Aldehyde oxidase distribution in haltere discs of homoeotic bithorax mutants in Drosophila melanogaster.

Summary Distribution of the enzyme aldehyde oxidase in transformed haltere discs from the homoeotic bithorax series of mutants was investigated by histochemical means. The bithorax (bx) mutant, which transforms the anterior part of the haltere into an alterior with blade, possesses in the haltere disc an aldehyde oxidase staining pattern similar to that of the anterior side of the wing disc. The postbithorax (pbx) mutant, which transforms the posterior haltere into a structure resembling the posterior wing blade, reveals an aldehyde oxidase staining pattern in the haltere disc characteristic of the posterior side of the wing disc pouch. When both (bx ³ (pbx) mutants are present the haltere develops into a metathoracic wing. It is shown here that the transformed haltere disc closely resembles the previously established pattern in the wing disc with respect to aldehyde oxidase distribution. Change in the pattern of aldehyde oxidase in bithorax mutants signals alteration in gene expression which at least for this particular enzyme correlates well with the morphological transformation from haltere to wing. A possible correlation between pattern of enzyme activity and developmental compartmentalization has been discussed.     

Aldehyde oxidase distribution in the imaginal discs of some diptera

Summary In the imaginal discs ofMusca domestica, Drosophila melanogaster, D. simulans, D. hydei, andZaprionus spec. the enzyme aldehyde oxidase (AO) appeared in a clear-cut pattern. In the leg and eye-antennal discs of these species this pattern shows a high degree of conformity, while that of the wing and haltere discs is species-specific.No aldehyde oxidase activity was detected in the imaginal discs ofCalliphora erythrocephala, Phormia regina orLucilia cuprina, but the discs of these species are characterized by grossly similar patterns of 5-nucleotidase. Since the other species studied lack this enzyme, the two enzymes may perform similar functions in the morphogenesis of the discs.The coincidence of the sharp boundary of the AO pattern in the leg and wing discs ofD. melanogaster with the boundary between the anterior and posterior disc compartments gives a strong indication for the existence of analogous compartments in other discs showing a similar sharply bounded AO pattern. Compartmentalization may be considered a general phenomenon which occurs in discs of all segments and is not restricted toD. melanogaster. From the changes in the AO pattern during disc development it can be deduced that the localisation of this enzyme is regulated by supracellular determination involving positional information.     

Influence of homoeotic genes on the aldehyde oxidase pattern in imaginal discs of Drosophila melanogaster

In a study of the regulation of enzyme patterns in imaginal discs the aldehyde oxidase pattern was determined for some homoeotic mutations of D. melanogaster. Earlier indications that suggested that this pattern follows the determinitive state of compartments within imaginal discs were confirmed by the aldehyde oxidase (AO) pattern of both the wing and haltere discs from en¹; bx³, en¹; pbx, and en¹; bx³ pbx larvae and the antennal discs from Antp^73b and ss^a larvae. We additionally analyzed whether AO activity depended on the determinative state of an entire compartment or was expressed autonomously in clones. Homozygous engrailed clones were induced by mitotic recombination. From the AO clones found in normally negative areas of the posterior compartment it was concluded that enzyme activity depended upon the determinative state of the cells and was not a function of the compartment as a whole. The results are described with reference to a scheme in which compartmental and subcompartmental selector genes are thought to determine a binary code on which AO patterns depend.     

Expression of phenotypes in a temperature-sensitive allele of the apterous mutation in Drosophila melanogaster

A temperature-sensitive allele of the apterous (ap) locus of Drosophila melanogaster has been used to examine the phenotypes produced by this mutation, which include wing, mesonotal, and haltere deficiencies, precocious adult death, and nonvitellogenic oocyte development. When raised at 15°C, homozygous ap^ts78j adults have nearly wild-type wing morphology except for patches of missing triple-row bristles and posterior wing margin deficiencies. With the exception of two missing bristles, the dorsal mesonotum and the haltere appear as wild-type. Increasing deficiency of structures derived from the wing and haltere imaginal discs results from increasing culture temperature, and at 29°C, the wing blade, many dorsal mesonotal bristles, and the haltere are absent. The temperature-sensitive period in development for these deficient phenotypes extends from late-second to mid-third instar. Despite extensive deficiencies seen after ap^ts78j larvae are heat-pulsed at 29°C, no duplication of the notal structures is evident, a common response of other mutants having extensive wing deficiencies. When raised at 29 or 25°C, ap^ts78j adults are short-lived, and females show nonvitellogenic oocyte development. At 22°C, however, adults are long-lived, and females are vitellogenic and lay fertile eggs. A sharp temperature-sensitive period for both phenotypes is located during the first 24 hr of pupal development. The application of a juvenile hormone mimic, ZR-515, restored vitellogenesis to ap^ts78j females raised at 25°C but was unable to rescue them from precocious death.     

