation of wingless-dependent scutellar bristles without repressing wingless expression. Together, these experiments provide additional evidence that the NURD complex is involved in repression of Wnt target genes (Kon, 2005).

The lethality of p66 mutants is caused by misregulation of ecdysone-regulated genes during larval stages. If p66 was involved in repression of ecdysone-induced targets such as E74 and DHR3, then loss of p66 should lead to ectopic gene expression. In contrast, it was found that ecdysone-induced genes are not activated, suggesting that the role of p66 is more complex and indirect. In microarray studies of ecdysone response, 44% of genes that changed expression were repressed. It is speculated that repression of one or more of these genes is required for ecdysone-induced expression and that p66 is required for this process (Kon, 2005).

An additional connection between the NURD complex and ecydsone response is made through Bonus (bon), the Drosophila homolog of TIF1. TIF1 was identified as a protein that interacts with HP1, a heterochromatin-associated protein. The NURD complex may be involved in histone modification to allow HP1 binding. Similar to p66 mutants, bon mutants die during pupal stages due to misregulation of ecdysone-induced genes. Furthermore, E74 expression is also reduced in bon mutants. Thus it is speculated that p66 and the NURD complex may be involved in regulation of ecdysone response through HP1-mediated repression (Kon, 2005).

p66 mutations can affect both wg and ecdysone-induced gene expression. Furthermore, in cell culture reporter assays, it was found that expression of p66 inhibits activation of a TCF reporter by ß-catenin, of an SRE reporter by the M1 receptor, and of an NF-AT reporter by high levels of intracellular calcium. In addition, two human p66 homologs, hp66alpha and hp66ß, function as transcriptional repressors when tethered to a promoter, suggesting that transcriptional repression is a shared activity of p66 proteins. However, p66 did not repress an albumin luciferase reporter, implying that expression of p66 does not cause a defect in general transcription. Therefore, it is concluded that p66 can repress multiple signaling pathways, and it is hypothesized that this repression is mediated by recruitment of the NURD histone deacetylase complex. This conclusion is also supported by previous reports that expression of p66 can change the localization of MBD3, a component of the NURD complex (Kon, 2005).

Although overexpression of p66 can repress multiple signaling pathways, no loss-of-function phenotypes were detected that would be consistent with this hypothesis. In Drosophila, relatively mild and tissue-specific phenotypes for repressors have also been found for Pangolin/dTCF and naked cuticle. With respect to Pangolin/dTCF, this could be due to its dual role as both a repressor and an activator, but in other cases, a lack of phenotypes is possibly due to redundancy among parallel pathways (not necessarily among related genes). This suggestion is in analogy to the SynMuv genes in C. elegans. Animals mutant in either a synMuvA or a synMuvB gene alone have a normal vulva; animals mutant for both a synMuvA gene and a synMuvB gene have a multi-vulval (Muv) phenotype. Indeed, in the C. elegans vulva, p66 functions in a redundant pathway (Kon, 2005).

P66 is present when NURD complex is purified from Xenopus oocytes and is found to be associated with the NURD complex in mammalian cells in association with the methyl DNA-binding protein MBD2, as part of the MeCP1 complex. Is p66 a component of the NURD complex or an accessory factor? WdMi-2 and p66 co-purify, suggesting that they form a complex. However, if all of the complex members participate in the same processes, then the corresponding mutants should also have the same phenotypes. To ascertain whether p66 and rpd3 (HDAC homolog) function together is difficult since rpd3 also participates in the Sin3 complex. Thus, it is likely that rpd3 will display a wider range of phenotypes than a mutant of the NURD complex alone (Kon, 2005).

However, the p66 mutant can be compared to the other NURD complex mutant characterized in Drosophila, dMi-2. dMi-2 is required for oogenesis and for cell viability. rpd3 is also likely important for oogenesis; a hypomorphic rpd3 allele produces very few eggs. In contrast, p66 mutant germlines produce normal embryos, and p66 mutant clones survive in third instar imaginal discs. dMi-2 zygotic mutants die during larval stages, which coincides with the lethality of strong allele combination of p66 mutants. It is possible that dMi-2 may also be required for ecdysone response in larval stages. Since the p66 mutant phenotype does not completely mimic Mi-2 mutant phenotypes, it is suggested that p66 is not a core component of the NURD complex, but could have a regulatory function. However, it is equally possible that differences in allelic strength or perdurance of maternal contributions obscures the full range of phenotypes on the several components (Kon, 2005).

These results are further supported by experiments demonstrating that dMi-2 and the Drosophila MBD2/3 protein do not colocalize in nuclei. dMi-2 is distributed ubiquitously in embryonic nuclei, while Drosophila MBD2/3 is localized in a speckled pattern. This result suggests that Drosophila MBD2/3 is not an integral component of all dMi-2 complexes (Kon, 2005).

