Separable functions of wingless in distal and ventral patterning of the Tribolium leg
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The gene wingless (wg) in Drosophila is an important factor in leg development. During embryonic development wg is involved in the allocation of the limb primordia. During imaginal disk development wg is involved in distal development and it has a separate role in ventral development. The expression pattern of wg is highly conserved in all arthropods (comprising data from insects, myriapods, crustaceans, and chelicerates), suggesting that its function in leg development is also conserved. However, recent work in other insects (e.g. the milkweed bug Oncopeltus fasciatus) argued against a role of wg in leg development. We have studied the role of wg in leg development of the flour beetle Tribolium castaneum. Using stage-specific staggered embryonic RNAi in wild-type and transgenic EGFP expressing enhancer trap lines we are able to demonstrate separable functions of Tribolium wg in distal and in ventral leg development. The distal role affects all podomeres distal to the coxa, whereas the ventral role is restricted to cells along the ventral midline of the legs. In addition, severe leg defects after injection into early embryonic stages are evidence that wg is also involved in proximal development and limb allocation in Tribolium. Our data suggest that the roles of wg in leg development are highly conserved in the holometabolous insects. Further studies will reveal the degree of conservation in other arthropod groups.
KeywordsWingless Leg development Arthropods Appendage evolution Tribolium castaneum
The gene wingless (wg) is mainly known for its role in segment polarity specification during body segmentation in Drosophila (reviewed in Sanson 2001). However, wg is also an important factor in Drosophila leg development (e.g., Cohen et al. 1993; Struhl and Basler 1993; Campbell et al. 1993). The leg primordium in Drosophila is already specified in the embryo. At embryonic stage 11 the primordium of the leg is still combined with the wing primordium in the so-called thoracic limb primordium (Cohen et al. 1993). Wg is required for the specification of the entire thoracic limb primordium where it activates the Distal-less (Dll) gene via the early Dll enhancer 304 (Cohen et al. 1993; Kubota et al. 2003; McKay et al. 2009). This early function of Wg is repressed on the dorsal side by Decapentaplegic (Dpp) signaling and on the ventral side by epidermal growth factor receptor signaling (Goto and Hayashi 1997; Kubota et al. 2000), thus restricting the thoracic limb primordium to the ventral–lateral side. At embryonic stage 14 the cells of the wing primordium downregulate Wg signaling and separate from the leg primordium (Cohen et al. 1993). The cells of the leg primordium subdivide further into proximal and distal cells. Wg is also involved in this process by activating the late enhancer LT ("leg trigger") of Dll in the distal cells, whereas the proximal cells do not require wg function at this stage (McKay et al. 2009; Estella and Mann 2008; Estella et al. 2008). This function of wg is separate from the early wg function as evidenced by the fact that Wg cooperates with Dpp rather than being repressed by it (Held et al. 1994; Diaz-Benjumea et al. 1994; Penton and Hoffmann 1996; Jiang and Struhl 1996; Theisen et al. 1996; Lecuit and Cohen 1997; Theisen et al. 2007). Finally, wg is then required for the specification of ventral fate in the legs (Struhl and Basler 1993; Theisen et al. 1996; Brook and Cohen 1996; Johnston and Schubiger 1996). This function, however, appears to be mechanistically different from the previous function where Wg protein forms a morphogen gradient. The ventral function of wg is local and does not require Wg protein to form a morphogen gradient (Diaz-Benjumea and Cohen 1994; Wilder and Perrimon 1995; Theodosiou et al. 1998). Instead, Wg might interact with other local factors to specify ventral fate through its target gene H15 (Wilder and Perrimon 1995). In summary, wg has three temporally separate and mechanistically different functions during the development of the Drosophila leg. The earliest function specifies and allocates the entire leg primordium including the proximal cells. By contrast, the later function in organizing pattern formation along the proximal–distal axis concerns only distal cells and the proximal cells do not require wg at this stage. The ventral role, finally, is temporally and functionally separate from the proximal–distal role.
