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EVOLUTION & DEVELOPMENT 6:5, 310–324 (2004)Analysis of nubbin expression patterns in insectsHua Li and Aleksandar Popadić Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA Author for correspondence (email: apopadic@biology.biosci.wayne.edu)SUMMARY Previous studies have shown that the genenubbin (nub) exhibits large differences in expression patternsbetween major groups of arthropods. This led us tohypothesize that nub may have evolved roles that areunique to particular arthropod lineages. However, in insects,nub has been studied only in Drosophila. To further explore itsrole in insects in general, we analyzed nub expressionpatterns in three hemimetabolous insect groups:zygentomans (Thermobia domestica, firebrat), dyctiopterans(Periplaneta americana, cockroach), and hemipterans(Oncopeltus fasciatus, milkweed bug). We discovered threemajor findings. First, observed nub patterns in the ventralcentral nervous system ectoderm represent a synapomorphy(shared derived feature) that is not present in otherarthropods. Furthermore, each of the analyzed insectsexhibits a species-specific nub expression in the centralnervous system. Second, recruitment of nub for a role in legsegmentation occurred early during insect evolution.Subsequently, in some insect lineages (cockroaches andflies), this original role was expanded to include joints betweenall the leg segments. Third, the nub expression in the headregion shows a coordinated change in association withparticular mouthpart morphology. This suggests that nubhas also gained an important role in the morphologicaldiversification of insect mouthparts. Overall, the obtaineddata reveal an extraordinary dynamic and diverse pattern ofnub evolution that has not been observed previously for otherdevelopmental genes.INTRODUCTIONArthropods are the most diverse and successful animalphylum, encompassing a spectacular range of morphologicaldiversity. This extraordinary diversity was made possible bytheir modular body design, a key feature of which is thedivision of bodies into separate modules (segments). In termsof macroevolutionary trends, arthropod evolution has beencharacterized by fusion of segments into discrete functionalunits. This process, tagmosis, resulted in the establishment ofdistinct morphological and functional body regions, such asthe head, thorax, and abdomen in insects. At the same time,the identity of a particular segment is influenced by the typeand function of its appendages. These two processes, theestablishment of distinct body regions and appendagediversification, are mutually interdependent and togetheraccount for a large portion of the morphological diversityin arthropods. Thus, elucidating the molecular mechanismsthat have contributed to these two trends is key tounderstanding morphological evolution in arthropods.In the past decade, largely through the evolutionarydevelopmental studies of homeotic (Hox) genes, we havestarted to gain insight into the molecular basis of tagmosis(Raff 1996; Carroll et al. 2001; Wilkins 2002). Because of theirrole in establishing segmental identities along the anteroposterioraxis, Hox genes can serve as useful molecular markers310to study patterns of regional differentiation leading to theformation of distinct body regions (Carroll 1995; Gellon andMcGinnis 1998; Popadić et al. 1998a; Hughes and Kaufman2002). The emerging view from these studies indicates thatchanges in both the expression patterns and functionaldomains of Hox genes have played an important role inestablishing distinct body plans that characterize the fourmajor extant arthropod groups (Cohen 1993; Carroll et al.2001). In addition, alterations of homeotic gene expressionpatterns are also associated with the morphological divergenceof specific appendages. For example, changes inproboscipedia patterning are tightly linked with the transformationof chewing to sucking mouthparts in insects (Rogerset al. 2002), whereas Ultrabithorax variations can becorrelated with the morphological evolution of the anteriorthoracic legs in mallacostracan crustaceans (Averof andCohen 1997). However, much less is known about the roleof other nonhomeotic genes in diversification of arthropodappendages. A number of recent studies indicate that nubbin,or pdm-1, may be a good candidate for such a role.Nubbin (nub) belongs to the POU homeodomain genefamily; hence, it is also referred to as a pdm (POU domainprotein) (Billin et al. 1991). POU domain genes aredevelopmental regulators involved in a number of differentprocesses such as cell migration, proliferation, lineage,neurogenesis, and the differentiation of specific structures& BLACKWELL PUBLISHING, INC.