How might p66 function? In mammalian cells, human p66 protein is associated only with the NURD complex as part of the MeCP1 complex, which additionally contains methylated DNA-binding activity through the MBD2 protein. Similarly, the Xenopus NURD complex, which copurifies with p66, in contrast to mammalian NURD complexes, also has methylated DNA-binding activity. The NURD complex, through Mi-2, interacts with the zinc-finger proteins Hunchback, Tramtrak, and Ikaros. It is hypothesized that p66, also a zinc-finger-containing protein, functions similarly to these transcription factors to recruit the NURD complex to methylated DNA (Kon, 2005).

This hypothesis is supported by experiments that demonstrate that human p66 interacts with MBD2 and MBD3 in vitro. Furthermore, overexpression of p66 can change localization of MBD3. On the basis of these results, it is hypothesized that p66 may function as a link between the NURD complex and the methylated DNA-binding proteins MBD2 and MBD3. It is proposed that p66 recruits the NURD complex to mediate methylation-mediated silencing. Since both DNA methyl transferases and DNA-methylated binding proteins exist in Drosophila, it is likely that there is some vestige of a methylation system in Drosophila, although the function is unknown at this time (Kon, 2005).

Effects of Mutation or Deletion

A screening was carried out to identify components of the wingless signal transduction pathway; the screen sought dominant suppressors of the rough eye phenotype, caused by a transgene that drives ectopic expression of wingless during eye development. Essentially such a screen would identify cellular components that when mutated would fail to transduce signals from ectopically produced Wingless. Four strong suppressors were found that fall into two complementation groups. Two are recessive alleles of armadillo and two are recessive alleles of a locus on the fourth chromosome, designated as pangolin. A stronger allele of pan was isolated which shows strong genetic interactions when trans-heterozygous with either of the two isolated armadillo mutations, causing adult phenotypes characteristic of reduced wg activity. These results suggest that pan, like arm, encodes a component of the wingless transduction pathway, and also raise the possiblity that these components may physically interact (Brunner, 1997).

Adults mutant for pan show crippled or absent antennae and legs, and the wing is transformed into a duplicated notum. There is a loss of dorso-ventral polarity in the leg pattern. Wing notches are found in 20% to 30% of adults carrying one allele. The abdominal structure is altered with sternite structures affected in all animals. Typically, sternite bristles are lost and sternite tissues are replaced by pleura. Thus embryos mutant for pan display a segment polarity phenotype in which the larval epidermis forms a lawn of ventral denticles and lacks naked cuticle between the segmental denticle belts, similar to armadillo mutants (Brunner, 1997).

A constitutively active form of Armadillo was expressed in wild type flies and results in a naked phenotype in which the denticles normally positioned in the anterior portion of each segment are replaced by naked cuticle. In pan mutants no naked cuticle due to active Armadillo overexpression is observed, thus showing that in the absence of PAN, ARM activity is blocked and cannot cause a biological wingless response in vivo (Brunner, 1997).

An N-terminal deletion mutant of pangolin was ectopically expressed in flies. Ubiquitous expression of pangolin results in a segment polarity phenotype. In the most severe transgenics, the denticle belts are also narrowed in the dorsal-ventral axis. A decay in the stripes of engrailed expression is apparent in these mutants during late stage 9 (van de Wetering, 1997).

When a constitutively active from of armadillo is expressed in pangolin mutants, its action is largely inhibited. Such double mutants resemble pangolin mutants in that they have alternating denticles and naked cuticle, with portions of the lateral naked cuticle converted to denticles. However, they often have regions of naked cuticle intruding into the normal denticle belt at the ventral midline (van de Wetering, 1997).

The mutation ciD is mutant for two neighboring loci, cubitus interruptus and pan. In situ hybridication experiments with pan probes reveal that in ciD/+ heterozygote embryos, pan transcripts are detected in a pattern indistinguishable from that of ci transcripts, providing additional evidence that the molecular lesion of ciD disrupts both genes. It is a curious coincidence that pan and ci are adjacent genes, as ci encodes a transcription factor that is essential for transducing all examples of Hedgehog signaling, whereas the present evidence suggests an equivalent role for Pan in Wingless signal transduction. The fact that the ciD mutation abolishes both activities also calls for a reassessment of genetic epistasis experiments in which this allele is used to assay the relationship between Hedgehog and Wingless signaling (Brunner, 1997)