The determination of aldehyde oxidase activity patterns in the wing discs of Drosophila melanogaster

Summary The pattern of aldehyde oxidase (AO) activity was determined in wing discs of Drosophila melanogaster larvae homozygous for the mutants apt ⁷³ⁿ, Beaded, and vestigial (vg) in order to determine if reduction in field size in the pouch could be related to alterations of the wild-type AO pattern, as suggested by the Kauffman (1978) hypothesis. The pattern in wild-type discs was resolved into six areas for comparison with mutant discs. vg discs developed at 25° C showed restriction of the pattern into a small area on the anterior side of the disc, and comparison of vg and wild-type prepupal wings allowed positive identification of the AO pattern elements which remained. AO patterns in vg wing discs grown at 27°, 29°, and 31° C were progressively more complete and similar to wild-type, reflecting the reduction in cell death in discs grown at higher temperatures. These results show that cell loss during the third instar in vg development at 25° C is responsible for the alteration of the AO pattern, rather than field size reduction, and that determination of the pattern must take place much earlier than the time of its first appearance during the third larval instar, and before cell death in vg discs begins. Thus mutants acting at earlier stages will be necessary for further tests of the Kauffman hypothesis.     

Components of positional information in the developing wing margin of the Lyra mutant of Drosophila

Summary A number of parameters characteristic of the wing margin precursor in imaginal discs of Drosophila are known: the zone of non-proliferating cells or ZNC (O'Brochta and Bryant 1985), aldehyde oxidase (AO) and other enzyme staining patterns (Sprey et al. 1982), E1C antigen localization in a narrow band along the margin (Piovant and Lena 1988). To test our hypothesis that such parameters, and others, act in concert to determine margin identity and the positional information that specifies the bristles and hairs appropriate to the anterior, posterior and distal margins, we have examined these parameters in the dominant mutant Lyra, in which much of the anterior and posterior margins is missing. After establishing that Lyra phenotype is already evident in the early pupal wing, we tested the known imaginal disc parameters and found that only Mab E1C (Piovant and Lena 1988) distribution differs from wild type, suggesting that E1C antigen may be a component of positional information. Sibatani's (1983) model for specification of positional information (PI) applied to wing discs predicts the Lyra adult wing shape as well as the reduced distribution of E1C antigen in Lyra wing discs. The model is based on the assumption that specification of positional information depends on interactions of multiple, independent factors. Clonal analysis with shaggy (Simpson et al. 1988 and Ripoll et al. 1988) indicates that factors in addition to E1C antigen contribute to margin PI in Lyra wings and should allow us to test the multi-component hypothesis further.     

Evolutionary origin of the insect wing via integration of two developmental modules

SUMMARY Insect wing is a key evolutionary innovation for insect radiation, but its origins and intermediate forms are absent from the fossil record. To understand the ancestral state of the wing, expression of three key regulatory genes in insect wing development, wingless (wg), vestigial (vg), and apterous (ap) was studied in two basal insects, mayfly and bristletail. These basal insects develop dorsal limb branches, tracheal gill and stylus, respectively, that have been considered candidates for wing origin. Here we show that wg and vg are expressed in primordia for tracheal gill and stylus. Those primordia are all located in the lateral body region marked by down‐regulation of early segmental wg stripes, but differ in their dorsal–ventral position, indicating their positions drifted within the lateral body region. On the other hand, ap expression was detected in terga of mayfly and bristletail. Notably, the extensive outgrowth of the paranotal lobe of apterygote bristletail developed from the border of ap‐expressing tergal margin, and also expressed wg and vg. The data suggest that two regulatory modules involving wg–vg are present in apterygote insects: one associated with lateral body region and induces stick‐like dorsal limb branches, the other associated with the boundary of dorsal and lateral body regions and the flat outgrowth of their interface. A combinatorial model is proposed in which dorsal limb branch was incorporated into dorsal–lateral boundary and acquired flat limb morphology through integration of the two wg–vg modules, allowing rapid evolution of the wing.     