Li and PopadicŁEvolution of nub expression in insects 313Fig. 1. Sequence comparisons and phylogenetic relationships of the arthropod Pdm/Nub orthologs. The names of sequences that weregenerated by this study are highlighted in bold. (A) Alignment of amino acid sequences of the Pdm/Nub and Vvl orthologs from differentarthropod species. The POU domains and homeodomains of both genes have unique amino acids that can serve as reliable diagnosticmarkers for homology assignment. The amino acid differences between Pdm and Vvl genes are highlighted in gray. Portions of the POUdomain and the homeodomain within the nub and vvl fragments are marked above the sequences. Arrows mark the sequences of primers usedin this study. The sequence corresponding to the in situ probe is marked with a bar. (B) Neighbor-joining tree depicting the sequencerelationships of the firebrat, cockroach, and milkweed bug Pdm genes to other PDM/NUB/OCT family members (class II POUhomeodomainproteins) and to members of the related Vvl genes (class III POU-homeodomains). This unrooted tree shows a clearassignment of the three insect nub genes to the PDM/NUB/OCT family with a strong bootstrap support (100%). The internal nodes in thePDB/NUB/OCT family are not significantly resolved. All sequences except those of the three insect species (Oncopeltus, Periplaneta, andThermobia) were acquired from GenBank; for accession numbers see Materials and Methods. Dm, Drosophila melanogaster; Of,themilkweed bug Oncopeltus fasciatus; Pa, the cockroach Periplaneta americana; Td, the firebrat Thermobia domestica; Ps, the woodlousePorcellio scaber; Pc, the crayfish Procambarus clarkii; Af, the brine shrimp Artemia franciscana; St, the spider Steatoda triangulosa; Cs, thespider Cupiennius salei; Hs, human Homo sapiens; Sp, the sea urchin Strongylocentrotus purpuratus.

314 EVOLUTION & DEVELOPMENT Vol. 6, No. 5, September^October 2004Fig. 2. The expression patterns of nub in Thermobia domestica (firebrat) embryos. Below the images of each stage are correspondingmagnified insets of both the head and thoracic appendages (A’, B’, C’, and D’, respectively) and of the posterior abdominal region (A’’, B’’,C’’, and D’’, respectively). Earlier stages were not stained. An, antenna; Mn, mandible; Mx, maxilla; Lb, labium. T1–T3, thoracic legs;A5–A10, abdominal segments. Arrowheads were used to indicate staining pattern in head appendages, whereas arrows point to theexpression in thoracic appendages. Stars indicate the nonspecific staining in pleuropodia (appendages of segment A1).(Fig. 2C’, arrows). In the mid-ventral neuroectoderm, nubexpression was expanded laterally to include an additionalcluster of cells on both sides of the embryo. This expansionwas most noticeable in the thoracic region and in the posteriorabdomen in segments A5–A8 (Fig. 2C’’). In addition, thetrend toward fusion of the two mid-ventral regions continued

Li and PopadicŁin the posterior abdomen. The final modulation of the firebratpattern occurred toward the end of dorsal closure (Fig. 2D).Although nub continued to be absent in the antennae, it wasstrongly expressed around the base of the mandibles. It wasalso expressed in the basal and proximal regions of themaxillae and in most of the labial appendages (Fig. 2D’). Inthe legs, there were three distinct domains of nub expression: aproximal region corresponding to the future coxal segment, amiddle region, and in the distal tip. In the mid-ventral region,nub expression was restricted to cells in the center of eachsegment; lateral expression completely disappeared (compareFig. 2C’’ and Fig. 2D’’). In summary, firebrat embryos werecharacterized by a distinct and dynamic nub expression patternquite different from those observed in other arthropods.Embryonic expression pattern of nub inPeriplaneta americana (cockroach)UsingaclonedPa nub cDNA fragment for whole-mount insitu hybridization, we also studied nub mRNA expressionpatterns in embryos of this species. Cockroaches belong toone of the more basal insect orders (Blattodea) and undergotypical hemimetabolous development. However, comparedwith those of Thermobia, Periplaneta embryos begin legsegmentation at a much earlier stage. Thermobia embryoniclegs become segmented quite late, around the time of dorsalclosure, when these appendages have almost reached theirfinal length. In contrast, Periplaneta appendages undergosegmentation much earlier, during germ-band extension,around the time when legs just begin to elongate.At early stages of Periplaneta development, two featurescharacterized nub expression pattern in this species. First,there was strong distinct nub expression in the appendages. Infirebrat embryos, nub