A test was performed to see whether nkd is regulated by Wg activity using gain- and loss-of-function experiments. In wg mutant embryos, nkd transcription initiates normally but is markedly reduced by stage 11. nkd transcript accumulates to higher levels and more broadly across the segment in nkd mutant embryos, presumably owing to the lack of negative feedback that Nkd protein normally provides to its own Wg-dependent expression. nkd expression is enhanced when Wg is ubiquitously expressed. Misexpression of either Wg or an activated form of the wg-signal transducer Armadillo (UAS-Arm S10) in wing, leg, haltere and antennal imaginal discs results in similar patterns of ectopic nkd transcription. ArmS10-induced nkd transcript obeys sharp boundaries consistent with a cell-autonomous induction of nkd by Wg (Zeng, 2000).
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).
Wnts and bone morphogenetic proteins (BMPs) are signaling elements that are crucial for a variety of events in animal development. In Drosophila, Wingless (Wg, a Wnt ligand) and Decapentaplegic (Dpp, a BMP homolog) are thought to function through distinct signal transduction pathways and independently direct the patterning of the wing. However, recent studies suggest that Mothers against Dpp (Mad), the key transducer of Dpp signaling, might serve as a node for the crosstalk between these two pathways, and both positive and negative roles of Mad in Wg signaling have been suggested. This study describes a novel molecular mechanism by which Dpp signaling suppresses Wg outputs. Brinker (Brk), a transcriptional repressor that is downregulated by Dpp, directly represses naked cuticle (nkd), which encodes a feedback inhibitor of Wg signaling, in vitro and in vivo. Through genetic studies, this study demonstrates that Brk is required for Wg target gene expression in fly wing imaginal discs and that loss or gain of brk during wing development mimics loss or gain of Wg signaling, respectively. Finally, it was shown that Dpp positively regulates the expression of nkd and negatively regulates the Wg target gene Distal-less (Dll). These data support a model in which different signaling pathways interact via a negative-feedback mechanism. Such a mechanism might explain how organs coordinate inputs from multiple signaling cues (Yang, 2013).
This study has shown that Brk directly represses nkd expression. The direct repression of nkd by Brk is underscored by three observations. First, a Brk site was identified in the intronic region of nkd, which Brk physically occupies in vitro. Second, ChIP analysis shows that Brk binds a DNA region near this Brk site in embryos in a manner inversely related to Wg activity. Third, reporter analysis in Kc cells indicates that Brk represses Arm-dependent activation of an intronic WRE containing this Brk site, but only when the Brk site is intact. In addition, genetic analyses has shown that the repression of nkd by Brk is functionally significant. In the developing wing, it was found that the loss of brk de-represses nkd and downregulates Wg target proteins, such as Dll and Sens. Conversely, ectopic brk inhibits nkd expression and markedly enhances Dll expressio. Furthermore, removal of nkd prevents the loss of Dll in brk clones whereas co-expression of nkd abolishes the expanded Dll caused by ectopic brk. In adult wing, the loss and gain of brk phenotypically resembles the loss and gain of Wg signaling, respectivel. Consistent with a repressive role of Dpp cascade on brk, it was found that ectopic Dpp signaling enhances nkd and inhibits Wg signaling). These results support a model in which Dpp signaling increases the expression of Nkd, a Wg inhibitor, by the downregulation of Brk, and thereby inhibits the Wg outputs. In another words, nkd might fall into a class of Dpp targets, which are de-repressed upon the activation of Dpp signaling. This study has thus uncovered a previously unsuspected molecular mechanism underlying the interaction between Wg and Dpp signaling pathways in Drosophila wing development (Yang, 2013).
Until recently, little has been known about the cross-interaction between Wg and Dpp signaling in Drosophila wings, in spite of the fact that the fly wing has served as an excellent model system for the dissection of the molecular basis of these signaling transduction pathways. This is in contrast to Drosophila leg imaginal discs, in which mutual repression between Wg and Dpp signaling has long been suspected. However, several studies have indicated that manipulation of Dpp signaling levels in the wing sometimes leads to phenotypes resembling those caused by loss or gain of Wg activity. Notably, ectopic Dpp signaling increases notches in the wing, which is characteristic of reduced Wg signaling. However, the underlying mechanism for this effect of Dpp is not clear. Recently, independent research groups have suggested that Mad, the key effector of Dpp signaling, might play a role in the regulation of Wg target gene expression in fly wings. The molecular basis for their findings has mainly been the physical interaction between Mad and TCF, similar to the findings in mammals, in which several Smad proteins interact with members of the lymphoid enhancer binding factor 1/TCF family of DNA-binding HMG box transcription factor. It remains to be determined whether the role of Mad is direct or indirect because the reporter assays in these studies were performed with TOPFlash or similar constructs in mammalian cell culture, which might not always accurately represent the complicated situation of the in vivo regulation of Wg target genes. Furthermore, manipulation of Mad expression in wing discs influences Dll expression in different directions. Although these intriguing discrepancies can be explained by the physical interaction between Mad and TCF, the current model offers an alternative interpretation based on the negative regulation of nkd by Brk, which might suggest an indirect role of Mad in Wg signaling. For example, the current model could provide an explanation for the previous finding that ectopic Dpp signaling, caused by Mad, Medea, TkvQD, etc., results in notched wings (Yang, 2013).
The role of Brk in Wg signaling has been previously documented in Drosophila. It has been suggested that brk is able to antagonize Wg signaling based on the activity of a midgut-specific Ubx reporter gene in which physical interactions among Brk, Teashirt and CtBP have been described. In leg discs, Wg signaling may directly repress Dpp morphogen expression via an Arm-TCF-Brk complex, offering a direct model for the cross-talk between Wg and Dpp. However, the current studies have indicated a positive role for Brk in Wg signaling through an indirect action. In addition to the repression of Dpp targets, the roles of Brk in Wg signaling described in these different models exemplify the pleiotropic actions of brk throughout development and might provide the molecular basis for tissue-specific consequences of developmental signaling pathways (Yang, 2013).
nkd was first identified as a Drosophila segment-polarity gene, mutation of which gives rise to major deficits in fly embryonic development. Its expression appears to be universally induced by Wg in fly embryos and larval imaginal discs. It is interesting that although the loss of nkd in embryos has an effect similar to gain of wg, decreased nkd function in fly wings shows little impact. However, none of the nkd alleles used in these studies has been well characterized at the molecular level. Given the complexity of nkd transcriptional regulation, it could be that these mutant forms of nkd still possess residual function in the wing. Alternately, overexpression of nkd blocks ectopic Wg signaling in the eyes and generates PCP phenotypes in the wing through a direct interaction with Dsh. Consistent with these observations, this study found that loss of brk can cause a dramatic increase of nkd expression in certain areas of the wing imaginal disc, leading to wing notches and PCP defects. The current findings suggest that nkd may indeed play roles, at a certain level, in both canonical and noncanonical Wg signaling in fly wings. However, a closer examination of nkd function in fly wings is needed (Yang, 2013).
In conclusion, this study found that Brk influences Wg signaling by directly repressing nkd expression and could serve as a node for cross-talk between the Wg and Dpp signaling pathways. Wnt-BMP cross-interactions have been implicated in many developmental and disease processes). For example, a Wnt-BMP feedback circuit mechanism is important for inter-tissue signaling dynamics in tooth organogenesis in mouse. The findings may therefore add new insights into cell differentiation and human cancer (Yang, 2013).
Wnt/ß-catenin signals orchestrate cell fate and behavior throughout the animal kingdom. Aberrant Wnt signaling impacts nearly the entire spectrum of human disease, including birth defects, cancer, and osteoporosis. If Wnt signaling is to be effectively manipulated for therapeutic advantage, how Wnt signals are normally controlled must first be understood. Naked cuticle (Nkd) is a novel and evolutionarily conserved inducible antagonist of Wnt/ß-catenin signaling that is crucial for segmentation in Drosophila. Nkd can bind and inhibit the Wnt signal transducer Dishevelled (Dsh), but the mechanism by which Nkd limits Wnt signaling in the fly embryo is not understood. This study shows that nkd mutants exhibit elevated levels of the ß-catenin homolog Armadillo but no alteration in Dsh abundance or distribution. In the fly embryo, Nkd and Dsh are predominantly cytoplasmic, although a recent report suggests that vertebrate Dsh requires nuclear localization for activity in gain-of-function assays. While Dsh-binding regions of Nkd contribute to its activity, a conserved 30-amino-acid motif, separable from Dsh-binding regions, was identified that is essential for Nkd function and nuclear localization. Replacement of the 30-aa motif with a conventional nuclear localization sequence rescued a small fraction of nkd mutant animals to adulthood. This studies suggest that Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal (Waldrop, 2006; full text of article).
This study reports a structure–function analysis of Drosophila Nkd. The finding that nkd mutants have elevated Arm/ß-catenin levels concomitant with broadened domains of Wg target gene expression is consistent with prior reports of Nkd targeting Dsh, an enigmatic Wnt signal transducer that acts upstream of ß-catenin degradation. Although Wnt-signal-induced Dsh accumulation has been observed in cultured cells, transgenic mice, and some cancers, and recent studies indicate that Dsh, like ß-catenin, can be degraded by the ubiquitin–proteasome pathway, the current data show that Nkd does not attenuate Wnt signaling in the embryo by significantly altering steady-state Dsh levels or distribution. If Nkd promotes Dsh degradation in the fly embryo, as has recently been proposed on the basis of overexpression of mammalian Nkd in cultured cells, it must act only on a subset of Dsh, perhaps the fraction engaged in signaling. Consistent with this idea, rare, punctate Nkd/Dsh colocalization was observed in embryonic ectodermal cells (Waldrop, 2006).
