Animals

Two-spotted cricket, Gryllus bimaculatus, was used in this study. To obtain clear results for whole-mount immunostaining, we used a mutant with white eyes (autosomal recessive; gwhite) (Fig. 1A), kindly provided by I. Nakatani of Yamagata University. During late embryogenesis and just after molt, their bodies become more transparent than in usual.

Fig. 1

Adults and an egg of a mutant two-spotted cricket, Gryllus bimaculatus (gwhite) with white eyes. (A) Adult male (left) and female (right). (B) Egg. Slightly pointed pole is anterior (upper side). Scale bars = 1 cm (A), 1 mm (B).

Rearing of crickets

All nymphs and adults of G. bimaculatus were reared at 27 ± 1 °C with humidity of 70 ± 2% under a 10L: 14D photoperiod, and fed on crashed dog-food and artificial rabbit-food. Under these conditions, nymphs molt every week, and the adults emerge after the 8th molt. Generation time of the cricket is about two months. The 1st–8th instar nymphs were maintained in plastic cases of 10 × 10 × 12 cm, and the adults were in cases with 15 × 15 × 20 cm. Each case contains two cotton wool-plugged plastic tubes for supplying water, and several crumpled papers as a shelter. In the case rearing the adults, a pile of moist and folded tissue papers (about 1 cm in height) was used for females to lay eggs during the dark period. Tissue papers for laid eggs were exchanged with new ones every day, and freshly laid eggs were collected with forceps. The eggs were placed separately on moist tissue papers and incubated under the same conditions as employed for rearing adult crickets (27°C and 70% humidity). Nymphs hatched out from the eggs on 13th day of incubation were transferred to the rearing cases.

Observation of embryogenesis

To observe position and form of cricket embryos within the egg at each stage, eggs were soaked in 30% bleach for 5 min and removed their chorion. Dechorionated eggs were washed thoroughly in phosphate buffered saline (pH 7.4) and then observed in the saline under dark-field illumination, using stereo-microscope. For observation of the embryo morphology, embryos with yolk masses were taken out from the eggs, and then the yolk masses were removed carefully with tungsten needles. The embryos were mounted on microscope slides and observed their detail features under a bright-field microscopy with the Nomarski optics.

Immunohistochemistry

To examine the localization of engrailed (en) and Distal-less (Dll) proteins in developing cricket embryos, we used a monoclonal antibody (anti-en protein), namely MAb 4D9, purchased from the Developmental Studies Hybridoma Bank (Baltimore, MD, USA) (Patel et al., 1989b), and a polyclonal antibody (anti-Dll protein), kindly provided by Dr. Sean Carroll (University of Wisconsin) (Panganiban et al., 1995).

Immunostaining was performed as described previously for grasshopper embryos by Patel et al. (1989b). All embryos at stages 5–12 were stained as whole-mount preparation. To detect en proteins, the MAb 4D9 supernatant was used at 1:3 dilution and the secondary antibody, peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch Lab), was used at a dilution of 1:100. On the other hand, the Dll antibody was used at a dilution of 1:200 and the secondary antibody, peroxidase-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Lab), was used at a dilution of 1:400. To observe the expression domain precisely, the stained embryos were dehydrated in ethanol, placed in xylene, then embedded in paraffin and serially sectioned at 5 μm thickness. Both stained whole embryos and sections were mounted on microscope slides and then observed under a Nomarski differential-interference microscope.

Definition of developmental stages in Gryllus bimaculatus

Although outline of the embryogenesis of G. bimaculatus has already reported by Miyamoto and Shimozawa (1983), detailed embryonic structures have not been described. Here, general aspects of morphologies of the developing embryos and metathoracic limbs are briefly illustrated in Fig. 2. Most of the eggs are hatched on the 13th day ± 12 hr under incubation conditions of 27 ± 1°C and 70 ± 2% humidity. We divided total developmental span into 16 stages based on morphological features of developing embryos and appendages, as summarized in Table 1. The correspondence between stages and morphologies are shown in Fig. 2.

Fig. 2

Illustrations showing embryogenesis in G. bimaculatus. Numbers indicate stages. (A) Whole embryos (ventral view). (B) Metathoracic appendages. Every appendage in G. bimaculatus first appears as a bud. An, antenna; Cer, cercus; Cl, claw; Cx, coxa; E, anteroposterior length of embryo; Ey, eye; FL, proximodistal length of femur in metathoracic leg; Fm, femur; G, gnathal segments; L, proximodistal length of metathoracic leg; Lb, labium; Lm, labrum; Mn, mandible; Mx, maxilla; P, pleuropodium Pco, protocorm; Pcp, protocephalon; Pl, prothoracic legs; S, tibial spur; St, stomodium; Ta, tarsus; Ti, tibia; Tr, trochanter.

