BIOLOGICAL OVERVIEW

The term pangolin refers to a long tailed, sticky-tongued tropical Old World (Asia and Africa) mammal; it feeds chiefly on ants. The pangolin's body is covered with large, flat, imbricated horny scales; it somewhat resembles the New World armadillo in terms of its feeding habits and its employment of a curled up, hedgehog-like defensive posture. For these reasons, the gene pangolin is most appropriately named, calling to mind both Armadillo and Hedgehog, two proteins discussed below in connection with Pangolin function.

Pangolin is one of Drosophila's more recently sequenced segment polarity genes. The discovery of the involvement of an HMG-domain in vertebrate Wnt signaling (Wnts are homologs of Wingless) encouraged a search for a similar HMG-domain protein in Drosophila. The vertebrate HMG-domain proteins Lef-1 and XTcf-3 have been shown to physically interact with ß-catenin, a homolog of Drosophila Armadillo. HMG-domain proteins are presumptive transcription factors; evidence from Xenopus suggests that during Wnt signaling the HMG protein interacting with ß-catenin ( XTcf-3) is transported as a complex into the nucleus, where it can act as a transcription factor (Behrens, 1996, Huber, 1996 Molenaar, 1996). Both ß-catenin and Armadillo serve as downstream components of the Wnt/Wingless signal transduction pathway; this pathway is involved in vertebrate mesoderm formation, neural differentiation and limb patterning, and in Drosophila, is involved in similar functions but is best known as a segment polarity gene.

Isolated as a homolog of the vertebrate HMG-domain proteins described above, Pangolin is able to physically interact with vertebrate ß-catenin. Mutations of pan cause a segment polarity phenotype similar to that of wingless mutation (Brunner, 1997). In an unexpected association, Pangolin is found immediately adjacent to the gene cubitus interruptus, a zinc finger transcription factor that targets wingless, decapentaplegic and patched (Brunner, 1997). The two genes have a head-to-head orientation (van de Wetering, 1997). CI acts in the hedgehog pathway.

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

One mode of action for Wingless and Wnts is suggested by the observation that the LEF-1/ß-catenin complex binds to an E-cadherin promoter fragment. Dring primitive streak formation in mouse embryos, embryonic ectodermal cells, which represent a true epithelial cell layer, give rise to mesoderm. During primitive streak formation, some ectodermal cells lose E-cadherin expression and express LEF-1. It is tempting to speculate that a complex of LEF-1 and ß-catenin is involved in down-regulating E-cadherin, resulting in an altered cell adhesion accompanying a cell fate transformation (Huber, 1996).

Coop functions as a corepressor of Pangolin and antagonizes Wingless signaling

Wingless (Wg) signaling regulates expression of its target genes via Pangolin and Armadillo, and their interacting cofactors. In the absence of Wg, Pangolin mediates transcriptional repression. In the presence of Wg, Pangolin, Armadillo, and a cohort of coactivators mediate transcriptional activation. This study uncovered Coop (Corepressor of Pan) as a Pangolin-interacting protein. Coop and Pangolin form a complex on DNA containing a Pangolin/TCF-binding motif. Overexpression of Coop specifically represses Wg target genes, while loss of Coop function causes derepression. Coop is shown to antagonize the binding of Armadillo to Pangolin, providing a mechanism for Coop-mediated repression of Wg target gene transcription (Song, 2010).

