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.     

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.     

Retrotransposon-induced ectopic expression of the Om(2D) gene causes the eye-specific Om(M) phenotype in Drosophila ananassae

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci, which are scattered throughout the genome. Om mutations are all semidominant, neomorphic, nonpleiotropic, and associated with the insertion of a retrotransposon, tom. We have found that the Om(2D) gene encodes a novel protein containing histidine/proline repeats, and is ubiquitously expressed during embryogenesis. The Om(2D) RNA is not detected in wild-type eye imaginal discs, but is abundantly found in the center of the eye discs of Om(2D) mutants, where excessive cell death occurs. D. melanogaster flies transformed with the Om(2D) cDNA under control of the hsp70 promoter display abnormal eye morphology when heat-shocked at the third larval instar stage. These results suggest that the Om(2D) gene is not normally expressed in the eye imaginal discs, but its ectopic expression, induced by the tom element, in the eye disc of third instar larvae results in defects in adult eye morphology.     

Isocitrate dehydrogenase in D. melanogaster imaginal discs: pattern development and alteration by homoeotic mutant genes

The distribution of the soluble form of NADP+-dependent isocitrate dehydrogenase (ICDH) was examined in Drosophila melanogaster imaginal discs. Development of the enzyme patterns and the specific transformations of the patterns by homoeotic mutants were studied. ICDH pattern formation was followed in eye-antennal discs and wing discs from the late 2nd instar stage through 3rd instar and 8 hours into prepupal development. The patterns formed gradually in both disc types. The most interesting pattern developed in the eye portion of the eye-antennal disc complex. ICDH distribution as well as staining intensity correlated well with differentiation of the ommatidia. The spatial distribution of ICDH within the discs was under genetic control. The patterns reflected the state of determination of the disc. When the presumptive tissue type was transformed via mutant homoeotic genes to different determinative states, the ICDH pattern likewise transformed to the pattern characteristic of the newly acquired structure.     

Protein synthetic patterns during differentiation of imaginal discs in vitro.

Leg, wing, and eye-antennal imaginal discs of Drosophila melanogaster were obtained by dissection from late-third instar larvae and cultured in vitro in Schneider's medium containing β-ecdysone. Differentiation of adult structures was obtained in more than 90% of all cultures. Differentiation was somewhat slower than normal in vivo development, but synchronous, repeatable, and reasonably completed structures were obtained. Our initial efforts at analyzing the molecular events of imaginal disc differentiation in culture have been to study the protein synthetic pattern which occurs throughout the culture period. Discs were pulse labeled with [³⁵S]methionine, and the proteins were separated by SDS-gel electrophoresis. Analysis of the synthetic pattern was done by autoradiography of these gels using X-ray film. In all three disc types, pronounced changes in protein synthetic patterns occurred throughout the culture period. These changes appeared to be under strict temporal control. Although disc-specific differences could be seen, a comparison of the three discs types revealed a striking similarity in the changes which occurred in the patterns of protein synthesis during the 5-day culture period. In general, the protein synthetic patterns of different imaginal discs at the same period during differentiation showed greater similarities than the patterns of a single disc type at different periods. These results are consistent with a view of differentiation as a tightly controlled program of gene activation and deactivation operating throughout the differentiation process.     

Patterns of protein synthesis in the imaginal discs of Drosophila melanogaster: a comparison between different discs and stages

High-resolution two dimensional gel electrophoresis has been used to study the patterns of protein synthesis in imaginal discs of Drosophila melanogaster. In this paper we first compare the patterns of protein synthesis in wing, haltere, leg 1, leg 2, leg 3 and eye antenna imaginal discs of late third instar larvae. We have detected only quantitative changes: differences in 17 proteins among the different imaginal discs. In addition, we have analysed the variations in pattern of proteins in the wing disc of the last larval stage and early pupae as well as in wing discs cultured in vivo for 6 days. Variations in these patterns affect more than 20% of the proteins and involve both qualitative and quantitative changes. Some of the changes may correspond to protein phosphorylation. Correlations of these changes between discs and through development are also discussed. Correspondence to: F. Santaren     

