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.     

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.     

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.     

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.     

Aldehyde oxidase activity in the tumorous-head strain of Drosophila melanogaster

Gross aldehyde oxidase activity from the egg-stage through 10-day-old adults and distribution of the enzyme in eye-antennal imaginal discs in third instar larvae were determined for the tumorous-head strain of Drosophila melanogaster. Aldehyde oxidase activity of several laboratory strains was measured for comparative purposes. Aldehyde oxidase activity was 100% higher during embryogenesis in tuh(ASU) eggs than in Oregon-R-C eggs. A second period of elevated aldehyde oxidase activity was observed during metamorphosis where tuh(ASU) pupae averaged 65% more enzyme activity than Oregon-R-C. Therefore, during determination and differentiation of the eye-antennal imaginal disc, the tuh(ASU) strain possesses a high aldehyde oxidase activity. Wild-type Drosophila melanogaster antennal imaginal discs are aldehyde oxidase positive, whereas attached eye imaginal discs are apparently aldehyde oxidase negative. A sample of eye-antennal imaginal discs from tuh(ASU) third instar larvae revealed that either one or both eye discs of 64% of the larvae were aldehyde oxidase positive. Aldehyde oxidase activity may be correlated with the homoeotic transformation in parts of the eye disc.     

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.     

Autonomous cellular differentiation of homoeotic bithorax mutants of Drosophila melanogaster

Various mixtures of imaginal disc cells from wild-type and from homoeotic bithorax mutants have been studied in an in vivo Drosophila cell culture system. These mutants effect specific types of segmental transformations, e.g., bithorax-3 (bx³) transforms the anterior region of the metathorax (MT) into a region resembling the anterior mesothorax (MS), while postbithorax (pbx) transforms the posterior MT into a posterior MS-like region. In cell mixtures, wild-type haltere-disc cells segregate from wild-type wing-disc cells. On the other hand, bx³ and pbx haltere-disc cells integrate with wild-type cells derived from anterior and posterior regions, respectively, of wing discs. The behavior of these and other tested mutants of the bithorax series indicates in all cases studied that (1) the effects of the mutants are cell-autonomous, and (2) cellular affinities are determined by the genetic constitution rather than the segmental origin of the cells.     

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.     

Effects of engrailed in the genital disc of Drosophila melanogaster

engrailed has been postulated to be the “selector gene” involved in the establishment of the anterior-posterior compartment border in several imaginal discs and in at least the first two abdominal segments of Drosophila melanogaster. Our study of the effects of different mutant engrailed genotypes on genital disc development provided the following major results: All three terminal primordia (female and male genitalia, and analia) were affected. Different heteroallelic combinations showed different expressivities, and the three terminal primordia were differently affected by the same mutant genotype. The engrailed genotypes deleted specific elements of the adult terminalia without causing associated pattern duplications. The reduced morphology of the male engrailed genital disc was analogous to the pattern deletions observed in the adult terminalia. That the engrailed phenotype is stable was demonstrated by culturing in vivo intact and fragmented engrailed genital discs. Cell death was found in a significant number of mature male en²/en³ genital discs. The results are discussed in terms of the segmental organization of the genital disc and in terms of the “selector gene” function postulated for the engrailed locus. The interpretation that each terminal primordium has an anterior and a posterior compartment is presented and it is assumed that in the genital disc engrailed transforms posterior cells into anterior cells that do not develop, thereby causing the deficiency pattern of the engrailed phenotype.     

Establishment of compartments in the developing leg imaginal discs ofDrosophila melanogaster

Summary By X-irradiation ofM/M ⁺ embryos and larvae to induce mitotic recombination, clones ofM ⁺/M⁺ genotype were obtained (Fig. 1). Since such cells grow faster than the surroundingM/M ⁺-cells they can fill large areas within the compartments of an imaginal disc.The present studies concentrated mainly on the three leg discs. Clones were induced by doses of 1000 r at ages ranging from 3±0.5 h after oviposition to 144 h.All clones induced later than the blastoderm stage were absolutely restricted to either the anterior or the posterior compartment of a disc. The border between the anterior and posterior compartment runs as a straight line along the entire leg and at the distal end separates the two claws (Figs. 5, 6, 7). A further subdivision of the anterior compartment is indicated by clones initiated in the second larval instar (Fig. 11). A parallel subdivision could not be detected in the posterior compartment. Irradiation in the early third instar led to clones which were restricted to single longitudinal bristle rows and leg segments. But no clear-cut compartment borders could be found; in particular a proximo-distal separation appears to be absent.Among the 318 clones induced at the blastoderm stage eleven extended from the wing into the second leg (Fig. 8), or from the haltere into the third leg.With the exception of 3 clones that apparently occupied the anterior as well as the posterior compartment of a wing or a leg, all clones remained confined to either the anterior or the posterior compartment.Frequently clones overlapped left and right forelegs (Fig. 9). Intersegmental overlaps were not observed.The results show that the earliest compartment borders appear in all thoracic discs. This suggests that compartmentalization is a fundamental process common to all discs.Supported bySchweizerischer Nationalfonds Gesuch Nr. 3.480-0.75     

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.     

