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 <strong>Popadic</strong>Łin 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
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