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.     

Regulative interaction of mature imaginal disc Fragments with embryonic and immature disc tissues inDrosophila

Summary These experiments examined whether inDrosophila immature imaginal disc tissue and tissues from embryonic stages can influence pattern regulation in a disc fragment in the same way as can mature imaginal discs. Immature imaginal discs, or the cells of whole embryos, were mixed with a test fragment (presumptive notum) from a mature wing disc. The immature tissues in each mixture were genetically marked and had been heavily irradiated (25 Kr gamma) prior to mixing to prevent growth and maturation during subsequent culture in vivo. Alteration of the regulative behavior of the test fragment (that is, regeneration of wing) thus provided an assay for the communication of positional information by the immature tissues. The results suggest that this capacity arises well before competence to metamorphose, as early as the 16th hour of embryonic development, whereas prior to 16 h, essentially no stimulation of regeneration occurred. It is suggested that the imaginal disc (or presumptive disc) cells of the embryo may have been responsible for this early stimulatory capacity.     

Larval cells become imaginal cells under the control of homothorax prior to metamorphosis in the Drosophila tracheal system

In Drosophila melanogaster, one of the most derived species among holometabolous insects, undifferentiated imaginal cells that are set-aside during larval development are thought to proliferate and replace terminally differentiated larval cells to constitute adult structures. Essentially all tissues that undergo extensive proliferation and drastic morphological changes during metamorphosis are thought to derive from these imaginal cells and not from differentiated larval cells. The results of studies on metamorphosis of the Drosophila tracheal system suggested that large larval tracheal cells that are thought to be terminally differentiated may be eliminated via apoptosis and rapidly replaced by small imaginal cells that go on to form the adult tracheal system. However, the origin of the small imaginal tracheal cells has not been clear. Here, we show that large larval cells in tracheal metamere 2 (Tr2) divide and produce small imaginal cells prior to metamorphosis. In the absence of homothorax gene activity, larval cells in Tr2 become non-proliferative and small imaginal cells are not produced, indicating that homothorax is necessary for proliferation of Tr2 larval cells. These unexpected results suggest that larval cells can become imaginal cells and directly contribute to the adult tissue in the Drosophila tracheal system. During metamorphosis of less derived species of holometabolous insects, adult structures are known to be formed via cells constituting larval structures. Thus, the Drosophila tracheal system may utilize ancestral mode of metamorphosis.     

The dachsous gene, a member of the cadherin family, is required for Wg-dependent pattern formation in the Drosophila wing disc

The dachsous (ds) gene encodes a member of the cadherin family involved in the non-canonical Wnt signaling pathway that controls the establishment of planar cell polarity (PCP) in Drosophila. ds is the only known cadherin gene in Drosophila with a restricted spatial pattern of expression in imaginal discs from early stages of larval development. In the wing disc, ds is first expressed distally, and later is restricted to the hinge and lateral regions of the notum. Flies homozygous for strong ds hypomorphic alleles display previously uncharacterized phenotypes consisting of a reduction of the hinge territory and an ectopic notum. These phenotypes resemble those caused by reduction of the canonical Wnt signal Wingless (Wg) during early wing disc development. An increase in Wg activity can rescue these phenotypes, indicating that Ds is required for efficient Wg signaling. This is further supported by genetic interactions between ds and several components of the Wg pathway in another developmental context. Ds and Wg show a complementary pattern of expression in early wing discs, suggesting that Ds acts in Wg-receiving cells. These results thus provide the first evidence for a more general role of Ds in Wnt signaling during imaginal development, not only affecting cell polarization but also modulating the response to Wg during the subdivision of the wing disc along its proximodistal (PD) axis.     

Wg and Egfr signalling antagonise the development of the peripodial epithelium in Drosophila wing discs

Imaginal discs contain a population of cells, known as peripodial epithelium, that differ morphologically and genetically from the rest of imaginal cells. The peripodial epithelium has a small contribution to the adult epidermis, though it is essential for the eversion of the discs during metamorphosis. The genetic mechanisms that control the identity and cellular morphology of the peripodial epithelia are poorly understood. In this report, we investigate the mechanisms that pattern the peripodial side of the wing imaginal disc during early larval development. At this time, the activities of the Wingless (Wg) and Epidermal growth factor receptor (Egfr) signalling pathways specify the prospective wing and notum fields, respectively. We show that peripodial epithelium specification occurs in the absence of Wingless and Egfr signalling. The ectopic activity in the peripodial epithelium of any of these signalling pathways transforms the shape of peripodial cells from squamous to columnar and resets their gene expression profile. Furthermore, peripodial cells where Wingless signalling is ectopically active acquire hinge identity, while ectopic Egfr activation results in notum specification. These findings suggest that suppression of Wg and Egfr activities is an early step in the development of the peripodial epithelium of the wing discs.     