Coop (CG1621) was found in an attempt to identify novel Pan-interacting proteins by a mass spectrometry-based proteomic approach. It contains an N-terminal MADF domain and a C-terminal BESS domain. This architecture is conserved in 16 Drosophila proteins, one C. elegans protein, and one zebrafish protein. The putative dimeric BESS domain does not exist in mammals, and there is one uncharacterized MADF-containing protein in humans (ZSCAN29/ZNF690). Earlier studies on other members of this family indicated that they might be involved in transcriptional regulation. The interaction between Coop and Pan was confirmed in a series of pull-down experiments. In lysates of Drosophila Kc cells, Pan was coimmunoprecipitated by Coop. Consistent with a role in transcription, Coop was localized in the nucleus in these cells. To determine if this interaction was direct, GST pull-down experiments were performed. A GST fusion of Coop could pull down in vitro translated Pan. In the reverse experiment, Coop was pulled down by a Pan central fragment (247-362) harboring its DNA-binding domain, but not by a Pan N-terminal fragment (1-160). This interaction was further mapped to the C-terminal part of Coop (161-358), which contains its BESS domain (Song, 2010).

Since the DNA-binding domain of Pan may be involved in the interaction with Coop, it was asked whether Coop affected the DNA affinity of Pan. This question was addressed in a gel shift assay. Pan (247-362) induced the mobility shift of a DNA oligonucleotide containing a consensus Pan/TCF-binding site, but did not affect that of a control oligonucleotide in which the binding site was mutated. Coop alone did not bind to this DNA probe. However, in the presence of Pan, Coop induced a supershift of this oligonucleotide. This suggests that the interaction between Coop and Pan does not interfere with Pan binding to DNA. Instead, it suggests that Coop and Pan form a complex on Pan targets (Song, 2010).

To test whether the interaction between Coop and Pan affects Wg signaling in vivo, Coop was overexpressed in wing imaginal discs and expression of Wg targets was monitored, including Distal-less (Dll), senseless (sens), and Notum/wingful (wf). The expression of Coop in the dorsal compartment of wing discs by apterous-Gal4 (apGal4) resulted in the loss of Dll protein in this domain. This repression occurred at the transcriptional level, as the expression of a Dll-lacZ (DllZ) reporter transgene was similarly repressed in the same assay. The expression of Coop in the posterior compartment of wing discs by engrailed-Gal4 (enGal4) also repressed Dll expression, but in a different pattern: Dll staining was lost in the domain away from the dorsal-ventral (D/V) boundary, and was only weakened in the domain close to the D/V boundary. The Wg morphogen is expressed at the D/V boundary and forms a concentration gradient in the wing disc. It is possible that the observations reflected a dosage-dependent effect of Coop on different levels of Wg signaling. To test this idea, Coop was also expressed at lower levels, either in clones or in the center of wing discs by spalt enhancer-Gal4 (salEGal4). Indeed, the repression of Dll expression was observed preferentially in the domain with lower levels of Wg signaling. These results implied that Coop functions to antagonize incoming Wg signaling (Song, 2010).

sens is a high-threshold target of Wg in wing discs and is expressed in two to three rows of cells flanking the D/V boundary. The expression of sens starts at the center of the wing disc and extends to the periphery at late larval stage. The expression of Coop by enGal4 resulted in the repression of sens in the posterior compartment of wing discs, while the expression of Coop by apGal4 prevented Sens from extending to the periphery in the dorsal compartment. Consistent with observations with Dll, lower levels of Coop expression by act>CD2>Gal4 or salEGal4 did not affect sens expression (Song, 2010).

wf is another known Wg target gene. A 4-kb upstream element has been isolated from the wf locus, which responded to Wg signaling in cell culture. A lacZ transgene under its control (wfZ) mimicked wf expression in wing discs. The expression of Coop repressed this wfZ in wing discs and wf reporter in cultured Drosophila cells. In addition to Wg targets in the wing disc, the expression of Coop also repressed H15, a Wg target gene in the leg disc. Thus, in the case of the four Wg target genes analyzed, Coop functioned consistently as a negative transcriptional regulator (Song, 2010).