The Hedgehog (Hh) and Wingless (Wg) signaling pathways play important roles in animal development. The activities of the two pathways depend on each other during Drosophila embryogenesis. In the embryonic segment, Wg is required in anterior cells to sustain Hh secretion in adjacent posterior cells. In turn, Hh input is necessary for anterior cells to maintain wg expression. The Hh and Wg pathways are mediated by the transcription factors Cubitus interruptus (Ci) and Pangolin/TCF (Pan), respectively. Coincidentally, pan and ci are adjacent genes on the fourth chromosome in a head-to-head orientation. Genetic and in situ hybridization data indicate that ci^D is a mutation affecting both ci and pan. Whereas pan is expressed ubiquitously during embryogenesis, in ci^D mutant embryos pan is expressed in an intense, segmentally-repeated manner. This expression is intriguingly similar to that of the ci gene and it occurs both in homozygous and heterozygous mutant embryos. Moreover, it was found that in ci^D/ci^D homozyogous mutants, the typical striped expression of ci is absent; instead, ci transcripts are dispersed uniformly at low levels, like pan transcripts in wild type embryos. Molecular analysis reveals that the ci^D allele is caused by an inversion event that swaps the promoter regions and the first exons of the two genes. The ci gene in ci^D is controlled by the ubiquitous pan promoter and encodes a hybrid Ci protein that carries the N-terminal region of Pan. The predicted Ci fusion protein product consists of the first 246 amino acids of Pan fused in frame to the Ci protein, of which the first 13 N-terminal amino acids are missing. The N-terminal domain of Pan has previously been shown to bind to the beta-catenin homolog Armadillo (Arm), raising the possibility that Wg input, in addition to Hh input, modulates the activity of the hybrid CiD protein. Indeed, Wg signaling induces the expression of the Hh target gene patched (ptc) in ci^D animals. Evidence is provided that this effect depends on the ability of the CiD protein to bind Arm. Genetic and molecular data indicate that wild-type Pan and CiD compete for binding to Arm, leading to a compromised transduction of the Wg signal in heterozygous ci^D/+ animals and to a dramatic enhancement of the gain-of-function activity of ci^D in homozygous mutants. Thus, the Hh and the Wg pathways are affected by the ci^D mutation, and the CiD fusion protein integrates the activities of both (Schweizer, 1998).

It has been proposed that during embryogenesis ci^D functions as a gain-of-function allele of ci. This is largely based on the striking finding that CiD can substute for Hh protein in driving expression of Hh-responsive genes. Specifically, in homozygous ci^D mutant embryos, the ptc, wg and gooseberry genes are expressed in wider stripes, when compared to wild type, even in the absence of active Hh protein. However, homozygous ci^D mutant embryos can be rescued to adult by introducing a duplication of the pan/ci genomic region (unpublished). Thus it is unlikely that the observed phenotypes can be ascribed merely to the dosage of CiD protein. Rather, it appears to be critical that no wild-type Pan protein accompanies the CiD protein in ci^D homozygous embryos. This allows all Arm protein that accumulates in response to Wg to be unrestrained to bind to, and activate the CiD protein. It is predicted that the expression of an N-terminally complete Pan protein -- even if its DNA-binding activity has been impaired -- would abolish the extreme gain-of-function effect of CiD in homozygous mutant embryos. Thus it must be the simultaneous gain and loss of Arm binding sites by Ci and Pan, respectively, that confers these unusual properties to the ci^D allele. Together with the observation that CiD interferes in a dominant-negative manner with Wg signaling, these results illustrate that the levels of free and accessible Arm protein critically determine the output of Wg signaling in wild-type and the output of CiD signaling in mutant situations (Schweizer, 1998 and references).

The signaling mediated by Arm requires not only its cytoplasmic accumulation but also the activity of the DNA-binding protein Drosophila TCF known as Pangolin (Pan). Arm directly interacts with Pangolin through its central Armadillo repeats, a region that is critical in Arm's ability to induce cell death. Pangolin function in an APC-like mutant background cannot be completely eliminated; however, flies heterozygous for two mutant alleles of pangolin, pan^ciD and pan¹³, were examined to determine if Pangolin is required in the activation of cell death that results from Apc loss. pan^ciD is mutant for two loci, ci and pan; it behaves as both a hypomorphic allele of ci and a null allele of pan. In flies heterozygous for pan^ciD, some retinal neurons are rescued from cell death in the Apc mutant. As was seen with ectopic Zw3 expression, the rescued cells are detected solely at the apical surface of the eye. The same rescue is found in Apc mutants that are heterozygous for the null allele pan¹³. These findings suggest that elevated Arm levels activate a cell death pathway via the Arm/Pangolin complex (Ahmed, 1998).