Several Nkd constructs with mutant or deleted Dsh-binding regions possessed a reduced but still substantial nkd rescue activity. Perhaps NkdΔR1S/GFPC, lacking both Dsh-binding regions, is able to target Dsh in vivo (and hence rescue a nkd mutant) by virtue of overexpression, through other low-affinity Nkd/Dsh-binding regions, or by as yet uncharacterized proteins that bridge Nkd to Dsh. Consistent with these possibilities, some NkdΔR1S/GFPC/Dsh colocalization was also observed (Waldrop, 2006).
Three independent lines of investigation—evolutionary sequence comparisons, sequencing of lethal nkd alleles, and transgenic nkd rescue assays—pinpointed a 30-aa motif, separable from Dsh-binding regions, that is crucial for fly Nkd activity and nuclear localization. The comparable positions, identical sequence length, and similar predicted structure of insect and mammalian 30-aa motifs suggests that the family of Nkd proteins may inhibit Wnt signaling through a common mechanism. Given the small size and presumably simple α-helical structure of the 30-aa motif, it is unlikely to possess intrinsic catalytic activity but, in addition to its weak NLS activity, it could serve as a protein-docking motif (Waldrop, 2006).
In addition to several reports that have documented nucleo-cytoplasmic shuttling of ß-catenin, Axin, and APC, it is noteworthy that two recent reports revealed a potential role for Fz and Dsh in the nucleus. In response to Wg signaling at the fly neuromuscular synapse, the Fz2 C terminus was detected in puncta of postsynaptic muscle nuclei although not in ectodermal nuclei, so this report's significance to Nkd's action in ectoderm is unclear. Xenopus Dsh has a separable NLS and nuclear export sequence (NES), with the former required for 'signaling activity' in gain-of-function assays. However, a vertebrate Dsh construct with a mutant NES exhibited increased nuclear accumulation but no activity increase relative to that of wild-type Dsh, arguing against nuclear Dsh concentration—at least when it is overexpressed—being rate limiting for activity. Intriguingly, Dsh NES and NLS motifs seem to be conserved in D. melanogaster Dsh, but their significance remains to be investigated (Waldrop, 2006).
These data extend the still rudimentary knowledge of Nkd action in the fly embryo. The epistatic relationship between wg and nkd suggests that, in the absence of Wg ligand, the low levels of Nkd in a wg mutant (because Wg normally upregulates nkd transcription) inhibit spontaneous ligand-independent signaling through the Wnt receptor complex. Wg exposure promotes Arm accumulation and induction of target genes, including en, hh, and nkd. Nkd, synthesized in the cytoplasm, accumulates and targets an uncharacterized fraction of cytoplasmic Dsh. However, Nkd/Dsh binding alone is apparently insufficient to limit Wg signaling during stages 10–11, since Nkd uses its 30-aa motif to inhibit Arm accumulation, restrict Wg-dependent gene expression, and access the nucleus. Although it is possible that the 30-aa motif is required in the cytoplasm, and that the ability of the 30-aa motif to confer nuclear access is a consequence rather than a cause of activity, three lines of evidence support a nuclear role for Nkd: (1) a subpool of Nkd normally accumulates in the embryonic nuclei after stage 10; (2) the 30-aa motif, distinct from the Dsh-binding sequence, is necessary for both nuclear localization and activity and is sufficient to increase the activity of mouse Nkd1 when expressed in the fly; and (3) a heterologous NLS increased nuclear localization and nkd rescue activity of NkdΔ30aa (Waldrop, 2006).
While these experiments strongly suggest a role for Nkd in the nucleus, they do not reveal the nature of that role. Likewise, lacking insight into how Dsh transmits Wnt signals into the nucleus, the experiments thus far reveal neither the relevant subcellular location(s) of Nkd action nor a molecular mechanism by which Nkd inhibits Dsh activity. The punctate Nkd/Dsh colocalization that was observed in embryonic cytoplasm, and rarely, in nuclei, is consistent with Nkd either affecting Dsh nucleo-cytoplasmic transport or impinging directly on the chromatin of Wnt-responsive genes. The inability to observe increased nuclear Nkd or Dsh after treatment with a nuclear export inhibitor suggests that nuclear export of Nkd (and possibly Dsh) in the fly embryo (1) does not occur (e.g., if each protein were degraded in the nucleus following import); (2) occurs over a longer time period relative to proteins such as Lines that can rapidly shuttle between nucleus and cytoplasm; (3) is independent of CRM-1; or (4) like the presumed Nkd/Dsh interaction, involves only a fraction of the total pool of each protein. Future experiments will be required to distinguish among these possibilities (Waldrop, 2006).
The four-domain structure of both insect and vertebrate Nkd's argues that there once existed an ancient 'core' mechanism by which Nkd engaged Dsh to inhibit Wnt signaling. However, given the sequence divergence between insect and mammalian Nkds, their current mechanisms may share little similarity beyond Dsh binding. Recently, PR72 and PR130, two alternatively spliced B'' subunits of the multi-subunit enzyme protein phosphatase 2A (PP2A), were shown to associate with mammalian Nkd and to modulate its inhibitory effect on ectopic Wnt signaling. Since Dsh is phosphorylated by kinases such as CK1, CK2, and Par1 following Wnt stimulation, recruitment of phosphatases to Dsh by Nkd represents an attractive hypothesis to explain the inhibitory action of Nkd on Wnt signaling via Dsh. Consistent with this possibility, Nkd, PP2A, and Dsh kinases co-immunoprecipitated with vertebrate Dsh. However, unlike the vertebrate Nkd/PR72 interaction, thus no direct interactions by Y2H were detected between the fly PR72 homolog (CG4733) and full-length fly Nkd or any of the regions in Nkd, in particular the 30-aa motif, that are crucial for activity. Thus, regulation of Nkd activity by PR72/PR130 may be a derived, vertebrate-specific phenomenon—analogous in some ways to the effect of mammalian Nkd2 but not Nkd1 on intracellular TGF-α trafficking that may be distinct from the mechanism by which Nkd regulates Wg signaling in Drosophila (Waldrop, 2006).
In Drosophila, nkd is crucial for shaping gradients of Wnt activity, but is this role conserved in vertebrates? Mouse nkd genes are expressed during embryogenesis in dynamic patterns reminiscent of known Wnt gradients. A recent report described nkd1 mutant mice with a targeted deletion of exons 6 and 7 (encoding the EFX domain) but allowing in-frame splicing between exons 5 and 8, resulting in expression of a residual Nkd1 protein very much analogous to the NkdΔR1S/GFPC construct that lacks Dsh-binding sequences but retains three conserved motifs. Given that nkd1 is more broadly expressed than nkd2 during mouse development, it was surprising that nkd1–/– mice are viable and fertile, even though mutant mouse embryo fibroblasts show elevated Wnt reporter activity and homozygous male mice exhibit a sperm maturation defect. Although genetic redundancy between nkd1 and nkd2 could account for these observations, the results suggest an alternative hypothesis, namely that the residual protein produced in the reported nkd1 mutant mice, like EFX-deleted NkdΔEFX/GFPC and NkdΔR1S/GFPC constructs, has significant activity in vivo, despite the observation that a mutant mNkd1 protein lacking the EF hand is defective at blocking Wnt signaling in cultured cells. Resolution of this quandary awaits an investigation of strong loss-of-function mutations in each mammalian nkd gene. Given the broad involvement of Wnt/ß-catenin signaling in mammalian development and cancer, coupled with the similar loss-of-function phenotypes of fly nkd, axin, and apc homologs, it is hoped that these studies guide future investigations of vertebrate Nkd proteins as regulators of Wnt signaling and candidate tumor suppressor genes (Waldrop, 2006).
The Wnt-signaling cascade is required for several crucial steps during early embryogenesis, and its activity is modulated by various agonists and antagonists to provide spatiotemporal-specific signaling. Naked cuticle is a Wnt antagonist that itself is induced by Wnt signaling to keep Wnt signaling in check. Little is known about the regulation of this antagonist. It has been shown that the protein phosphatase 2A regulatory subunit PR72 is required for the inhibitory effect of Naked cuticle on Wnt signaling. The present study shows that PR130, which has an N terminus that differs from that of PR72 but shares the same C terminus, also interacts with Naked cuticle but instead functions as an activator of the Wnt-signaling pathway, both in cell culture and during development. PR130 modulates Wnt signal transduction by restricting the ability of Naked cuticle to function as a Wnt inhibitor. These data establish PR130 as a modulator of the Wnt-signaling pathway and suggest a mechanism of Wnt signal regulation in which the inhibitory activity of Naked cuticle is determined by the relative level of expression of two transcripts of the same protein phosphatase 2A regulatory subunit (Creyghton, 2006).
Precise control of Wnt/β-catenin signaling is critical for animal development, stem cell renewal, and prevention of disease. In Drosophila, the naked cuticle (nkd) gene limits signaling by the Wnt ligand Wingless (Wg) during embryo segmentation. Nkd is an intracellular protein that is composed of separable membrane- and nuclear-localization sequences (NLS) as well as a conserved EF-hand motif that binds the Wnt receptor-associated scaffold protein Dishevelled (Dsh), but the mechanism by which Nkd inhibits Wnt signaling remains a mystery. This study identified a second NLS in Nkd that is required for full activity and that binds to the canonical nuclear import adaptor Importin-α3. The Nkd NLS is similar to the Importin-α3-binding NLS in the Drosophila heat-shock transcription factor (dHSF), and each Importin-α3-binding NLS required intact basic residues in similar positions for nuclear import and protein function. These results provide further support for the hypothesis that Nkd inhibits nuclear step(s) in Wnt/β-catenin signaling and broaden the understanding of signaling pathways that engage the nuclear import machinery (Chan, 2008).
