Morphometric analysis of thoracic muscles in wildtype and in bithorax Drosophila.код для вставкиСкачать
THE ANATOMICAL RECORD 226:373-382 (1990) Morphometric Analysis of Thoracic Muscles in Wildtype and in Bithorax Drosophila M. DAVID EGGER, SUZAN HARRIS, BONNIE PENG, ANNE M. SCHNEIDERMAN, AND ROBERT J. WYMAN Department of Anatomy, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635 (M.D.E., S.H., B.P.); Department of Biology, Yale University, New Haven, Connecticut 06511 (A.M.S., R.J.W.) ABSTRACT The tergotrochanteral (TTM) “jump” muscles in the second (T2) and third (T3) thoracic segments of the fruit fly, Drosophila melanogaster, were analyzed morphologically and morphometrically in wildtype (Canton-S)and bithorax mutants (abx b 2 pbx I DfT3RIP2). In the transformed T3 segments of mutant flies, the TTMs were greatly increased in fiber number (330% of wildtype), length (141%), and volume (460%), thus manifesting both hyperplasia and hypertrophy. In contrast, TTMs in the “untransformed” T2 segments of mutant flies were both hypoplastic and hypotrophic, in that significant decreases in fiber number (93% of wildtype), length (go%), and volume (80%) were observed. Two relationships emerged from analysis of the morphometric data: 1)Although the fiber numbers and volumes of the transformed T3 TTMs in bithorax flies were greatly increased, the total combined volumes of the TTMs in T2 + T3 remained approximately the same in bithorax compared to wildtype flies. 2) The changes in TTM volumes in bithorax flies compared to those in wildtype were proportional to the relative changes in fiber numbers times the relative changes in muscle lengths. These observations suggest that the genes of the bithorax complex influence the number and the length of tubular muscles fibers of the TTMs, but do not significantly affect the mean cross-sectional areas of these fibers. Fibrillar muscle fibers, which are not found at all in T3 segments in wildtype flies, were observed in the transformed T3 segments of bithorax mutants in 11 of 18 cases (61%),but typically as wisps, not in complete muscles. We suggest that, in the T3 segment of the bithorax flies, the relative differences between the massive transformation of tubular TTMs vs. the minimal appearance of fibrillar muscles may be related, in part, to the relative availability of muscle precursors. The bithorax complex (BX-C) is a much-studied group of homeotic genes in the fruit fly, Drosophila melanogaster. If BX-C genes malfunction, posterior body segments are transformed. In resultant mutant flies, transformed posterior segments appear similar to more anterior segments (Lewis, 1978, 1982; Ayala and Kiger, 1984). A striking manifestation of BX-C transformation is that the mutant fly may have two pairs of wings, one pair each projecting from the cuticle of the second (T2) and the third (T3) thoracic segments (Fig. 11, in contrast to the wildtype fly, which has only one pair of wings projecting from T2. Most anatomical observations on BX-C phenotypes have been made on epidermal structures (cuticle). In this paper, we extend the anatomical observations of four-winged BX-C mutants to a quantitative examination of muscles in T2 and T3. The muscles of the thorax in the fly are principally of two basic types (Miller, 1950): 1) tubular muscles, which constitute most of the muscles of the thorax, including the tergotrochanteral muscles (TTMs) which are the “jump” muscles that provide the power to the extension of the legs, and 2) fibrillar muscles, which are found only in insects, and which are characteristic 0 1990 WILEY-LISS, INC. of the indirect flight muscles (see, for instance, Tiegs, 1954; Pringle, 1957; Elder, 1975; Smith, 1984; for the muscular anatomy of the thoracic segments in wildtype flies, see Zalokar, 1947; and Miller, 1950; for the anatomy of the thoracic exoskeleton, see Ferris, 1950). Some qualitative observations have already been made of the musculature in four-winged BX-C mutants. Ferrus and Kankel (1981) found only tags of muscles in the transformed third thoracic segment (T3) of the bithorax mutant, bx? pbx I DfT3)P9. In no case did they find muscles representative of T2 in T3. Schneiderman et al. (1987) repeated the observations of Ferrus and Kankel (1981) on muscular transformations in T3, but used the bithorax mutant, (abx b 2 pbxl DfT3R)P2) (Lewis, 1980a,b, 1982), which results more reliably in a four-wing phenotype than does the mutant studied by Ferrus and Kankel(l981). Schneiderman et al. (1987) estimated that the TTMs in T2 of bithorax Received August 2, 1988; accepted June 30, 1989. 