The wg gene was the first segment polarity gene that has been shown to have a conserved expression pattern in long-germ and short-germ insects (Nagy and Carroll 1994). In the meantime, wg has been isolated and studied in a number of other arthropods including different insect species (e.g. Jockusch et al. 2000; Niwa et al. 2000; Dearden and Akam 2001; Miyawaki et al. 2004), as well as myriapods (Hughes and Kaufman 2002; Prpic 2004; Janssen et al. 2004), crustaceans (Duman-Scheel et al. 2002; Nulsen and Nagy 1999; Prpic 2008), chelicerates (Damen 2002; Prpic et al. 2003), and an onychophoran (Eriksson et al. 2009). These data show that the expression pattern of wg in all arthropods is highly conserved suggesting conserved functions. Surprisingly, however, functional tests have so far provided no support for highly conserved functions. Especially the three separate roles of wg in leg development (allocation, distal, and ventral development) seem not to be conserved. In the milkweed bug Oncopeltus fasciatus and the cricket Gryllus bimaculatus wg is necessary for normal body segmentation, but does not seem to have a role in leg development (Angelini and Kaufman 2005a; Miyawaki et al. 2004). These two species belong to more basal hemimetabolous insect groups and Angelini and Kaufman (Angelini and Kaufman 2005b) have therefore suggested that the role of wg in appendage development evolved only later in the holometabolous insects. Previous data from Tribolium castaneum indicated that in this holometabolous insect wg is indeed required for appendage allocation, because legs were absent after wg RNAi, but a role of wg in distal and ventral development remained unclear (Ober and Jockusch 2006). We have therefore studied the temporal requirement of wg in Tribolium leg development using staggered stage-specific RNAi. Our results show that in Tribolium wg has three consecutive roles in allocation, distal, and ventral development, like in Drosophila. Our data suggest that the origin of these functions in insect evolution must predate the split between Diptera and Coleoptera.
Materials and methods
Parental and embryonic staggered RNAi
Parental RNAi was performed as described previously (Bucher et al. 2002). The pupae were injected with wg dsRNA with a concentration of 3,000 ng/µl and incubated at 32°C until eclosion. Eclosed females were mated with wild-type males and the eggs of two consecutive egg lays (24 h each) were collected. For embryonic RNAi, eggs were collected directly after egg deposition and incubated at 25°C until injection (4, 8, 12, and 18 h after egg laying). Injections were performed with beveled borosilicate needles using a micromanipulator and a FemtoJet injection controller (Eppendorf). Injected embryos were incubated at 25°C until hatching or (for in situ expression analysis) until germ band retraction. Removal of the wg mRNA below the level of detection was confirmed in each case by whole mount in situ hybridization using a wg probe.
Embryo fixation and in situ hybridization
Embryos for in situ hybridization were dechorionized with a 50% solution of DanKlorix (Colgate-Palmolive) in water and fixed with 4% formaldehyde in a mixture of PEMS (0.1 M Pipes, 2 mM MgSO4, 1 mM EDTA; pH = 6.9) and heptane. Vitelline membranes were removed by methanol shock and subsequent shearing through a syringe needle (19 G gauge). Whole-mount in situ hybridization detection of expression of enhanced green fluorescent protein (EGFP) mRNA was performed as described previously (Prpic et al. 2001). After in situ hybridization embryos were fixed with 4% formaldehyde in phosphate-buffered saline with 0.02% Tween-20, pH = 7.4 and embedded in 80% glycerol for microscopy.
Microscopy and imaging
Analysis of the cuticle markers (leg bristles) was performed with a laser scanning microscope (Zeiss LSM 510). Cuticles and embryos from the in situ hybridizations were observed with differential interference contrast microscopy (Zeiss Axioplan-2). Images were captured with an Intas digital camera and were subjected to adjustment of brightness, contrast, and color values using Adobe Photoshop image processing software (Version 7.0 for Apple Macintosh).