was mostly absent from the appendages(compare Figs. 2A and 3A). Second, there was weakexpression in the middle and lateral region of the abdominalsegments. The abdominal pattern was characterized by anascending gradient in the posterior direction from A1 to A10,resulting in the strongest nub expression being in theposterior-most abdominal segments (Fig. 3, A and B). Thisis reminiscent of abdominal nub expression in firebratembryos, which was also distinguished by an ascendinggradient in the posterior direction (compare Figs. 2A and 3A).As cockroach development progressed, nub expressioncontinued to be dynamic, with distinctly different patterns inthe head and thoracic appendages (Fig. 3C). A high level ofnub appeared in the mid-ventral region, starting at the labialsegment and ending at the segment A1 (Fig. 3C). The earlierfaint expression in the abdomen faded, and only the strongmost posterior abdominal expression persisted (Fig. 3D). Inlater stages of development, when the embryos began dorsalclosure, the expression of nub in the appendages continued tochange (Fig. 3E). Expression disappeared in the antennae andEvolution of nub expression in insects 315mouthparts and was restricted to several clusters of nubexpressingcells in the legs. The most distinct feature of thislate pattern was strong and elaborate mid-ventral neuroectodermalexpression, from the labial to the segment A4 (Fig.3E). This CNS pattern was not uniform and encompassed awider mid-ventral segmental region in the thorax and a morenarrow area in the abdomen. At this time, posteriorabdominal expression was restricted to A11 (Fig. 3E).In the head region, from early to late stages, nub wasabsent from antennae. As limb buds began to elongate, nubwas expressed throughout the maxillae and labium (Fig. 3F)and in a diffuse spot in the mandible (Fig. 3G). By 50% ofembryogenesis, mandibular expression disappeared (Fig. 3H).Maxillary and labial expression continued to be similar andresolved into a distinct ‘‘rings and sock’’ pattern, with two‘‘rings’’ in the middle and a ‘‘sock’’ at the tip of both maxillaryand labial palps (Fig. 3H). In addition, diffuse nub expressionwas also observed in the proximal portion of the maxillaryand labial appendages, corresponding to the future lacinia(glossa) and galea (paraglossa). As development progressed,only cells in two rings in the maxillary and labial palpscontinued to express nub (Fig. 3I). Finally, by the end ofdorsal closure, nub expression in all head appendages wascompletely lost (Fig. 3J).Highly specific nub expression was also observed in thethoracic legs. At early stages, when the leg limb buds justbegan to elongate, three bands of nub expression were seen inthe distal half of each leg (Fig. 3K). In the proximal to distaldirection, we labeled these bands as 1 (most proximal, withthe narrowest and weakest level of expression), 2 (located inthe middle, with the highest level of expression), and 3 (mostdistal, similar in intensity to 1). As appendages continued toelongate, a new band (4) of nub expression appeared in thedistal portion of the leg (Fig. 3L). At about 50% stage, thecockroach legs underwent segmentation, and it was at thistime that a fifth band of nub (5) appeared in the distal portionof the appendage (Fig. 3M). At this stage, the spatial andtemporal organization of nub expression coincided preciselywith the establishment of distinct leg segments: the boundarybetween body wall and coxa (band 1), between coxa andtrochanter (band 2), between trochanter and femur (band 3),between femur and tibia (band 4), and between tibia andtarsus (band 5). As the embryo began dorsal closure, the legsbecame fully segmented and the coxa became enlargedcompared with the other segments (Fig. 3N). Although nubexpression in each segment was still detectable, the previouslystrongly stained bands started to fade in an anterior (band 5)or in posterior direction (bands 3 and 4). The exceptions werebands 1 and 2, which continued to display a strong signal.Toward the late stages of embryogenesis, legs were fullyelongated and only clusters of cells continued to express nub(Fig. 3O). On the ventral side of the coxa, there was distinctand strong nub expression in the posterior part and weaker

