318 EVOLUTION & DEVELOPMENT Vol. 6, No. 5, September^October 2004The CNS pattern in Oncopeltus was also different, with nubexpressingcells localized at each side of the central midline(Fig. 4B). As the appendages continued to elongate, there wascontinuing strong expression in the brain and in the headappendages but weakening of signal in the legs (Fig. 4C). Inthe mid-ventral region, whereas most of nub-expressing cellswere still in two mid-lateral columns, some cells started toexpress nub in the central midline of the mandibular segment(Fig. 4C). As a consequence, the CNS expression pattern inthis segment took an appearance of the letter ‘‘x.’’ The thirdkey feature of milkweed nub expression was observed duringlate development (Fig. 4, D and E), with all segments in themid-ventral region exhibiting this ‘‘x’’-like pattern.The most distinguishing feature of the Oncopeltus patternwas its dynamic and complex expression in the headappendages. Very early in development when limb buds werejust being formed, nub was strongly expressed in the ocularand brain region and in all head appendages (Fig. 4F). Forthe first time noted in hemimetabolous insect embryos, nubwas expressed in the antennae. Slightly later, at 20% ofdevelopment, the signal in the head limb buds became muchweaker (compare the staining in head and thoracic limb budsin Fig. 4A). However, the signal in the brain and ocularregion remained strong. At 25% of development, antennalexpression disappeared completely and the signal in themandibles became weak and diffuse (Fig. 4G). Maxillaryexpression remained strong and localized in the centralportion of the maxillary limb buds, whereas labial expressiondifferentiated into two diffuse spots (Fig. 4G, arrows). Thus,at this stage, each head appendage exhibited a unique nubpattern. By 30% of development, nub expression reappearedin antennae, but only in their ventral region (Fig. 4 H).Mandibular and maxillary expressions were similar, with adiffuse ventrally localized signal. nub expression in the labialappendages was weak at this stage and was restricted to theirdistal region. At 35–40% of development, antennal expressionencompassed the distal portion of these appendages (Fig. 4I).The mandibular and maxillary appendages exhibited anincrease in nub expression that extended throughout theirventral portions. In addition, a strong signal appeared towardthe dorsolateral sides of the maxillary segment. Expression inthe labial appendages also increased and remained localizedventrally in their distal regions. Finally, as the embryounderwent dorsal closure, the nub pattern changed again (Fig.4J). Expression in the antennae disappeared, the mandiblesand maxillae retained their strong ventral signal, and the fusedlabial appendages exhibited weaker expression in their distalregion. Overall, milkweed bug embryos were characterized bya complex pattern in the head region that had several uniquefeatures. First, Oncopeltus exhibited an expression in theantennae (in contrast to firebrats and cockroaches). Second,the antennal pattern was dynamic, with multiple appearancesand disappearances of nub. Third, in milkweed bugs it was themandibles and maxillae that showed a similar pattern,whereas in cockroaches and firebrats maxillae and labiumwere similar.In Oncopeltus embryos, the timing of leg segmentation wasintermediate compared with that of firebrat and cockroachembryos: It started later than in the cockroach but earlierthan in firebrats. In other words, at comparable middevelopmentalstages, milkweed bug embryos have fewerdistinct leg segments than cockroach embryos. At the sametime, firebrat embryonic legs exhibited no visible segmentationat all. As shown in Fig. 4A, all thoracic leg budsexpressed nub-during early germ-band extension. As theselimb buds elongated, a diffuse spot appeared at the base of thelegs nearest the body wall (Fig. 4 K, star). There were alsothree bands of nub-expressing cells located at proximal,middle, and distal leg regions (Fig. 4 K, arrowheads).Although there were no discernible leg segments at this stage,the proximal and middle bands roughly corresponded to thelocation of the coxa–trochanter/femur and femur–tibiaboundaries. However, as