Table 1

Embryogenesis in Gryllus bimaculatus

Expression patterns of engrailed in body segments are conserved

We used a monoclonal antibody 4D9 to observe expression of engrailed (en) in G. bimaculatus embryos at stages 5–12. The en expression patterns are shown in Figs. 3 and 4. At stage 5, the en expression patterns were segmentally repeated stripes in the gnathal and thoracic segments (Fig. 3A). In addition, a stripe of en also appears in a region becoming the first abdominal segment (Fig. 3A). With the caudal extension of the embryo, the number of abdominal stripes increases and finally sixteen stripes were observed in the fully segmented embryo (Fig. 3C). These stripes are localized in the posterior compartment of every segment. No stripe of en was detected in the most caudal segment, i.e., the eleventh abdominal segment (Fig. 4D), whereas the clear en stripe was found in the posterior compartment of the tenth abdominal segment (Figs. 3C, D and 4D). In progress of development, the gnathal and thoracic stripes become wide. In the thoracic terga, en stripes are much wider than those in abdomens at stage 12 (Fig. 3F). The intensity of expression in the segments becomes maximum at stage 11 (Fig. 3D). From this stage, several en-positive cells are regularly arranged at the mid-ventral region of every body segment (Fig. 3H) and in the head (Fig. 3G). Since the en expression during neurogenesis has been reported in many organisms including arthropods, annelids, and chordates (Patel et al., 1989b), the en-positive cells may become a subset of neuroblasts, ganglion mother cells and neurons in the central nervous system. The distribution of en-positive cells during neurogenesis in G. bimaculatus closely resembles that in the grasshopper, Schistocerca americana (Patel et al., 1989b; Condron et al., 1994).

Fig. 3

Expression patterns of engrailed in developing embryos. (A–F) Whole embryos at stage 5 (A), stage 7 (B), stage 9 (C), stage 11 (D), and stage 12 (E, F). en stripes were observed in the posterior margin of each segment. (G) The head region at stage 12 where four pairs of patches of en positive cells are arranged at both sides. (H) The ventral side of abdomen (the 4th and 5th abdominal segments) at stage 11 where en positive cells are regularly arranged at every segments. Scale bars = 0.5 mm (A–F), 100 μm (G), 50 μm (H).

Fig. 4

Expression patterns of engrailed in developing appendages. (A-C) Mesothoracic appendages of embryos at stage 7 (A), stage 9 (B), and stage 11 (C). en is expressed at the posterior compartment as in body segments. (D-E) Cerci of embryos at stage 8 (D) and stage 9 (E). At stage 11, en expression was also observed in the apodeme of the femur (arrowhead) (C). At stage 8, expression in cercus appears without connecting any en stripes in the body (D). Subsequently, the expression domain is expanded and connected with abdominal 10th stripe (A10) at dorsal (arrowhead) (E). (F) Cross section of E. The expression domain occupies about one third region of circumference. Similar pattern was also observed in other appendages (data not shown). Scale bars = 100 μm.

Expression patterns of engrailed in the posterior compartments of appendages

The en gene is also expressed in developing appendages such as the antenna (Fig. 3F), mandible, maxilla, labium, and thoracic legs (Fig. 4A-C), but not in the developing labrum. During elongation of these appendages, en expression is limited to the posterior compartment of each appendage. At stage 7 when each appendage bud begins to elongate laterally, the segmental en stripe extends in the posterior portion of each appendage (Figs. 3B and 4A). In thoracic leg buds, the expression in the proximal region is more intense than that in the distal region (Fig. 4A, B). At stage 11, en is also expressed in the femoral apodeme in each leg (Fig. 4C). Although no stripe of en is detected in the eleventh abdominal segment of embryo as above mentioned, the en expression is observed in developing cercus which is the appendage of the eleventh abdominal segment (Figs. 3C-F and 4D-F). With elongation of the cercus, the expression extends proximally and connects to the en stripe of the tenth abdominal segment (Fig. 4E). In this stage, the orientation of the cercus changes toward the posterior direction along the anteroposterior axis of the body, so that en appears expressed in the dorsal region of the cercus (Fig. 4E). A transverse section of the immunostained cercus reveals that en-positive cells are localized in the posterior one third (Fig. 4F). The similar expression pattern was also observed in transverse sections of other immunostained developing appendages (data not shown).