316 EVOLUTION & DEVELOPMENT Vol. 6, No. 5, September^October 2004Fig. 3. The expression pattern of nub in cockroach Periplaneta americana embryos. (A–E) Early nub expression is localized specifically in theappendages and posterior abdomen, and only during late development does it extend into the mid-ventral region. (F–J) Highermagnification inserts of nub expression in the head appendages. (K–O) A series of magnified T2 leg inserts showing the dynamic nature ofnub expression during leg development. An, antenna; Mn, mandible; Mx maxilla; Lb, labium; T1–T3, thoracic legs; A3–A10, abdominalsegments. Leg segments: Cx, coxa; Tr, trochanter; Fe, femur; Ti, tibia; Ts, tarsus. Stars indicate the nonspecific staining in pleuropodia(appendages of the A1 segment).expression anteriorly. Two additional clusters of nub-expressingcells were observed in the trochanter, although at a verylow level. There was also novel nub expression in the tipof each leg.Embryonic expression patterns of nub in ahemipteran Oncopeltus fasciatusTo study hemipteran nub mRNA expression, we used thecloned Of Pdm cDNA fragment for whole-mount in situhybridization. Hemipterans represent one of the phylogenetically‘‘younger’’ hemimetabolous insect orders, comparedwith Zygentoma (firebrats) and Blattodea (cockroaches).Thus, studies of milkweed bug embryos provide an importantdata point, allowing us to infer whether the trends in nubexpression observed in more basal insect groups extend tohemipterans as well.Three key features characterized the nub expression inOncopeltus embryos (Fig. 4, A–E). First, during earlydevelopment, strong nub expression was localized primarily

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 PopadicŁEvolution 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.

320 EVOLUTION & DEVELOPMENT Vol. 6, No. 5, September^October 2004Theotherkeytrendintheevolutionofnub expression inarthropods is in its expansion from the periphery of theembryo (appendages) to the mid-ventral region. In bothchelicerates and crustaceans, there is complete absence of nubin the center of the embryo (Fig. 5, A and B). In insects,however, mid-ventral expression in neuroectoderm is one ofthe most noticeable aspects of nub expression. Intriguingly,each insect species exhibits a unique species-specific CNSpattern (Fig. 5, C–E). Functional studies in Drosophila showthat nub is part of a gene regulation cascade specifying theidentity of developing neuroblasts in the CNS (Yeo et al.1995; Brody and Odenwald 2000; Isshiki et al. 2001). Thesefindings, combined with our observations, suggest that nubexpression in the CNS is unique to the insects and hascontinued to evolve toward more elaborate spatial andtemporal patterns that coincide with diversification of insectmorphology.Expression patterns within insects are dynamicand species specificAs depicted in Fig. 5, nub expression patterns exhibitedsubstantial differences between and within the major arthropodclasses. However, in groups such as spiders andcrustaceans, these differences are rather static (Fig. 5, A andB). More specifically, once the pattern of expression isestablished, it does not change during development. Incontrast, our data clearly indicate that variation is a keyfeature of nub expression within insect embryos. The bestillustration of this variation is provided by dynamic temporaland spatial changes of nub expression in the CNS andantennae.If one compares the timing of its expression in the CNSversus appendages, it is evident that the onset of nubexpression within the CNS is highly variable. In firebratembryos, nub is first expressed in the CNS and is subsequentlydetected in the appendages. In cockroach embryos, theearliest expression of nub is detected in the appendagesfollowed by the CNS. And in milkweed bug embryos,expression of nub in appendages and CNS occurs simultaneouslyat a very early stage. In addition to this temporalvariation, nub also exhibits spatial variation in its pattern. nubexpression in the CNS was found to have a unique speciesspecificpattern for each insect species studied. In firebrats,nub is expressed in groups of cells aligned along the midline ofeach segment in a uniform pattern that persists throughoutdevelopment. In the cockroach, nub expression appears in anirregular triangular pattern encompassing a wider region inthe thoracic than in the abdominal segments. Once established,this pattern does not change from early to latedevelopment. In contrast, CNS expression of nub in milkweedbugs is very dynamic. In early developmental stages, nub isexpressed in cells aligned along either side of the midline, withexpression excluded from any of those cells within themidline. As development proceeds, cells within the midlineform an ‘‘x’’ pattern of expression in each segment, beginningin T1 and ending at the posterior abdominal segment.Antennal expression represents another example of ahighly variable nub pattern. In embryos of basal insect groupssuch as firebrats and cockroaches, nub is absent in antennae(Fig. 5, C and D). This observation is also consistent withthose on spiders and crustaceans. No nub expression