The cell biology of Drosophila wing metamorphosis in vitro

Summary We have examined the metamorphosis of the wing imaginal disc of Drosophila during culture in vitro in the continuous presence of 20-hydroxy ecdysone (0.1 g/ ml). We find that the sequence of cellular changes in the wing blade during culture closely match those occurring in situ, involving two periods at which the dorsal and ventral surfaces are joined only by cell processes containing trans-alar microtubule arrays. Good pupal and imaginal cuticle secretion is found in this system.     

Multiple roles of the gene zinc finger homeodomain-2 in the development of the Drosophila wing

The gene zfh2 and its human homolog Atbf1 encode huge molecules with several homeo- and zinc finger domains. It has been reported that they play important roles in neural differentiation and promotion of apoptosis in several tissues of both humans and flies. In the Drosophila wing imaginal disc, Zfh2 is expressed in a dynamic pattern and previous results suggest that it is involved is proximal–distal patterning. In this report we go further in the analysis of the function of this gene in wing development, performing ectopic expression experiments and studying its effects in genes involved in wing development. Our results suggest that Zfh2 plays an important role controlling the expression of several wing genes and in the specification of those cellular properties that define the differences in cell proliferation between proximal and distal domains of the wing disc.     

Temporal and spatial windows delimit activation of the outer ring of wingless in the Drosophila wing

Extracellular signalling molecules play many roles in the development of higher organisms. They are used reiteratively in different tissues and stages, but the response of the receiving cells is controlled in a context dependent manner. The pattern of expression of the signalling molecule Wingless/WNT in Drosophila is extraordinarily complex. We have studied the mechanism that controls its expression and function in the outer ring of the Drosophila wing hinge. Our findings indicate that wingless expression is controlled by a dual mechanism: its initial activation requires the product of zinc finger homeodomain 2 and is subsequently repressed by the product of the gene complex elbow/no ocelli. This tight regulation restricts the activation of wingless temporally and spatially. Later in development, wingless expression is maintained by an autoregulatory loop that involves the product of homothorax. We have analyzed the phenotype of a wingless allelic combination that specifically removes the outer ring, and our results show that Wingless is required to promote local proliferation of the wing base cells. Thus, cell proliferation in the proximal-distal axis is controlled by the sequential activation of wingless in the inner ring and the outer ring at different stages of development.     

Model for the regulation of size in the wing imaginal disc of Drosophila

For animal development it is necessary that organs stop growing after they reach a certain size. However, it is still largely unknown how this termination of growth is regulated. The wing imaginal disc of Drosophila serves as a commonly used model system to study the regulation of growth. Paradoxically, it has been observed that growth occurs uniformly throughout the disc, even though Decapentaplegic (Dpp), a key inducer of growth, forms a gradient. Here, we present a model for the control of growth in the wing imaginal disc, which can account for the uniform occurrence and termination of growth. A central feature of the model is that net growth is not only regulated by growth factors, but by mechanical forces as well. According to the model, growth factors like Dpp induce growth in the center of the disc, which subsequently causes a tangential stretching of surrounding peripheral regions. Above a certain threshold, this stretching stimulates growth in these peripheral regions. Since the stretching is not completely compensated for by the induced growth, the peripheral regions will compress the center of the disc, leading to an inhibition of growth in the center. The larger the disc, the stronger this compression becomes and hence the stronger the inhibiting effect. Growth ceases when the growth factors can no longer overcome this inhibition. With numerical simulations we show that the model indeed yields uniform growth. Furthermore, the model can also account for other experimental data on growth in the wing disc.     

spalt-induced specification of distinct dorsal and ventral domains is required for Drosophila tracheal patterning

Morphogenesis of the Drosophila tracheal system relies on different signalling pathways that have distinct roles in specifying both the migration of the tracheal cells and the particular morphological features of the primary branches. The current view is that the tracheal cells are initially specified as an equivalent group of cells whose diversification depends on signals from the surrounding cells. In this work, we show that the tracheal primordia are already specified as distinct dorsal and ventral cell populations. This subdivision depends on the activity of the spalt (sal) gene and occurs prior to the activity of the signalling pathways that dictate the development of the primary branches. Finally, we show that the specification of these two distinct cell populations, which are not defined by cell lineage, are critical for proper tracheal patterning. These results indicate that tracheal patterning depends not only on signalling from surrounding cells but also in the different response of the tracheal cells depending on their allocation to the dorsal or ventral domains.     