Whether Coop generally affects transcription, or specifically affects Wg signaling was tested. The effect of Coop on the expression of Notch targets wg and cut was examined. The expression of Coop by enGal4 or other drivers did not affect expression of either gene, confirming that the effect observed on Wg targets was not due to the interference in the upstream level of Wg signaling. Then Coop was expressed by apGal4, which strongly repressed Wg targets, and the expression of other non-Wg targets was monitored. Lgs plays a key role in transducing Wg signal. No effect on lgs expression by Coop was observed. Similarly, the expression of Coop had no effect on the Hedgehog (Hh) target gene decapentaplegic (dpp), or the Dpp targets optomotor blind (omb) and spalt. Similarly, hh, patched (ptc), and en expression were unaffected. In this study it was noticed that overexpression of Coop had a weak negative effect on the Gal4/UAS system. It is therefore possible that Coop expression was reduced, leading to an underestimation of the strength of Coop's activity. Whatever the case, the effect does not compromise any of these conclusions, as none of the targets monitored in this study was driven by the Gal4/UAS system. Taken together, these results suggest that the repressive effect of Coop is probably mediated by its interaction with Pan, and not an indirect effect on wg expression or components of the Wg signaling cascade. Importantly, Coop does not appear to affect Hh or Dpp signaling, suggesting its effect on the Wg pathway is fairly specific (Song, 2010).

In the next step, it was asked whether Coop was required for the proper regulation of Wg target gene expression. The effect of Coop RNAi on the basal transcriptional levels of Wg target genes was examined in Drosophila Kc cells. For comparison, RNAi against Groucho or CtBP, two known corepressors of the Wg pathway, was also performed. Surprisingly, the knockdown of Coop mRNA in Kc cells by dsRNA treatment was very inefficient. The levels of most mRNAs could be knocked down to 10%-15% after 4 d; however, the levels of Coop mRNA remained at 60%-70% after 4 d and 40%-50% after 7 d. Even so, under this condition, the knockdown of Coop mRNA caused a twofold to threefold increase in Arm-independent basal expression of Wg target gene nkd and CG6234, similar to the effect observed with Groucho RNAi, but slightly weaker than the effect seen with CtBP RNAi. In addition, the combination of Coop and CtBP RNAi showed an additive effect (Song, 2010).

Whether reducing Coop function affected the activation of Wg target genes was examined in vivo. This was tested by expressing dsRNA against Coop in the wing disc and monitoring expression of Wg targets. Coop is expressed ubiquitously in imaginal discs. The knockdown of Coop enhanced expression of Dll and wf, suggesting that endogenous Coop also affects Wg-mediated target activation. Starting from an enhancer P element (EP) line, two Coop alleles (hereafter referred to as coop) were generated that encode truncated proteins. coop mutant flies seemed normal, but showed enhanced Wg signaling in a sensitized background: Loss of Coop enhanced a rough eye phenotype caused by ectopic Wg signaling (sev-Wg). In coop mutant clones, moderately enhanced expression of Dll was also observed in the wing disc. This is consistent with in vivo RNAi results, indicating that Coop is a repressor of Wg target genes. Although coop clones had a weaker effect than RNAi, this difference might be due to the perdurance of Coop protein. The RNAi was induced earlier than the clones of coop, thus probably eliminating Coop more thoroughly (Song, 2010).

There are 15 sequence homologs of Coop in Drosophila, and it is possible that one or several of them also had similar roles in Wg signaling. Some Coop family members were examined, and overexpression of CG6854 and Adf1 were found to also repressed Wg signaling in Drosophila cultured cells. As a next step, the possibility that CG6854 or Adf1 acts like Coop in vivo was examined. Overexpression of CG6854 strongly repressed expression of Dll and sens. However, in contrast to Coop, it also repressed expression of wg and targets of other pathways, suggesting the repressive effect of CG6854 may be less specific than that of Coop. Adf1 behaved like CG6854. Taking these results together, it is proposed that, unlike Coop, Adf1 and CG6854 are not specific repressors of the Wg pathway (Song, 2010).