Alterations in the cell cycle of Drosophila imaginal disc cells precede metamorphosis

Flow cytometric analyses of imaginal disc and brain nuclei of Drosophila melanogaster have been made throughout the third larval instar. In wing, haltere, and leg discs the proportion of cells in the G₂M phase of the cell cycle (tetraploid cells) increases with larval age. In contrast, in the eye disc and in brain the proportion of tetraploid cells, already low at the outset of the instar, declines further. Measurement of growth rates for disc and brain tissue during the same developmental period was carried out by the cell counting procedure of Martin (1982). Our results are consistent with the conclusion that imaginal discs grow exponentially with an apparent doubling time of 5–10 hr from the resumption of cell division (in the first or second larval instar) until about 95 hr, when the apparent doubling time increases. Cell numbers increase until at least 5 hr after formation of white prepupae (122 hr), but during the preceding 10 hr the rate of increase is low. Thus, for wing and leg discs, but not for the eye disc and brain, the declining growth rate is associated with an increase in the proportions of tetraploid cells. In conjunction with cell counts and flow cytometry, fluorometric determination of disc DNA content at 112 hr indicated that the diploid DNA content of imaginal disc nuclei is 0.45 pg.     

Retrotransposon-induced ectopic expression of the Om(2D) gene causes the eye-specific Om(M) phenotype in Drosophila ananassae

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci, which are scattered throughout the genome. Om mutations are all semidominant, neomorphic, nonpleiotropic, and associated with the insertion of a retrotransposon, tom. We have found that the Om(2D) gene encodes a novel protein containing histidine/proline repeats, and is ubiquitously expressed during embryogenesis. The Om(2D) RNA is not detected in wild-type eye imaginal discs, but is abundantly found in the center of the eye discs of Om(2D) mutants, where excessive cell death occurs. D. melanogaster flies transformed with the Om(2D) cDNA under control of the hsp70 promoter display abnormal eye morphology when heat-shocked at the third larval instar stage. These results suggest that the Om(2D) gene is not normally expressed in the eye imaginal discs, but its ectopic expression, induced by the tom element, in the eye disc of third instar larvae results in defects in adult eye morphology.     

Actinomycin D-induced phenocopies in Drosophila melanogaster and their relevance to the time of gene action

Drosophila melanogaster larvae of a wild-type and a mutant stock, cultured in an axenic, chemically defined medium, were treated for one day with different concentrations of actinomycin D at different stages of development. Phenocopies affecting various organs of the adult occurred in different frequencies and in different patterns depending on the age at treatment. Assuming that the induced phenocopies were due primarily to the inhibition of DNA-dependent RNA synthesis by actinomycin D, the differential phenocopy effect indicates that: (1) Many genes which affect the differentiation of imaginal discs are activated in the third larval instar. (2) The developmental timing of gene activation in the third instar differs for various genes within a imaginal disc and in different imaginal discs.Submitted in partial fulfillment of the requirements for the Ph. D. degree. Supported by U.S. Public Health Service Grant GM1 1537 to I. H. Herskowitz.     

An eye imaginal disc-specific transcriptional enhancer in the long terminal repeat of thetom retrotransposon is responsible for eye morphology mutations ofDrosophila ananassae