Dpp signalling is a key effector of the wing-body wall subdivision of the Drosophila mesothorax

During development, the imaginal wing disc of Drosophila is subdivided along the proximal-distal axis into different territories that will give rise to body wall (notum and mesothoracic pleura) and appendage (wing hinge and wing blade). Expression of the Iroquois complex (Iro-C) homeobox genes in the most proximal part of the disc defines the notum, since Iro-C(-) cells within this territory acquire the identity of the adjacent distal region, the wing hinge. Here we analyze how the expression of Iro-C is confined to the notum territory. Neither Wingless signalling, which is essential for wing development, nor Vein-dependent EGFR signalling, which is needed to activate Iro-C, appear to delimit Iro-C expression. We show that a main effector of this confinement is the TGFbeta homolog Decapentaplegic (Dpp), a molecule known to pattern the disc along its anterior-posterior axis. At early second larval instar, the Dpp signalling pathway functions only in the wing and hinge territories, represses Iro-C and confines its expression to the notum territory. Later, Dpp becomes expressed in the most proximal part of the notum and turns off Iro-C in this region. This downregulation is associated with the subdivision of the notum into medial and lateral regions.     

Egfr/Ras pathway mediates interactions between peripodial and disc proper cells in Drosophila wing discs

All imaginal discs in Drosophila are made up of a layer of columnar epithelium or the disc proper and a layer of squamous epithelium called the peripodial membrane. Although the developmental and molecular events in columnar epithelium or the disc proper are well understood, the peripodial membrane has gained attention only recently. Using the technique of lineage tracing, we show that peripodial and disc proper cells arise from a common set of precursors cells in the embryo, and that these cells diverge in the early larval stages. However, peripodial and disc proper cells maintain a spatial relationship even after the separation of their lineages. The peripodial membrane plays a significant role during the regional subdivision of the wing disc into presumptive wing, notum and hinge. The Egfr/Ras pathway mediates this function of the peripodial membrane. These results on signaling between squamous and columnar epithelia are particularly significant in the context of in vitro studies using human cell lines that suggest a role for the Egfr/Ras pathway in metastasis and tumour progression.     

Apposition of <Emphasis Type="Italic">iroquois</Emphasis> expressing and non-expressing cells leads to cell sorting and fold formation in the <Emphasis Type="Italic">Drosophila</Emphasis>imaginal wing disc

Background  

The organization of the different tissues of an animal requires mechanisms that regulate cell-cell adhesion to promote and maintain the physical separation of adjacent cell populations. In the Drosophila imaginal wing disc the iroquois homeobox genes are expressed in the notum anlage and contribute to the specification of notum identity. These genes are not expressed in the adjacent wing hinge territory. These territories are separated by an approximately straight boundary that in the mature disc is associated with an epithelial fold. The mechanism by which these two cell populations are kept separate is unclear.     

The Dorsocross T-box transcription factors promote tissue morphogenesis in the Drosophila wing imaginal disc

The Drosophila wing imaginal disc is subdivided into notum, hinge and blade territories during the third larval instar by formation of several deep apical folds. The molecular mechanisms of these subdivisions and the subsequent initiation of morphogenic processes during metamorphosis are poorly understood. Here, we demonstrate that the Dorsocross (Doc) T-box genes promote the progression of epithelial folds that not only separate the hinge and blade regions of the wing disc but also contribute to metamorphic development by changing cell shapes and bending the wing disc. We found that Doc expression was restricted by two inhibitors, Vestigial and Homothorax, leading to two narrow Doc stripes where the folds separating hinge and blade are forming. Doc mutant clones prevented the lateral extension and deepening of these folds at the larval stage and delayed wing disc bending in the early pupal stage. Ectopic Doc expression was sufficient to generate deep apical folds by causing a basolateral redistribution of the apical microtubule web and a shortening of cells. Cells of both the endogenous blade/hinge folds and of folds elicited by ectopic Doc expression expressed Matrix metalloproteinase 2 (Mmp2). In these folds, integrins and extracellular matrix proteins were depleted. Overexpression of Doc along the blade/hinge folds caused precocious wing disc bending, which could be suppressed by co-expressing MMP2RNAi.     