is foundin the first two prosomal segments (cheliceral and pedipalpal)in spiders. Similarly, nub is absent from the antennal 1segment of crustaceans (Abzhanov and Kaufman 2000).These observations indicate that lack of nub expression in theanterior-most appendages is an ancestral arthropod feature.However, in more derived hemimetabolous insects such asmilkweed bugs, we observed nub in this new domain.Moreover, its expression undergoes dramatic temporal andspatial change during development (Fig. 4, F–J). Early on,nub is strongly expressed throughout the antennae. Then, asthe appendages start to elongate, its expression is lost. Asdevelopment of appendages continues, nub accumulationreappears again in a cluster of cells at the posterior-distalportion of antennae and then shifts to their most distal partand continues to be strongly expressed during dorsal closure.Toward the end of development, its expression disappearsagain. nub accumulation has also been detected in the eye/antennal disc of Drosophila larvae (Dick et al. 1991;Rodriguez Dd Ddel et al. 2002), further suggesting this novelpattern to be of relatively recent origin and to be sharedamong phylogenetically more derived insect groups.This study revealed that nub function may be evolvingmore rapidly than that of homeotic or other leg patterninggenes such as Dll and dac (Panganiban et al. 1995; Popadic´ etal. 1998b; Prpic et al. 2001; Hughes and Kaufman 2002).Also, the most dynamic temporal and spatial changes inexpression occurred in milkweed bugs, a relatively derivedhemimetabolous insect. Additional dynamic changes in nubpattern were observed in firebrat and cockroach embryos,especially in legs and mouthparts. These observations pose anintriguing question as to what level of developmentalvariation actually exists in nature. Whereas traditional viewssupport relatively high levels of conservation in developmentalpathways (Raff 1996), the present analysis of nubsuggests that variation may be more prevalent than previouslythought.Role of nub in insect leg segmentationMainly based on insights from leg development in Drosophila,it is now known that the Notch (N) signaling pathway plays afundamental role in the process of segmental boundaryformation in legs (de Celis et al. 1998; Rauskolb et al. 1999;Casares and Mann 2001). In flies, the actual joint formation is

Li and PopadicŁmediated by establishing distinct rings of N expression inthese regions of the leg. nubbin is also expressed in a series ofconcentric rings in third instar Drosophila leg discs (Andersonet al. 1995; Averof and Cohen 1997), and its mutantexpression results in shortened and gnarled legs (Cifuentesand Garcia-Bellido 1997). Mutant clones of Notch also causeloss of nub expression in Drosophila legs. Conversely, ectopicexpression of nub is induced within clones of cells expressingactivated Notch (Rauskolb et al. 1999). Thus, in Drosophilalegs, nub is positively regulated downstream of Notch.Whereas this information indicates that nub is also involvedin leg patterning in Drosophila, in addition to its previouslydescribed roles in CNS and wing development, the questionremains as to when this acquisition of leg function occurred.As shown in Fig. 5A, in spider embryos nub is localized toa single distal band on all walking legs. In crustacean embryoswith joint-less trunk appendages such as Artemia franciscana,no expression of nub has been detected (Averof and Cohen1997). However, in crustaceans with segmented endopods(main leg branches), nub is localized to a series of rings thatcorrespond to joints in the distal leg region (Abzhanov andKaufman 2000; Damen et al. 2002). These findings indicatethat nub may have a partial role in leg segmentation inchelicerates and crustaceans, mainly during the establishmentof distal leg segments. However, as is obvious from Fig. 5, Aand B, formation of most leg joints in those groups has to beunder the control of other genes.As summarized in Fig. 5C, there are two dominant bandsof nub expression in firebrat legs, a proximal band and a middistalband. The locations of these bands roughly correspondto the positions of the future coxa–trochanter and femur–tibiaboundaries, respectively. But leg segmentation in firebratsoccurs very late in development when it is technically verydifficult to perform the in situ experiments (due to thedeposition of embryonic cuticle). This prevents us frominferring the complete pattern of nub in this species and torelate it to the formation of joints. Leg development inPeriplaneta embryos is quite different, and clearly identifiableleg segments can be recognized by mid-stages of embryogenesis.In this species, the appearance of five nub stripes