374 M.D. EGGER ET AL. Fig. 1. Photograph of a bithorax mutant (abx bn-?pbxIDfl3R)P.Z') fly (Drosophila melanogaster). Fig. 2. Low-power electron micrograph of a cross-section through a wildtype tergotrochanteral muscle, in the metathorax (third thoracic segment). This muscle has eight tubular fibers, one of which has been outlined. F, the outlined tubular fiber; My, one of the many myofibrils included in each muscle fiber; N, centrally placed nucleus within a tubular fiber; Tr,tracheole. THORACIC MUSCLES IN BITHORAX DROSOPHILA mutants were unchanged compared to wildtype, but that in T3, the TTMs of mutants were greatly increased in volume. Furthermore, Benson and King (1987), studying the same mutant a s Schneiderman e t al. (1987), found, unlike Ferrus and Kankel (1981)) strands of well-formed indirect flight muscles in the transformed T3. Our chief aims were 1)to extend the analysis of the tubular muscles to the quantitative level, principally by analyzing the effects of the genetic transformations on i) the number of muscle fibers, ii) the lengths, and iii) the volumes of the TTMs; 2) to determine, with respect to the transformation of the T3 TTMs, whether the increase in muscular volume reported by Schneiderman et al. (1987) was due to hypertrophy, to hyperplasia, or to some combination of the two; and 3) to determine whether the transformation included fibrillar muscles, which in wildtype flies are found in T2, but not in T3. METHODS Our wildtype flies were of the Canton-S strain. The parental strains of the bithorax flies were (abx bX3pbxl T M l ) and (Df13R)P2 I T M l ) . Crosses were made with flies that had been isolated as pupae, just prior to eclosion. Flies were maintained in vials containing a mixture of agar, yeast, white cornmeal, molasses, mold inhibitor, and water. Room temperature was thermostatically maintained between 23-26°C. Flies selected for morphometry were late pupae on the brink of eclosion (Bainbridge and Bownes, 1981, stage P15 (i)).The sexes of all pupae were noted; some pupae were weighed. All of our samples contained a n equal number of males and females. The data were analyzed taking sex into consideration. In order to make the rearing conditions as equivalent a s possible, we selected as many pupae as possible from the same vial. We typically removed pupae only during the first few days when mature pupae were observable in a fresh vial. The bithorax flies that we chose for analysis were those that appeared to be the most completely transformed. On the other hand, the flies of the parental strains removed for analysis from the same vials as the bithorax flies (“littermate” controls) appeared grossly indistinguishable from wildtype. Independent analysis of the two parental strains indicated that they did not differ from each other morphometrically. Furthermore, with only one exception (Table 2), the parental strains did not differ significantly from wildtype on any measure. Each pupa was dissected in ice-cold Ringer’s solution. The proboscis, legs, wings, and much of the abdomen were cut off, taking care not to pull, stretch, or distort the head. A few small holes were pierced into the dorsal thorax and in the eyes. Then the tissue was immersed in ice-cold fixative (3% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.4)) for 3-18 hr, followed by three rinses in 0.1M sodium cacodylate buffer containing 0.2M sucrose (pH 7.4). Postfixation was in 1%osmium tetroxide in 0.1M sodium cacodylate buffer (pH 7.4) for 2 h r at 4“C, followed by dehydration throuph a n ethanolic series and embedment in EMbed812 (Electron Microscopy Sciences). Semithin sections (0.8 bm) were examined by light microscopy (LM), 375 while ultrathin sections were examined by electron microscopy (EM) (Philips CM12). For measuring the volumes of the TTMs in T2 and T3, every 20th horizontal section through the fly was photographed (LM), and the cross-sectional area of the muscle of interest was determined by using a Bioquant morphometric system. The width of the endomysium, as well a s extramuscular spaces, appeared almost negligible in the muscle sections on which the crosssectional area of the muscles were measured, for