A growing body of evidence indicates that the traditionally 'cytoplasmic' Wnt signal transducers Axin, Apc, and Dsh also act in the nucleus. In the cytoplasm, Apc promotes β-catenin degradation and regulates the cytoskeleton, but nuclear Apc can recruit transcriptional corepressors to Wnt target genes and, like Axin, escort β-catenin from the nucleus. In light of recent evidence that Axin/Dsh oligomers cross-link Wnt-bound receptors at the plasma membrane during signal activation, it remains unclear whether the nuclear roles for Axin or Dsh are similar to their cytoplasmic functions or whether they, like Apc, have novel nuclear functions (Chan, 2008).
Nkd is also a conserved Wnt signal regulator whose subcellular localization initially suggested a cytoplasmic site of action. These studies have shown that fly Nkd is composed of discrete motifs that confer membrane localization and binding to Dsh, as well as two NLSs. In addition to Nkd targeting an uncharacterized fraction of Dsh in the cytoplasm and/or at the plasma membrane, these data strongly support a nuclear role for Nkd, but whether Nkd inhibits Wnt signaling by altering nucleo-cytoplasmic transport of critical signaling components, such as Dsh or Arm, or by acting on the chromatin of Wnt target genes remains to be elucidated (Chan, 2008).
Genetic epistasis can be a powerful method to infer the regulatory logic of signal transduction cascades. Because Nkd is a 'side-regulator' whose loss-of-function phenotype is dependent on intact Wg signaling, double-mutants between nkd and dsh or arm did not help discern at which level Nkd inhibits the linear Wg signaling pathway. However, Nkd overexpression suppressed the gain-of-Wg signaling phenotype caused by overexpression of Dsh but not that caused by overexpression of an N-terminally deleted and hence degradation-resistant Arm/β-catenin; taken together with the observation that Nkd binds Dsh, epistasis experiments have led to the conclusion that Nkd acted at the level of Dsh and not 'downstream' of Arm/β-catenin in Wg signaling. However, in view of the present data, Nkd might also act in the nucleus at or above the level of Arm/β-catenin. Unfortunately, overproduction of wild type Arm is without phenotypic consequence, presumably because of an excess capacity of the β-catenin 'destruction complex' to degrade ectopic Arm, thus preventing the making further conclusions at present about the epistatic relationship between Nkd and endogenous, degradation-sensitive Arm/β-catenin (Chan, 2008).
Despite the deletion of Dsh-binding sequences in otherwise wild type Nkd having only a minor effect on cuticle rescue activity, the present experiments further support the hypothesis that the Nkd/Dsh interaction is important for Nkd to inhibit Wg signaling. However, the experiments thus far do not clarify how the interaction is regulated in vivo or whether it must occur in the cytoplasm, nucleus, or both locations. Nevertheless, several lines of evidence indicate that a Nkd/Dsh interaction in the cytoplasm and/or near the plasma membrane is important for Nkd function: First, both proteins are predominantly cytoplasmic and/or membrane-associated. Second, punctate cytoplasmic Nkd/Dsh colocalization can be observed in embryos and in salivary gland. Third, the Dsh-binding EFX motif fused to GFP was predominantly cytoplasmic. Fourth, deletion of Dsh-binding sequences in Nkd promoted nuclear localization, consistent with Dsh anchoring Nkd in the cytoplasm. Fifth, deletion of both Nkd NLSs eliminated nuclear localization whether or not Dsh-binding sequences were present, but Dsh-binding sequences were required for Nkd activity (Chan, 2008).
How Nkd acts on Dsh in the cytoplasm to inhibit Wg signaling is not known. One possibility is that Nkd sequesters Dsh away from Fz and/or Axin during signal activation, freeing Axin to regenerate β-catenin destruction complexes. Alternatively, Nkd might target 'activated' Dsh, possibly the pool of Dsh bound to the Wnt receptor complex, for degradation; consistent with Nkd targeting only a fraction of Dsh is the minimal colocalization of the two proteins in embryos as well as the lack of any obvious changes in Dsh levels in nkd mutants. Since Nkd can block the gain-of-Wg signaling phenotypes caused by overexpression of the Dsh kinase CK1, a third possibility is that Nkd blocks CK1-dependent phosphorylation of Dsh via a steric mechanism, although the relationship between Dsh phosphorylation status and activity remains unclear. Future experiments should clarify this issue, because each of these hypotheses makes distinct predictions about phosphorylation status and associated proteins in a native Nkd/Dsh complex (Chan, 2008).
These studies also provide several lines of evidence that the Nkd/Dsh interaction is not sufficient for Nkd to inhibit Wg signaling, and that binding in the nucleus might also be required to fully antagonize Wg signaling. First, the Dsh-binding regions of Nkd when overexpressed blocked phenotypes induced by Dsh overexpression but had no nkd rescue activity. Second, (fly) Nkd and (vertebrate) Dsh have NLSs, although it is not yet known whether fly Dsh acts in the nucleus. Third, rare punctate Nkd/Dsh nuclear colocalization can be observed by confocal microscopy in fly embryos. Fourth, the SV40-NLS increased NkdΔ30aa/GFPC activity when Dsh-binding sequences were intact but reduced activity when Dsh-binding sequences were deleted. The possibility cannot be ruled out that the activity of NkdΔ30aaNLS/GFPC, some of which remains outside the nucleus despite the strong heterologous NLS, is due to cytoplasmic Nkd/Dsh interactions. Similarly, NkdΔR1SΔ30aaNLS/GFPC, which was exclusively nuclear in embryos, might lack activity because of its inability to bind and be retained by Dsh in the cytoplasm. While one must be cautious when inferring site(s) of protein action from subcellular localizations, these studies collectively suggest that fly Nkd is required at multiple locations in Wg-receiving cells (Chan, 2008).
The Nkd-D6 motif has been subject to intense selection pressure, as it is identical in Nkd from D. pseudoobscura, a fly species that diverged from D. melanogaster approximately one billion generations ago. Similarly, the 30 aa NLS is part of a 58 aa motif, and the EFX is part of a 91 aa motif, that are also identical in the two Drosophila species. Using yeast two hybrid, Nkd-EFX residues have been identified that are either dispensable or critical for NkdEFX/DshbPDZ interactions, suggesting that interactions between the EFX motif and proteins other than Dsh might enforce strict motif conservation. Although each NLS contributes to Nkd activity and nuclear localization, heterologous NLSs did not fully replace the function of each Nkd NLS in rescue assays, and in both cases in this work, a heterologous NLS was deleterious to protein function. Absolute conservation of each of these motifs implies that both the tertiary structure and every square angstrom of each motif's surface are necessary for species survival. These experiments suggest that each Nkd motif is required for distinct thresholds and/or duration of Wg signal inhibition: the N-terminal and 30 aa motifs were required for reduction of Arm levels by stage 10, whereas the Dsh-binding EFX and Importin-α3-binding D6 motifs were dispensable to reduce Arm levels but were required, either directly or indirectly, to fully repress en and/or wg transcription by stage 11. Since the deletion of two highly conserved motifs (EFX and D6) preserved the mutant Nkd protein's ability to reduce Arm levels during stage 10, it seems unlikely that these motifs will be shown to possess an intrinsic catalytic activity. The hypothesis is therefore favored that Nkd acts as an inducible protein scaffold, with each of the conserved motifs able to bind additional protein(s). Perhaps there exist distinct Nkd-complexes depending on the subcellular compartment, state of signal activation, or time following signal initiation (Chan, 2008).
Alignment of the Importin-α3-binding NLSs in Nkd and dHSF revealed several conserved residues. Interestingly, the dHSF-NLS has been shown to be bifunctional, suppressing dHSF trimerization in the absence of heat-shock, and in response to heat or other stress conferring Importin-α3-dependent dHSF nuclear translocation and transcriptional induction of heat-responsive genes such as hsp70. These data suggest that the Nkd D6-NLS is also bifunctional, conferring Importin-α3-dependent nuclear localization as well as possibly binding nuclear protein(s) that repress Wg target gene transcription in some cells through stages 10-11. While non-import (presumably scaffolding) functions for Importin-αs have been inferred from phenotypes observed with importin-α deficiency in flies and worms, all of the current experiments support the hypothesis that the Nkd/Importin-α3 interaction promotes nuclear localization. The central region of Importin-α consists of 10 alpha-helical 'Arm' repeats (so named because they were first identified in the Drosophila Arm protein) stacked to form a banana-shaped molecule, the concave side of which harbors a groove that binds basic residues within NLSs. At present, it is not possible based on primary sequence to predict which Importin-α a given NLS will bind, although both the NLS and its three-dimensional context (i.e., adjacent sequence) have been demonstrated to contribute to NLS/Importin-α specificity. Future experiments will investigate whether the residues conserved between Nkd and dHSF represent a consensus Importin-α3-specific binding motif (Chan, 2008).
Vertebrate Nkds have a conserved 30 aa motif between the EFX and C-terminal histidine-rich regions, but whether the vertebrate proteins act in the nucleus like fly Nkd is not known. In this regard, no obvious difference was observed between the subcellular localizations of mouse Nkd1 fused to C-terminal GFP (mNkd1GFPC) vs. a similar construct that lacks the 30 aa motif (mNkd1Δ30aa/GFPC) when the proteins were produced in cultured mammalian cells. However, no obvious difference was observed between fly Nkd and NkdΔ30aa localizations in Drosophila S2 cells, but the differences in localization and function of these two constructs when produced in nkd mutant embryos were dramatic. These findings illustrate the importance of investigating the subcellular localizations of mutant proteins in a native environment that lacks the endogenous wild type protein. It might therefore be interesting to examine the subcellular localization of vertebrate Nkd constructs in nkd-mutant mice or zebra fish just as has been done in Drosophila. More importantly, future experiments must address the critical question of how Nkd antagonizes Wnt/β-catenin signaling in each of the subcellular compartments to which it localizes (Chan, 2008).