Optic morphology (Om) mutations ofDrosophila ananassae are semidominant, neomorphic and nonpleiotropic, map to at least 22 loci scattered throughout the genome, and are associated with the insertion of thetom retrotransposon. Molecular and genetic analyses have revealed that eye morphology defects ofOm mutants are caused by the ectopic or excessive expression ofOm genes in the eye imaginal discs of third instar larvae. It is therefore assumed that thetom element carries tissue-specific gene regulatory sequences which enhance expression of theOm genes. In the present study, we examined whether or not the long terminal repeats (LTR) of thetom element contain such an eye imaginal disc-specific enhancer, usingD. melanogaster transformants containing alacZ gene ligated to thetom LTR. Analyses oflacZ gene expression in the eye imaginal discs of third instar larvae of 18 independently established transformant lines showed that thetom LTR was capable of enhancinglacZ expression in all the transformant lines, but the degree of enhancement varied between lines. In addition, the effect of thetom LTRlacZ gene evidently changed when thetom LTR construct was relocated to different chromosomal positions. On the basis of these findings, it is hypothesized that ectopic and excessive expression of theOm genes in the eye imaginal discs is induced by an eye imaginal disc-specific enhancer present in thetom LTR, the effect of which may be subject to chromosomal position effects.     

Autonomous differentiation of the tumorous-head phenotype in Drosophila melanogaster

Summary Transformed areas derived from mature imaginal eye discs of the tumorous-head (tuh) mutant of Drosophila melanogaster were transplanted either into larval hosts (metamorphosis test) or into adult females (long-term in vivo culture). These disc fragments showed characteristic morphologic and enzymatic (aldehyde oxidase (aldox) positive) differences in comparison to a similar region in wildtype eye discs.The tissues derived from the central portion of the tuh eye disc which would normally give rise to eye facets transformed predominantly into homoeotic structures of the abdominal region of the fly. Posterior abdominal tergites arose in 88% of the transplants, of which 7% also possessed genital tissue. In addition, 10% showed duplicated vibrissae with no accompanying homoeotic alteration and 2% differentiated into unidentifiable structures.Our preliminary results from long-term cultures have shown the capacity of the tuh transformed area to grow in vivo and to maintain its differentiation potential. This kind of approach therefore provides an opportunity to follow transdetermination properties of a homoeotically altered tissue.In the present study we demonstrate that during larval life, the presumptive region of the tuh transformed area can be removed from the surrounding unaffected eye disc tissue. From the autonomous differentiation of the tuh phenotype we conclude that the homoeotic change is cell-intrinsically expressed and that the aldox positive areas in the tuh eye discs signal an altered state of determination. Leave of absence: Department of Biological Sciences, Florida Technological University, Orlando, Florida 32816, USA     

Patterns of protein synthesis in imaginal discs of Drosophila melanogaster.

Patterns of polypeptide synthesis in wing, leg and eye-antenna imaginal discs and in whole larvae of wild-type and and mutant Drosophila melanogaster have been examined using two-dimensional polyacrylamide gel electrophoresis and autoradiography. After 2 hr of labeling with 35S during the third larval instar, the synthesis of more than 318 polypeptides has been detected in imaginal discs. Of these, 268 are present in similar amounts in all three disc types. The remaining polypeptides detected in the three imaginal disc types fall into two categories: those unique to a particular disc type, and those specific for a particular pair of disc types. These results are discussed in relation to the spectrum of gene expression in imaginal discs.     

Development of the temperature-sensitive homoeotic mutant ophthalmoptera of Drosophila melanogaster

The mutant stock eyeless-ophthalmoptera of Drosophila melanogaster develops normally at 17°C, but the eye produces an outgrowth of wing tissue at 29°C. High temperature is effective in altering phenotype only if applied during the second instar. This demonstrates a genetically controlled developmental process acting during mid-larval life which determines whether cells of the optic imaginal disc embark upon a developmental pathway leading to wing or to eye.     