the wildtype, parental, and bithorax mutant flies alike. Accordingly, the total volumes of the muscles were calculated without correcting for the slight contributions of extramuscular tissues or spaces. Muscle length was reconstructed by counting and adding together the number of cross sections on which the muscle could be seen, having calculated that the distance between each section was 16 pm (20 x 0.8 Fm). Virtually all observed muscle fibers extended for the entire length of the muscles they comprised. Individual muscle fibers were counted from the LM cross sections. No corrections were made for shrinkage. To test the reliability of the method, the volumes of the muscles from several flies were independently deter- Fig. 3. Higher-power electron micrograph of a cross-section through a tubular fiber from a tergotrochanteral muscle in the mesothorax (second thoracic segment) of a wildtype fly. Long arrow points to glycogen granules, G ; arrowheads indicate border of muscle fiber; Mi, mitochondrion; My, myofibril; N, nucleus. 376 M.D. EGGER ET AL. Fig. 4. Light micrograph of a horizontal section (0.8 pm) through the thorax of a late pupa, just before eclosion, of a wildtype female fly (Canto&). The upper, large outlined muscles are the tergotrochanteral muscles in the mesothorax (T2 TTMs). The lower, small outlined muscles (indicated by arrows) are the tergotrochanteral muscles of the metathorax (T3 TTMs). The heavy broken line indicates the posterior boundary ofT2. At this horizontal level, most ofthe exoskeleton of T3 lies ventral to the section. C, cardia; DLM, dorsal longitudinal muscle; DVM, dorsoventral muscle. Fig. 5. Light micrograph of a horizontal section (0.8 pm) through the thorax of a late pupa, just before eclosion, of a female bithorax fly (ubx br-7 pbxiDfT3RIP2). This section is cut in a more dorsal plane than the section in Figure 4. The upper, large outlined muscles are the tergotrochanteral muscles in the mesothorax (T2 TTMs). The lower, small outlined muscles (indicated by arrows) are the tergotrochanteral muscles in the transformed metathorax (T3 TTMs). The heavy broken line indicates the boundary, a t this level, between T2 and T3. DLM, dorsal longitudinal muscle; DVM, dorsoventral muscle. 378 M.D. EGGER ET AL. TABLE 1. Wildtype (Canton-S)tergotrochanteral muscles (TTMs) T2 T3 Fiber Length Volume Fiber Length Volume No. (pm) (10-6 pm3) No. (pm) (10-6 pm3) Male (n = 6) x 28.1 SD 2.9 Female (_n= 6 ) X 28.3 SD 2.2 519 44 4.27 1.01 7.9 0.3 279 36 0.326 0.066 539 28 4.77 0.70 7.8 0.4 265 33 0.322 0.078 mined by two different investigators. The measurements were typically within a fraction of a percent of each other. Because no systematic right-left differences were observed in any of the data, measurements from paired muscles of each fly were pooled and analyzed as total values for a pair of muscles/fly by analysis of variance. Differences between group means were assessed by a Newman-Keuls test (Winer, 1971). RESULTS In the second thoracic (T2) and third thoracic (T3) segments of wildtype (Canton-S) and bithorax (abx b 2 pbxlDfT3R)P2) mutant flies (Fig. l ) , we examined the fiber numbers (number of individual muscle fibersl muscle), overall lengths, and total volumes of the tergotrochanteral muscles (TTMs) (Figs. 2, 3). The morphometric observations were made on horizontally sectioned wildtype (Fig. 4) and bithorax (Fig. 5) flies. Similar observations were made on the bithorax parental strains [(DfT3R)P2ITMl) and (abx b$ pbxlTMl)] (Tables 1, 2). TABLE 2. Percentage comparisons of wildtype with bithorax mutants and parental strains for tergotrochanteral muscles (TTMs) (all values for wildtype defined as = 100%) T2 T3 Fiber Fiber No. Length Volume No. Length Volume (n) (16) (18) (18) (16) (18) (18) Bithorax' 93 90 330 80 141 460 (n) (20) (20) (20) (20) (20) (20) 99 101 108 106 101 Parental' 94 Yabx bx? pbxlDfl3RiP2). All the values in this row of the table differ significantly from wildtype (P <0.05). '(abx bx? pbxITM1) and (Dfl3R)P2ITMl). The only value in this row significantly different from wildtype is that for T2 TTM fiber number (P<0.05). In summary, the T2 TTMs of our bithorax flies had only 93% as many fibers, 90% of the length, and only 80% a s much volume as in the wildtype flies. Such changes are not due to possible differences in overall body size between bithorax and wildtype flies, because