Affinity-purified anti-Nkd antisera made against either of two parts of the protein detect a segmentally repeated cytoplasmic distribution very similar to the embryonic RNA pattern. No staining is detected in nkd7H16 mutant embryos, and high-level ubiqitous expression is seen in heat-shocked P[Hs-nkd] embryos. Nkd antibody immunoprecipitates from embryonic protein extracts a protein that runs at a slightly higher relative molecular mass than its predicted. Consistent with the lack of a maternal requirement for nkd (DiNardo, personal communication to Zeng, 2000), the transcript is absent from maternally derived embryonic RNA from 0-2 h AEL (Zeng, 2000).
nkd transcription is initiated in embryos during the late cellular blastoderm stage in broad anterior and posterior domains, in a manner reminiscent of gap genes. During early germ-band extension (stage 8-9), nkd transcription is nearly ubiquitous, with higher RNA levels in the 2-3 cell rows posterior to the Hh/En stripe that require nkd to limit Hh/En production. At this stage, Wg protein is evenly distributed on both sides of the stripe of cells that express wg RNA. During full germ-band extension, nkd expression is most abundant anterior to, and lower just posterior to, the Hh/En stripe. nkd RNA is lower still in the Hh/En-producing cells. Hh signaling in the Hh/En cells excludes Wg protein during this time, resulting in asymmetric Wg distribution with an anterior bias. nkd mutants do not develop this anterior bias of Wg, indicating that nkd may be required for hh to exclude Wg from Hh/En cells. Nonetheless, after embryonic stage 10, Wg protein and nkd RNA are found together in many tissues. nkd RNA and wg RNA are expressed in overlapping patterns in imaginal discs and other larval tissues, with nkd domains being slightly broader than those of wg (Zeng, 2000).
nkd is an embryonic lethal recessive zygotic mutation that produces multiple segmentation defects, the most prominent of which is the replacement of denticles by excess naked cuticle. This phenotype is also seen in embryos exposed to excess Wg, as well as in embryos lacking both maternal and zygotic contributions from any of three genes that antagonize Wg: zeste-white3/glycogen synthase kinase 3beta (zw3/gsk3beta), D-axin and D-Apc2. In nkd embryos, hh and en transcripts initiate normally but accumulate in broad stripes, including cells further from the source of Wg, which suggests that these cells are hypersensitive to Wg. Next, a stripe of new wg transcription appears just posterior to the expanded Hh/En stripe. This extra wg stripe requires both wg and hh activity and is required for the excess naked cuticle seen in nkd mutants. The death of cells producing Hh/En contributes to the marked shortening of nkd mutant cuticles (Zeng, 2000).
nkd loss-of-function clones were induced in imaginal discs and adult structures using two strong nkd alleles, nkd7H16 and nkd7E89, and one moderately severe allele, nkd9G33, all of which are embryonic lethal. nkd alleles were originally generated in the genetic background of a weak allele of the pair-rule gene hairy (h1). h1 clones give rise to ectopic wing-vein bristles and thoracic microchaetes. Furthermore, nkd and h genetically interact: nkd, h1/hnull is lethal, whereas h1/h null is viable (A. Martinez-Arias, personal communication to Zeng, 2000). Therefore clones were generated of the strong allele nkd7E89 from which h1 had been removed. In many tissues where Wg signals control pattern, including the wing, thorax, abdomen, haltere and eye, phenotypically normal nkd clones are seen. h 1, nkd7H16, but not h +, nkd7E89 or h1, nkd9G33 clones give rise to a rough eye phenotype and loss of wing margin bristle phenotype that may be due to h-nkd interactions (Zeng, 2000).
To test whether nkd clones arise at biased locations, clones were scored in adult legs marked with the bristle marker yellow. nkd and control clones appeared with similar frequency in each leg quadrant. The expression of Wg target genes in nkd clones was examined to look for subtle changes in gene expression that might be compatible with normal tissue patterning. Distalless (Dll) is distributed in a broad gradient centered on the margin of the wing disc. Induction of nkd clones results in no apparent alteration in the Dll expression gradient within, or adjacent to, multiple clones. No changes in cytoplasmic Arm accumulation, an indicator of Wg activity, are noted within or adjacent to nkd clones (Zeng, 2000).
A screen was carried out to identify genes interacting with Armadillo, the Drosophila homolog of ß-catenin. Two viable fly stocks have been generated by altering the level of Armadillo available for signaling. Flies from one stock overexpress Armadillo (Armover) and, as a result, have increased vein material and bristles in the wings. Flies from the other stock have reduced cytoplasmic Armadillo following overexpression of the intracellular domain of DE-cadherin (Armunder). These flies display a wing-notching phenotype typical of wingless mutations. Both misexpression phenotypes can be dominantly modified by removing one copy of genes known to encode members of the wingless pathway. This paper identifies and describes further mutations that dominantly modify the Armadillo misexpression phenotypes. These mutations are in genes encoding three different functions: establishment and maintenance of adherens junctions; cell cycle control, and Egfr signaling (Greaves, 1999).
Mutations have been characterized in 17 genes (26 deficiencies) that interact with Armover and/or Armunder. Interaction strength varies from deficiency to point mutation, suggesting that several genes in the original deficiencies could have contributed to, or modified, the interaction. Only for 7 of the 17 genes have interactions been identical between the point mutation and the corresponding starting deficiency. The 17 genes were sorted into four groups. Group 1 consists of wingless pathway genes: Four known members of the pathway were identified: wg, dsh, zw3, and nkd. All interact in the direction expected (wg, dsh, and sgg/zw3 had already been tested prior to the screen). naked (nkd) has also been identified as a suppressor of Armunder and an enhancer of Armover; however, these interactions are much weaker than those seen for zw3M11 (Greaves, 1999).
Among the interactors identified was naked (nkd), a mutant that has long been associated with excess Wg activity. The embryonic phenotype of nkd mutants is characterized by an excess of naked cuticle, just like that of sgg/zw3 mutants or embryos overexpressing Wg. In the case of sgg/zw3, this phenotype clearly follows from overactivation of the pathway, irrespective of whether endogenous wg is present or not. In contrast, wg/nkd double mutants resemble the wg single mutant, suggesting that nkd is upstream of wg. More precisely, since nkd mutants have enlarged stripes of Engrailed [and concomitant Hh] expression, nkd has been proposed to be a negative regulator of Engrailed expression. Broader hh expression in nkd embryos (as a result of widened engrailed expression) is thought to induce ectopic stripes of wg expression; this would cause the naked cuticle phenotype. However, in wing imaginal discs, wg expression is not controlled by engrailed or hh and therefore the finding that nkd modifies the Armover and Armunder phenotypes in the wing implies a more widespread role for nkd in Wg signaling. Maybe the absence of nkd function renders cells more responsive to Wg. This would explain why endogenous Wg is required for the nkd phenotype to arise. It would also be consistent with the genetic interactions that are detected in the wing. Note that, so far, no function has been ascribed to nkd in disc development (Greaves, 1999).
Wg and Wnt molecules tightly associate with membrane and extracellular matrix and appear not to be readily soluble. Thus, it is unlikely that these proteins freely diffuse through extracellular spaces. Rather, Wg appears to be transported via active cellular processes. This phenomenon was first demonstrated using the shibirets (shits) mutation to block endocytosis. shi encodes the fly dynamin homolog, a GTPase required for clathrin-coated vesicle formation. Rather than the broad, punctate Wg protein distribution normally found over several cell diameters on either side of the wg-expressing cells, shi mutant embryos show high level accumulation of Wg around the wg-expressing cells. Structure/function analysis of the Wg molecule further supports the idea that active transport of the ligand is essential. Four mutations within wg have been isolated that specifically disrupt Wg transport without abolishing signaling activity. These mutant molecules generate a restricted response within the segment, as assayed by both cuticular pattern elements and molecular events. Homozygous mutant embryos produce naked cuticle but little denticle diversity, and show narrowed domains of Wg protein distribution and Arm stabilization. Three of these four mutations are single amino acid substitutions; each affects a residue that is highly conserved throughout the Wnt family, suggesting that ligand transport may be an important general aspect of Wnt function (Moline, 1999 and references).
To assess the functional consequences of this broad Wg distribution, a means has been devised to perturb endocytosis in spatially restricted domains within the embryo. A transgene expressing a dominant negative form of shibire (shi), the fly dynamin homolog, was constructed. When this transgene is expressed using the GAL4-UAS system, Wg protein distribution within the domain of transgene expression is limited and Wg-dependent epidermal patterning events surrounding the domain of expression are disrupted in a directional fashion. These results indicate that Wg transport in an anterior direction generates the normal expanse of naked cuticle within the segment and that movement of Wg in a posterior direction specifies diverse denticle cell fates in the anterior portion of the adjacent segment (Moline, 1999).
Interfering with posterior movement of Wg rescues the excessive naked cuticle specification observed in naked (nkd) mutant embryos. It is proposed that the nkd segment polarity phenotype results from unregulated posterior transport of Wg protein and therefore that wild-type Nkd function may contribute to the control of Wg movement within the epidermal cells of the segment (Moline, 1999).
