Wnt6 is required for maxillary palp formation in Drosophila
© Doumpas et al.; licensee BioMed Central Ltd. 2013
Received: 24 July 2013
Accepted: 23 September 2013
Published: 3 October 2013
The Erratum to this article has been published in BMC Biology 2015 13:100
Wnt6 is an evolutionarily ancient member of the Wnt family. In Drosophila, Wnt6 loss-of-function animals have not yet been reported, hence information about fly Wnt6 function is lacking. In wing discs, Wnt6 is expressed at the dorsal/ventral boundary in a pattern similar to that of wingless, an important regulator of wing size. To test whether Wnt6 also contributes towards wing size regulation, we generated Wnt6 knockout flies.
Wnt6 knockout flies are viable and have no obvious defect in wing size or planar cell polarity. Surprisingly, Wnt6 knockouts lack maxillary palps. Interestingly, Wnt6 is absent from the genome of hemipterans, correlating with the absence of maxillary palps in these insects.
Wnt6 is important for maxillary palp development in Drosophila, and phylogenetic analysis indicates that loss of Wnt6 may also have led to loss of maxillary palps on an evolutionary time scale.
KeywordsDrosophila Wnt6 Maxillary palps
During animal development, tissue growth is tightly controlled, leading to adults of defined sizes and proportions. Tissue growth is regulated by a combination of patterning cues, which give each tissue a specific identity and hence size, and environmental cues, which are sensed through nutrient responsive pathways and act to proportionately scale the whole animal [1–4]. Despite intense interest in understanding how tissue growth is controlled, the underlying molecular mechanisms are only partly understood. The Drosophila wing has become one model system that is frequently used to study this problem. Growth of the fly wing is promoted via signals emanating from the anterior/posterior (A/P) and dorsal/ventral (D/V) compartment boundaries, such as Dpp and wingless respectively . Although wingless appears to be the main growth-promoting signal emanating from the D/V boundary, a second Notch-induced signal at the D/V boundary also non-autonomously induces wing growth . The wingless paralog Wnt6 is also expressed at the D/V boundary . Therefore, we decided to test whether Wnt6 might constitute this second signal.
Drosophila is one of the model systems in which Wnt signaling and function have been most intensively studied. Drosophila has seven Wnt genes. Of these, the best understood is wingless, the founding member of the class. Wingless has a myriad of functions during development. One function is to pattern epidermal cells to form repetitive patterns of naked cuticle and denticle belts, small tooth-like structures used for traction during larval crawling [7, 8]. Wingless is also necessary for development of all imaginal discs – the tissues resident in the larva which will give rise to adult tissues during metamorphosis. For instance, in the wing disc, early expression is responsible for specifying the wing primordium whereas later expression sets up the D/V axis of the wing [8, 9]. The remaining Wnts are comparatively less well studied; nonetheless, some functions are known for four of the remaining Wnts: Wnt3 is involved in axon guidance as well as salivary gland migration, Wnt2 regulates salivary gland migration, tracheal development and testis morphogenesis, Wnt8 is part of the Toll/Dorsal signaling network which both specifies the D/V axis of the embryo and participates in the immune response, whereas Wnt4 is critical for the regulation of cell motility during ovarian morphogenesis (reviewed in ). The functions of Wnt6 and Wnt10, however, are not known.
Wnt6 function has been studied in Xenopus, where it was found to be expressed in tissues close to and inside the developing heart, where it regulates heart organogenesis . Since, to our knowledge, Wnt6 mutant flies have not been reported, we generated Wnt6 knockout flies. We find that Wnt6 knockout flies, however, do not have growth defects in the wing. Instead, they completely lack maxillary palps. Together with antenna, maxillary palps are one of two olfactory epithelia in Drosophila. Recent studies suggest maxillary palps might also be involved in taste enhancement . The function of maxillary palps as an olfactory organ is well conserved throughout insects. For instance, mosquitos use maxillary palps to smell CO2, which is used for host seeking behavior . Hence, since Wnt6 knockout flies lack maxillary palps, they might constitute a useful tool for studying olfaction and behavior [13, 15].
Wnt6 knockout flies lack maxillary palps
Wnt6 can activate canonical Wnt signaling and is required for proper positioning of wing margin chemosensory bristles
Wnt6 is expressed in the maxillary palp anlage
Evolutionary loss of Wnt6 correlates with loss of maxillary palps
Wnt6 appears to have a specific role during Drosophila development, promoting maxillary palp formation. This is surprising, given that Wnt6 is quite ancient and present in most bilaterians [25, 26]. One possible interpretation is that the Wnt6 function might be redundant in most parts of the animal, perhaps due to overlapping expression with wingless, whereas Wnt6 expression in the maxillary palp might have been acquired in insects in a non-redundant fashion. This specific function in promoting maxillary palp formation might serve as a useful tool for studying the contribution of maxillary palps to olfaction and behavior. The maxillary palp contribution is currently assayed by surgical removal of the palps, whereas this could now also be accomplished genetically .