our measurements of body weights of wildtype and bithorax flies revealed no statistically significant differences. In fact, the bithorax flies tended to be heavier (Table 3). TergotrochanteralMuscles (TTMs) in T3 Analysis of variance of data for the T3 TTMs (see Tables 1 and 2) revealed that, with respect to fiber number, males had more fibers than the females (P<0.005), there were statistically significant effects of sex x genotype (P<0.005), and the genotypes differed (P<O.OOl). With respect both to muscle lengths and volumes, there were no significant sex differences or sex x genotype interactions, but the genotypes differed (P<O.OOl). More specifically for the T3 TTMs, in bithorax flies, Tergotrochanteral Muscles (TTMs) in T2 the mean number of fibers (26.0)-more than three Analysis of variance with respect to fiber number times the mean number of T3 TTM fibers in wildtype revealed no difference between the sexes for the T2 flies (7.9) or in flies of the parental strains (8.51-was TTMs, nor were there any significant sex x genotype almost identical to the mean number of fibers in their interactions. However, there was a statistically signif- T2 TTMs. The mean length of the T3 TTMs in bithorax icant (P<0.05) effect of genotypes for the T2 TTMs, in flies (382 pm) was significantly (P<0.05) longer than that the mean number of fibers counted in bithorax that in wildtype flies (272 pm), or than in flies of the flies (26.1), a s well as the mean number seen in flies of parental strains (288 pm), which did not differ signifthe parental strains (26.41, were significantly less icantly from wildtype. The mean volume of the T3 (P<0.05) than the mean number of fibers counted in TTMs in bithorax flies (1.49 x 106pm3) was more than four times greater than in wildtype flies (0.32 x 106 wildtype flies (28.2). Analysis of variance of data for muscle lengths and pm3) or in flies of the parental strains (0.33 x 106 volumes revealed that for the T2 TTMs, the females pm3). Thus, in bithorax flies, the volumes of the T3 had longer and larger muscles than the males TTMs were 40% of volumes of the T2 TTMs, compared (P<O.Ol). This was expected because females are larger to 7% in wildtype and parental strains, a relative inthan males, and, most likely, muscle length is directly crease of almost six-fold. An analysis of variance of the [T2 TTMs + T3 TTMs] influenced by the size of the thoracic cavity. We also found that the genotypes differed (P<0.005), in that muscle volume/f ly revealed that there was apparently the mean length of the T2 TTM muscles in bithorax no effect of genotype, suggesting that this volume flies (476 pm) was significantly shorter (P<0.05) than might be conserved in wildtype versus bithorax flies, in the wildtype flies (529 pm) or in flies of the parental even though the total number of [T2 TTMs + T3 strains (523 pm). With respect to volume, the mean TTMsl muscle fibers in bithorax flies was almost twice volume was significantly (P<O.Ol) smaller in bithorax that in wildtype flies. That is, in bithorax flies, the flies (3.61 x 106 pm3) than in wildtype (4.52 x 106 TTMs in T2 + T3 are markedly increased in total numpm3) or in flies of the parental strains (4.58 x 106 ber of muscle fibers, but not increased in total volume (Fig. 6). This constancy in the total volume of the TTMs pm3). Mean Volumes of Tergot rochanteral Muscles In Second (T2) and Third ( T 3 ) Thoracic Segments 6.0 1. 5.0 379 . . 1 .i c3 E 0 4.0 r K Q) E 3.0 - 3 >o 2.0 1.o 0 Wild Type N = 12 Parental Strains N=20 Bithorax (abx bX3 pbx I Df (313)~ 2 ) N=18 Fig. 6. Histograms ofthe total volumes ofthe tergotrochanteral muscles ("Ms) in T2 and T3 of flies of wildtype (Canton-S), bithorax mutants (abx bn3 pbxlDff3R)P2j and of the parental strains (abx bx? pbxlTM1) and (Dfl3RjP2ITMlj of the bithorax mutants. The areas of the histograms filled with parallel lines indicate the mean total volumes of the T2 WMs; the dotted areas of the histograms indicate the mean total volumes of the T3 "Ms; the capped lines in the histograms indicate estimated standard deviations. TABLE 3. Weights just prior to eclosion (mg) (Stage P15 (i) of Bainbridge and Bownes, 1981) fibers making up the muscles were constant, so that changing the total number of fibers by a certain percentage would be equivalent to changing the cross-sectional area of the whole muscle by the same percentage. If one