Having established that Coop has a defined role in Wg signaling, in contrast to CG6854 and Adf1, the mechanism by which Coop represses Wg target genes was examined. As the interaction between Arm and Pan is essential for activation of Wg target genes, whether Coop functions by preventing this process was tested. In cultured Drosophila Kc cells, Pan and Arm were overexpressed in the absence or presence of overexpressed Coop, and Pan was coimmunoprecipitated via Arm. The presence of Coop greatly reduced the amount of Pan coimmunoprecipitated. Similarly, when Pan was coimmunoprecipitated via Coop, the presence of overexpressed Arm also prevented this interaction. These results suggest that the binding of Coop to Pan and the binding of Arm to Pan are mutually exclusive (Song, 2010).

As Coop can interact with a domain in Pan that is conserved in other TCFs, whether Coop could also affect Wnt signaling was examined. Interestingly, activation of a Wnt signaling reporter was repressed by ectopic expression of Coop in HEK293T cells. It is likely that Coop achieved this by interfering with the conserved interaction between β-catenin and TCF. These results indicate a way in which Wnt signaling could be additionally regulated by a functional homolog of Coop. Since BESS domain proteins apparently do not exist in mammals, it is postulated that a Coop-like repressor function is carried out by a TCF-interacting protein that is not necessarily structurally related to Coop. Proteomic analysis of TCF interaction partners may help to identify functional homologs of Coop in mammals (Song, 2010).

It has been shown in vitro that β-catenin and TLE1 compete for binding to Lef1. By competing for an overlapping binding site adjacent to the DNA-binding domain of Lef1, TLE1 prevents the recruitment of β-catenin. This study also shows that Coop and Arm also compete to bind Pan. Thus, the levels of nuclear β-catenin/Arm, determined by the levels of Wnt/Wg signaling, decide the transcriptional activity of TCF/Lef/Pan. This dosage-dependent, reversible mechanism helps to shape Wnt/Wg gradient-induced expression of downstream targets (Song, 2010).

Ectopic Wnt signaling, transduced via interaction between β-catenin and TCF, is often detected in human cancers. Several β-catenin-binding proteins, including ICAT and Chibby, can interfere with the interaction of these two proteins. This study reports the identification of Coop as another potential blocker of the β-catenin-TCF interaction. As β-catenin has divergent functions in more than Wnt signaling, TCF-binding proteins may help to specifically decrease the transcriptional outputs of ectopic Wnt signaling. Given the specific effect of Coop in the Wg pathway, it is believed Coop may function as a specific inhibitor of Arm-Pan interaction in Drosophila. Further studies to map the Coop-Pan interaction may uncover novel ways to prevent the interaction between β-catenin and TCF (Song, 2010).

Regulation of anisotropic tissue growth by two orthogonal signaling centers

The Drosophila wing has served as a paradigm to mechanistically characterize the role of morphogens in patterning and growth. Wingless (Wg) and Decapentaplegic (Dpp) are expressed in two orthogonal signaling centers, and their gradients organize patterning by regulating the expression of well-defined target genes. By contrast, graded activity of these morphogens is not an absolute requirement for wing growth. Despite their permissive role in regulating growth, this study shows that Wg and Dpp are utilized in a non-interchangeable manner by the two existing orthogonal signaling centers to promote preferential growth along the two different axes of the developing wing. The data indicate that these morphogens promote anisotropic growth by making use of distinct and non-interchangeable molecular mechanisms. Whereas Dpp drives growth along the anterior-posterior axis by maintaining Brinker levels below a growth-repressing threshold, Wg exerts its action along the proximal-distal axis through a double repression mechanism involving the T cell factor (TCF) Pangolin (Barrio, 2020).