these leg segments became visible(Fig. 4L), nub expression subsequently disappeared except fora distal band in the tibial/tarsal segment (Fig. 4L). As legelongation continued, this band first became restricted to theventral side (Fig. 4M) and then became diffuse (Fig. 4N).Generally speaking, nub expression in milkweed bug legs isdistinct, encompassing both conserved and novel aspects.Furthermore, the observed leg pattern was also partiallyassociated with leg segmentation, but not at the level seen incockroaches. Whereas nub was localized at every leg joint inPeriplaneta embryos, its expression could only be associatedwith only three leg segments in Oncopeltus (consistent with thelater completion of leg segmentation in this species).DISCUSSIONFrom spiders to insects, the global evolution ofnub expression patterns in arthropod embryosnub is one of the few developmental genes for which extensivecomparative data are available. Among arthropods, nubexpression has been examined in chelicerates, includingspiders and horseshoe crabs (Abzhanov and Kaufman 2000;Damen et al. 2002), and in several crustacean species (Averofand Cohen 1997; Abzhanov and Kaufman 2000; Gibert et al.2002). Within insects, this gene was studied only in Drosophila(Lloyd and Sakonju 1991; Anderson et al. 1995; Isshiki et al.2001). Because higher flies have a highly derived mode ofdevelopment and a relatively recent phylogenetic origin, it isunclear whether the pattern observed in Drosophila isrepresentative of all insects. With our analysis of anapterygote and two hemimetabolous insects, it is now possibleto consider the evolution of nub expression in arthropods ingeneral.
Li and <strong>Popadic</strong>ŁEvolution of nub expression in insects 319Fig. 5. A cladogram summarizing evolution of nub expression patterns in arthropod embryos. (A) In the spider Steatoda triangulosa, nubexpression is restricted to the legs. (B) In the crustacean Porcelio scaber (woodlouse), the nub domain is extended to include the antenna 2segment. (C–E) The species-specific nub patterns within the insect lineage. Light blue depicts early expression in the legs of milkweed bugs.Striped boxes reflect subsequent modulations of CNS expression in each insect. Chelicerates: Ch, chelicerae; Pdp, pedipalp; L1, legs 1.Crustaceans: A1, first antenna; A2, second antenna; Mx1, maxillae 1; Mx2, maxilae2; T1/mxp, first trunk limb/maxilliped; all other labelsare as used in previous figures.AsshowninFig.5,nub expression is class specific andsometimes even species specific. Nonetheless, all examinedarthropods share a common expression pattern in appendages,indicating that nub was originally an ‘‘appendage’’gene. In chelicerates, generally thought to be basal arthropods,nub is localized exclusively to the walking legs and otherleg-derived structures (Abzhanov and Kaufman 2000; Damenet al. 2002). In the spider Steatoda triangulosa (Fig. 5A), asingle band of nub expression was detected in the tarsus of allprosomal legs. This basic chelicerate pattern was substantiallyaltered in the crustacean Porcelio scaber (Fig.5B).First,nubexpression spread anteriorly into the head region. Note thatthis head expression is incomplete, encompassing some butnot all the segments. Second, although nub is still restricted tothe distal leg segments, it is expressed in a set of rings (insteadof in a single band as in spider embryos). This refinement ofthe nub expression continues even further in insect embryos.In firebrat and cockroach embryos (Fig. 5, C and D), therewas no expression in the antennal and mandibular appendages.However, milkweed bug embryos exhibited a strongantennal and mandibular staining (Fig. 5E). Thus, the spreadof nub expression in the anterior direction is complete ininsects and now includes all the head segments. In addition,insect embryos also exhibit a further proximal expansion ofnub expression in the legs (Fig. 5, C–E). Whereas nub islocalized only in the distal leg segments in spiders andcrustaceans, its expression in insects encompasses proximalleg regions as well. This is particularly pronounced incockroaches (Fig. 5D), in which nub is expressed in all legsegments.
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