Expression patterns of Distal-less depend on the type of appendages

We used a polyclonal antibody to observe expression of Distal-less (Dll) in G. bimaculatus embryos at stages 5∼12. Stained embryos are shown in Figs. 5 and 6. Dll expression was specifically observed in developing appendages. The initial expression was found in the metathoracic appendage bud at stage 5 (Fig. 5A). By stage 8, Dll is expressed in the primordia of labrum, antenna, maxilla, labium, thoracic legs, and preuropodium (Fig. 5B). In addition, Dll expression was also observed in the apical region of the cercus at stage 9 (Fig. 5C). As shown in Fig. 6A, initial expressions in the maxillary and labial buds are limited to the distal half regions. These regions become the telopodites defined as the distal shafts of the arthropod limbs (Snodgrass, 1935). The similar expression pattern was also observed in the thoracic appendage buds. However, no Dll expression region was found only in mandibular buds (Fig. 6A). These expression patterns are consistent with those reported for Drosophila melanogaster, Precis coenia (Panganiban et al., 1994), and Thermobia domestica (Popadić et al., 1996).

Fig. 5

Expression patterns of Distal-less in whole embryos. Each embryo is at stage 5 (A), stage 8 (B), stage 9 (C), and stage 11 (D). Dll expression was initially observed at primordium of metathoracic appendage (A). At stage 8 (B), Dll is expressed in growing appendages excepting mandible (Mn). The expression was also defined in cercus (Cer) at stage 9 (C). With the elongation, each appendage exhibits distinct expression pattern (C, D). A1, appendage of first abdominal segment; An, antenna; Lb, labium; Lm, labrum; Mx, maxilla; T1-T3, appendages of first -third thoracic segments. Scale bars = 250 μm.

Fig. 6

Expression patterns of Distal-less in different appendage types. (A) Gnathal appendages at stage 8. Dll expressions were observed in distal regions of the maxilla (Mx) and the labium (Lb). No expression domain was observed only in the mandible (Mn). (B) Maxilla at stage 11. The expression were observed at the maxillary palpus (Mp), galea (G) and lacinia (Lc). The expression pattern in the maxillary palpus closely resembles that in leg, showing in C. (C) A mesothoracic leg at stage 11. Intense expression domains were observed in middle and distal regions. (D) An antenna at stage 11. The expression domain corresponds to the telopodite of the antenna. (E) A cercus at stage 11. The expression is restricted only in the distal region. Fl, flagellum; Fm, femur; Ta, tarsus; Ti, tibia; Pdc, pedicel; Scp, scape. Scale bars = 50 μm (A), 100 μm (B-E).

With further elongation and segmentation of the appendages, the Dll expression becomes intense and then the expression pattern changes, depending on the appendage type (Fig. 5C, D). In thoracic legs, intense expressions were observed in the distal and middle regions (Fig. 6C). We found that the distal region becomes tarsus and a part of tibia in future, whereas the middle region corresponds to the entire region of femur. The similar expression pattern was also observed in the labial and maxillary palpus (Fig. 6B, C). Dll is also expressed in two endites of the maxilla, the galea and lacinia (Fig. 6B). Interestingly, the expression is restricted to the distal region of each endite, as observed in early gnathal limb buds.

In other types of appendages such as the labrum, antenna, and cercus, no change in Dll expression patterns was observed with elongation of the appendages (Fig. 5D), although the expression patterns in the appendages are more or less different from each other. In elongating antenna, Dll is expressed in most of the distal region corresponding to future pedicel and flagellum of the antenna, but not in the basal narrow region probably becoming the scape segment (Fig. 6D). The entire region of the growing labrum and the apical region of the cercus also consist of Dll-positive cells (Figs. 5D and 6E).

Expression patterns of en and Dll are conserved in developing antenna and legs of Drosophila and G. bimaculatus

In the insect appendage formation, two types of developmental process are known: One is seen in holometabolan insects such as Drosophila. The appendages are formed as imaginal discs in the larval body. The other type is known in hemimetabolan insects: The appendages are initially formed as buds in the early embryo. In G. bimaculatus, the type of the appendage formation belongs to the latter, in which imaginal discs are not formed during appendage development (Fig. 2B). We compared the expression patterns of en and Dll in the developing antenna and legs of G. bimaculatus with those in the corresponding imaginal discs of Drosophila as shown in Fig. 7. In the Drosophila embryo, Dll expression patterns are different between antenna and leg discs, whereas the en expression in the posterior compartment is similar between them. The differences between the antenna and leg buds were also observed in G. bimaculatus (Fig. 7). Therefore, the expression patterns of en and Dll in the developing antenna and legs closely resemble those in the Drosophila imaginal discs. The similarities of en and Dll expression patterns between the two organisms imply that common molecules may be involved in development of insect antenna and legs and that their functions may be common in various insects in spite of different mode of appendage development. Since the en and Dll expression patterns in the crustaceans have been reported to resemble closely those in insects (Patel et al., 1989a; Panganiban et al., 1995), these genes may function commonly in arthropod appendage formation.