The Drosophila cubitus interruptus (ci) gene encodes a sequence-specific DNA-binding protein that regulates transcription of Hedgehog (Hh) target genes. Activity of the Ci protein is posttranslationally regulated by Hh signaling. In animals homozygous for the ci^D mutation, however, transcription of Hh target genes is regulated by Wingless (Wg) signaling rather than by Hh signaling. ci^D is shown to encode a chimeric protein composed of the regulatory domain of dTCF/Pangolin (Pan) and the DNA binding domain of Ci. Pan is a Wg-regulated transcription factor that is activated by the binding of Armadillo (Arm) to its regulatory domain. Arm is thought to activate Pan by contributing a transactivation domain. A constitutively active form of Arm potentiates activity of a CiD transgene and coimmunoprecipitates with CiD protein. The Wg-responsive activity of CiD could be explained by recruitment of the Arm transactivation function to the promoters of Hh-target genes. It is suggestrd that wild-type Ci also recruits a protein with a transactivation domain as part of its normal mechanism of activation (Von Ohlen, 1999).

Epistasis analysis was carried out to position Adenomatous polypopsis coli tumor suppressor homolog 2 (Apc2) with respect to other components of the signal transduction pathway. wg; Apc2^DeltaS double mutant embryos (with Apc2^DeltaS mutant mothers) show a partial rescue of the wg phenotype, with restoration of the normal diversity of cuticular pattern elements and small expanses of naked cuticle, suggesting that Apc2 is downstream of wg. There are two possible explanations for the fact that the double mutant does not show the same phenotype as the Apc2 single mutant: either Apc2^DeltaS is not null, or the negative regulatory machinery remains partially active in the absence of Apc2. If Apc2^DeltaS is not null, it was reasoned that repeating the epistasis test with Apc2^DeltaS in trans to a deficiency removing Apc2 (Df(3R)crb87-4) might further reduce Apc2 function, producing a double mutant phenotype more similar to that of Apc2^DeltaS alone. However, when this was done, there was no change in the double mutant phenotype, suggesting that Apc2^DeltaS may be genetically null for this function. Other components of the Wg signal transduction pathway act downstream of Apc2. Embryos maternally and zygotically mutant for both dishevelled (dsh) and Apc2 show a phenotype indistinguishable from the dsh single mutant, as do embryos maternally mutant for both dsh and Apc2 that are zygotically dsh/Y; Apc2^DeltaS/Df(3R)crb87-4. Likewise, arm; Apc2 and Apc2; dTCF double mutants (derived from Apc2 homozygous mothers) are indistinguishable from arm or dTCF single mutants. Thus, dsh, arm, and dTCF all act genetically downstream of Apc2; this was expected for arm and dTCF, but was surprising for dsh (McCartney, 1999).

The murine Nemo homolog Nlk has been implicated in regulating Wnt signaling by repressing the Arm/ß-Catenin-TCF complex, a component of Wingless signaling. Whether such an interaction occurs in flies during wing vein formation was examined. The extra vein phenotypes observed in nmo mutant wings are very similar to those that have been previously described as resulting from overexpression of Armadillo and both vertebrate ß-catenin and plakoglobin in the wing. In addition, ectopic veins are produced as a result of ectopic wg and dsh expression. Constitutively active Armadillo (UAS-Arm^s10) expressed using the 1348-Gal4 driver leads to the formation of moderate ectopic veins emanating from the PCV, in addition to more severe ectopic veins along LII. These are both regions of the wing that are sensitive to nmo mutations and where similar ectopic veins are observed in nmo^adk. The phenotypes seen with overexpression of wg and arm are consistent with the theory that Nemo is a negative regulator of Wingless signaling since loss of nemo mimics extra veins seen with overexpression of arm and wg (Verheyen, 2001).

Overexpression of both wildtype mouse Lef-1 (a TCF homolog) and a constitutive repressor form of dTCF (Pangolin) results in dominant negative phenotypes. The constitutive repressor form of dTCF (UAS-dTCFDeltaN) is unable to bind Arm and represses wg-dependent gene expression. These findings suggest that expression of wildtype dTCF (UAS-dTCFwt) somehow interferes with a wingless-targeted transcription factor in a dominant negative way. Consistent with this, it is found that ectopic expression of UAS-dTCFwt using vestigial-Gal4 results in defects in the posterior wing margin, a phenotype seen with loss of wg signaling. Ectopic expression of the UAS-dTCFDeltaN using the 1348-Gal4 driver is lethal. However, homozygosity for nmo^adk is able to rescue the lethality and the flies that emerged had reduced ectopic wing veins. This finding can be interpreted by taking into account the dual roles TCF plays in the nucleus. In nmo^adk mutants, the negative regulation of endogenous dTCF may be reduced, leading to more wg-dependent signaling and the induction of extra veins similar to those seen with constitutive Arm expression. Expression of UAS-dTCFDeltaN in the nmo^adk background most likely interferes with the de-repressed endogenous dTCF and block the induction of extra veins seen in nmo^adk (Verheyen, 2001).