assumes such a cylindrical model for the measured muscles, then i t is possible to calculate a n approximate mean cross-sectional area (as well a s a n approximate mean diameter) for the T2 and the T3 TTMs. (af -- Vtn x L, where af is the approximate "mean cross-sectional area" of a muscle fiber, n = number of fibers, L = length of the muscle, V = volume of the muscle. This "mean cross-sectional area" is a computed, rather than a measurable, quantity, because the individual fibers were not actually cylindrical, but fusiform. Also, the cross-sectional perimeters were distorted from true circles due to packing and other factors. This calculation does not include a correction for the proportion of the total muscle cross-sectional area due to extracellular space.) For the T2 TTMs, the mean calculated crosssectional fiber area was 303 pm2 in the wildtype and 291 pm2 in the bithorax flies. (The corresponding calculated diameters were 19.6 and 19.2 pm.) For the T3 TTMs, the mean calculated cross-sectional fiber area was 149 pm2 in wildtype and 150 pm2 in bithorax flies. WildtvDe Bithorax' 0.60 0.77 (n = 6) Ma_le X (n = 11) Female x 0.73 (n = 15) 0.79 (n = 7) '(abx bn3 pbxlDff3R jP2). in T2 + T3 cannot reflect any limiting of space for insertion on the cuticle or within the thoracic cavity, because the total volume enclosed by the T2 + T3 exoskeleton is clearly markedly increased in bithorax mutants compared to wildtype flies. Another relationship emerged by comparing percentage changes in fiber number, length, and volume in the T2 and in the T3 TTMs of bithorax mutants vs. wildtype. In both these muscles, the observed changes in volume were proportional to the changes in fiber number times the changes in overall length. If these muscles were true cylinders, this would be exactly the relationship expected if the cross-sectional areas of the 380 M.D. EGGER ET AL. (The corresponding calculated diameters were both 13.8 Fm). In view of the marked differences in the sizes of the muscles, especially between the T3 TTMs in wildtype vs. bithorax flies (the mean volume in the bithorax flies was 460% that of the wildtype), the similarity of these parameters is truly striking, suggesting that the genes of the BX-C which produced our mutants affected the number and length of tubular muscles fibers of the TTMs but not the mean cross-sectional areas of these fibers. Note that the mean calculated cross-sectional areas of the fibers of the TTMs in T3 were much smaller (about 50%) than those in T2, in both wildtype and bithorax flies. In other words, in spite of massive transformation in the bithorax flies, the individual T3 TTM fibers in some sense retained their identity as T3 fibers (though their number and lengths were significantly increased). That is, the T3 TTMs were incompletely transformed; they were phenotypically T2 in fiber number but remained phenotypically T3 in mean fiber diameter. Another way of describing this transformation is that the T3 TTMs in the bithorax flies were increased both as a result of hyperplasia in the number of the muscle fibers, and as a result of hypertrophy, manifested as a n increase in fiber length. While the increased fiber length may have been a n obligatory effect of the markedly increased distance between the sites of origin and insertion that resulted from the increased size of the transformed T3 cuticle, the increased fiber number most likely resulted from a genetic change induced in the transformed tubular T3 TTMs. Fibrillar Muscles In the T2 of both wildtype and bithorax flies, the conspicuous indirect flight muscles, such a s the dorsal longitudinal muscles (DLMs) and the dorsoventral muscles (DVMs), are composed of fibrillar fibers (Fig. 7). We found no fibrillar muscle fibers in T3 of wildtype flies, but of the 18 bithorax flies in which we surveyed T3 for fibrillar muscle, we found some fibrillar fibers in 11 flies (61%), typically in small wisps of muscle (Fig. 8). However, we found no consistently transformed fibrillar muscle in T3 that clearly resembled the T2 DLMs and DVMs, in marked contrast to our observations of the tubular T3 TTMs, which were invariably present and markedly enlarged in the bithorax flies. With respect to our studies of fibrillar muscle in the transformed metathorax, our observations fell between those of Ferrus and Kankel(l981) and those of Benson and King (1987), with the