A high-resolution morphogenetic map of the second-leg basitarsus inDrosophila melanogaster

Summary The lineages of cells on the second-leg basitarsus ofDrosophila melanogaster were analyzed by examining gynandromorphs andMinute mosaics. Bracts lie proximal to bristles on the adult basitarsus, yet bract precursor cells were found to originate lateral to bristle precursor cells. In 6 of the 8 longitudinal rows of bristles on this segment, the bract cells arise ventral to the bristle cells; in the others they arise dorsally. The lateral cell origins are interpreted as reflecting a pattern of lateral cell movements associated with evagination of the leg disc. An unusual discrepancy was observed in the relative frequencies of male vs. female bracts and bristles in gynandromorphs. The discrepancy suggests that there is a cell-autonomous sexual difference in either the time at which cells begin moving during evagination or the speed with which they move.On the basis of the results, it is reasoned that the bristle pattern of the basitarsus does not originate in its final form. Prior to evagination, the bristle cells of each row are apparently closer together than in the final pattern, and the rows are farther apart. Evidence is presented which suggests that the bristle cells of each row may originally be arranged in a jagged line which is later straightened by cell movements.The two locations where the anterior/posterior compartment boundary of the second leg passes through the basitarsus were found to vary relative to the bristle pattern. If this boundary is assumed to be a fixed line of positional values, then the extent of the observed variability — which is estimated to be ± 1 or 2 cell diameters — provides a measure of the precision of patterning around the circumference.     

Changes in the distribution of gap junctions inDrosophila melanogaster wing discs during the third larval and early pupal stages of development

Summary Developmental changes in the distribution of gap junctions in early, mid and late third larval stage wing discs and in pupariation+6 h and pupariation+24 h stage wing discs fromDrosophila melanogaster were analyzed by quantitative electron microscopy. Gap junctions occur in all 12 intradisc regions examined in each of the five developmental stages. Their distribution is non-random and changes during development which suggests that they are developmentally regulated. The gap junctions are not static structures, rather they grow and regress during development. The changes tend to be gradual ones without sudden increases or decreases. Gap junctions continuously form and grow in size throughout the third larval stage and during the first 6 h following pupariation. Their surface density, number, percent of the lateral plasma membrane area, and absolute area as well as the lateral plasma membrane surface density all increase during this time. Between pupariation+ 6 h and pupariation+24 h all but one of these parameters decrease indicative of gap junctional breakdown. Gap junctions are most numerous and change least during development in the apical cell regions where intercellular contacts are close and stable. They change most in the basal cell regions where intercellular contacts tend to be looser and change during development. The most dramatic change is in the absolute area which increases by a factor of 23 between the early third larval stage and pupariation+24 h. At pupariation the rate of gap junction growth undergoes a transient increase before the phase of disassembly begins. Developmental changes in gap junction surface density are closely coupled with changes in the lateral plasma membrane surface density which suggests that these may be coregulated. Evidence from mutants suggests that when the number and density of gap junctions fail to increase in proportion to lateral plasma membrane growth, wing disc development will be abnormal. Our results support the idea that some minimum gap junction density is required for normal development and that this must increase as development proceeds. The results are consistent with the notion that gap junctions are involved in pattern formation and growth control and are discussed with respect to the acquisition of competence for metamorphosis, disc growth, disc morphogenesis and changes in the hormonal environment.     

Evolution of nested genes with special reference to cuticle proteins inDrosophila melanogaster

Summary One of the pupal cuticle protein (PCP) genes has been found within an intron of aDrosophila housekeeping gene (theGart locus) that encodes three enzymes involved in the purine pathway. This intronic gene has been described as a gene within a gene, and the gene is now called a “nested” gene. Because the intronic PCP gene has sequence similarity with the larval cuticle protein (LCP) gene, it may have been derived from one of the LCP genes or their ancestral gene. We have studied possible phylogenetic relationships among these five genes by comparing nucleotide sequences of four LCP genes with that of the PCP gene. The results obtained suggest that the PCP gene may have originated from an ancestral gene before duplication of the LCP genes occurred. Using the number of synonymous (silent) substitutions, we then estimated the divergence time between the PCP gene and the LCP genes to be about 70 million years (Myr). The divergence time estimated is much larger than that for the sibling species ofD. melanogaster (about 2.5 Myr), indicating that the “nested” gene structure can be seen not only inDrosophila melanogaster, but also in other distantly relatedDrosophila species.     