kurtz, a novel nonvisual arrestin, is an essential neural gene in Drosophila

The kurtz gene encodes a novel nonvisual arrestin. krz is located at the most-distal end of the chromosome 3R, the third gene in from the telomere. krz is expressed throughout development. During early embryogenesis, krz is expressed ubiquitously and later is localized to the central nervous system, maxillary cirri, and antennal sensory organs. In late third instar larvae, krz message is detected in the fat bodies, the ventral portion of the thoracic-abdominal ganglia, the deuterocerebrum, the eye-antennal imaginal disc, and the wing imaginal disc. The krz(1) mutation contains a P-element insertion within the only intron of this gene and results in a severe reduction of function. Mutations in krz have a broad lethal phase extending from late embryogenesis to the third larval instar. The fat bodies of krz(1) larva precociously dissociate during the midthird instar. krz(1) is a type 1 melanotic tumor gene; the fat body is the primary site of melanotic tumor formation during the third instar. We have functionally rescued these phenotypes with both genomic and cDNA transgenes. Importantly, the expression of a full-length krz cDNA within the CNS rescues the krz(1) lethality. These experiments establish the krz nonvisual arrestin as an essential neural gene in Drosophila.     

Regulation of Nadp-Malic Enzyme in the Eye-Antennal Disc of D. melanogaster/D. simulans Hybrids: Evidence for cis- and trans-Regulation

Pattern regulation of malic enzyme (ME) distribution in D. melanogaster/D. simulans (mel/sim) hybrid eye-antennal discs was investigated. Both cis- and trans-regulation of the spatial distribution pattern was observed within the eye portion of the disc complex. D. simulans possesses gene(s) that operate in trans in the hybrids to suppress ME staining along the morphogenetic furrow, a region that always stains in D. melanogaster. ME structural genes of both species were expressed in cis within the ommatidial preclusters and clusters of the hybrids. Malic enzyme was not expressed elsewhere in the eye disc of either species. Restoration of the D. melanogaster furrow pattern element occurred in partial hybrids that were homozygous for the D. melanogaster 3R where the structural gene resides. Therefore, a dominant gene(s) in the D. simulans 3R suppresses the D. melanogaster furrow pattern, while a recessive gene(s) in the D. melanogaster 3R restores the pattern when the trans-suppressor is removed. These conclusions agree with those found for regulation of aldehyde oxidase distribution in D. melanogaster/D. simulans hybrid wing discs.     

Specific cell ablation in Drosophila using the toxic viral protein M2(H37A)

The expression of toxic viral proteins for the purpose of eliminating distinct populations of cells, while leaving the rest of an organism unaffected, is a valuable method for analyzing development. Using the Gal4-UAS system, we employed the M2(H37A) toxic ion channel of the influenza-A virus to selectively ablate the Drosophila eye-antennal imaginal discs, hemocytes, dorsal vessel and nervous tissue and comparatively monitored the effects of expressing the apoptosis-promoting protein Reaper in identical cell populations. In this report, we demonstrate the effectiveness of M2(H37A)-mediated ablation as a new means to selectively eliminate cells of interest during Drosophila development.Key words: cell ablation, dorsal vessel, Drosophila, eye imaginal disc, hand, lamellocytes, M2 toxin, reaper     

Analysis of metamorphosis in Drosophila melanogaster: Characterization of giant,an ecdysteroid-deficient mutant

The mutant allele giant of Drosophila melanogaster affects the timing and the level of increase in ecdysteroid titer normally occurring at puparium formation. The third larval instar is extended by 4 days in phenotypically “giant” individuals during which the imaginal discs mature slower than normal and finally take on the folding pattern characteristic of maturity at a time when normal individuals have already formed puparia. After puparium formation, development occurs at the same rate in giant and wild-type animals. Feeding 20-hydroxyecdysone at 94 hr after oviposition allows giant larvae to develop at the same rate as wild-type larvae and to produce normal-sized adults (although at 94 hr the imaginal discs of giant lack much of the folding pattern of mature discs). Radioimmunological determination of ecdysteroid titers in giant and normal individuals indicates that the peak of ecdysteroid activity associated with puparium formation is lower in giant and occurs 4 days later than normal. These results indicate that giant is an ecdysteroid-deficient mutant with major effects on metamorphosis. Unlike previously reported ecdysteroid-deficient mutants, however, giant larvae eventually develop into adults and may be induced to undergo complete metamorphosis at the same time as wild type by feeding 20-hydroxyecdysone.     

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.