former observing much less, and the latter somewhat more complete transformations of normally metathoracic (T3) structures toward the mesothoracic (T2). Ferrus and Kankel (1981) examined a different genotype, (bx? pbxlDfl;3)P9), which would be expected to produce less completely transformed phenotypes than the triple mutation that we examined (abx b 9 pbxlDfl;3R)P2). Benson and King (19871, however, did examine the same genotype that we studied. They obtained light microscopic and scanning electron micrographs of transformed DVM fibrillar muscle fibers in T3 that appear quite similar to those found in T2 of wildtype. Fig. 7. Electron micrograph of a cross-section through a fibrillar fiber from a dorsoventral indirect flight muscle in the mesothorax (T2) of a wildtype fly. Arrow indicates glycogen granules, G; Mi, mitochondrion; My, myofibril. DISCUSSION Do BX-C Gene Effects Respect Parasegmental Boundaries ? In fruit flies, the BX-C genes are crucial for the organization of the second and third thoracic (T2, T3) and first eight abdominal segments (Al-A8) (Lewis, 1978, 1982). It has been suggested that each gene is predominantly responsible for specifying the identity of a portion of a segment or parasegment (Lewis, 1978; Martinez-Arias and Lawrence, 1985; Lawrence, 1988). The cellular localizations of BX-C gene products provide indirect molecular biological support for this model (e.g., Beachy e t al., 1985; Canal and Ferrus, 1987; Hooper, 1986; Wedeen e t al., 1986; White and Wilcox, 1985). However, anatomical evidence that the cuticle is always transformed within segmental or parasegmental boundaries is somewhat weak (see, e.g., Cole and Palka, 1982). In addition, Peifer and Bender (19861, who studied a variety of bithorax mutants, observed a decrease in the size of the mesonotum (anterior T2 cuticle) of their most extremely transformed bx mutants. According to a standard model of BX-C function (Morata et al., 19861, bx mutants should affect sequences which code for, or regulate the production of, product(s) 38 1 THORACIC MUSCLES IN BITHORAX DROSOPHILA model. We extended the observations on the bithorax mutants to the musculature, in which we found a reduction in TTM volume, length, and fiber number in T2-that is, anterior to the regions affected by the mutated genes according to the parasegmental model of gene action. Does Cuticular Development Determine Musculature? Does Muscular Development Determine Cuticle? Fig. 8. Light micrograph of a horizontal section (0.8 pm) through the thorax of a late pupa, just before eclosion, of a male bithorax fly (abx b*7 pbxlDff3RIPZ). The thick broken line indicates the boundary between the second thoracic (T2) and third thoracic (T3) segments. The thin broken line outlines a cross-section through a fibrillar muscle fiber, F. The solid line outlines the transformed metathoracic tergotrochanteral muscle (T3 TTM). A portion of a mesothoracic dorsoventral muscle (DVM) is visible. Note that in wildtype flies, fibrillar muscles are not found in T3. The T3 fibrillar muscle (F)in this section occupies a position somewhat similar to that of a DVM in the T2 of wildtype flies. This T3 fibrillar muscle (F) extended virtually throughout the dorsoventral extent of the T3 cavity, about 430 pm in length. With respect to the possible developmental interactions of cuticle and muscle, Ferrus and Kankel (1981) concluded that the epidermis does not control muscular development, because they failed to find in their bithorax mutants any muscles representative of T2 in T3. They also concluded that muscles do not play a dominant role in producing cuticular folding. The transformed T3 in their bithorax mutants appeared to form a relatively normal notum (the dorsal thoracic cuticle found in T2 in wildtype), although the underlying musculature was largely absent. That normal cuticular development can occur in the absence of muscle was confirmed by Costello and Wyman (1985). The question of the dependence of muscular determination on cuticular form was also taken up by Lawrence and Johnston (1984, 1986). Lawrence (1982) had demonstrated that, unlike the cuticle, the muscles of the thoracic segments are not subdivided into anterior and posterior compartments. By observing the location of a male-specific muscle in mosaics, Lawrence and Johnston (1984) were able to demonstrate that muscle can develop independently of