2D gene expression parameters of wing imaginal disc of Drosophila for developmental analysis

 By using high resolution two-dimensional (2D) gel electrophoresis coupled with computer-analysis we have established a quantitative Drosophila wing imaginal disc protein database of third instar larvae as a reference to be used for comparative purposes in genetic studies. A general catalogue integrated by 1,184 ³⁵S-methionine-labelled polypeptides from wing imaginal disc has been obtained. The level of expression for all the proteins has been quantitatively determined. The quantitative reproducibility of the analysis system has been estimated and all the controls studied as database reference to interpret the results of experiments with mutant discs. One example, corresponding to iro ¹ mutation, has been used to show how some of the changes observed with mutant discs clearly extend out of the limits defined by the controls. This enables us to generate comparative parameters for the study of proliferation, morphogenesis and differentiation of Drosophila and opens the possibility of rapidly defining the nature and quantity of changes in patterns of gene expression in developmental genetic studies. Received: 21 June 1996 / Accepted: 27 September 1996     

The tumour suppressor gene l(2)giant discs is required to restrict the activity of Notch to the dorsoventral boundary during Drosophila wing development

During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule fringe (fng). The activity domain will be established symmetrically at each side of the boundary of Fng-expressing and non-expressing cells. Here, I present evidence that the Drosophila tumour-suppressor gene lethal (2) gaint discs (lgd) is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development.     

Drosophila Tbx6-related gene, Dorsocross, mediates high levels of Dpp and Scw signal required for the development of amnioserosa and wing disc primordium

Regional differentiation along the dorsoventral (DV) axis of the Drosophila embryo primarily depends on a graded BMP signaling activity generated by Decapentaplegic (Dpp) and Screw (Scw). We have identified triplicated Dpp and Scw target genes Dorsocross1, 2 and 3 (Doc1, 2, 3) that have a conserved T-box domain related to the vertebrate Tbx6 subfamily and act redundantly to induce dorsal structures. Doc genes are expressed in the dorsal region in the early blastoderm. After gastrulation, newly expressed Doc appears in a segmental pattern in the ectoderm. This expression correlates spatially with the second phase of Dpp expression in the ectoderm. Doc expression in the early blastoderm is abolished in either dpp or scw mutant embryos, whereas the ectodermal segmented expression depends only on Dpp. Inactivation of Doc genes with RNAi dramatically affected the development of amnioserosa and wing disc primordia, both of which depend on high levels of BMP signaling, although leg disc primordium, which depends on low levels of BMP, remained intact. Doc1 mRNA expressed in Xenopus embryos induced ventral mesoderm, suppressed activin-induced events and induced Xvent genes, which are analogous to the effects of native Tbx6 and its upstream regulator, BMP-4. These results suggest that the Tbx6 subfamily act in the BMP signaling pathway required for embryonic patterning in both animals.     

The gene fl(2)d is required for various Sxl-controlled processes in Drosophila females

Summary In Drosophila melanogaster, the gene Sex-lethal (Sxl) controls the processes of sex determination, dosage compensation, oogenesis and sexual behaviour. The control of Sxl is by alternative splicing of its primary RNA. We have identified a gene, female-lethal-2-d (fl(2)d), which is needed for the female-specific splicing of Sxl RNA and which also has a vital function independent of Sxl. Here we analyse other aspects of the gene fl(2)d. Specifically, we have analysed the effect of the temperature-sensitive mutation fl(2)d ¹ on the viability of adult flies homozygous for this mutation. We have found that the viability of the mutant females is reduced, while that of the mutant males is not affected. In addition, the capacity of the mutant females to be inseminated is considerably reduced, whilst all the mutant males are able to inseminate females. These effects on females are suppressed by Sxl ^M1. However, the fat body cells of fl(2)d ¹ homozygous females are able to synthesize yolk proteins at the restrictive temperature. We have also carried out, in males, a clonal analysis of fl(2)d ², a mutation lethal in both sexes. We have found that the clones are fully viable. We conclude that the gene fl(2)d seems to be necessary during the adult life of females for the processes that require Sxl ⁺ activity. Moreover, the Sxl-independent vital function of fl(2)d seems to be required in both sexes only during larval development. Offprint requests to: L. Sánchez     

Puromycin enhances 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster

The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the early and late proteins. However, when puromycin was applied after hormone treatment, only the late proteins were induced. The possible implication of these observations is discussed.     