Arrangement of bristles as a function of bristle number on a leg segment inDrosophila melanogaster

Summary The arrangement of bristles on a leg segment of the fruitflyDrosophila melanogaster was studied in various mutants that have abnormal numbers of bristles on this segment. Eighteen mutations at six different genetic loci were analyzed, plus five double or triple mutant combinations. Recessive mutations at theachaete-scute locus were found to affect distinct groups of bristles:achaete mutations remove mechanosensory bristles, whereasscute mutations remove mainly chemosensory bristles. Mechanosensory bristles remain uniformly spaced along the longitudinal axis unless their number decreases below a certain threshold, suggesting that spacing is controlled by cell interactions that cannot function when bristle cells are too far apart. Above a certain threshold, bristle spacing and alignment both become irregular, perhaps due to excessive force from these same interactions. Chemosensory bristles occupy definite positions that are virtually unaffected by removal of individual bristles from the array. Extra chemosensory bristles develop only near the six normal sites. At two of the six sites the multiple bristles tend to exhibit uniform longitudinal spacing — a property confined to mechanosensory bristles in wild-type flies. To explain the various mutant phenotypes the following scheme is proposed, with different mutations directly or indirectly affecting each step: (1) spots and stripes are demarcated within the pattern area, (2) one bristle cell normally arises within each spot, multiple bristle cells within each stripe, (3) incipient bristle cells inhibit neighboring cells from becoming bristle cells, and (4) the bristle cells within each stripe become aligned to form rows and then repel one another to generate uniform spacing.     

Segmental organisation of the tail region in the embryo of Drosophila melanogaster

Summary The segmental organisation of the tail region in the embryo of Drosophila melanogaster, which is defined here as the epidermal region posterior to the boundary between abdominal segments A7 and A8, has been investigated by means of ultraviolet (UV) laser fate-mapping and phenotypic analysis of embryonic mutants that alter the segmental pattern of the larval cuticle. Wild-type embryos were irradiated in the presumptive tail region with a UV- laser microbeam of 20 m diameter at the blastoderm stage. The ensuing defects were scored in the cuticle pattern of the tail region of the first-instar larva, which is described in detail in this paper. The spatial distribution of defect frequencies was used to construct a blastoderm fate-map of the cuticle structures of the larval tail region. The segmental origin of the larval tail structures was inferred from the phenotypic analysis of segmentation and homoeotic mutants, which revealed pattern repetition throughout the embryonic tail region corresponding to four segment anlagen, A8 to A11, and a non-segmental telson. These data enabled the transformation of the blastoderm fate-map of cuticle structures into a map of tail segment anlagen. The tail anlage occupies about 10% of the egg length (EL), bounded by segment A7 anteriorly at 20% EL and by the proctodaeum posteriorly at 10% EL, as measured from the posterior pole. The anlagen of segments A8 and A9 appear to be narrow dorso-ventral strips of blastoderm cells similar to the anlagen of the trunk segments, whereas the anlagen of A10 and A11 are smaller and produce fewer pattern elements. The telson is represented in the cuticle by the tuft which derives from a very dorsal posterior position. The antero-posterior axis of the entire tail anlage appears curved upward posteriorly. Differences in the mode of development between tail and trunk segments are discussed, as are similarities of larval and imaginal tail development in Drosophila. Comparison with tail development in other insects suggests that, during evolution, the transition from semi-long-germ to long-germ development modified the organisation of the tail region without affecting its primary subdivision into metameric units.     