Fig. 7

Schematic illustration of engrailed and Distal-less expression patterns in Drosophila imaginal discs and cricket limb buds. Each gene expression domain found in cricket is similar to that in Drosophila in spite of different developmental mode. A, anterior; D, distal; P, posterior; Pr, proximal.

Dll is expressed in the telopodite of each appendage

In the early development in G. bimaculatus, the similarities in Dll gene expression patterns were found among different appendage types except the mandible: Dll is expressed in the distal region corresponding to the telopodite of each appendage. Since the morphological analysis of the Drosophila mutant lacking the Dll activity suggested that formation of the telopodite is regulated by Dll (Cohen and Jürgens, 1989), the similarities in Dll expression patterns suggest that the initial formation of all appendages except the mandible may occur by determination of the telopodite of the appendage by Dll. In addition, en expression in the posterior compartment is conserved pattern among different appendage types. Therefore, common expression patterns of the Dll and en genes in the early appendage formation in spite of different appendage type may support a hypothesis that every appendage type was evolved from a type of limb of a myriapod-like ancestor, which was originally proposed by Snodgrass (1935). He also assumed that in the insect mandible, the telopodite region is absent and consists of only the coxopodite. It is interesting to note that expression of Dll was not observed in the cricket mandible. Furthermore, no expression of Dll gene in the mandible has been also observed in the lepidopteran insect, Precis coenia (Carroll, 1994; Panganiban et al., 1994). These facts support his assumption and suggest that mandible may evolve differently from other appendages in the insect. On the other hand, we found that expression of en was not detected in the labrum, although Dll is expressed in it. Thus, we assume that although the labrum is originally derived from an appendage, its posterior compartment may be somehow degenerated.

Late expression patterns of Dll depend on the structure of the appendage

We found that expression domains of the Dll genes become defined finely in each appendage type with the progress of morphogenesis in the appendages. For instance, since the expression domain of the Dll gene was found to correspond to the telopodite region in each appendage, we found that the boundary between coxopodite and telopodite corresponds to the articulation region of coxa-trochanter of G. bimaculatus in the leg (Fig. 6C) or to the scape-pedicel articulation region in the antenna (Figs. 6D and 8). The Dll expression patterns in the maxillary palpus (Fig. 6B) also suggest that the maxillary palpus corresponds to the telopodite, as speculated previously (Snodgrass, 1935; Machida, 1981; Bitsch, 1994). In addition, Dll expression was also found in the distal regions of the galea and lacinia, which are two endites in the maxilla, showing that they also bear the telopodite elements. Thus, we concluded that the galea and lacinia may be homologous with other appendages.

Fig. 8

Illustrations showing engrailed (en; red) and Distal-less (Dll; green) expression domains both in cricket antenna and leg. en is expressed in the corresponding posterior compartment of the antenna and leg buds. Dll expression domains correspond to pedicel (Pdc) and flagellum (Fl) in antenna. Space between two intense expression domains of Dll in leg correspond to the region including the femur (Fm) -tibia (Ti) articulation. Cx, coxa; Scp, scape; Ta, tarsus; Tr, trochanter.

In cricket embryos, from stage 9 the Dll expression patterns begin to change with the elongation and segmentation, depending on the type of appendages (Fig. 5C, D). Final expression patterns of Dll are classified into three types. First, the expression was expanded entire region of the appendage as seen in the antenna (Fig. 6D). Second, the expression was observed in a distal region of the appendage as in the cercus (Fig. 6E). Third, as in the leg, intense expression was observed in distal and middle regions (Fig. 6C). The space between the intensely expressed regions corresponds to the position of the prospective femur-tibia articulation (Fig. 8). The maxillary and labial palpus, whose expression pattern of Dll belongs to the third type, have an articulation corresponding to the femurtibia, indicating high homology with the leg. In the maxillary palpus of G. bimaculatus, the articulation between the 2nd and 3rd segment from the distal corresponds to the femurtibia articulation in the leg. On the other hand, in the first and second types, since the space observed in the third type was not found, an articulation corresponding to the femur-tibia may be absent. Thus, we considered that the Dll expression patterns in the late appendage development are specific for the appendage type and that these expression patterns may be closely related to the segmentation patterns, especially participated in the positioning of the articulation between the femur and the tibia or between corresponding segments of the appendages.