Self-refinement of Notch activity through the transmembrane protein Crumbs: modulation of gamma-secretase activity

Cell interactions mediated by Notch family receptors have been implicated in the specification of tissue boundaries. Tightly localized activation of Notch is crucial for the formation of sharp boundaries. In the Drosophila wing imaginal disc, the Notch receptor is expressed in all cells. However, Notch activity is limited to a narrow stripe of cells along the dorsal–ventral compartment boundary, where it induces the expression of target genes. How a widely expressed protein becomes tightly regulated at the dorsal–ventral boundary in the Drosophila wing is not completely understood. Here, we show that the transmembrane protein Crumbs is involved in a feedback mechanism used by Notch to refine its own activation domain at the Drosophila wing margin. Crumbs reduces the activity of the γ-Secretase complex, which mediates the proteolytic intracellular processing of Notch. These results indicate a novel molecular mechanism of the regulation of Notch signal, and also that defects in Crumbs might be involved in similar abnormal γ-Secretase complex activity observed in Alzheimer's disease.     

In vitro culture of Drosophila imaginal disc cells: aggregation, sorting out, and differentiative abilities

This paper describes the aggregation in vitro of cells dissociated from imaginal discs and demonstrates the sorting out of undifferentiated cells from different imaginal discs and from differently determined regions of the same imaginal disc, as well as the abilities of such cells to undergo pattern reconstruction when injected into larvae. Dissociated cells begin to aggregate by 1.5 hr of rotation. By 5 hr of rotation, large aggregates of loosely associated cells appear. By 18 hr the aggregates have condensed and taken on a characteristic epithelial structure. To study sorting out in undifferentiated cells, we combined a histochemical stain for acid phosphatase with the use of the acid phosphatase null mutant acphn-11. We performed cell mixing experiments with 0-2 (prospective notum) and 2-8 (prospective wing) fragments, with the A and P (prospective anterior and posterior) fragments of the dorsal mesothoracic disc and with mixtures of cells from ventral prothoracic and dorsal mesothoracic discs. We found that prospective anterior and posterior dorsal mesothoracic cells do not sort out, but that prospective notum and wing and leg and wing cells do. The results from differentiated implants are consistent with those from undifferentiated mixes.     

Live Cell Imaging of Butterfly Pupal and Larval Wings In Vivo

Butterfly wing color patterns are determined during the late larval and early pupal stages. Characterization of wing epithelial cells at these stages is thus critical to understand how wing structures, including color patterns, are determined. Previously, we successfully recorded real-time in vivo images of developing butterfly wings over time at the tissue level. In this study, we employed similar in vivo fluorescent imaging techniques to visualize developing wing epithelial cells in the late larval and early pupal stages 1 hour post-pupation. Both larval and pupal epithelial cells were rich in mitochondria and intracellular networks of endoplasmic reticulum, suggesting high metabolic activities, likely in preparation for cellular division, polyploidization, and differentiation. Larval epithelial cells in the wing imaginal disk were relatively large horizontally and tightly packed, whereas pupal epithelial cells were smaller and relatively loosely packed. Furthermore, larval cells were flat, whereas pupal cells were vertically elongated as deep as 130 μm. In pupal cells, many endosome-like or autophagosome-like structures were present in the cellular periphery down to approximately 10 μm in depth, and extensive epidermal feet or filopodia-like processes were observed a few micrometers deep from the cellular surface. Cells were clustered or bundled from approximately 50 μm in depth to deeper levels. From 60 μm to 80 μm in depth, horizontal connections between these clusters were observed. The prospective eyespot and marginal focus areas were resistant to fluorescent dyes, likely because of their non-flat cone-like structures with a relatively thick cuticle. These in vivo images provide important information with which to understand processes of epithelial cell differentiation and color pattern determination in butterfly wings.     