Tissue-specific and substrate-specific detection of aldehyde and pyridoxal oxidase in larval and imaginal tissues of Drosophila melanogaster

The substrate specificities of aldehyde and pyridoxal oxidases in Drosophila melanogaster have been determined with a variety of aliphatic and aromatic aldehydes. This analysis has led to the discovery that 2,4,5-trimethoxy-benzaldehyde is a specific substrate for pyridoxal oxidase, as based on the histochemical distribution of oxidase activity, the absence of enzymatic activity in the lpo ¹strains, and the dosage dependence on the number of lpo ⁺genes present. The tissue-specific localization of aldehyde oxidase (AO) and pyridoxal oxidase (PO) in the larval and adult structures showed that AO was present in all the major internal organs of the larvae and adults, including brain, imaginal discs, Malpighian tubules, digestive system, and reproductive structures. Pyridoxal oxidase is present in many of the same structures which possess AO, but is missing from the cardia, crop, imaginal discs, ovarian follicle cells, paragonia, pericardial cells, and wreath cells. The only structure which possesses PO but lacks AO is the larval salivary gland. These histochemical differences in AO and PO distribution were also confirmed by enzymatic analysis of the activities present in homogenates of ovaries, paragonia, and salivary glands. The general pattern of enzyme expression appears to be established during embryogenesis and maintained throughout the life of the individual.This work was supported by NIH Grants AG01975 and GM27866.This paper is dedicated to Professor Donald F. Poulson, Yale University, a pioneer in Drosophila developmental genetics.     

Mutations in tumour suppressor genes,l(2)gl andl(2)gd,alter the expression ofwingless inDrosophila

To understand the roles of two well known tumour suppressor genes.l(2)gl andl(2)gd in normal imaginal disc development inDrosophila, we have initiated a study to examine effect of mulations of these genes on the expression of genes involved in the patterning of the imaginal discs. In this study we show that the expression ofwingless, theDrosophila orthologue of the mammalian oncogeneWnt, is affected in the imaginal discs ofl(2)gl ⁴ andl(2)gd ¹ mutant individuals. In the tumourous wing imaginal discs froml(2)gl mutant larvae, the pattern ofwingless expression was progressively disrupted with an increase in the area of expression, Tumourous wing imaginal discs froml(2)gd homozygous individuals exhibited progressive broadening and extension of the wingless expressing domains. We suggest thatl(2)gl andl(2)gd might be involved in regulating post embryonic expression ofWingless.     

An ultrastructural and developmental analysis of the corpus allatum of juvenile hormone deficient mutants ofDrosophila melanogaster

Summary The ultrastructure of the corpus allatum of theapterous mutantsap ⁴ andap ^56f ofDrosophila melanogaster during larval-pupal-adult metamorphosis and adult life was correlated with the gland's ability to synthesize juvenile hormone in vitro. During the early wandering period of the third instar of both mutants, a high concentration of smooth endoplasmic reticulum, mitochondria and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. Juvenile hormone biosynthesis by the glands is high at that time and, in fact, only slightly lower than that of wild type glands. In contrast to the wild type gland, the cells of the pupal and pharate adult corpus allatum of both mutants contains highly electron dense mitochondria with tubular cristae but no whorls of smooth endoplasmic reticulum nor glycogen clusters. The frequency and size of the lipid droplets, putatives depots of the juvenile hormone precursors, in cells of theap ^56f gland is a function of the insect's age, but both are lower than in wild type gland cells. Juvenile hormone biosynthesis by both mutant glands remains at the basal level when compared to increased synthesis by the wild type gland. The frequency and density of lipid droplets in cells of theap ⁴ corpus allatum are much lower than in theap ^56f glands. During adult life, the ultrastructural profile of theap ^56f corpus allatum is similar to that of the wild type gland although the in vitro production of juvenile hormone by the former is much lower than that of the wild type gland. The ultrastructural features of the adult corpus allatum ofap ⁴ homozygotes reveal precocious degeneration and support the view that this non-vitellogenic mutant is a juvenile hormone deficient mutation.     

Developmental Analysis of the Wing Disc in the Mutant Engrailed of DROSOPHILA MELANOGASTER

The engrailed (en) mutation leads to the transformation of the posterior structures of the dorsal mesothoracic disc into those characteristic of the anterior region of the same disc. Similar posterior-anterior duplications have been detected in dorsal as well as ventral structures of all the thoracic segments. —Genetic combinations of en with other pattern mutants have shown their synergistic effect on the posterior wing pattern.—A clonal analysis of the en wing disc shows that en affects its development in a characteristic way. The genetic change, by induced mitotic recombination, of en⁺ into en cells is followed by the corresponding transformation, except when it takes place some cell divisions prior to differentiation.—The en posterior wing disc cells show positive affinities with normal anterior wing disc cells in aggregates.—The mode of action of the en⁺ locus controlling wing disc development is discussed.     