Using en-Gal4-driven shiD expression to reduce posterior movement of Wg suppresses the phenotype of the segment polarity mutation, naked. nkd mutant embryos secrete denticle belts that have essentially normal denticle type diversity but that are replaced to varying degrees by naked cuticle. This excess naked cuticle depends upon Wg activity levels. The wg;nkd double mutant shows no naked cuticle across the ventral region; reducing the dosage of wg in a nkd mutant restores denticle belts. Thus wild-type nkd gene function appears to be involved in limiting Wg signaling activity within the segment. Consistent with this idea, Wg target genes become ectopically expressed in nkd mutant embryos. The en expression domain expands 2-3 cell diameters during stage 9, and an ectopic stripe of wg expression arises at stage 10, in the row of cells posterior to this expanded en domain. The posterior expansion of en expression suggests that nkd might play a role in restricting the movement of Wg protein in a posterior direction. Indeed, when nkd mutant embryos are generated that express shiD at moderate levels in the en domain, there is a dramatic reduction in the amount of naked cuticle specified. These embryos are very similar in appearance to wild-type embryos in which en-Gal4 drives shiD expression, except that the nkd mutant head defect is not fully rescued. en-Gal4-driven shiD expression also prevents the ectopic activation of en expression in nkd mutants. The stripes of en expression in the thoracic and abdominal segments are restored to the normal width, although some expansion is still observed in the head segments (Moline, 1999).
Since wild-type Wg signaling activity is required for stabilization of en expression, En stripes of normal width indicate that sufficient functional Wg contacts both rows of en-expressing cells to produce normal target gene regulation. This result demonstrates that expression of shiD does not interfere with Wg signal transduction and supports the idea that moderate level shiD expression reduces, but does not eliminate, transport of Wg across the affected domain. In contrast, embryos expressing high level shiD in the en domain show a narrowed stripe of En antibody staining, suggesting that Wg can no longer traverse the first row of en-expressing cells to stabilize en in the second row. However, because of the severe effects of a more complete endocytotic block, these embryos do not secrete cuticle properly and so the effects on cuticle pattern are not interpretable (Moline, 1999).
During early stages of wild-type embryogenesis, Wg protein can be detected at high levels in cells both anterior and posterior to the wg-expressing row of cells. Diversity of denticle types, as well as stabilization of en expression in the adjacent cells, are specified by Wg activity during these early stages of embryonic development. By mid-stage 10, when Wg is no longer required for denticle specification or en stabilization, the Wg protein distribution shifts and Wg appears to be excluded from the en-expressing cells. This exclusion is not observed in nkd mutants at the same stage. Rather, Wg protein continues to be detected in cells on either side of the wg-expressing row of cells and the levels become substantially higher due to the ectopic stripe of wg expression. These results suggest that nkd gene function may play a role in the posterior restriction of Wg protein that occurs during stage 10. Hence the mutant phenotype is rescued dramatically when this restriction is produced artificially, by expressing shiD in the en-expressing row of cells. All stage 11 and 12 embryos derived from this cross show posterior restriction of Wg protein, indicating that the nkd homozygotes do not exhibit excess posterior movement of Wg under these conditions. It is suspected that, in wild-type embryos, this restrictive function is not limited to the en-expressing cells. If this were the case, then one would expect to observe excess naked cuticle replacing denticle belts when wg+ is expressed in the en domain. Instead, en-Gal4-driven wg+, either alone or when co-expressed with shiD, does not produce substantial amounts of ectopic naked cuticle. Thus, it seems likely that some ability to restrict posterior Wg movement during later stages is shared by the rows of cells at the anterior of each segment (Moline, 1999).
It is believed that this analysis of Wg transport by perturbing endocytosis is physiologically relevant because a similar inhibition of transport can be produced by overexpressing Dfz2, the cognate receptor for Wg. It is presumed that these effects result from sequestering ligand, because pattern defects are observed only when Wg levels are limiting. No change from the wild-type cuticle pattern is detected when Dfz2 is driven at ubiquitous high levels of expression with E22C-Gal4. However, in embryos heterozygous for a null mutation of wg, significant pattern defects are observed at a frequency of 60%. Ectopic denticles appear in the domain of cells that normally secrete naked cuticle, similar to what is observed in segments where anterior Wg transport is perturbed by shiD. These pattern defects caused by Dfz2 overexpression are accompanied by a restricted Wg protein distribution and by a narrowed domain of Arm stabilization. However, it is not possible to directly compare Dfz2 with shiD in this experiment. E22C-Gal4-driven expression of shiD, even with UAS lines that express at low levels, results in cell death and failure to secrete cuticle, as was the case with the original shits mutation at restrictive temperature (Moline, 1999).
During Drosophila embryogenesis, a genetic cascade establishes repeating developmental units, the parasegments, along the anterior-posterior axis. Anterior and posterior boundaries of parasegments are defined by narrow stripes of cells expressing the segment polarity genes engrailed and wingless, respectively. Through single and double mutant analysis, genetic interactions regulating the precise activation of engrailed and wingless in alternate parasegments are described. The pair-rule gene odd-skipped and the segment polarity gene naked are both required to restrict engrailed expression. odd-skipped represses expression of fushi tarazu, a known activator of engrailed. naked prevents activation of engrailed by fushi tarazu, without affecting fushi tarazu expression. engrailed expression is thus limited to narrow stripes of cells at the anterior boundaries of these parasegments. wingless expression is regulated by both odd-skipped and the pair-rule gene paired. odd-skipped represses wingless expression, while paired restricts the domain of expression of odd-skipped. wingless expression is thus allowed in narrow stripes of cells at the posterior boundaries of these parasegments. Accurate expression of engrailed and wingless is also required for cells within each parasegment to assume their proper positional identity. In odd-skipped mutants, the positional identities of particular cells are changed, creating mirror-image duplications of the body pattern. A model is presented describing how the altered expression patterns of fushi tarazu, engrailed, and wingless generate the mutant phenotype (Mullen, 1995).
Mutations in the segment polarity gene nkd result in increased cell death that appears in a striped pattern in the abdominal epidermis. The cells that die are in the extreme posterior of the segment where en is expressed. The segment borders are shallow and in some cases run together between adjacent segments. TUNEL was used to localize the cell death in nkd mutants relative to en expression in order to show that the majority of the increased cell death is in the en-expressing cells. The nkd embryos have about a 6-fold increase in apoptosis in En cells as compared to similar staged wild-type embryos. The domain of En expression is expanded anteriorly in nkd mutants, which is where the increased cell death is located. There is also a slight increase in cell death throughout the segment (Pazdera, 1998).
wg embryos are substantially smaller than wild-type embryos. This difference in size is due in part to the smaller size of cells that underlie denticle belts versus naked cuticle and also because wg embryos experience considerably greater cell death than wild-type embryos. nkd appears to partially suppress this cell death. The wg;nkd doubly mutant embryo is slightly larger than the wg single mutant, with a larger field of denticles that shows more pronounced segmental modulation of denticle orientation and deeper segmental indentation of the denticle belt lateral margin. This deeper indentation suggests that wg does not specify the naked cuticle at the lateral margin of wild-type denticle belts. There also appears to be an indeterminate denticle type specified in addition to the row-5-type denticles; thus this may be an exception to the rule that loss of wg activity always results in uniformity of denticle type (Bejsovec, 1993).
The effect of nkd on the wg cuticle phenotype requires wild-type activity of ptc and en, but not hh. The suppression is not apparent in the wg ptc;nkd triple mutant nor in the wg en;nkd triple mutant, both of which are as small as the wg single mutant. In contrast, wg;nkd hh triple mutants are large and also show indeterminate denticle types similar to those seen in wg;nkd mutants. The wg;nkd suppression is not observed in wg;nkd double mutants that are also heterozygous for en. Therefore the gene dosage of en is crucial for altering the wg;nkd cuticle pattern. Since the decay of en expression in wg;nkd mutants is indistinguishable from that of wg single mutants, the suppression is not mediated through stabilized en expression. The suppression depends on the same early transient expression of en that is responsible for the segmental denticle polarity reversals observed in wg single mutants. This effect may be enhanced in the absence of nkd activity to give the more exaggerated segmental modulation in the wg;nkd double mutant. Thus, the effect of transient en expression on polarity may be separate and distinct from its effect on wg;ptc denticle morphology. The effect on polarity is independent of nkd gene activity, since it is still observed (and is, in fact, enhanced) in wg;nkd double mutants, whereas the effect on denticle morphology appears to be dependent on nkd gene activity. Loss of hh activity makes the wg mutant phenotype more severe. The wg;hh double mutant is smaller than wg or hh single mutants and the cuticle is poorly differentiated. This phenotype may result from increased cell death. In any case, the wg;nkd increase in embryo size is epistatic to the wg;hh decrease in embryo size. wg;nkd hh triple mutantsshow suppression of both the wg and hh mutant phenotypes (Bejsovec, 1993).
wingless activity is required to maintain its own expression. In wg homozygotes that make detectable but non-functional protein, Wg protein is activated by pair-rule gene action at the onset of gastrulation (stage 8; 3 hours of development), but it decays in all segments during the extended germ band stages 10 and 11 (5.5-7 hours). wg activity therefore has an autocatalytic effect that is essential for its own continued transcription. Since Wg protein is secreted, this autocatalytic effect may not be restricted to those cells initially expressing wg. Wg protein entering neighboring cells may have the potential to activate wg expression de novo in these cells. Based on analysis of wg expression in embryos homozygous for ptc or nkd, it is proposed that the autocatalytic effect can extend 3 to 4 cells anterior or posterior to the wild-type wg-expressing domain (Bejsovec, 1993).