Wingless mutation inDrosophila melanogaster

A temperature sensitive lethal allele of thewingless locus ofDrosophila melanogaster together with previously studied lethal and viable alleles in this locus, has been used to study some properties of this locus. These studies show the existence of two lethal phases for thewingless lesion; one during embryogenesis and another during pupation. By growing embryos with temperature sensitivewingless lesion at the permissive temperature and letting the larvae develop at non-permissive temperature, a large-scale cell death and subsequent regeneration were seen to occur in the mutant wing discs. This cell death followed by regeneration alters the normal developmental potential of the wing disc. Disc transplantation experiments show that these discs are incapable of differentiating into wing blade structures.     

Sensitive periods for abnormal patterning on a leg segment inDrosophila melanogaster

Summary The development of a leg segment of the fruitflyDrosophila melanogaster was analyzed in order to determine whether the orderliness of the segment's bristle pattern originates via waves of cellular interactions, such as those that organize the retina. Fly development was perturbed at specific times by either teratogenic agents (gamma rays, heat shock, or the drug mitomycin C) or temperature-sensitive mutations (l(1)63, l(1) Notch^ts1, orl(1) shibire ^ts1 ), and the resulting abnormalities (e.g., missing or extra structures) were mapped within the pattern area. If bristles develop in a linear sequence across the pattern, then they should show sensitivity to perturbations in the same order, and wavefronts of cuticular defects should result. Contrary to this prediction, the maps reveal no evidence for any directional waves of sensitivity. Nevertheless, other clues were uncovered as to the nature and timing of patterning events. Chemosensory bristles show earlier sensitivities than mechanosensory bristles, and longer bristles precede shorter ones. The types and sequence of cuticular abnormalities imply the following stages of bristle pattern development: (1) scattered inception of bristle mother cells, each surrounded by an inhibitory field, (2) alignment of the mother cells into rows, (3) differential mitoses, (4) assignment of cuticular fates to the mitotic progeny, (5) polytenization of the bristle cells, (6) fine-tuning adjustments in bristle spacing, and (7) signalling from bristle cells to adjacent epidermal cells, inducing them to form bracts.     

Segmental organisation of the head in the embryo of Drosophila melanogaster

Summary Embryos of Drosophila melanogaster were irradiated in the presumptive head region with a UV-laser microbeam of 20 m diameter at two developmental stages, the cellular blastoderm and the extended germ band. The ensuing defects were scored in the cuticle pattern of the head of the first-instar larva, which is described in detail in this paper. The defects caused by irradiating germ band embryos when morphologically recognisable lobes appear in the head region were used to establish the segmental origin of various head structures. This information enabled us to translate the spatial distribution of blastoderm defects into a fate map of segment anlagen. The gnathal segments derive from a region of the blastoderm between 60% and 70% egg length (EL) dorsally and 60% and 80% ventrally. The area anterior to the mandibular anlage and posterior to the stomodaeum is occupied by the small anlagen of the intercalary and antennal segments ventrally and dorsally, respectively. The labrum, which originates from a paired anlage dorsally at 90% EL, is separated from the remaining head segments by an area for which we did not observe cuticle defects following blastoderm irradiation, presumably because those cells give rise to the brain. The dorsal and lateral parts of the cephalo-pharyngeal skeleton appear to be the only cuticle derivatives of the non-segmental acron. These structures derive from a dorso-lateral area just behind the putative brain anlage and may overlap the latter. In addition to the segment anlagen, the regions of the presumptive dorsal pouch, anterior lobe and post-oral epithelium, whose morphogenetic movements during head involution result in the characteristic acephalic appearance of the larva, have been projected onto the blastoderm fate map. The results suggest that initially the head of the Drosophila embryo does not differ substantially from the generalised insect head as judged by comparison of fate map and segmental organisation.     