Negative regulation ofUltrabithorax expression byengrailed is required for proper specification of wing development inDrosophila melanogaster

In both vertebrates and invertebrates, homeotic selector genes confer morphological differences along the antero-posterior axis. However, insect wing development is independent of all homeotic gene functions, reflecting the ground plan of an ancestral pterygote, which bore wings on all segments. Dipteran insects such asDrosophila are characterized by a pair of wings in the mesothoracic segment. In all other segments, wing development is essentially repressed by different homeotic genes, although in the metathorax they are modified into a pair of halteres. This necessitates that during development all homeotic genes are to be maintained in a repressed state in wing imaginal discs. In this report we show that (i) the function of the segment polarity geneengrailed (en) is critical to keep the homeotic selector geneUltrabithorax (Ubx) repressed in wing imaginal discs, (ii) normal levels of En in the posterior compartment of haltere discs, however, are not enough to completely repressUbx, and (iii) the repression ofUbx byen is independent of Hedgehog signalling through which the long-range signalling ofen is mediated during wing development. Finally we provide evidence for a possible mechanism by whichen repressesUbx. On the basis of these results we propose thaten has acquired two independent functions during the evolution of dorsal appendages. In addition to its well-known function of conferring posterior fate and inducing long-range signalling to pattern the developing appendages, it maintains wing fate by keepingUbx repressed.     

Yolk-polypeptide synthesis and uptake in ovaries of the Apterous4 mutant of Drosophila melanogaster

Summary The non-vitellogenic ovaries from homozygous apterous ⁴ mutants of Drosophila melanogaster synthesise yolk-proteins when cultured in vitro, which accounts for the ability of ap ⁴/ap ⁴ ovaries to become vitellogenic in a male-host environment. However, when ap ⁴/ap ⁴ ovaries are transplanted into D. arizonensis females, a large proportion of the ovarian yolk proteins are of the arizonensis type, indicating that ap ⁴/ap ⁴ ovaries are also able to take up yolk proteins in a suitable female-host environment.     

Developmental compartments in the Drosophila melanogaster wing disc

Distribution of the enzyme aldehyde oxidase (AO) within the pouch of the mature wing disc is precise and differential. General locations of compartmental boundaries have been identified by fate mapping and studies of AO distribution. The suspected locations of the boundaries were verified by analyzing the distribution of AO-negative cells within an AO-stained background in gynandromorphs and in X-ray-induced clones of AO-negative cells. The anterior/posterior border appeared slightly anterior to the junction of the AO⁺ anterior presumptive wing surfaces and AO^? posterior wing surfaces. A narrow band of AO⁺ cells extending proximodistally on both presumptive wing surfaces belongs to the posterior compartment. Two dorsal/ventral (dor./vent.) restrictions were found. The dor./vent. restriction equivalent to the dor./vent. border found in the adult wing was located at the ventral most edge of the AO-stained presumptive wing margin. A second restriction which was less strictly obeyed was found on the dorsal edge of the wing margin. We conclude that the whole presumptive wing margin is part of the dorsal compartment. Within the anterior wing margin an intensively stained oval was also found to be clonally restrictive. Therefore, territories were found within the prospective wing margin for which no such features have been identified in the adult Drosophila melanogaster wing.     

The wings ofBombyx mori develop from larval discs exhibiting an early differentiated state: a preliminary report

Lepidopteran insects present a complex organization of appendages which develop by various mechanisms. In the mulberry silkworm,Bombyx mori a pair of meso- and meta-thoracic discs located on either side in the larvae gives rise to the corresponding fore- and hind-wings of the adult. These discs do not experience massive cell rearrangements during metamorphosis and display the adult wing vein pattern. We have analysed wing development inB. mori by two approaches, viz., expression of patterning genes in larval wing discs, and regulatory capacities of larval discs following explantation or perturbation. Expression of Nubbin is seen all over the presumptive wing blade domains unlike inDrosophila, where it is confined to the hinge and the wing pouch. Excision of meso- and meta-thoracic discs during the larval stages resulted in emergence of adult moths lacking the corresponding wings without any loss of thoracic tissues suggesting independent origin of wing and thoracic primordia. The expression of wingless and distal-less along the dorsal/ventral margin in wing discs correlated well with their expression profile in adultDrosophila wings. Partially excised wing discs did not showin situ regeneration or duplication suggesting their early differentiation. The presence of adult wing vein patterns discernible in larval wing discs and the patterns of marker gene expression as well as the inability of these discs to regulate growth suggested that wing differentiation is achieved early inB. mori. The timings of morphogenetic events are different and the wing discs behave like presumptive wing buds opening out as wing blades inB. mori unlike evagination of only the pouch region as wing blades seen inDrosophila.     

