434 of dac expression is present as in the wild-type legs (Fig. 4f, g). The gene homothorax (hth) is in the wild type expressed in the proximal area of all appendages (Fig. 4h), but the distal extension of expression is different in the different appendage types (Angelini and Kaufman 2004). Gene expression is restricted to the proximal third of the legs, but is expressed in the proximal two thirds of the appendage in antennae, mandible, and labium and fills the proximal half of the maxilla (Fig. 4h). This proportional pattern is identical in Sp8/9 RNAi animals although the legs are shorter than in the wild type (Fig. 4i). A conserved role in appendage axis elongation Previous work in Drosophila has shown that the D-Sp1 gene is expressed in the thoracic limb primordia in the embryo and in the leg imaginal discs in the larva (Wimmer et al. 1996; Schöck et al. 1999; Estella et al. 2003). In the leg discs, D-Sp1 is expressed in concentric rings that roughly correspond to the position of the future joints between the leg segments. This expression pattern is very similar to the late expression of Sp8/9 in the legs of the beetle T. castaneum. In this insect species, Sp8/9 is expressed in up to four segmental rings that lie at a similar location in the legs as the expression rings of the Serrate (Ser) gene, which encodes the ligand of the Notch (N) receptor (Beermann et al. 2004). RNAi experiments with Sp8/9 have shown that the podomeres are severely shortened and sometimes fused, and that the number of Ser rings is also reduced. Based on these data, Beermann et al. (2004) suggested that Sp8/9 is involved in the control of allometric growth of the individual leg segments, probably by interfering with the Notch pathway, which is known to control allometric podomere growth in Drosophila and other arthropod species (de Celis et al. 1998; Rauskolb and Irvine 1999; Bishop et al. 1999; Prpic and Damen 2009). It has been noted previously that the process of leg segment growth is tightly linked with the process of joint formation, because both processes are regulated by the Notch pathway and its targets (Milán and Cohen 2000), and this would then also explain the observed podomere fusions. Our results in Oncopeltus support an evolutionarily conserved role of Sp8/9 in the control of allometric podomere growth. First, the Sp8/9 expression pattern in the legs after full germband elongation is very similar to the pattern in Tribolium and consists of several rings adjacent to the constrictions of the future leg joints. Second, after RNAi, all leg segments are still present but severely shortened compared to the wild-type legs. In addition, the podomere fusions observed in the legs and in the antennae are compatible with the notion that the Notch pathway is also affected, because similar podomere fusion phenotypes Dev Genes Evol (2009) 219:427–435 are observed in Drosophila when members of effectors of the Notch pathway are impaired (e.g., de Celis et al. 1998; Rauskolb and Irvine 1999; Bishop et al. 1999). No evidence for a role of Sp8/9 in appendage specification There is some evidence in Drosophila that D-Sp1 has a role in leg specification by activating Dll expression in the embryonic leg primordia together with Wg and Dpp signaling and Dll autoregulation (Estella et al. 2003; McKay et al. 2009). However, this evidence is not conclusive because the experiments were not able to discriminate between the effect of D-Sp1 and the effect of the neighboring gene btd. Beermann et al. (2004) suggest that Dll is a target gene of Sp8/9 in Tribolium, although Dll is still expressed in the legs after Sp8/9 RNAi. In summary, the evidence for a role of Sp8/9 in leg specification and Dll activation in the insects studied so far is inconclusive. Our results with Sp8/9 RNAi in Oncopeltus provide no evidence for a role of Sp8/9 in appendage specification or Dll activation. All appendages are present in Sp8/9 RNAi animals and the legs consist of all podomeres including the distal claws. This suggests that the specification of the appendages and their overall proximal–distal patterning is not disrupted. This is further evidenced by the expression of the proximal–distal marker genes Dll, dac, and hth in Sp8/9 RNAi animals. Although the legs in these animals are much shorter than in the wild type, the expression of Dll, dac, and hth is proportionally identical to the wild-type expression, indicating that proximal, medial, and distal fates are present. These data also suggest that Dll, dac, and hth expression is not dependent on activation by Sp8/9. We note, however, that we found very weak residual expression of Sp8/9 after Sp8/9 RNAi (data not shown), and thus we cannot exclude the possibility that the phenotypes we obtained do not represent the loss-of-function (null) phenotype. Acknowledgments NDS would like to thank Paul Z. Liu for introducing her to Oncopeltus work and Nipam H. Patel for support and advice. This work has been supported by an Education Abroad Program Grant and a Fulbright Travel Grant (both to NDS), the European Community’s Marie Curie Research Training Network ZOONET under contract MRTN-CT-2004-005624 (to EAW) and by a DFG Emmy Noether Program grant PR 1109/1-1 (to NMP). Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References Angelini DR, Kaufman TC (2004) Functional analyses in the hemipteran Oncopeltus fasciatus reveal conserved and derived aspects of appendage patterning in insects. Dev Biol 271:306–321