Evidence for myosin heterogeneity inDrosophila melanogaster

Summary Electrophoresis of myosin extracts from larvae and adult tissues ofDrosophila melanogaster under non-dissociating conditions indicate that two of the bands seen are myosins. They stain for Ca²⁺ ATPase activity and when cut and re-run under dissociating conditions are found to contain a myosin heavy chain that co-migrates with rabbit skeletal muscle myosin heavy chain. One of the forms of myosin seen is found primarily in extracts from the leg. The other is common to the adult fibrillar flight muscles and the larval body wall muscles.The electrophoretic evidence for two myosin types is strengthened by the histochemical demonstration of two myofibrillar ATPases on the basis of their lability to acid or alkali preincubation. The myofibrillar ATPase in the leg and the Tergal Depressor of the Trochanter (TDT) are shown to be relatively acid labile and alkali stable. The larval body wall muscles and the adult fibrillar flight muscles have an ATPase which is acid stable and alkali labile. This distribution of the two myofibrillar ATPase coincides with that predicted by electrophoresis of extracts from whole tissue and also locates the two myosins to specific muscle types.     

An assembly zone antigen of the insect cuticle

The assembly zone is a morphologically distinct region in the insect integument that lies between the epidermis and its principal secretory product, the lamellate cuticle. Despite its central location in the process of cuticle formation, little is known about its structure or function. Using various antisera we have shown that in Drosophila melanogaster larvae and pupae the assembly zone is antigenically distinct from the overlying lamellate cuticle. This observation suggests that this region does not contain lamellae in the process of assembling but rather is a stable and permeable matrix through which lamellar components travel in the process of cuticle formation. Curiously an antigen present in the assembly zone was also contained in the moulting gel, indicating a heretofore unsuspected chemical relationship between these two materials.     

Contribution of the geneextramacrochaetae to the precise positioning of bristles inDrosophila

We examine the effect of mutations in theextramacrochaetae (emc) gene on the positioning of macrochaetes on the notum ofDrosophila. We show that, inemc mutants, most of the precursor cells appear earlier than in wild-type individuals, consistent with an antagonizing effect ofemc on the action of the proneural genesachaete andscute. We also show that reducingemc function affects the position of three bristles and/or of their precursors, but has no marked effect on the positioning of the other bristles.     

Development of thoracic curvature inDrosophila melanogaster

Summary The influence of muscle development on thorax morphogenesis has been investigated inDrosophila melanogaster. The development of an indirect flight muscle, the dorsal longitudinal muscle (DLM), has been thought to be responsible for the formation of the distinct thoracic curvature. Using aDrosophila mutant (sr/Df(3)sr) in which the DLM is completely missing, we have shown that a normally curved thorax still is produced. Such results indicate that an external structure (epidermis) is capable of developing wholly independent of an absent internal structure (muscle).     

Molecular cloning of three cDNAs encoding putative larval cuticle protein expressed differentially after larval ecdysis from the mulberry longicorn beetle, Apriona germari

Three cDNAs encoding putative larval cuticle protein (LCP) were cloned from the mulberry longicorn beetle, Apriona germari. The three cDNA sequences were 309 bp, 396 bp and 408 bp in length, encoding 103, 132 and 136 amino acid residues, respectively. The predicted molecular masses for these LCPs were approximately 9.2 kDa (AgLCP9.2), 12.3 kDa (AgLCP12.3) and 12.6 kDa (AgLCP12.6). Pairwise identity among AgLCP9.2, AgLCP12.3 and AgLCP12.6 were relatively low. Each AgLCP contained a type-specific consensus sequence identifiable in other insect cuticle proteins. The deduced amino acid sequence of AgLCP9.2 is most similar to Bombyx mori LCP18 and those of AgLCP12.3 and AgLCP12.6 are both most similar to B. mori LCP17. Northern blot analysis revealed that the three AgLCPs showed epidermis-specific expression. The expression profile of AgLCPs after larval ecdysis revealed by Northern blot analysis that the high-level mRNA expression of AgLCPs was detected on the first day of larval ecdysis for AgLCP9.2, on the fifth day for AgLCP12.3 and from the first day of larval ecdysis to the fifth day after larval ecdysis for AgLCP12.6, demonstrating that AgLCP mRNAs are differentially expressed in epidermis after larval ecdysis.     