Newly eclosed flies have wings that are highly folded and compact. Within an hour, each wing has expanded, the dorsal and ventral cuticular surfaces bonding to one another to form the mature wing. To initiate a dissection of this process, two mutant phenotypes were examined. (1) The batone (bae) mutant blocks wing expansion, a behavior that is shown to have a mutant focus anterior to the wing in the embryonic fate map. (2) Ectopic expression of protein kinase A catalytic subunit (PKAc) using certain GAL4 enhancer detector strains mimics the batone wing phenotype and also induces melanotic 'tumors'. Surprisingly, these GAL4 strains express GAL4 in cells, which seem to be hemocytes, found between the dorsal and ventral surfaces of newly opened wings. Ectopic expression of Ricin A in these cells reduces their number and prevents bonding of the wing surfaces without preventing wing expansion. It is proposed that hemocytes are present in the wing to phagocytose apoptotic epithelial cells and to synthesize an extracellular matrix that bonds the two wing surfaces together. Hemocytes are known to form melanotic tumors either as part of an innate immune response or under other abnormal conditions, including evidently ectopic PKAc expression. Ectopic expression of PKAc in the presence of the batone mutant causes dominant lethality, suggesting a functional relationship. It is proposed that batone is required for the release of a hormone necessary for wing expansion and tissue remodeling by hemocytes in the wing (Kiger, 2001).

Comparison of the effects of Ricin A and of PKAc on wing maturation indicates that ectopic PKAc does not simply inactivate hemocytes. Instead, it appears to substitute one normal function of hemocytes for another. Rather than carry out phagocytosis and ECM synthesis, hemocytes enter into an innate immune response in which lamellocytes are differentiated and crystal cells melanize target cells. Evidently, aggregation of lamellocytes within the wing blade interferes with wing expansion, and loss of normal hemocyte function interferes with bonding of dorsal and ventral surfaces. The observation that the effect of ectopic PKAc on the wing is suppressed by overexpression of Pan, the Drosophila homolog of mammalian blood cell transcription factors (lymphocyte enhancer-binding factor 1 and T cell factor), suggests that ectopic PKAc inhibits (or represses synthesis of) Pan, which in turn inhibits Wingless target gene expression. This conclusion is strengthened by the observation that ectopic expression of UAS-dCBP(nej+) using GAL4-30A produces phenotypes similar to those caused by ectopic PKAc. Pan is bound and its transcriptional activity inhibited by dCBP. Expression of PanDeltaN, a dominant-negative inhibitor of Wingless target gene expression, elicits what seems to be a massive induction of the cellular innate immune response. Thus, the Wingless signal transduction pathway may be involved in regulating a choice between the innate immune response and the apoptotic/ECM response (Kiger, 2001).

The dominant-lethal interaction between ectopic PKAc and bae is intriguing. When and how death occurs needs closer examination, as does the cellular focus of bae activity. What role PKAc normally plays in regulating hemocyte behavior remains to be investigated. The association of a wing phenotype with altered hemocyte behavior should provide a means of identifying additional genes involved in hemocyte function during wing maturation (Kiger, 2001).

Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. Essential roles of Wingless in patterning the leg imaginal disc are described. (1) Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it has previously been shown that Wingless signal transduction is not active in the proximal leg domain in larvae. (2) Downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, evidence is provided that Dll restricts expression of a proximal leg-specific gene expression. It is proposed that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages (Kubota, 2003).

To confirm whether Wg signaling is required cell autonomously for leg disc development, the dominant-negative forms of Drosophila TCF (DTCFdeltaN) or Drosophila axin (Daxin) were expressed in the limb primordia. The Dll-Gal4 driver, which is turned on in the limb primordium at stage 11 and continues to be active in leg and wing discs, was used. In Dll-GAL4 embryos carrying UAS-DTCFdeltaN or UAS-Daxin, the overall size of leg discs was reduced. Expression of Esg was preferentially reduced in the dorsal side. The drastic reduction of Dll mRNA and protein in distal leg cells in the arm^H8.6 mutants as well as in DTCFdeltaN- and Daxin-expressing embryos demonstrate that Wg signaling is required for both proximal and distal leg cells. In arm mutants, Hth-expressing cells expand to the distal domain. This observation suggests that, upon loss of Wg signaling, prospective leg disc cells lose their identity and adopt the fate of trunk ectoderm. However, disc-specific reduction of Wg signaling does not affect wing disc formation, although the Dll-Gal4 driver is active in the wing primordium. It is concluded that late function of Wg signaling promotes formation of the leg disc with a higher requirement in the proximal domain, but is dispensable for wing disc formation (Kubota, 2003).