A map of surface cuticle markers for the legs of T. castaneum
The present map of cuticular markers identifies several markers—distributed over most podomeres—that do not display individual variation. These markers are useful morphological landmarks for phenotypic analysis of leg phenotypes in the larva. The remaining markers are present in the majority of wild-type specimens and may be used for phenotypic analysis if the low amount of individual variation is taken into account. Only few markers, especially campaniform sensillae, display higher rates of individual variation and should not be used for phenotypic analysis.
Parental RNAi with Tc-wg
The layed eggs were incubated further and the rate of hatching was recorded. In the uninjected controls around half of the eggs were empty shells (hatching rate first egg lay, 61%; second egg lay, 42%; Fig. 3c). The percentage of empty egg shells increased in the GFP dsRNA-injected animals, and finally increased to over 95% in wg dsRNA injected animals (Fig. 3c). All eggs that reached hatching in both controls and also in the experiment gave rise to wild-type larvae. These data indicate that pRNAi with wg is leading to reduced female productivity (probably by interfering with gonad development or oogenesis), is embryonically lethal and the few obtained wild-type larvae in the wg dsRNA-injected animals likely are escapers.
Embryonic RNAi effects after staggered injections
Since the pRNAi experiments lead to either empty egg shells or wild-type cuticles, we turned to staggered embryonic RNAi (eRNAi). eRNAi circumvents the problem of effects already during gonad formation or oogenesis, and by staggering the injections the effects that lead to early embryonic lethality can also be excluded.
The distal phenotypes of the 18-h injections were similar to the "nonpareille" phenotypes of the 12-h injections, but were weaker in most cases. The weakest cases had all typical podomeres including the claw, but the femur was elongated and thinner than in the wildtype and the tibiotarsus showed ectopic constrictions (Fig. 7d). The intermediate phenotype had normal coxa and trochanter, but a shortened tibiotarsus, no claw and a single ectopic constriction in the femur (Fig. 7b), and the most severe phenotypes were "nonpareille" phenotypes with pearl-shaped tibiotarsus and rounded ectopic subdivisions of the femur, and the trochanter seems to be fused entirely to the coxa (Fig. 7c).
Evidence for a role of wg in leg allocation in Tribolium
Our experiments with pupal injections were not able to reveal a function of wg in leg development. Despite good eclosion and survival rates, the females that eclosed from pupae injected with wg dsRNA had a very low productivity and from the few eggs almost no larvae hatched; those larvae that did hatch were wildtype. The low productivity suggests that wg might have a role already during oogenesis. The low hatching rate indicates that wg has functions early in development that lead to early embryonic lethality and the few wild-type larvae must be escapers that by chance did not receive wg dsRNA.
Early injections into eggs lead to similar effects. Most of the eggs of the 4- and 8-h injections were empty or contained irregular cuticular structures similar to those observed by Bolognesi et al. (2008). The few larvae that developed showed severe phenotypes indicating problems with several developmental processes including segmentation. The legs of these larvae were severely malformed. The defects we have observed are very similar to those reported by Ober and Jockusch (2006). These authors also found segmentation defects and severely truncated or missing legs after wg RNAi. The specimens figured in their Fig. 2d–g are very similar to our results after early injections after 4 and 8 h. The specimen figured in their Fig. 2i shows shortened and severely malformed appendages, which is also very similar to what we have found in the early injections. Ober and Jockusch (2006) have interpreted these phenotypes as support for a role of wg in appendage allocation. However, because in all of these specimens the segments are also malformed, it is unclear whether the leg defects are true leg developmental defects or secondary defects because of the defective segment formation.
We avoided interfering with the early functions of wg in body segmentation by applying later injections after 12 and 18 h. The most severe leg phenotypes in these injections are seen in the 12-h injections: the legs are malformed and all parts of the leg are affected including the coxa. This is very similar to the early function of wg in leg allocation in Drosophila when Wg is required for activating the 304 enhancer of Dll in all cells of the leg primordium including proximal (coxal) cells (Cohen et al. 1993; McKay et al. 2009). We thus interpret the severe malformations in 12-h injections as evidence for a role of Tribolium wg in the specification and allocation of the entire leg primordium.