Isolation and characterization ofgrandchildless-like mutants inDrosophila melanogaster

Summary Two temperature-sensitive sex-linkedgrandchildless (gs)-like mutations (gs(1)N26 andgs(1)N441) were induced by ethylmethane sulphonate inDrosophila melanogaster. They complemented each other and mapped at two different loci (1–33.8±0.7 forgs(1)N26 and 1–39.6±1.7 forgs(1)N441), which were not identical to those of any of thegs-like mutants reported in earlier work.Homozygous females of the newly isolated mutants produced eggs that were unable to form pole cells and developed into agametic adults. Competence of the embryos to form pole cells was not restored by wild-type sperm in either mutant; that is, the sterility caused by these mutations is controlled by a maternal effect.Fecundity and fertility ofgs(1)N26 females were low, and their male offspring showed a higher mortality than that of female offspring, causing an abnormal sex ratio. The frequency of agametic progeny was 93.1% and 55.8%, when the female parents were reared at 25° C and 18° C, respectively. In eggs produced by thegs(1)N26 females reared at 25° C, the migration of nuclei to the posterior pole was abnormal, and almost no pole cell formation occurred in these egg. Furthermore, half of these eggs failed to cellularize at the posterior pole. When the females were reared at 18° C, almost all of the eggs underwent complete blastoderm formation, and in half of these blastoderm embryos normal pole cells were formed.In the other mutant,gs(1)N441, the fecundity and fertility of the females were normal. The agametic frequency in the progeny was 70.8% and 18.6% when the female parents were reared at 25° C and 18° C, respectively. In the eggs laid by females reared either at 25° C or at 18° C, the migration of nuclei to the periphery and cellularization proceeded normally; nevertheless, in the majority of the embryos no pole cell formation occured at the stage when nuclei penetrated into the periplasm. When the females were reared at 18° C, some of the embryos from these females formed some round blastoderm cells with cytologically recognizable polar granules and nuclear bodies, which are attributes of pole cells. The temperature sensitive period ofgs(1)N441 was estimated to extend from stage 9 to 13 of King's stages of oogenesis.     

Cell interactions in the generation of chaetae pattern inDrosophila

Summary We have already shown that theachaetae-scute complex (AS-C) ofDrosophila is regulated by two genes,hairy andextramacrochaetae. Using mutants in these genes, we have analysed how different levels of expression of AS-C affect the pattern of chaetae. The results indicate that the spatial distribution of chaetae results from cell interactions, probably by a mechanism of lateral inhibition. The results are discussed in view of the different theories of pattern formation.     

Probing the evolution of senescence inDrosophila melanogaster withP-element tagging

Natural populations host a wealth of genetic variation in longevity and age-specific schedules of reproduction. This variation provides critical information for inferring the evolutionary origin of senescence. Patterns of mutational effects on age-specific fecundity and survival provide additional insight to distinguish alternative models of senescence. In this study,P-elements bearing thewhite minigene were inserted at random into a common genetic background, generating lines ofD. melanogaster with single, stable transposon inserts. A series of 48 single-P-element lines revealed statistically significant heterogeneity in both longevity and fecundity. Longevity and early fecundity were only weakly positively correlated (r=0.286,P=0.0398). Both the pooled sample and 30 of the individual lines exhibited a leveling of age-specific mortality at advanced ages, in opposition to the classical demographic models. To the extent that these mutational effects are representative of naturally-occurring mutations in heterogeneous populations, this result presents a problem for the evolutionary theory of senescence. Natural selection is inefficient at removing deleterious mutations that are expressed only at late ages, and selection may not differentiate between mutations whose effects on longevity are post-reproductive. A leveling of the mortality rate would also be seen if mutations whose expression is delayed until very late simply do not occur. A simulation of mutation-selection balance among the 48P-element tagged lines shows that the mean longevity declines monotonically with increasing mutation rate, consistent with the mutation-accumulation model.     