Two orthogonal signaling centers, corresponding to the AP and DV compartment boundaries and expressing the Dpp and Wg morphogens, regulate growth and patterning of the developing wing along the AP and PD axes, respectively. Whereas graded activity of these morphogens defines the spatial location of longitudinal veins and sensory organs that decorate the adult wing along these two axes, their graded activity is not an absolute requirement for its growth-promoting role. Despite the non-instrumental role of Wg and Dpp gradients in regulating tissue size, this study presents evidence that these two morphogens control the size of the adult wing along two orthogonal axes by mediating the growth-promoting activities of compartment boundaries in a non-interchangeable manner through the use of morphogen-specific molecular mechanisms. While Dpp regulates growth along the AP axis by maintaining the levels of the transcriptional repressor Brinker below a growth-repressing threshold, Wg regulates growth along the PD axis by counteracting the activity of TCF as a transcriptional repressor. At the time TCF was molecularly identified in flies, it was shown that clones of cells mutant for TCF are poorly recovered in the primordium of the wing pouch and proposed to be a consequence of TCF promoting proliferative growth. However, later studies identified cell competition as the mechanism to eliminate cells with steep differences in Wg signaling in the wing primordium. The Warts-Hippo signaling pathway governs organ size in animals, and the upstream regulators include the atypical cadherins Fat and Dachsous. Surprisingly, inactivation of the Warts-Hippo signaling pathway was unable to rescue the tissue size defects caused by morphogen depletion. These data indicate that for wing blade cells to grow along the PD and AP axes, cells need first to lose TCF and Brinker, and it is proposed that Hippo signaling can then modulate the amount of growth of those cells in which these two repressors are not active or expressed. The experimental data are consistent with a model whereby a minimal amount of signaling from the two morphogens, sufficient to maintain the activity levels of the two transcriptional repressors below a growth-repressing threshold, regulate the physical size of the adult wing primordium along the AP and PD axes. The mechanistic similarities of how Dpp and Wg morphogens, their gradients, and their range of activity regulate the patterning and growth of the fly wing are remarkable and might shed light on the role of morphogens in regulating proliferative growth and patterning in vertebrates (Barrio, 2020).

Experimental conditions in developing wings in which proliferation rates are either increased or reduced have shown that a perfectly normal-sized wing can be obtained with fewer or more cells. Similarly, experimental randomization of the orientation of cell divisions in the growing wing primordium can give rise to well-shaped adult wings. These results suggest that the ability of compartment boundaries, and their dedicated morphogens, to drive anisotropic growth and regulate the width and length of the adult wing blade does not rely only on the control of cell division or oriented cell divisions. Several experimental data indicate that it is the range of the morphogen and not the total amount of it that regulates the physical size, and not the number of cells, of each axis. How do Wg and Dpp regulate growth preferentially along a certain axis and not the other? Restricted expression of these two morphogens along the two existing orthogonal boundaries does not appear to be essential as their ability to drive anisotropic growth is still observed when they are ubiquitously overexpressed in all wing cells. The experimental data indicate that the capacity of Wg and Dpp to drive anisotropic growth relies on the existence of morphogen-specific and non-interchangeable molecular mechanisms mediating their growth-promoting activities and the requirement of the presence of the two of them to drive growth. In this regard, each morphogen promotes growth only along a particular axis, as the distance to the source of the other morphogen has to be maintained to get sufficient levels of the two of them to promote wing growth. The data also indicate that the Wg gradient contributes to orient growth along the PD axis. However, this contribution does not appear to play an essential role since well-shaped elongated wings can be obtained upon uniform expression of Wg (Barrio, 2020).