C-terminal-binding protein directly activates and represses Wnt transcriptional targets in Drosophila

Regulation of Wnt transcriptional targets is thought to occur by a transcriptional switch. In the absence of Wnt signaling, sequence-specific DNA-binding proteins of the TCF family repress Wnt target genes. Upon Wnt stimulation, stabilized β-catenin binds to TCFs, converting them into transcriptional activators. C-terminal-binding protein (CtBP) is a transcriptional corepressor that has been reported to inhibit Wnt signaling by binding to TCFs or by preventing -catenin from binding to TCF. This study shows that CtBP is also required for the activation of some Wnt targets in Drosophila. CtBP is recruited to Wnt-regulated enhancers in a Wnt-dependent manner, where it augments Armadillo (the fly β-catenin) transcriptional activation. CtBP is required for repression of a subset of Wnt targets in the absence of Wnt stimulation, but in a manner distinct from previously reported mechanisms. CtBP binds to Wnt-regulated enhancers in a TCF-independent manner and represses target genes in parallel with TCF. The data indicate dual roles for CtBP as a gene-specific activator and repressor of Wnt target gene transcription (Fang, 2006).

CtBP has previously been identified as a repressor of Wnt signaling, as measured by TCF reporter genes in cultured cells. Consistent with this, CtBP was identified in an overexpression screen via its ability to suppress Wg and Arm action in the developing eye. In wing imaginal discs, CtBP overexpression also inhibited the Wg target Senseless (Sens). Consistent with this overexpression data, the reduction of CtBP in cultured cells via RNAi is also consistent with a role for CtBP in repressing some Wnt targets (Fang, 2006).

The working model for CtBP repression of Wnt target gene expression holds that CtBP binds to the same area of the nkd and CG6234 loci as TCF, but this binding is TCF-independent. Consistent with this, knock down of CtBP and TCF or gro synergistically derepresses nkd expression. No synergism was seen with TCF/gro double depletions. The RNAi and ChIP data together favor a model where CtBP acts in parallel with TCF/Gro to repress nkd expression in the absence of Wg stimulation. Because CtBP has no detectable ability to bind nucleic acids, it is assumed that unknown DNA-binding protein(s) recruit CtBP to the WRE (Fang, 2006).

The existing models for CtBP antagonism of Wnt signaling cannot explain the data. TCF-independent recruitment of CtBP to WREs is not consistent with work suggesting direct binding of CtBP to TCF. The alternative mechanism, where a CtBP/APC complex diverts Arm/β-catenin away from TCF, also is inconsistent with the results. In this model, the activation of nkd expression after CtBP RNAi treatment would be dependent on TCF and arm. Because the derepression of nkd occurred when both CtBP and TCF were depleted and was not affected when arm was also inhibited, this model is not favored to explain the effects of CtBP depletion on nkd expression. These distinct mechanisms for CtBP repression are not mutually exclusive and may all occur in some contexts (Fang, 2006).

There is a qualitative difference in the amount of derepression found between the two Wg targets studied in Kc cells. Depletion of CtBP and TCF/gro causes a large (20- to 30-fold) increase in nkd basal expression, but has a much more modest (<3-fold) effect on CG6234. These differences may reflect a fundamental difference in the way TCF/Gro and CtBP act on various Wnt targets in unstimulated cells, but it is equally likely that the surrounding cis-elements in these targets have a strong influence on the degree of derepression that can be observed (Fang, 2006).

In addition to defining a novel mechanism for CtBP repression of Wg targets, strong evidence is provided for CtBP playing a role in Wg-mediated transcriptional activation. In the wing imaginal discs, loss of CtBP resulted in a lag in Wg-dependent activation of Sens and a reduction in Dll expression. In cultured Kc cells, CtBP depletion caused a two- to three-fold reduction in the ability of Wg to activate CG6234 expression. The ability of Gal4-Arm chimeras to activate a Gal4 reporter gene is also highly dependent on CtBP levels. In all these contexts, CtBP is not absolutely required for Wg signaling, but is necessary for maximal activation of Wg/Arm transcriptional activation (Fang, 2006).

The positive effect of CtBP on Wg signaling is direct, as judged by ChIP. Assuming that ChIP is measuring the degree of occupancy of CtBP on the chromatin, and not simply antigen accessibility, Wg stimulation promotes the association of CtBP with the CG6234 WRE. This increase in CtBP binding is not observed in TCF-depleted cells. Gal4-Arm recruits endogenous CtBP to a UASluc reporter. Taken together, these data support a model where TCF/Arm recruits CtBP to Wg targets. No binding between Arm and CtBP has been detected by co-immunoprecipitation, suggesting that another factor(s) may act as an adaptor between CtBP and the Arm bound to TCF (Fang, 2006).

