Dev Genes Evol (2009) 219:427–435 expression in the thoracic legs becomes slightly non- homogeneous (Fig. 2d), indicating the emergence of iterated rings in the legs at later stages. At fully retracted germband stages, the expression pattern in the appendages is more restricted (Fig. 2e). In the antenna, Sp8/9 is restricted to a large ventral patch at the distal end and a smaller dot dorsal to it. The expression in the mandible has ceased and the expression in the maxilla is very weak. In the labium, Sp8/9 expression is largely confined to a faint ring near the middle of the appendage. In the thoracic legs, the expression is seen in three rings. These expression rings are located proximally adjacent to the constrictions that mark the presumptive joints between the leg segments (podomeres; note that not all future podomeres are yet developed at this stage of development) (Fig. 2f). Functional analysis of Sp8/9 using RNAi In order to establish the function of Sp8/9 during development of Oncopeltus, we have applied parental RNAi (Liu and Kaufman 2004a). The results of these injections are summarized in Table 1. In wild-type embryos, shortly before hatching, the appendages lie flat on the body and reach the posterior end of the abdomen (Fig. 3d, e). In Sp8/9 RNAi embryos, the appendages are severely shortened. They stick out (Fig. 3a) and do not reach further than the middle of the body (Fig. 3a, b). In wild-type hatchlings the appendages are long and composed of several segments (podomeres) (Fig. 3f). The rostrum is a complex of four appendages (Fig. 3g). The labrum is thin and sharply pointed, the mandibles and the maxillae are long and thread-like, and the labium consists of four segments (Fig. 3g). The rostrum of the Sp8/9 RNAi animals is malformed (Fig. 3h). The labium is shortened; the distal segments are fused, bent, and enlarged at the tip. Therefore, the filiform mandibles and maxillae protrude from the labium at the distal end, while normally they are entirely ensheathed by it. The overall morphology of the mandibles, maxillae, and the labrum is normal in Sp8/9 RNAi animals, but they are shorter than in the wild type (Fig. 3h). The wild-type antennae consist of a basal antennifer and four antennal segments (Fig. 3i). The antennae are severely affected in all Sp8/9 RNAi animals (Fig. 3c, j). The 431 antennifer and the first segment of the antenna are roughly identical in size and shape to the wild type, but the three distal antennal segments are entirely fused and the antennal appendage as a whole is severely shortened. In about half of the cases, the fused distal antennal portion displays small ectopic outgrowths (arrow in Fig. 3j). The wild-type thoracic legs consist of a short coxa, a trochanter which is closely attached to the femur, a tibia, and a two-segmented tarsus with two claws (Fig. 3k). The legs of Sp8/9 RNAi animals are much shorter than in the wild type (Fig. 3c, l, m). All leg segments are present but some of them are fused together. The tarsal segments are always fused, but they can still be distinguished because constrictions indicating the rudimentary joints are still present. The trochanter and femur are also always fused. In some speci- mens, the proximal podomeres are so severely malformed and fused that they cannot be distinguished anymore (Fig. 3m). In contrast to these joints, the joints between femur and tibia and between tibia and tarsus are always present. Expression of Distal-less, dachshund, and homothorax in Sp8/9 RNAi embryos Since the results of the RNAi experiments indicated a role of Sp8/9 in appendage development, we next studied the expression of leg developmental genes expressed at different positions along the proximal–distal leg axis. The gene Distal-less (Dll) is expressed in the distal portion of all appendages except for the mandible (Fig. 4a, b) (Angelini and Kaufman 2004). In the head appendages, Dll is expressed in the tip of the labrum, the entire distal portion of the antenna, and the tips of maxilla and labium (Fig. 4a). The thoracic legs show two separate domains of Dll expres- sion: a large distal domain (“sock”) and a ring of expression proximal to it (Fig. 4b). The antennae are shorter in Sp8/9 RNAi embryos, but they express Dll in their distal region (Fig. 4c). In addition, Dll is expressed in the distal tips of labrum, maxilla, and labium in Sp8/9 RNAi embryos (Fig. 4c). The thoracic legs are much shorter in Sp8/9 RNAi embryos than in the wild type, but they show a “ring and sock” pattern of Dll expression similar to the wild-type legs (Fig. 4d). The dachshund (dac) gene is in the wild-type expressed in a medial ring in the thoracic legs, and in Table 1 Parental RNAi with Sp8/9 Sp8/9 pRNAi Control (injection buffer) WT n (%) 4 (2.4%) 104 (95%) Unspecific phenotype n (%) Appendage phenotype n (%) 6 (3.6%) 5 (5%) 160 (94%) 0 (0%) Total n 170 109 Given are the total numbers (n) of specimens in each category. WT stands for wild-type specimens after injection. Unspecific phenotypes were specimens that showed some development, but had died before any discernible morphology was formed. These phenotypes had a similar frequency in the Sp8/9 pRNAi and in the control injections. The appendage phenotype was obtained only in the Sp8/9 pRNAi