Separate functions of wg in distal and ventral development
In the "candy cane" and "nonpareille" phenotypes in the 12- and 18-h injections the coxa is not affected indicating that at this stage of development wg is not required for the development of the proximal leg parts anymore. The distal leg parts, however, are malformed and the distal-most portion, the claw, is missing in most specimens. This distal function is very similar to the distal function in Drosophila leg imaginal precursors where wg is required for the development of all leg portions distal to the coxa by activating the LT enhancer of Dll (Cohen et al. 1993; Estella et al. 2008; McKay et al. 2009). The extra constrictions in the femur or the tibiotarsus in the strong and weak "nonpareille" phenotypes, respectively, are unexpected and their origin is unclear. The normal number of constrictions (future joints) in Drosophila is specified by the action of the Notch pathway (de Celis et al. 1998; Rauskolb and Irvine 1999; Bishop et al. 1999) which is regulated by the leg gap genes and these are regulated by Wg signaling in conjunction with Dpp signaling (Lecuit and Cohen 1997; Estella and Mann 2008). It is currently unknown whether a similar cascade exists in Tribolium, but this is very likely because the role of the Notch pathway in leg segmentation is also conserved in the spider Cupiennius salei, a basally branching arthropod (Prpic and Damen 2009). Thus, it is possible that interference with the distal function of wg disturbs the normal segmentation of the Tribolium legs leading to ectopic constrictions in the distal segments.
The "ventral-less" phenotype appeared only in the 18-h injections. In these legs all segments are normal except that the ventral-most tissue is lacking. This ventral function appears to be separate from the distal function since the "ventral-less" phenotype does not show any distal defects. Again this is very similar to the ventral function of wg in Drosophila where Wg does not act as a concentration-dependent morphogen, but rather acts locally on the ventral side probably in conjunction with other ventrally expressed factors (Diaz-Benjumea and Cohen 1994; Wilder and Perrimon 1995; Theodosiou et al. 1998).
In summary, our results indicate three separate functions of wg in leg development of Tribolium, and these functions also are temporally separated. The early function of wg is the specification of the entire leg primordium. This is supported by the strong leg defects after the 12-h injections that affect all leg parts including the coxa. Later injections cannot interfere with normal coxa development (the "candy cane" and "nonpareille" phenotypes in which distal leg portions are affected, but the coxa is normal) suggesting that at this stage wg has a role in the formation of the entire distal leg portion, but not in the proximal cells anymore. Future studies have to reveal whether this distal function is performed by wg as a morphogen in conjunction with dpp as the second morphogen as it is the case in Drosophila (e.g. Lecuit and Cohen 1997). Finally, the late function of wg is the specification of ventral leg cells. This function is independent of the distal function, because in the "ventral-less" phenotype the formation of the proximal–distal axis is not affected. The fact that the effect is restricted to ventral leg cells is evidence that it does not involve a Wg gradient.
Our results indicate that the separate functions of wg in leg allocation, distal, and ventral leg development must have evolved before the split between Diptera and Coleoptera. Without further information from more basal insects or non-insect arthropods this suggests that the leg functions of wg have evolved in the last common ancestor of the holometabolous insects and thus earlier than suggested by Angelini and Kaufman (2005b). In order to exactly pinpoint the origin of the involvement of wg in arthropod leg development, the function of wg has to be studied in additional holo- and hemimetabolous insect species and also in other arthropod groups where functional data are currently lacking.
We thank Gregor Bucher, Ernst A. Wimmer, and Johannes Schinko for providing us with the enhancer trap lines Goe-04609 and Goe-12407 from the GEKU insertional mutagenesis screen. We especially thank Martin Klingler for his support and advice. We also thank the members of our group for stimulating discussions and Marco Winkler for technical assistance. This work has been supported by the Deutsche Forschungsgemeinschaft (grant number PR 1109/1-1).
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