precedesany visible demarcation of distinct leg regions (Fig. 5D). Onlyafterward does continuing nub expression begin to coincidewith the formation of segmental boundaries in the legs (Fig. 3,M and N). This coordination between the precise patterningof nub expression and leg segmentation in the cockroachembryos is highly indicative of a role in formation of joints.The observed pattern in Periplaneta is also reminiscent ofNotch signaling-regulated nub expression in Drosophila legdevelopment. These similarities suggest that regulation of legsegmentation in two widely divergent insect species may behomologous.The timing of leg segmentation in milkweed bug embryosis intermediate compared with firebrats and cockroaches.Evolution of nub expression in insects 321Proximal leg segments in Oncopeltus are established relativelyearly during germ-band extension, but segmentation of distalleg regions does not occur until late development. As inPeriplaneta, thelegpatternsofnub expression in Oncopeltusroughly correspond to the position of future joints and itsappearance precedes formation of segments. There are alsotwo main differences between observed nub patterns betweenmilkweed bugs and cockroaches. First, nub expression inOncopeltus legs is composed of only three bands (Fig. 5E)versus five bands in Periplaneta. Second, two of these bands inOncopeltus begin to fade much earlier (compared withcockroach). The possible explanation for the first discrepancyis that milkweed bug nub may be involved in formation ofsome, but not all, leg segments. The likely explanation for thesecond discrepancy is that nub mayplayaroleininitiatingtheformation of some leg joints in Oncopeltus but is not requiredfor its maintenance.Overall, our analysis suggests that recruitment of nub for arole in leg segmentation may have occurred early in insectevolution. In both insects and crustaceans, nub expression isassociated with establishment of some but not all legsegments. In insects, the primary association is with proximaland mid-leg segments. However, only the two most distalsegments may be involved in crustaceans. These findingsindicate that nub cannot be a necessary element in theregulation of overall leg segmentation in arthropods. Rather,the observed expression patterns suggest that this gene wasindependently recruited in insects and crustaceans for apossible role in development of specific leg joints. Subsequently,in some insect lineages (cockroaches and flies) thisoriginal role was expanded to include joints between all legsegments.Role of nub in differential development of headappendagesThe insect head has a modular organization and is composedof the three pregnathal (ocular, antennal, and intercalary) andthree gnathal segments (mandibular, maxillary, and labial). Apair of appendages grows from each gnathal segment andforms the future mouthparts (Brusca and Brusca 1990).Diversity in organization of the insect head is best representedby the variation in structure and morphology of themouthparts. There are two basic types of mouthparts:mandibulate, specialized for chewing and biting, andhaustellate, specialized for piercing and sucking (Matsuda1965). The mandibular type represents the ancestral form andis characteristic of members of most basal hexapod lineages(Zygentoma, Orthoptera, Blattoidea, etc.). Its main feature isthat the mandibles function as ‘‘jaws,’’ whereas the maxillaryand labial limbs form branched appendages that arestructurally identical until the midline fusion of the latter(Fig. 6A, left). In the haustellate type, it is the mandibular and

322 EVOLUTION & DEVELOPMENT Vol. 6, No. 5, September^October 2004Fig. 6. Evolving pattern of nub expression in insect mouthparts. (A) Diagrams of two basic types of mouthparts in insects. In mandibulatetype (left), the mandibles function as jaws and are structurally different from both maxillae and labium. The latter two segments arestructurally and functionally very similar, but the labium appendages fuse to form a lower lip. In the haustellate type (right), the mandiblesand maxillary laciniae form wire-like stylets. In contrast, the labium forms a stylet-enclosing sheath. (B) Differential expression of nub (darkblue) in embryonic insect head appendages is associated with particular mouthpart morphology. In embryos of biting and chewing insects(Thermobia and Periplaneta), nub expression is similar in the maxillary and labial appendages. nub is generally not expressed in themandibles except transiently. However, in the haustellate type (Oncopeltus), mandibular expression is similar to the maxillary pattern.Expression in the labium occurs only in the distal region of the appendage. Light blue