Arm has transcriptional activation activity in both the N- and C-terminal portions of the protein. CtBP overexpression or RNAi depletion greatly effects the activity of the N-terminal half of Arm but has no effect on the C-terminal portion. Consistent with this, the N-terminal portion can recruit CtBP to a reporter gene, but not the C-terminus. Other factors that have been linked to the N-terminal portion of Arm include Lgs and Pygo and the ATPases Pontin and Reptin. It may be that CtBP acts in concert with one or more of these factors (Fang, 2006).

CtBPs have strong sequence similarity with D2-hydroxyacid dehydrogenases. hCtBP1 is a functional dehydrogenase and point mutations blocking CtBP1 dehydrogenase activity inhibit its ability to interact with binding partners and act as a transcriptional corepressor. However, another group found that similar mutations had no effect on the ability of CtBP to repress transcription. In this report, mutation of two residues (D290A and H312T) predicted to be essential for catalytic activity had no effect on the ability of fly CtBP to potentiate Gal4-Arm transcriptional activation. Further complicating the issue is data from experiments expressing the fly CtBP fused to Gal4DBD in mammalian cells. In some cells, Gal4-CtBP activated a UAS reporter, while the same reporter was repressed in other cell lines. Interestingly, conversion of CtBP's catalytic histidine to glutamine abolished transcriptional activation, but not repression. The heterologous nature of these experiments and the differences in the assays employed may explain the discrepancy between these studies, and further experiments will be needed on endogenous targets to determine how much dehydrogenase activity of CtBP contributes to repression and activation of Wnt targets (Fang, 2006).

Although CtBP is required for maximal activation of CG6234 expression and a Gal4-Arm-dependent reporter gene, Wg activation of nkd does not appear to require CtBP. The basis for this gene-specific requirement for CtBP is not clear. CtBP is recruited to the nkd WRE in a Wg-dependent manner, similar to what was observed for CG6234. It may be that CtBP is required for nkd activation, but this is masked by its role in repressing nkd expression. This hypothesis could be tested if were possible to separate CtBP's activator and repressor activities (Fang, 2006).

The requirement for CtBP in Wnt transcriptional activation may have been previously overlooked due to its well-characterized role as a co-repressor. For example, mouse embryos that lack CtBP2 have axial truncations and reduced Brachyury (T) expression that is reminiscent of Wnt3a mutants. These results suggest that the activating role for CtBP in Wnt signaling that was identified is evolutionarily conserved (Fang, 2006).

SoxNeuro acts with Tcf to control Wg/Wnt signaling activity

Wnt signaling specifies cell fates in many tissues during vertebrate and invertebrate embryogenesis. To understand better how Wnt signaling is regulated during development, genetic screens were performed to isolate mutations that suppress or enhance mutations in the fly Wnt homolog, wingless (wg). This study finds that loss-of-function mutations in the neural determinant SoxNeuro (also known as Sox-neuro, SoxN) partially suppress wg mutant pattern defects. SoxN encodes a HMG-box-containing protein related to the vertebrate Sox1, Sox2 and Sox3 proteins, which have been implicated in patterning events in the early mouse embryo. In Drosophila, SoxN has been shown to specify neural progenitors in the embryonic central nervous system. This study shows that SoxN negatively regulates Wg pathway activity in the embryonic epidermis. Loss of SoxN function hyperactivates the Wg pathway, whereas its overexpression represses pathway activity. Epistasis analysis with other components of the Wg pathway places SoxN at the level of the transcription factor Pan (also known as Lef, Tcf) in regulating target gene expression. In human cell culture assays, SoxN represses Tcf-responsive reporter expression, indicating that the fly gene product can interact with mammalian Wnt pathway components. In both flies and in human cells, SoxN repression is potentiated by adding ectopic Tcf, suggesting that SoxN interacts with the repressor form of Tcf to influence Wg/Wnt target gene transcription (Chao, 2007).

SoxN downregulates the Wg/Wnt pathway to reduce target gene expression. Downregulation is a crucial process because it sensitizes the signal response to allow rapid on/off switching and also keeps the system off in cells that are not actively responding to signal. Many genes have been shown to negatively regulate Wg/Wnt pathway activity through the destabilization of Arm/beta-catenin. Far fewer are known to exert negative regulatory effects downstream of Arm. The vertebrate Sox proteins -- Sox9, XSox3, XSox17α and XSox17ß -- as well as Chibby, a conserved nuclear factor, antagonize Wg/Wnt signaling by binding to Arm/beta-catenin and preventing it from partnering with Tcf to activate target gene expression. SoxN, however, does not bind beta-catenin in cell-culture assays, and does not share strong homology with the C-terminal sequences through which vertebrate Sox proteins bind this protein. Furthermore, SoxN function is not influenced by Arm levels. No difference was observed in SoxN-mediated TOPflash repression when cells were induced by co-transfection with a constitutively stabilized beta-catenin versus with Wnt-induced medium. Instead, both TOPflash and genetic experiments indicate that SoxN function depends on Tcf and Gro, its co-repressor (Chao, 2007).