The role of nutrition in creation of the eye imaginal disc and initiation of metamorphosis in Manduca sexta

With the exception of the wing imaginal discs, the imaginal discs of Manduca sexta are not formed until early in the final larval instar. An early step in the development of these late-forming imaginal discs from the imaginal primordia appears to be an irreversible commitment to form pupal cuticle at the next molt. Similar to pupal commitment in other tissues at later stages, activation of broad expression is correlated with pupal commitment in the adult eye primordia. Feeding is required during the final larval instar for activation of broad expression in the eye primordia, and dietary sugar is the specific nutritional cue required. Dietary protein is also necessary during this time to initiate the proliferative program and growth of the eye imaginal disc. Although the hemolymph titer of juvenile hormone normally decreases to low levels early in the final larval instar, eye disc development begins even if the juvenile hormone titer is artificially maintained at high levels. Instead, creation of the late-forming imaginal discs in Manduca appears to be controlled by unidentified endocrine factors whose activation is regulated by the nutritional state of the animal.     

A position-specific cell surface antigen in the drosophila wing imaginal disc

The antibody produced by the hybrid cell line DK.1A4 recognizes an antigen present initially on all the epithelial cells of the D. melanogaster wing imaginal disc. This antigen becomes progessively restricted to cells in the dorsal region of the disc during the final larval instar. The presence of the antigen does not correlate with the specific adult structures to which the cells will eventually contribute, but rather with the position of the cells in the disc. In late discs, the line bounding the region in which the antigen persists corresponds to the boundary between the dorsal and ventral compartments as revealed by a clonal analysis of the undifferentiated disc. Together, these data suggest that the antigen's disappearance may be specific to the cells of the ventral compartment of the wing disc.     

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.     

Absence of distal to proximal intercalary regeneration in imaginal wing discs of Drosophila melanogaster.

The fate of an imaginal disc cell of Drosophila can be affected by the associations and interactions that it has with other cells in the disc. A fragment of an imaginal disc, not regenerating under conditions allowing a complementary fragment to do so, can be stimulated to regenerate by interactions with cells of the complementary fragment [Haynie, J. L., and Bryant, P. J. (1976) Nature (London)259, 659–662]. We report here that one nonregenerating fragment of an imaginal wing disc cannot be stimulated to regenerate by interactions with cells from other parts of the disc. This fragment, containing the anlagen of the distal wing, fails to regenerate proximally when combined with a proximal fragment even though this association stimulates some proximal fragments to regenerate distally. We suggest that this may be a phenomenon similar to that observed in cockroach legs by H. Bohn (1970, Wilhelm Roux Arch. Entwicklungsmech. Organismen165, 303–341), in which proximal regeneration from grafted distal leg segments proceeds only to a limited extent. We consider the possibility that there exist reiterated sets of positional information arranged concentrically in the wing disc.     

Neuronal specificity and its development in the Drosophila wing disc and its derivatives

The imaginal wing disc of flies gives rise to the adult wing blade and dorsal thorax (notum). A great deal has been learned in recent years about the process of neurogenesis in this disc; a number of genes that play crucial roles in the formation of sensory mother cells and in the differentiation of the sensory organs have been identified and their roles defined. Given this extensive background of developmental genetics, it has seemed profitable to summarize what is known about the end-products of neural development, the adult sensory organs. Discussed are their physiological function and role in behavior, the pathways followed by their axons in the CNS, and both genes and epigenetic processes that might play some role in the later stages of neural development and in adult function. The highly individual characteristics of certain of the sensory organs is emphasized, both in the context of their adult roles and as a challenge for future studies in developmental genetics. © 1993 John Wiley & Sons, Inc.     

Chironomus tentans epithelial cell lines sensitive to ecdysteroids, juvenile hormone, insulin and heat shock

A new culture medium, ZW, and the preparation of an extract of adult Drosophila, FX, are described, which for the first time allow the in vitro proliferation of normal Drosophila cells in the absence of undefined heterologous components. Cells from 6-hour-old Drosophila embryos can extensively differentiate and/or proliferate in ZW supplemented with FX and insulin. Cells isolated from wing discs of 90–120-hour-old larvae require ecdysterone for proliferation in ZW, in addition to FX and insulin. Explanted ovaries, testes, genital discs and intact or halved wing discs of 100-hour-old larvae grow in the same medium, at least in part due to cell proliferation. High concentrations of ecdysterone prevent differentiation and/or proliferation of cells from embryos and from wing discs and cause the lysis of most isolated imaginal disc cells grown in vitro, while cuticular differentiations are induced in wing discs and disc fragments grown in vitro.