In ptc mutant embryos, wg expression rapidly expands during stages 8 and 9, resulting in a wg transcription domain that usually extends 3 cells anterior to the initial wg stripe. This expansion requires functional wg activity. Using a wg allele, which produces detectable but non-functional gene product, a wg;ptc doubly mutant strain was constructed. No expansion of the mutant wg expression domain is observed at stages 8 and 9, when such expansion would be detected in the ptc single mutant. Furthermore, wg expression decays during the extended germ band stages, just as it does in the wg single mutant. This indicates that wg activity is required for the expansion of its own expression domain in ptc mutants, as well as for maintenance of its own expression. Therefore, wild-type ptc activity blocks the autocatalytic effects of wg activity in the cells anterior to the wg transcription domain and thus prevents inappropriate wg expression in these cells. For instance, ptc may play a role in restricting Wg protein transport or uptake so that cells anterior to the endogenous wg stripe are not exposed to levels of secreted Wg protein that will trigger the autocatalytic activity (Bejsovec, 1993).
Inappropriate wg expression is also observed in nkd embryos. An ectopic stripe of wg gene product is detected in stage 10 (mid-extended germ band) embryos, in cells that would give rise to the posterior edge of the denticle belt. As in the ptc situation, this ectopic wg expression depends on wg activity, since this expression is not observed in wg;nkd mutant embryos that produce detectable wg product. The normal and ectopic wg stripes in nkd single mutants are separated by an expanded domain of en-expressing cells. This en activity must block wg expression because in the absence of en activity, wg is expressed in these cells. In the en;nkd double mutant, wg protein and RNA extend 3 to 4 cells posterior to the endogenous wg stripe; this expanded domain may include the row of cells that would express wg ectopically in the nkd single mutant. Thus, as with ptc, wild-type nkd activity may restrict the potential for secreted wg product to activate its own expression in neighboring cells (Bejsovec, 1993).
In the ptc en;nkd triple mutant, wg is transcribed in all cells of the segment. Initially, the expression pattern resembles that of ptc mutants, with anterior expansion, but by stage 10 wg RNA is uniformly expressed throughout the segment. It is concluded that regardless of their initial pair-rule input, all epidermal cells in the ventral portion of the segment can potentially express wg. In ptc and nkd mutant embryos, wg expression does not depend on input from the en-expressing cells for its maintenance. In ptc;en, en;nkd and ptc en;nkd mutant embryos, wg expression does not decay as it does in en mutants. Therefore in the wild-type situation, en-expressing cells may act to maintain wg expression in the neighboring stripe of cells by counteracting some property conferred by ptc and nkd gene activity. For example, a signal from these cells may override an inhibitory effect of ptc and nkd on wg autoregulation within the wg-expressing cells (Bejsovec, 1993).
The nkd single mutant phenotype shows essentially wild-type denticle belts that are variably eliminated and replaced with naked cuticle. This inappropriate specification of naked cuticle requires wg activity. In the wg;nkd double mutant, no naked cuticle is observed across the ventral region. Furthermore, in nkd mutants that are heterozygous for wg, there is less ablation of denticle belts and the embryos appear more like wild-type. Therefore the extent of naked cuticle produced in nkd mutant embryos depends critically on the gene dosage of wg. Even greater suppression of the nkd phenotype is obtained by reducing the dosages of en and hh, as well as wg. en expression is also affected in nkd mutant embryos. The en expression domain is expanded 2 to 3 cells in a posterior direction during stages 8 and 9. It has been argued that the expanded en domain prevents activation of wg in the cells immediately posterior to the endogenous wg stripe. This exclusion of wg expression, which would reduce the total amount of naked cuticle specification activity in the segment, may account for the residual denticle belts observed in nkd single mutants. This view is consistent with the phenotype of the en;nkd double mutant, which is more severe than the nkd single mutant phenotype. Only naked cuticle and no other pattern elements are visible on the ventral surface. This severe naked phenotype is not simply a failure to differentiate cuticular structures, because dorsal pattern elements differentiate normally. wg activity is entirely responsible for this enhanced naked phenotype. Removing wg activity, in the wg;en;nkd triple mutant, restores a lawn of denticles to the ventral surface. The phenotype of the ptc en;nkd triple mutant is similar to that of the en;nkd double mutant, where only naked cuticle and no other pattern element is visible. Like en;nkd, this pattern is due to wg activity, since denticles are restored to the cuticle pattern in wg;ptc en;nkd mutants (Bejsovec, 1993).
Removal of ptc activity enhances the nkd mutant phenotype. The ptc;nkd double mutant produces only naked cuticle, with no ventral denticles visible. nkd mutants heterozygous for ptc also produce a completely naked cuticle. Since reducing ptc levels enhances the severity of the nkd phenotype, the wild-type ptc gene product probably functions to decrease the wg naked cuticle specification activity. The uniform naked cuticle produced by ptc;nkd double mutants suggests that all cells in the segment are exposed to wg. Eliminating wg activity, in the wg ptc;nkd triple mutant restores denticles. As in nkd single mutants, the en expression domain is expanded in the ptc;nkd double mutant. Consequently wg expression is blocked in these cells, so that the wg expression domain in ptc;nkd mutants is similar to that of ptc single mutants. However, the boundaries of Wg protein distribution, which are very sharply defined in ptc and ptc;en double mutants, appear to be less sharply defined in ptc; nkd mutants. This suggests that in the absence of nkd activity, Wg protein might be transported more effectively to parts of the segment outside of the wg expression domain. This enhanced distribution might also explain the apparently lower levels of Wg protein in the wg domain of ptc;nkd double mutants, as compared to ptc single mutants. In ptc and ptc en mutants, nkd gene activity restricts Wg protein movement and thus it accumulates to high levels in the wg transcription domain. Although the antibody preparations do not reveal detectable levels of Wg protein in cells outside of the wg domain of ptc;nkd double mutants or of nkd single mutants, these cells are exposed to levels of wg activity sufficient to specify naked cuticle. It is concluded that the antibody preparations cannot detect low levels of Wg protein that are functionally active by genetic criteria. Therefore, since the simplest means of restricting wg activity is by directly regulating Wg protein distribution, it is proposed that both ptc and nkd may act by altering movement or uptake of the Wg protein (Bejsovec, 1993).
The expression pattern of nkd is not known, but genetic data show that it is required in the cells posterior to the wg stripe. In the absence of nkd gene product, these cells inappropriately express wg when en activity is not present to repress it. When en activity is present, the en expression domain expands in a posterior direction. This expansion depends on wg activity, because expansion is delayed in nkd mutants that are heterozygous for wg and it is absent in wg;nkd double mutants. It is proposed that the en domain expands in response to inappropriate wg signaling, possibly due to increased Wg protein movement, and that wg activity extending to the line of cells posterior to the en-competent domain then induces wg expression in an autocatalytic fashion. This model demands only that wild-type nkd gene activity restricts wg activity; nkd need not act as a repressor of en expression. This model also explains a puzzling result obtained in experiments where deregulated wg response is produced in all cells of the segment. Embryos that carry a heat-shock wingless construct, which provides ubiquitous wg expression, show expansion of the en expression domain. A similar en expansion is observed in mutants for the zeste white 3 gene, which acts as a negative regulator of the wg response pathway. Both situations produce a phenotype identical to that of the ptc;nkd double mutant: expanded en expression and uniform naked cuticle specification. The model presented here would predict this expansion of the en domain as a consequence of uniform response to wg signal across the segment. The extent of cells competent to express en in response to wg signal may be defined by earlier pair-rule gene action. The width of the en domain in nkd and ptc;nkd mutants varies in a pair-rule fashion (Bejsovec, 1993).
nkd also appears to modulate wg activity in cells anterior to the wg stripe. nkd mutants that are heterozygous for ptc show an anterior expansion of wg expression similar to the ptc;nkd double mutant, whereas ptc heterozygotes normally would show a wild-type pattern of wg expression. This unexpected ptc/+;nkd phenotype corresponds with the production of a cuticle indistinguishable from ptc;nkd naked cuticle secreted by all cells of the segment. Therefore when nkd is absent, a reduction in the amount of ptc activity in the anterior cells may allow wg autoactivation in those cells. The ptc phenotype is subject to nkd dosage effects as well. ptc homozygotes that are heterozygous for nkd show a ptc-like pattern that is disrupted by greater expanses of naked cuticle. Thus, while both ptc and nkd are recessive mutations, each shows a dominant effect in homozygotes of the opposite mutation. This indicates that the dosage of each of these two gene products is critical for correct distribution of wg activity within the segment. ptc encodes a transmembrane molecule that is localized to the cell surface. Both ptc and nkd can influence pattern in the absence of wg activity; therefore, it is proposed that these molecules alter some fundamental property of the cell membrane in which they are inserted. These cell surface properties may contribute to pattern in the absence of wg, but have their major effect on pattern via the wg signaling pathway. It is possible that these molecules restrict wg activity by regulating Wg protein transport and/or endocytosis. For instance, ptc and nkd may alter membrane properties such that endocytosis of the Wg protein is less efficient, thereby restricting the Wg protein distribution in wild-type embryos. Alternatively, they may be endocytosed as part of the membrane and provide tags that target the Wg-containing intracellular vesicles directly to the lysosome. In this way, Wg might be degraded before it can trigger a response in the cell, and it would not be transported effectively to cells further away from the endogenous wg-expressing stripe of cells. During wild-type patterning, Wg protein distribution is dynamic and has different functional consequences. Between 4 and 6 hours, Wg protein is present in cells on either side of the wg stripe; this corresponds with the time during which wg acts to specify the diverse denticle types in the segment and to stabilize en expression in the cells posterior to the wg stripe. After 6 hours, Wg is distributed in a graded fashion in the cells anterior to the wg stripe; this corresponds to the time during which wg specifies naked cuticle identity. It is striking that Wg protein is detected at high levels in these anterior cells only after the time when their ptc expression levels decrease. Therefore it is proposed that the ptc and nkd molecules might play key roles in regulating the dynamic pattern of Wg protein distribution, which is crucial for the development of wild-type pattern (Bejsovec, 1993).