One way to explain these observations is that SoxN contributes to the assembly or stability of the Tcf repressor complex on DNA. The consensus-sequence recognition for HMG domains in the Sox and Tcf families is reported to be similar, although XSox3 and XSox17ß fail to bind a consensus Tcf DNA sequence. It is shown that SoxN does not compete for Tcf-binding sites as a means of repressing target gene transcription, but the data support a model in which SoxN might bind DNA elsewhere or might bind Tcf sites transiently to initiate or stabilize the assembly of a repressor complex (Chao, 2007).

A similar model may explain the results from Xenopus that showed that XSox3-mediated repression does not require interaction between XSox3 and beta-catenin. XSox3 strongly interferes with dorsal fate specification in Xenopus embryos and represses TOPflash-reporter activity in vitro. HMG-domain mutations render XSox3 inactive in embryos without affecting its interaction with beta-catenin or its repression in TOPflash assays. Thus, it is the DNA-binding domain, not the beta-catenin-interacting C-terminus, that is relevant to its in vivo function in dorsal determination in Xenopus. XSox3 represses the expression of the dorsal-specific Nodal-related gene Xnr5 through optimal core binding sequences adjacent to and partially overlapping with Tcf sites in the Xnr5 promoter (Zhang, 2003). By contrast, the fly SoxN shows no discrepancy between its behavior in TOPflash assays and its in vivo effects. This suggests that the synthetic Tcf-binding sites arranged in the TOPflash-reporter plasmid are sufficient to support SoxN repressor function (Chao, 2007).

Because adding Tcf-site competitor DNA does not diminish the repressive capacity of limiting amounts of SoxN, the role of SoxN in repression does not appear to be stoichiometric. Therefore, the idea is favored that Sox proteins may act in a catalytic fashion during repressor-complex assembly at Wnt target gene promoters, rather than forming a structural part of the repressor complex itself. It was not possible to detect direct binding of SoxN with either Tcf, Gro or Arm, raising the possibility that SoxN interacts with some as yet unidentified protein that chaperones assembly of the repressor complex. A SoxN-binding cofactor, SNCF, has been identified in Drosophila (Bonneaud, 2003), but this gene is expressed only in pre-gastrulation embryos. Because Wg signaling occurs exclusively post-gastrulation, and specification of naked cuticle begins more than 4 hours after gastrulation, it is not thought that SNCF is a likely candidate for mediating this aspect of SoxN function. Rather, it is likely to play a role in the neuronal specification events promoted by SoxN at earlier stages of embryogenesis (Chao, 2007).

It is curious that uniformly overexpressed SoxN represses Wg signal transduction in dorsal epidermal cells more severely than in ventral cells. This effect is evident in both cuticle pattern elements and in en expression, and is reminiscent of defects observed in the 'transport-defective' class of wg mutant alleles, which includes wg^NE2. These mutations restrict Wg-ligand movement ventrally to promote only local signaling response while simultaneously abolishing all dorsal signaling, suggesting a fundamental difference in ventral and dorsal cell response. Perhaps it is not a coincidence that the NC14 mutation was isolated in the wg^NE2 mutant background. Further analysis of SoxN function may help to determine the molecular basis for dorsoventral differences in Wg signal transduction (Chao, 2007).

The role of Pygopus in the differentiation of intracardiac valves in Drosophila

Cardiac valves serve an important function; they support unidirectional blood flow and prevent blood regurgitation. Wnt signaling plays an important role in the formation of mouse cardiac valves and cardiac valve proliferation in Zebrafish, but identification of the specific signaling components involved has not been addressed systematically. Of the components involved in Wnt signal transduction, pygopus (pygo), first identified as a core component of Wnt signaling in Drosophila, has not been investigated with respect to valve development and differentiation. This study took advantage of the Drosophila heart model to study the role of pygo in formation of valves between the cardiac chambers. Cardiac-specific pygo knockdown in the Drosophila heart was found to cause dilation in the region of these cardiac valves, and their characteristic dense mesh of myofibrils does not form and resembles that of neighboring cardiomyocytes. In contrast, heart-specific knockdown of the transcription factors, arm/beta-Cat, lgs/BCL9, or pan/TCF, which mediate canonical Wnt signal transduction, shows a much weaker valve differentiation defect. Double-heterozygous combinations of mutants for pygo and the Wnt-signaling components have no additional effect on heart function compared with pygo heterozygotes alone. These results are consistent with the idea that pygo functions independently of canonical Wnt signaling in the differentiation of the adult interchamber cardiac valves (Tang, 2014).

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date revised: 15 August 2020 

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