While the growth-promoting role of Dpp emanating from the AP compartment boundary has been experimentally validated and recently clarified, previous experimental characterization of the growth-promoting role of Wg emanating from the DV compartment boundary reached opposing conclusions. This study presents experimental evidence that Wg mediates the organizing activity of the DV boundary in terms of growth, as uniform expression of this morphogen rescues the extreme growth defects caused by the absence of a DV signaling center. Moreover, the data indicate that Wg is the main growth-promoting Wnt in the developing wing, the DV boundary is the main source of Wg driving proliferative growth of the primordium of the wing appendage, and boundary Wg regulates tissue growth and proliferation rates equally in distal and proximal regions of the developing wing appendage, throughout development and independently of its potential role as survival factor. This latter observation questions the proposal that Wg drives wing growth, at least in part, by promoting cell survival. This proposal was based on the ability of apoptotic inhibitors to rescue the poor recovery and growth of clones of cells unable to transduce the Wg signal, but cell competition was subsequently shown to be the mechanism used to eliminate cells with steep differences in Wg signaling. The experimental observation that even late depletion of Wg expression has an effect on wing size questions the proposal that continuous exposure to Wg is not an absolute requirement for wing cells to grow. Recently, a membrane-tethered form of the Wg protein was shown to be able to substitute for the endogenous Wg protein in producing normally patterned wings of nearly the right size. Either the activity of cellular extensions at a distance, higher stability of the membrane-tethered form of Wg, or emerging compensatory mechanisms should be able to facilitate or extend in time the exposure of all wing cells to the morphogen in the absence of secretion, thus fulfilling its continuous growth-promoting role (Barrio, 2020).

GENE STRUCTURE

The pan gene is located just upstream of and distal to cubitus interruptusand is transcribed in the opposite direction. It is a curious coincidence that pan and ci are adjacent genes, as ci encodes a transcription factor that is essential for transducing all examples of hedgehog signaling, whereas the present evidence suggests an equivalent role for PAN in wingless signal transduction (Brunner, 1997).

PROTEIN STRUCTURE

Amino Acids - 751

Structural Domains

The HMG-domain shows 88% identical residues with murine Lef-1. The N-terminal region of PAN is also conserved between PAN, Lef-1 and XTcf-3 (Brunner, 1997). Three regions of conservation are noted. First is the N-terminus, which in XTcf-3 and LEF-1 constitutes the ß-catenin interaction domain. Second is the high mobility group (HMG) box DNA-binding domain. Infrequently, an alternative exon encoding the second part of the HMG box is encountered. The alternative protein is termed dTCF-B. Third, a small region directly C-terminal to the HMG box is conserved among dTCF, TCF-1 and C. elegans pop-1 (van de Wetering, 1997).

A mammalian T cell factor 1 (TCF-1)-like protein from Drosophila, encoded by the pangolin (pan) locus consists of a DNA binding domain similar to that of other high mobility group proteins and a protein-protein interaction domain that binds beta-catenin (Armadillo in Drosophila) but it lacks a transcriptional activation domain. The pan locus spans approximately 50 kb and the mRNA results from the splicing of 13 exons. The open reading frame of pan is 2253 bp, encoding a putative 751 amino acid protein. The overall sequence identity, at the protein level, with murine TCF-1 and LEF-1 is 48% and 54% respectively. This identity is comparable to that seen between murine TCF-1 and LEF-1, which is 54%. The translation start site is at 414 bp. The HMG box is situated in the middle of the open reading frame, between nucleotide positions 1229 and 1453. This is followed by a stretch of 50 amino acids bearing striking similarity to human and mouse TCF-1 with almost 70% identity. This highly basic region just C-terminal to the HMG domain is conserved in TCF-1 and C. elegans POP-1 but severely truncated in LEF-1. This basic region is much less highly conserved compared with human and mouse LEF-1. There is a remarkable conservation of the exon/intron boundaries between the human and D. melanogaster genes, suggesting that they share a common ancestor. Chromosomal in situ hybridization locates pan to the base of chromosome 4, near the cubitus interruptus locus. pan and ci are separated by approximately 10 kb and are transcribed in opposite directions. Restriction map and sequence analyses confirm their close proximity. The small fourth chromosome undergoes little or no recombination and was previously reported to lack DNA polymorphisms; however, there are two DNA polymorphisms occurring in three combinations within the pan locus, demonstrating the presence of synonymous substitutions and the past occurrence of recombination. Evidence is presented suggesting that the protein encoded by pan is more similar to mammalian TCF-1 and Caenorhabditis elegans POP-1 than to mammalian LEF-1 (Dooijes, 1998).

date revised: 5 August 2023 

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