Chromosomal effects on peptidase activities inDrosophila melanogaster

The peptidase system inDrosophila melanogaster (dipeptidase-A, -B, and -C and leucine aminopeptidases G and P) was used as a model to study the effects of modifier genes on activity of enzymes with similar functions. A screen of X, second, and third chromosome substitution isogenic lines revealed the presence of activity modifiers for peptidases on all three chromosomes. Correlation analyses indicated that covariation between some of the peptidase activities is independent of genetic background, while others are associated with variable second chromosomes. Chromosome-specific effects onK _m ,V _max, and specific activity of partially purified peptidases were also detected. Moreover, a repeatable technique using anion-exchange column chromatography allowed the characterization of possibly two putative peptidic enzymes, glycyl-l-isoleucine-ase andl-leucyl-l-proline-ase, whose kinetic properties differ from the dipeptidases and the leucine aminopeptidases. These findings confirm the existence of activity modifiers for peptidases, much like other enzymes inDrosophila melanogaster. These studies were supported by grants from the National Institutes of Health (GM42-115-01A1), the Whitaker Foundation of the Research Corporation (C-2560), and the National Science Foundation (USE 8951018) to Kazuo Hiraizumi.     

A study of germinal mosaicism inDrosophila melanogaster

Summary Three-hundred and twenty fertile,pal-induced Y-chromosome mosaic males and females were obtained. Fractional analysis of the sons of 55 somatically mosaic flies that were also germinally mosaic tentatively suggests that the number of functional primordial germ cells inDrosophila melanogaster is variable and that it is seldom greater than 24. From the observed 0.17 frequency of germinal mosaicism it was estimated that the average number of pole cells at the end of blastoderm formation is 45. At present, the germ cells afford the only opportunity to compare genetic estimates of the number of blastoderm or primordial cells with available histological counts. The good agreement between them suggests that both the fractional and the mosaic frequency methods for estimating primordial or blastoderm cell numbers of various larval and imaginal anatomical structures provide reasonably close approximations of the actual values.     

Genomic imprinting of chromatin inDrosophila melanogaster

During gametogenesis, chromosomes may become imprinted with information which facilitates proper expression of the DNA in offspring. We have used a position effect variegation mutant as a reporter system to investigate the possibility of imprinting inDrosophila melanogaster. Genetic crosses were performed in which the variegating gene and a strong modifier of variegation were present either within the same parental genome or in opposite parental genomes in all possible combinations. Our results indicate that the presence of the variegating chromosome and a modifier chromosome in the same parental genome can alter the amount of variegation formed in progeny. The genomic imprinting we observed is not determined by the parental origin of the variegating chromosome but is instead determined by the genetic background the variegating chromosome is subjected to during gametogenesis.     

Mutations affecting phenol oxidase activity inDrosophila: quicksilver andtyrosinase-1

The complex enzyme phenol oxidase plays a major role in sclerotization and melanization of cuticle in insects. Production of active enzyme from the inactive proenzyme involves at least six protein components inDrosophila. We examine here the biochemical phenotype of two loci that affect phenol oxidase activity—quicksilver (qs; 1–39.5) andtyrosinase-1 (tyr-1; 2–54.5). Three mutations isolated by different procedures in three different laboratories are alleles at thequicksilver locus. The effects of these mutations have been monitored by means of enzyme assaysin vitro and in polyacrylamide gels and by measurement of catecholamine pool sizes. The activity of all three active enzyme components (A1, A2, and A3) is reduced inqs mutants. The activated enzyme of oneqs allele is thermolabile, while its activator is normal. Deletion and genetic mapping placetyr-1 nearpurple (pr; 2–54.5). Enzyme activity is reduced to 10% of normal but is not thermolabile and the activator is normal. The activity of all three A components is reduced. The diphenol oxidase activity in double mutant combinations shows that these mutations andDox-A2 (Pentzet al., 1986) affect this enzyme in different ways.B.C.B. was supported by National Institutes of Health Research Grant GM31217 and E.S.P. was supported by National Institutes of Health Research Grant GM19242 to T.R.F.W.