cuticular segmental identity. Subsequently, Hooper (1986), in studying the development of musculature in a variety of BX-C mutants of the Ultrabithorax group, found that epidermal transformations occurred in different segments than the muscular transformations, with the transformations of cuticle occurring about 1.5 segments anterior to the corresponding transformations in muscles. Our results, too, support the conclusion that muscular transformations can occur independently of cuticular changes. SUMMARY Anatomical evidence suggests that BX-C gene functions affect the morphology of cuticle (e.g., Costello and Wyman, 1985; Ferrus and Kankel, 1981; Lewis, 1978, 1982; Morata and Kerridge, 1980; Morata et al., necessary for the correct differentiation of the cuticle of 1986; Peifer and Bender, 1986), the nervous system anterior T3; abx should affect primarily posterior T2 (e.g., Benson and King, 1987; Burt and Palka, 1982; and anterior T3; and pbx should primarily affect pos- Green, 1981; Ghysen e t al., 1983, 1985; Jimenez and terior T3. Thus, Peifer and Bender’s (1986) observation Campos-Ortega, 1981; Schneiderman et al., 1987; of effects of bx on anterior T2 cuticle indicates a n ex- Strausfeld and Singh, 1980; Teugels and Ghysen, 1983, tension of bx effects beyond the hypothesized paraseg- 1985; Thomas and Wyman, 19841, and muscles (e.g., mental domain of action of that gene. Also, Peifer and Hooper, 1986; Lawrence and Johnston, 1984, 19861, Bender (1986) noted t h a t the differences which they though the interactions among the various tissues and observed in the halteres (which project from T3) and the segmental distributions of effects are not undernotal structures affected by abx or bx followed no clear stood. Although no set of homeotic mutations has been compartmental boundaries. They concluded that “the as much studied genetically and biochemically a s that effects of neither abx or bx coincide well with a single of the BX-C we are still far from understanding how parasegment or compartment.” They suggested that the BX-C genes control adult form. Our data, by proBX-C mutations may affect cell-by-cell regulation, viding quantitative analysis of phenotypes of major which could result in effects coinciding with neither BX-C mutations, contribute toward understanding in detail how alterations of functioning in three BX-C segments nor parasegments. Our data support those of Peifer and Bender (1986) genes may influence adult morphology. A major finding of this study was that, in the bithoin questioning the applicability of the parasegmental 382 M.D. EGGE:R ET AL. rax mutant which we examined, the major transformation of a tubular muscle (TTM in T3) occurred primarily as a hyperplastic transformation, which, in part, appeared to be compensated for by a corresponding hypoplastic change in the anterior neighboring segment (TTM in T2). ACKNOWLEDGMENTS This work was partially supported by a grant to M.D.E. from the Foundation of the University of Medicine and Dentistry of New Jersey (UMDNJ). LITERATURE CITED Ayala, F.J., and J.A. Kiger, Jr. 1984 Modern Genetics. Benjamird Cummings, Menlo Park, CA, pp. 554-563. Bainbridge, S.P., and M. Bownes 1981 Staging the metamorphosis of Drosophila melanogaster. J . Embryol. Exp. Morphol., 66.57-80. Beachy, P.A., S.L. Helfand, and D. Hogness 1985 Segmental distribu' tion of bithorax complex proteins during Drosophila development. Nature, 313545-551. Benson, A.J., and D.G. King 1987 Neuromuscular transformation in bithorax Drosophila melanogaster. Soc. Neurosci. Abstr., 13.251. Burt, R., and J . Palka 1982 The central projections of mesothoracic sensorv neurons in wild-tvoe DrosoDhila and bithorax mutants. Dev. Biol., 90t99-109. Canal, I., and A. Ferrus 1987 The expression of Ultrabzthorax (Ubx) during development of the nervous system of Drosophila. J . Neurogenet. 