Region-specific aldehyde oxidase activity in Tumorous-head eye discs of Drosophila melanogaster

Summary Histochemical staining for aldehyde oxidase in mature tumorous-head eye imaginal discs of Drosophila melanogaster reveals region-specific enzyme activity that normally is not found in wild type eye discs. Confined primarily to the central portion of the mutant disc is a morphologically distinct area that can be predicted to be the only aldehyde oxidase (aldox) positive tissue in the eye disc. Prior to staining, this area can be removed mechanically from the surrounding tissue and is characterized by smooth boundaries. The separated tissue stains for aldehyde oxidase whereas the remaining disc is aldox negative as in the wild type. We presume that the aldehyde oxidase positive region subsists in the primordium of the tumorous-head abnormality and propose that the appearance of this enzyme signals a change in the state of determination in the mutant disc.     

Positional information in imaginal discs transformed by homoeotic mutations

Summary Mutations of the bithorax complex result in segmental transformations in the thorax and abdomen ofDrosophila. The haltere discs from larvae homozygous forbx ³ orpbx are transformed so that the discs contain cells that will produce wing cuticle as well as cells that produce haltere cuticle. The pattern regulation behavior of these discs has been examined. The fate maps of the two discs were established, and then the regulative behavior of a number of fragments from both types of mutant discs was established by culturing the fragments in vivo prior to metamorphosis. The most important conclusion from this work is that the cells producing, haltere cuticle and wing cuticle within the same disc share the same positional information and that they communicate during pattern regulation.     

A mosaic analysis of the apterous mutation in Drosophila melanogaster

An analysis of three phenotypes expressed by the apterous-four (ap⁴) mutant of Drosophila melanogaster has been carried out in $\text{ap}{}^4\text{ap}{}^{\mathrm{+}}$ mosaic adults. The wing and haltere deficiency phenotype was found to be autonomous for entirely mutant structures. However, small patches of mutant anterior and posterior wing margin cells can exist in mosaic wings. Examples of duplicated and triplicated lengths of bristle rows were often found associated with the existing mutant patches. About 20% of the mosaics expressed the phenotype of precocious adult death, dying 30–40 hr after eclosion. The focus for this phenotype was located in the posterior region of the abdomen, and a strong correlation was found between expression of this phenotype and the presence of mutant Malpighian tubules. The focus for juvenile hormone deficiency in ap⁴ adults was located near that for precocious adult death; in fact, the two foci may be identical. It is suggested that defective functioning of ap⁴ adult Malpighian tubules results in abnormal hemolymph leading to early death. Inadequate juvenile hormone secretion could result indirectly from impaired glandular functioning attributable to abnormal adult hemolymph.     

Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations

Summary The ash-1 locus is in the proximal region of the left arm of the third chromosome of Drosophila melanogaster and the ash-2 locus is in the distal region of the right arm of the third chromosome. Mutations at either locus can cause homeotic transformations of the antenna to leg, proboscis to leg and/or antenna, dorsal prothorax to wing, first and third leg to second leg, haltere to wing, and genitalia to leg and/or antenna. Mutations at the ash-1 locus cause, in addition, transformations of the posterior wing and second leg to anterior wing and second leg, respectively. A similar spectrum of transformations is caused by mutations at yet another third chromosome locus, trithorax. One extraordinary aspect of mutations at all three of these loci is that they cause such a wide variety of transformations. For mutations at both of the loci that we have studied the expression of the homeotic phenotype is both disc-autonomous (as shown by injecting mutant discs into metamorphosing larvae) and cell autonomous (as shown by somatic recombination analysis). The original mutations which identified these two loci, although lethal, manifest variable expressivity and incomplete penetrance of the homeotic phenotype suggesting that they are hypomorphic. The phenotype of double mutants which were synthesized by combining different pairs of those original mutations manifest for two of the four pairs a greater degree of expressivity and slightly more penetrance of the homeotic transformations. This mutual enhancement suggests that the products of both loci interact in the same process. A third double mutant expresses a discless phenotype.Additional alleles have been recovered at both the ash-1 and the ash-2 loci. Some of these alleles as homozygotes or transheterozygotes express the wide range of transformations revealed first by double mutants. One of the alleles at the ash-1 locus when homozygous and several transheterozygous pairs can cause either the homeotic transformation of discs or the absence of those discs. The fact that these two defects, absence of specific discs and homeotic transformations of those same discs can be caused by mutations within a single gene suggests that the activity of the product of this gene is essential for normal imaginal disc cell proliferation. Loss of that activity leads to the absence of discs, whereas, reduction of that activity leads to homeotic transformations.