denotes transient nub expression in the mandibularsegment. Stripped ovals depict species-specific modulations of the labial and maxillary expressions in Thermobia and Periplaneta,respectively.maxillary appendages that are similar, whereas the labialappendages exhibit a completely different structure (Fig. 6A,right).Studies in firebrats, crickets, milkweed bugs, and flies haveshown that the basic programming and identity of the gnathalsegments are determined by three Hox genes: proboscipedia(pb), Deformed (Dfd), and Sex combs reduced (Scr) (Hughesand Kaufman 2002). Changes in pb expression have beendirectly associated with changes in mouthpart structure(Randazzo et al. 1991; Beeman et al. 1993; Rogers et al.2002). In insects with biting and chewing mouthparts, pb isexpressed in the labium and maxillae but not in the

Li and PopadicŁmandibles. However, in insects with piercing and suckingmouthparts, such as milkweed bugs, pb expression is mainlydetected in the labial appendages (Rogers and Kaufman1997). Thus, it has been proposed that loss of pb expression inthe maxillae was responsible for the transformation of themandibulate to the haustellate mouth type (Rogers et al.2002). More specifically, the loss of pb expression in milkweedbug embryos would ‘‘free’’ the maxillary appendages todiverge from their original labium-like phenotype. However,such change in pb regulation cannot account for thedevelopment of stylets in both maxillary and mandibularsegments. This suggests that other nonhomeotic genes areinvolved in the latter process.Consistent with the morphological diversification of headappendages, the sharpest difference of nub expression isobserved in this region. Patterns of expression show acoordinated change in association with particular mouthpartmorphology. In firebrat and cockroach embryos, which havetypical mandibulate mouthparts, nub is localized in themaxillary and labial appendages in a similar pattern (Fig.6B). This is particularly pronounced in Periplaneta embryos,which exhibit highly coordinated patterns of expression in thelabium and maxillae from early to late development. Such anobservation is consistent with the fact that these segments arestructurally very similar and differ from the mandibularsegment. Second, nub is generally not expressed in themandibles except locally and transiently in cockroaches(Fig. 6B). This suggests that in insects with biting andchewing mouthparts, mandibles were originally devoid of nubexpression.In milkweed bug embryos, which have the stylate–haustellate (sucking) mouthparts, nub is expressed in a verydifferent pattern. First, there is novel expression along themid-ventral region of the mandibular appendages. Second,this mandibular expression is similar to the maxillary pattern(Fig. 6B). Expression in the labium, however, is localized onlyin the distal portion of the appendage and exhibits acompletely different timing and pattern. This differentialexpression correlates with a change in the morphology of themaxillary and labial segments that is characteristic forpiercing and sucking mouthparts: Development and morphologyof the maxillary segment parallels that of themandibular segment. These observations provide the firstnonhomeotic gene example of a linkage between a change inexpression pattern and a corresponding change in themorphology of insect mouthparts. They also provide a strongindication that nub may function in the development ofhaustellate mouthparts.The emerging insight from this and other recent studiessuggests that the evolution of the maxillae and mandibles inOncopeltus and other hemipterans was governed by regulatorychanges at both higher (Hox genes such as pb and Dfd)and lower levels (genes such as nub) in the developmentalhierarchy. This underlines the critical importance of understandingthe functional roles and relationships between thesetwo classes of genes. For example, Dfd is expressed in themandibular segments in both Tribolium (which has mandibulatemouthparts) and in Oncopeltus (which has haustellatemouthparts). Functional studies have shown that in theabsence of Dfd, mandibles are transformed into antenna-likeappendages in these two insects (Brown et al. 2000; Hughesand Kaufman 2000). These findings show that Dfd orthologscontrol mandibular identity in both species. Yet the actualmorphology of mandibles in Tribolium and Oncopeltus is verydifferent, a result of distinct mandibulate and haustellatemodifications. Additional factors, either acting in parallelwith Dfd, or downstream targets, or independently of Dfd,must be involved in the establishment of these distinctmandibular morphologies. 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