Visual control of wing beat frequency in Drosophila

The present study shows that the wing beat frequency of Drosophila is visually controlled and modulated in response to different optomotor stimuli.Whereas rotational large field stimuli do not appear to modulate wing beat frequency, single rotating vertical stripes increase or decrease wing beat frequency when moving back-to-front or front-to-back, respectively. Maximal modulations occur at lateral stripe positions.Expansion stimuli eliciting the landing response cause a marked increase in wing beat frequency. Parameters of this frequency response depend in a graded fashion on certain stimulus properties, and the frequency response co-habituates with the landing response. Several results indicate that the frequency response is an integral component of the landing response, although it can also occur when the characteristic front leg extension is not observed. The complex spatial input integration underlying the frequency response and other motor components of the landing response cannot easily be explained by a system of large field integration units, but might indicate the existence of local expansion detectors.     

The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis

Niemann–Pick C (NPC) disease is a lethal neurodegenerative disorder affecting cellular sterol trafficking. Besides neurodegeneration, NPC patients also exhibit other pleiotropic conditions, indicating that NPC protein is required for other physiological processes. Previous studies indicated that a sterol shortage that in turn leads to a shortage of steroid hormones (for example, ecdysone in Drosophila) is likely to be the cause of NPC disease pathology. We have shown that mutations in Drosophila npc1, one of the two NPC disease-related genes, leads to larval lethal and male infertility. Here, we reported that npc1 mutants are defective in spermatogenesis and in particular in the membrane-remodeling individualization process. Interestingly, we found that ecdysone, the steroid hormone responsible for the larval lethal phenotype in npc1 mutants, is not required for individualization. However, supplying 7-dehydrocholesterol can partially rescue the male infertility of npc1 mutants, suggesting that a sterol shortage is responsible for the spermatogenesis defects. In addition, the individualization defects of npc1 mutants were enhanced at high temperature, suggesting that the sterol shortage may lead to temperature-sensitive defects in the membrane-remodeling process. Together, our study reveals a sterol-dependent, ecdysone-independent mechanism of NPC1 function in Drosophila spermatogenesis.     

Spalt major controls the development of the notum and of wing hinge primordia of the Drosophila melanogaster wing imaginal disc

The Drosophila wing and the dorsal thorax develop from primordia within the wing imaginal disc. Here we show that spalt major (salm) is expressed within the presumptive dorsal body wall primordium early in wing disc development to specify notum and wing hinge tissue. Upon ectopic salm expression, dorsally located second leg disc cells develop notum and wing hinge tissue instead of sternopleural tissue. Similarly, by salm over-expression within the wing disc, wing blade formation is suppressed and a mirror-image duplication of the notum and wing hinge is formed. In large dorsal clones, which lack salm and its neighboring paralogue spalt related (salr), the cells of the notum primordium do not grow; these dorsal cells are not specified as notum, hence no notum outgrowth develops. These results suggest that the zinc finger factors encoded by the salm/salr complex play important roles in defining cells of the early wing disc as dorsal body wall cells, which develop into a large dorsal body wall territory and form mesonotum and some wing hinge tissue, and in delimiting the wing primordium. We also find that salm activity is down-regulated by its own product and by that of the Pax gene eyegone.     

Analysis of morphogenetic movements in the development of the notum anlage of Drosophila melanogaster

Summary A comparison of the morphogenetic maps of the notum anlage of Drosophila melanogaster derived from the gynandromorph data and mosaics induced by somatic crossing-over during the first instar larval stage revealed that practically no major morphogenetic movements occur in the development of the anlage between the blastoderm and first instar larval stages and the adult stage. By comparing the morphogenetic map derived from gynandromorphs and the fate map derived from data on the transplantation of fragments of the mature wing imaginal disc, it was observed that no major morphogenetic movements occur in the notum anlage between the stages of the allocation of the disc and the mature disc. The results are consistent with the observations of other authors concerning the larval development of eye-antenna, wing and leg discs.     

The expression of PS integrins in Drosophila melanogaster imaginal disc cell lines

Summary Drosophila imaginal disc cell lines show a characteristic pattern of aggregation in culture, which appears to be due to cell-cell rather than cell-substrate interactions. We have examined the distribution of PS integrins in wing and leg cell lines, and find that these integrin homologues are expressed preferentially in aggregates. Cell sheets, small cell clumps and chains of cells express antigen at points of cell-cell contact only.