As previously noted , the Wnt6 gene is located directly adjacent to the wingless gene, raising the possibility that it arose as a genomic duplication of wingless. Accordingly, Wnt6 expression overlaps with that of wingless in numerous places . One possible reason for the overlapping expression patterns could be that Wnt6 expression is induced by wingless signaling; however, our data suggest this is not the case (Figure 3B). Instead, it is likely that they either share enhancer elements or that regulatory elements were also duplicated alongside the open reading frame. Since wingless and Wnt6 have similar expression patterns and presumably transcriptional regulation, and since the anti-wingless monoclonal antibody 4D4, the most widely-used in the field to detect wingless, appears to cross-react with Wnt6, some caution might be warranted in interpreting results with this antibody.
Given that Wnt6 is able to induce canonical wingless signaling in S2 cells (Figure 2A), we were surprised that Wnt6 is quite poor at inducing wingless signaling in the wing disc (Figure 2E-F’). Consistent with this observation, expression of UAS-Wnt6 with various GAL4 drivers such as patchedts-GAL4 (with GAL80ts) and nubbin-GAL4 cause pupal lethality; however, this does not yield obvious morphological defects in the resulting wings (not shown), suggesting that the lethality is likely due to expression in other parts of the body. In contrast, Wnt6 expression in the central nervous system, including the maxillary palp, induces obvious significant morphological effects. One possible explanation could be that a component required for Wnt6 signaling might be expressed at higher levels in the nervous system compared to wing discs.
The specific absence of Wnt6 from the aphid A. pisum and the plant lice D. citri, both belonging to the order Hemiptera, a group that has lost maxillary palps, suggests that Wnt6-loss could have been the underlying genetic alteration leading to this morphological change. In hemipterans, the mouthparts are modified to form a tube-like structure for piercing. The tube, formed by the labrum and labium, comprises piercing-sucking structures formed by the modified mandible and the maxilla . The evolutionary loss of the maxillary palps was one of many structural modifications leading to the specialized hemipteran mouthparts. The loss of the maxillary palps could have compromised the sense of smell in hemipteran ancestors, but this may have been compensated by the elaboration of sensory structures on the labium . The specific phenotype of the Wnt6 knock-out in Drosophila contrasts with the pleiotropic effects of other secreted signaling molecules including wingless. This means that the deletion of the gene, rather than tinkering with its regulatory regions, could have resulted in a subtle morphological change, the loss of the maxillary palp, contributing to the morphological evolution of the beak-like hemipteran mouthparts.
Although Wnt6 expression overlaps substantially with that of wingless, it appears to play a critical role in maxillary palp growth, but not wing growth. Phylogenetic analysis suggests that loss of Wnt6 also correlates with loss of maxillary palps on an evolutionary timescale.
All oligo sequences are listed in Additional file 3.
Generation of Wnt6KOand UAS-Wnt6 flies
Wnt6KO flies were generated by homologous recombination-based targeting using the 'ends-out’ strategy as previously described . Based on this strategy, 4 kb upstream and downstream flanks were amplified by PCR using the oligos described in Additional file 3, sequenced, and cloned into the pRK1 vector . Knockout flies were then back-crossed to the w1118 reference strain for five generations before studying. To generate UAS-Wnt6 flies, the Wnt6 ORF was amplified by PCR using the oligos listed in Additional file 3, and cloned into the EcoRI\NotI sites of pUAST .
Luciferase reporter assays
Luciferase reporter assays were based on the Promega pGL3 reporter system with a Wnt-responsive LEF7 firefly luciferase reporter and a renilla normalization control as previously described . Wingless and Wnt6 expression were achieved by co-transfecting pac-wg or pUAST-Wnt6 + pMT-GAL4/VP16, respectively .
S2 cells were grown in Express Five Serum Free Medium (Life Technologies, Carlsbad, California, USA) and transfected with Effectene (Qiagen, Venlo, Netherlands).
Antibodies used were mouse anti-wingless (4D4 Developmental Studies Hybridoma Bank); rat anti-Dll (Sean Carroll lab, R.M Bock Laboratories, 1525 Linden Drive, Madison, WI 53706) and guinea pig anti-sens .
The D. citri genome (Diaci1.1, 12× coverage) and transcriptome assemblies (Diaci_transcriptome_0.9) were downloaded from the International Asian Citrus Psyllid Genome Consortium website  and searched with TBLASTN with arthropod Wnt6 query sequences. The five top hits were re-blasted to the Uniprot and GenBank databases. D. citri Wnt1 is on the genomic scaffold scaffold5281.1|size8182|ref0095796|ref0108241.
planar cell polarity
Anti-wg antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa. ND is part of the Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS), University of Heidelberg.
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