Involvement of these molecules in the processing or interpretation of wg signal complicates the analysis of experiments examining ubiquitous wg transcription. It has been concluded that wg does not act as a graded signal because denticle diversity is generated when heat shock-wg is expressed uniformly in a wg mutant embryo. However, the results presented here indicate that the action of other segment polarity genes may contribute to the graded nature of wg signaling activity, and therefore uniformly transcribed wg may not actually produce uniform wg activity (Bejsovec, 1993).
fused is a segment polarity gene whose product is maternally required in the posterior part of each segment. To define further the role of fused and determine how it interacts with other segmentation genes, the phenotypes obtained by combining fused with mutations of pair rule, homeotic and other segment polarity loci were examined. When it was possible, the distribution of corresponding proteins in fused mutant embryos was also examined. fused-naked (fu;nkd) double mutant embryos display a phenotypic suppression of simple mutant phenotypes: both naked cuticle and denticle belts, which would normally have been deleted by one of the two mutants alone, were restored. In fused mutant embryos, engrailed and wingless expression is normal until germ band extension, but partially or completely disappears, respectively, during germ band retraction. In the fu;nkd double mutant embryo, en is expressed as in nkd mutant at germ band extension, but later this expression is restricted and becomes normal at germ band retraction. On the contrary, wg expression disappears as in fu simple mutant embryos. It is concluded that the requirements for fused, naked and wingless activities for normal segmental patterning are not absolute, and mechanisms are proposed by which these genes interact to specify anterior and posterior cell fates (Limbourg-Bouchon, 1991).
The Drosophila central nervous system derives from neural precursor cells, the neuroblasts (NBs), which are born from the neuroectoderm by the process of delamination. Each NB has a unique identity, which is revealed by the production of a characteristic cell lineage and a specific set of molecular markers it expresses. These NBs delaminate at different but reproducible time points during neurogenesis (S1-S5) and it has been shown for early delaminating NBs (S1/S2) that their identities depend on positional information conferred by segment polarity genes and dorsoventral patterning genes. Mechanisms have been studied leading to the fate specification of a set of late delaminating neuroblasts, NB 6-4 and NB 7-3, both of which arise from the engrailed (en) expression domain, with NB 6-4 delaminating first. No evidence is found for a direct role of hedgehog in the process of NB 7-3 specification. NB 7-3 normally requires Hh only for maintenance of Wg expression, which in turn leads to En maintenance. Evidence is presented to show that the interplay of the segmentation genes naked cuticle (nkd) and gooseberry (gsb), both of which are targets of wingless (wg) activity, leads to differential commitment to NB 6-4 and NB 7-3 cell fate. In the absence of either nkd or gsb, one NB fate is replaced by the other. However, the temporal sequence of delamination is maintained, suggesting that formation and specification of these two NBs are under independent control (Deshpande, 2001).
In the En domain Wg plays a role both in NB formation and NB specification. The homeodomain transcription factor En is a prerequisite for the formation of the NBs 6-4 and 7-3, because in its absence both NBs fail to form. Since Wg signaling is necessary for maintaining En expression, it is also essential for the formation of these two NBs. Hh is co-expressed in the En domain and En maintains Hh expression in rows 6 and 7, and Hh in turn is essential for Wg expression in row 5, thereby constituting a maintenance loop. Thus, for late NBs in row 6 and 7, the expression of En is crucial and Hh is required to maintain En expression via Wg. However, for the separate specification of NB 6-4 and NB 7-3, differential regulation of two Wg targets, nkd and gsb, is essential (Deshpande, 2001).
Wg is a diffusible molecule expressed in row 5 and acts on neighboring rows, which include rows 6 and 7. However, row 6 differs from row 7 because it expresses gsb, which is, as stated above, a target of Wg signaling. The fact that row 7 does not express gsb, despite being under the influence of Wg raises the question of how this differential regulation is brought about. In this work it is shown that Nkd is essential for this regulation. Nkd is a negative regulator of the Wg signal transduction pathway, itself being a target of this pathway. In the absence of Nkd, Gsb is derepressed, owing to Wg hyperactivity in row 7, leading to the generation of an ectopic NB 6-4 like fate. Thus, the distinct identities of NB 6-4 and NB 7-3 are brought about by the interplay of Gsb and Nkd. For NB 6-4 specification, Gsb is an essential factor. In the absence of Gsb NB 6-4 fails to be specified and instead takes the identity of NB 7-3 fate. Conversely, for NB 7-3 specification, a Gsb-free environment, which is created by the activity of Nkd, is essential. In summary, NB 6-4 needs the expression of Gsb and En, whereas NB 7-3 needs En but the absence of Gsb (Deshpande, 2001).
However, the fact that gsb as well as nkd are targets of Wg signaling makes it difficult to explain why gsb is repressed by nkd only in the posterior region of the En stripe. The posterior En domain is further away from the Wg source than the anterior En domain and therefore should receive a lower signaling input when compared with the anterior region. As a consequence, this should lead to higher Nkd activity in the anterior En cells, leading to a stronger Gsb repression in this region -- the opposite of what was observed. A careful analysis of the expression pattern on the transcriptional level does not give any obvious clues to solve this apparent paradox. During early germ band extension (stage 8-9) nkd transcription is nearly ubiquitous with higher RNA levels in the two to four cell rows posterior to the En stripe. At late phase of germ band extension, nkd expression is most abundant anterior to the En stripe and lower just posterior to the En-stripe. No significant difference between the anterior and posterior En domain could be detected. One explanation for the differential regulation of gsb could be that, owing to earlier pair rule gene activity of paired, the level of Gsb protein at the time of NB 6-4 delamination in the anterior En region is high enough to override repression by Nkd activity. Alternatively, a direct differential regulation of the two Wg targets that is due to the different levels of Wg signaling could be responsible for the observed regulatory differences. It could be that the regulation is such that the amount of Wg signaling within the En stripe causes a relatively homogenous level of nkd expression in this region. At the same time, the transcriptional activation of gsb could be more sensitive to Wg signaling levels, resulting in a very strong activation, especially near to the Wg-expressing cells. As a result, the relatively low Nkd activity in the whole En stripe might be able to inhibit gsb expression in the region of low gsb activation only: the posterior En domain. A hint that a differential regulation of Wg targets indeed exists comes from the Wg-dependent En regulation: it seems that a lower Nkd activity is sufficient to repress gsb but not to inhibit en expression. This conclusion was drawn from the finding that overexpression of nkd within the En stripe using an EnGal4 driver line leads to a selective repression of gsb with no obvious effect on en expression itself. Clearly, additional work has to be carried out to clarify these points (Deshpande, 2001).
Besides row 6 neuroectoderm, row 3 neuroectoderm also has the potential to generate an ectopic NB 7-3. It has been shown previously that in embryos mutant for ptc, neuroectodermal cells in the area of row 3 begin to express En and additional serotonergic neurons can be found in these mutant embryos, which suggests the presence of an ectopic NB 7-3 like fate. Additionally, when En is ubiquitously expressed, only row 3 has the ability to give rise to an ectopic NB 7-3 fate. In all cases, this occurs at the cost of row 3 NBs such as NB 3-3. It is thought that this might reflect that row 3 neuroectoderm, which is right in the middle of the segment, represents something like a 'ground state' in the neuroectoderm: in this area neither Hh nor Wg signaling may take place. Therefore the decision to specify late row 3 or late row 7 NBs seems to be only dependent on the absence or presence of En, respectively (Deshpande, 2001).
Previous work has indicated that genes expressed in proneural clusters are involved in specifying the individual fates of NBs that develop from these clusters. The finding that NB 6-4 and NB 7-3 can be mutually transformed while the sequence of birth does not change suggests that the mechanism for the timing of late NB delamination is independent from mechanisms that regulate NB identity. This might be reminiscent of early NBs. Initiation of S1 NB formation requires the activity of proneural genes that have been shown to be dependent on pair-rule genes. The identity of the NBs delaminating from these clusters, however, is dictated by the activity of segment polarity genes. Thus, the control of proneural gene expression that enables NB formation and the control of segmentation genes conferring NB identity occurs in parallel. At later stages, pair-rule gene expression vanishes and can no longer be responsible for NB formation. How is NB formation regulated in the following segregation waves? One possibility is that after the first segregation wave, NB formation and identity are more tightly linked; the finding that specific NBs like NB 4-2 are sometimes not transformed but missing in wg mutant embryos seems to support this idea. However, the finding that the transformed NB 6-4 and NB 7-3 are delaminating according to the 'old identity' shows that, at least in these cases, NB formation and specification is independent. The results favour the idea that the timing of the formation of proneural clusters within the neuroectoderm is generally independent of the segment polarity genes investigated here. This does not exclude permissive functions, such as those of En, which enable the proneural cluster formation as such. According to this hypothesis, intrinsic or extrinsic factors present in the position of the proneural cluster at the time of delamination govern the identities of the NBs. This might be not only true for the positional regulation of NB identity but also for the determination of NB identity along the temporal axis. Indeed, heterochronic transplantation experiments strongly support the possibility that one or more extrinsic factors exist that lead to stage specific NB identities. It will be a challenge for the future to identify these factors, and to investigate whether similar mechanisms exist in higher organisms (Deshpande, 2001).