Growth is required for cell competition in the imaginal discs ofDrosophila melanogaster

Summary Imaginal wing discs from late third-instar larvae were gammairradiated to induce clones of rapidly growingMinute ^– cells in a background of slowly growingMinute cells and culturedin vivo for periods up to 18 days. Clones in discs cultured for 16 to 18 days did not grow significantly larger than clones in uncultured controls, indicating that competition between populations of cells having potentially different mitotic rates does not occur in imaginal discs after their growth is completed.     

Aldehyde oxidase cross-reacting material in the Aldox n,cin, mal,and lxd mutants of Drosophila melanogaster

Rocket immunoelectrophoresis was used to estimate aldehyde oxidase cross-reacting material (AO-CRM) in larval hemolymph and adult fly extracts in mutants with reduced AO enzymatic activity. Hemolymph of larvae homozygous for Aldox ⁿ, which is a mutation of the presumed structural gene for AO, contains 30% of the wild-type CRM. The demonstration of AO-CRM in Aldox ⁿ larval hemolymph is surprising since this genotype has been reported to lack CRM. By contrast, adult Aldox ⁿ flies lack detectable CRM. The other AO-deficient mutants that were examined are cin, mal, and lxd; each has appreciable levels of CRM in both larval hemolymph and adult extracts. Detection of CRM in these mutants helps to clarify conflicting reports in the literature.This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to L.W.B.     

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.     

Effect of mutations in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> tumor suppressor <Emphasis Type="Italic">Merlin</Emphasis> on proliferation and differentiation of wing cells

Experiments on transplantation of wing imaginal discs homozygous for a mutation in the tumor suppressor gene Merlin have demonstrated that this mutation does not induce malignant tumors. Marking of the wing disc compartment borders by specific antibodies showed the absence of essential compartment border defects in case of the Merlin mutation. Drosophila melanogaster cells mutant for Merlin have shorter cell cycle than normal cells. Proliferation of imaginal discs lasts longer in case of the mutation. It is known that beginning from some moment of development, wing veins serve as clonal restriction lines that cannot be crossed by growing mosaic clones. We showed that the Merlin mutation leads to depression of vein clonal restriction property. This means that this gene is involved not only in the control of cell proliferation, but also in the control of cell mobility and adhesion.     

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.     

Minute mosaics caused by early chromosome loss

Summary Two genetic operations have been combined in order to ascertain whether there are differential proliferation rates in the syncytial nuclei and the blastoderm cells prior to the formation of the imaginal disc anlagen. Early chromosome loss caused by the mutantca ^nd has been associated with the generation ofMinute (M/M ⁺) genotypes in normal (M ⁺/M ⁺) zygotes or of non-Minute genotypes inMinute zygotes. The results indicate that there is no growth competition betweenMinute and non-Minute cells prior to the formation of the imaginal discs. Growth competition, however, leads later, during the proliferation phase of the discs, to the demarcation of compartment boundaries within imaginal discs.     

Correlation between adult transformation and aldehyde oxidase staining pattern in wing discs ofApterous genotypes inDrosophila melanogaster

Summary The aldehyde oxidase staining pattern in wing discs ofDrosophila melanogaster bearing the genotypesap ^blt /ap ^blt andap ^blt andap ^blt /ap ⁷³ⁿ showns changes from the wild-type pattern. Extensive areas of the presumptive dorsal posterior wing blade, which are normally unstained, have enzyme activity in these mutants. In wings of these genotypes, dorsal posterior structures are replaced by dorsal anterior wing structures. A strong correlation has been found between the frequencies of various staining patterns in the discs and the extent of transformation in the cuticular structures of the wing, which is consistent with the idea that aldehyde oxidase activity can be used as an indicator in the wing disc of this transformation. Unlike the homoeotic mutationengrailed, apterous has not been interpreted as a selector gene yet the work reported here shows thatapterous alleles can cause changes resembling those of theengrailed phenotype both in aldehyde oxidase staining behaviour and in the cuticular transformation.