4:161-177. Cole. E.S., and J . Palka 1982 The pattern of campaniform sensilla on the wing and haltere of Drosophila melanogaster and several of its homeotic mutants. J . Embryol. Exp. Morphol., 71:41-61. Costello, W.J., and R.J. Wyman 1985 Development of thoracic curvature in Drosophila melanogaster. Rouxs Arch. Dev. Biol., 194: 373-376. Elder, H.Y. 1975 Muscle structure. In: Insect Muscle. P.N.R. Usherwood, ed. Academic Press, New York, pp. 1-74. Ferris, G.F. 1950 External morphology of the adult. In: Biology of Drosophila. M. Demerec, ed. Hafner, New York (reprint: 1963, pp. 368-419. Ferrus, A., and D.R. Kankel 1981 Cell lineage relationships in Drosophila melanogaster: The relationships of cuticular to internal tissues. Dev. Biol., 85:485-504. Ghysen, A., L.Y. Jan, and Y.N. J a n 1985 Segmental determination in Drosophila central nervous system. Cell, 40:943-948. Ghysen, A., R. Janson, and P. Santamaria 1983 Segmental determination of sensory neurons in Drosophila. Dev. Biol., 99.7-26. Green, S.H. 1981 Segment-specific organization of leg motoneurones is transformed in bithorax mutants of Drosophila. Nature, 292: 152-154. Hooper, J.E. 1986 Homeotic gene function in the muscles of Drosophila larvae. EMBO J., 5t2321-2329. Jimenez, F., and J.A. Campos-Ortega 1981 A cell arrangement specific to thoracic ganglia in the central nervous system of the Drosophila embryo: Its behavior in homoeotic mutants. Rouxs Arch. Dev. Biol., 190:370-373. Lawrence, P.A. 1982 Cell lineage of the thoracic muscles of Drosophila. Cell, 29:493-503. Lawrence, P.A. 1988 The present status of the parasegment. Development [Suppl.], 104:61-65. & ,. Lawrence, P.A., and P. Johnston 1984 The genetic specification of pattern in a Drosophila muscle. Cell, 36:775-782. Lawrence, P.A., and P. Johnston 1986 The muscle pattern of a segment of Drosophila may be determined by neurons and not by contributing myoblasts. Cell, 45505-513. Lewis, E.B. 1978 A gene complex controlling segmentation in Drosoohila. Nature. 276.565-570. Lewjs, E.B. 19808 New mutants-D. melanogaster. Drosophila Inf. Serv.., .5.5:207-208. - -~Lew1s:E.B. 1980b Genetic control of body segmentation in Drosophila and Bombyx by homoeotic genes. Abstr. XVI Int. Congr. Ent., Kyoto, p. 161. Lewis, E.B. 1982 Control of body segment differentiation in Drosophila by the bithorax gene complex. In: Embryonic Development, Part A: Genetic Aspects. M.M. Burger and R. Weber, eds. Alan R. Liss, Inc., New York, NY, pp. 269-288. Martinez-Arias, A., and P.A. Lawrence 1985 Parasegments and compartments in the Drosophila embryo. Nature, 313:639-642. Miller, A. 1950 The internal anatomy and histology of the imago of Drosophila melanogaster. In: Biology of Drosophila. M. Demerec, ed. Hafner, New York (reprint: 1965), pp. 420-534. Morata, G., and S. Kerridge 1980 An analysis of the expressivity of some bithorax transformations. In: Development and Neurobiology of Drosophila. S. Siddiqi, P. Babu, L.M. Hall, and J.C. Hall, eds. Plenum, New York, pp. 141-154. Morata, G., E. Sanchez-Herrero, and J . Casanova 1986 The bithorax complex of Drosophila: An overview. Cell Differ., 18:67-78. Peifer, M., and W. Bender 1986 The anterobithorax and bithorax mutations of the bithorax complex. EMBO J., 5:2293-2303. Pringle, J.W.S. 1957 Insect Flight. Cambridge University Press, London. Schneiderman, A.M., M.L. Tao, and R.J. Wyman 1987 Transformation of identified muscle and its motorneuron in bithorax Drosophila. Soc. Neurosci. Abstr., 13.593. Smith, D.S. 1984 The structure of insect muscles. In: Insect Ultrastructure, Vol. 2. R.C. King and H. Akai, eds. Plenum Press, New York, pp. 111-150. Strausfeld, N.J., and R.N. Singh 1980 Peripheral and central nervous system projections in normal and mutant (Bithorax) Drosophila melanogaster. In: Development and Neurobiology of Drosophila. S. Siddiqi, P. Babu, L.M. Hall, and J.C. Hall, eds. Plenum, New York, pp. 267-290. Teugels, E., and A. Ghysen 1983 Independence of the numbers of legs and leg ganglia in Drosophila bithorax mutants. Nature, 304: 440 -442. Teugels, E., and A. Ghysen 1985 Domains of action of bithorax genes in Drosophila central nervous system. Nature, 314558-561. Thomas, J.B., and R.J. Wyman 1984 Duplicated neural structure in bithorax mutant Drosophila. Dev. Biol., 102:531-533. Tiegs, O.W. 1954 The flight muscles of insects-Their anatomy and histology; With some observations on the structure of striated muscle in general. Philos. Trans. R. Soc. Lond. [Biol.], 238:221347. Wedeen, C., K. Harding, and M. Levine 1986 Spatial regulation of antennapedia and bithorax gene expression by the polycomb locus in Drosophila. Cell, 44t739-748. White, R.A.H., and M. Wilcox 1985 Distribution of Ultrabithorm proteins in Drosophila. EMBO J., 42035-2043. Winer, B.J. 1971 Statistical Principles in Experimental Design. McGraw-Hill, New York. Zalokar, M. 1947 Anatomie du thorax de Drosophila melanogaster. Rev. Suisse Zool., 54:18-53.