In a genetic screen to identify mutations that suppress or enhance the mutant phenotype of the wg temperature-sensitive allele wgIL114, two EMS-induced mutations were isolated that subtly modify the wgIL114 mutant cuticle pattern. These modifier mutations, AR2 and DH15, fail to complement each other and thus identify a single complementation group, linked to wg on the second chromosome. These nonsense mutations are shown to disrupting the RacGAP50C locus. The two alleles produce identical phenotypes. Both mutations show no increase in severity when placed in trans to a deficiency for the region and so are likely to represent loss-of-function alleles (Jones, 2005). RacGap50C interacts genetically with nkd and appears to act at the same level or downstream of Axin in the control of Arm stabilization. The data indicate that RacGap50C probably does not act through Rac1 to negatively regulate Wg activity, nor are other GTPases likely to be involved in this aspect of epidermal patterning since the cuticle defects of mutant embryos can be rescued by a form of RacGap50C that lacks catalytic residues in the GTPase-activating domain. Moreover, previous work shows that other Rho family members are unlikely to be involved in Wg-mediated patterning. Overexpressing either constitutively active or dominant-negative Rho, Rac, or cdc42 transgenes disrupts dorsal closure but does not appear to affect ventral patterning. Loss of maternal Rho activity has been found to alter embryonic segmental pattern, but this is due to an early effect on establishing segmentation gene expression patterns. Ras activation through the EGF signaling cascade has been found to affect epidermal patterning, but in a way that counteracts Wg signaling. Thus a GTPase-activating protein would be expected to positively influence Wg-mediated patterning if it acted through Ras, rather than the negative influence observed for RacGap50C (Jones, 2005).
Juvenile hormone (JH) plays key roles in controlling insect growth and metamorphosis. However, relatively little is known about the JH signaling pathways. Until recent years, increasing evidence has suggested that JH modulates the action of 20-hydroxyecdysone (20E) by regulating expression of broad (br), a 20E early response gene, through Met/Gce and Kr-h1. To identify other genes involved in JH signaling, a novel Drosophila genetic screen was designed to isolate mutations that derepress JH-mediated br suppression at early larval stages. It was found that mutations in three Wnt signaling negative regulators in Drosophila, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), caused precocious br expression, which could not be blocked by exogenous juvenile hormone analogs (JHA). A similar phenotype was observed when armadillo (arm), the mediator of Wnt signaling, was overexpressed. qRT-PCR revealed that Met, gce and Kr-h1expression are suppressed in the Axn, slmb and nkd mutants as well as in arm gain-of-function larvae. Furthermore, ectopic expression of gce restored Kr-h1 expression but not Met expression in the arm gain-of-function larvae. Taken together, it is concluded that Wnt signaling cross-talks with JH signaling by suppressing transcription of Met and gce, genes that encode for putative JH receptors. The reduced JH activity further induces down-regulation of Kr-h1expression and eventually derepresses br expression in the Drosophila early larval stages (Abdou, 2011).
JH transduces its signal through Methoprene-tolerant (Met), Germ cell-expressed (Gce) and Krüppel-homolog 1 (Kr-h1) and the p160/SRC/NCoA-like molecule (Taiman in Drosophila and FISC in Aedes). The Drosophila Met and gce genes encode two functionally redundant bHLH-PAS protein family members, which have been proposed to be components of the elusive JH receptor. Both Met and gce mutants are viable and resistant to JH analogs (JHA) as well as to natural JH III. However, Met-gce double mutants are prepupal lethal and phenocopies CA-ablation flies. The Met protein binds JH III with high affinity. In Tribolium, suppression of Met activity by injecting double-stranded (ds) Met RNA causes precocious metamorphosis. Kr-h1 is considered as a JH signaling component working downstream of Met. In both Drosophila and Tribolium, Kruppel-homolog1 (Kr-h1) mRNA exhibits high levels during the embryonic stage and is continuously expressed in the larvae; then, it disappears during pupal and adult development. Kr-h1 expression can be induced in the abdominal integument by exogenous JH analog (JHA) at pupariation. Suppression of Kr-h1 by dsRNA in the early larval instars of Tribolium causes precocious br expression and premature metamorphosis after one succeeding instar. Thus, Kr-h1 is necessary for JH to maintain the larval state during a molt by suppressing br expression. Studies in Aedes, Drosophila and Tribolium have demonstrated that the p160/SRC/NCoA-like molecule is also required for JH to induce expression of Kr-h1 and other JH response genes. For example, Aedes FISC forms a functional complex with Met on the JH response element in the presence of JH and directly activates transcription of JH target genes (Abdou, 2011 and references therein).
In an attempt to isolate other genes involving JH signaling, a novel genetic screen was conducted, and mutations in were identified in three Wnt signaling component genes, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), induced precocious br expression, which was similar to a loss of JH activity. The evolutionarily conserved Wnt signaling pathway controls numerous developmental processes. The key mediator of the Drosophila Wnt pathway is Armadillo (Arm, the homolog of vertebrate β-catenin). When the Wnt signaling ligand, Wingless (Wg), is absent, the destruction complex is active and phosphorylates Arm, earmarking it for degradation. Upon Wg stimulation, the destruction complex is inactivated; as a result, unphosphorylated Arm accumulates in the cytosol and is targeted to the nucleus to stimulate transcription of Wnt target genes. Many players in the Wnt signaling pathway negatively regulate its activity. For example, Axin (Axn) is one of the main components of the destruction complex. Supernumerary limbs (Slmb) recognizes phosphorylated Arm and targets it for polyubiqitination and proteasomal destruction. Naked cuticle (Nkd) antagonizes Wnt signaling by inhibiting nuclear import of Arm. The current investigations reveal that the high activity of Wnt signaling in the Axn, slmb, and nkd mutants suppresses the transcription of Met and gce, genes encoding for putative JH receptors, thus linking Wnt signaling to JH signaling and insect metamorphosis for the first time (Abdou, 2011).
The 'status quo' action of JH in controlling insect metamorphosis is conserved in hemimetabous and most holometabous insects. However, the larval-pupal transition in higher Diptera, such as Drosophila, has largely lost its dependence on JH. For instance, in most insects, the addition of JH in larvae at the last instar causes the formation of supernumerary larvae. However, exogenous JH does not prevent pupariation and pupation in Drosophila, and instead disrupts the development of only the adult abdominal cuticle and some internal tissues. The molecular mechanisms underlying these differential responses to JH are not clear (Abdou, 2011).
Broad is a JH-dependent regulator that specifies pupal development and mediates the 'status quo' action of JH. In the relatively basal holometabolous insects, such as beetles and moths, JH is both necessary and sufficient to repress br expression during all of the larval stages. These studies revealed that JH is also required during the early larval stages in the more derived groups of the holometabolous insects, such as Drosophila, but it is not sufficient to repress br expression at the late 3rd instar. During the early larval stages, overexpression of the JH-degradative enzyme JHE, reduction of JH biosynthesis or disruption of the JH signaling always causes precocious br expression in the fat body. However, exogenous JHA treatment can not repress br expression in the fat body of late 3rd instar larvae. The molecular mechanism underlying the developmental stage-specific responses of the br gene to JH signaling remains to be clarified (Abdou, 2011).
As knowledge of signal transduction increases, the next step is to understand how individual signaling pathways integrate into the broader signaling networks that regulate fundamental biological processes. In vertebrates, Wnt signaling has been found to interact with different hormone signaling pathways to mediate various developmental events. For example, the Wnt/beta-catenin signaling pathway interacts with thyroid hormones in the terminal differentiation of growth plate chondrocytes and interacts with estrogen to regulate early gene expression in response to mechanical strain in osteoblastic cells. In insects, both Wnt and JH signaling are important regulatory pathways, each controlling a wide range of biological processes. This study reports that the Wnt signaling pathway interacts with JH in regulating insect development. During the Drosophila early larval stages, elevated Wnt signaling activity in the Axn, slmb, nkd mutants and arm-GAL4/UAS-armS10 flies represses Met and gce expression, which down-regulates Kr-h1 and causes precocious br expression in the fat body. Ectopic expression of UAS-gce in the arm-GAL4/UAS-armS10 larvae is sufficient for restoring Kr-h1 expression and then repressing br expression (Abdou, 2011).
Arm is a co-activator that interacts with Drosophila TCF homolog Pangolin (Pan), a Wnt-response element-binding protein, to stimulate expression of Wnt signaling target genes. In the absence of nuclear Arm, Pan interacts with Groucho, a co-repressor, to repress transcription of Wingless-responsive genes. Upon the presence of nuclear Arm, it binds to Pan, converting it into a transcriptional activator to promote the transcription of Wingless-responsive genes. It is proposed that Wnt signaling indirectly suppresses Met and gce expression by activating an unknown transcriptional repressor (Abdou, 2011).
JH signaling is well known to be a systemic factor that decides juvenile versus adult commitment. Wg is a morphogen that tissue-autonomously promotes proliferation and patterning during organogenesis. The current studies show that ectopically activating Wg signaling, either by mutations of negative regulators or by the ectopic expression of Arm, results in br derepression via loss of Met and Gce. How and why does the localized Wg signaling regulate the global JH signaling during insect development? It is hypothesized that though JH signaling activity is globally controlled by JH titer in the hemolymph, distinct tissues may response to JH with different sensitivity, which could be regulated by Wnt signaling-mediated Met and gce expression. Actually, it was found that precocious br expression is detectible in the fat body but not midgut of the Axn mutant 2nd instar larvae. This is one line of evidence to support that Wnt signaling regulates Met and gce expression in a tissue-specific manner (Abdou, 2011).
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date revised: 25 July 2912
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