Dynamic expression of a cell surface protein during rearrangement of epithelial cells in the Manduca wing monolayer.

The expression of cell surface protein 2F5 changes dynamically in space and time during morphogenesis of the Manduca wing pattern. Two cell types (generalized epithelial cells and scale precursors) rearrange within each of the two epithelial monolayers of the wing to form periodic rows of scale cells. These two monolayers also interact with each other during a brief period of adult development. Each cell type shows a different pattern of protein 2F5 expression during cell rearrangement and during interaction of the two wing monolayers. Before and after these morphogenetic movements of epithelial cells, the protein is expressed on only a small population of wing cells. In abdominal epithelia where scale cells are also present but are not arranged in periodic rows, the expression pattern of the surface protein is temporally and spatially very different. An earlier study (Nardi and Magee-Adams, Dev. Biol., 116, 278-290, 1986) had shown that basal processes only extend from epithelial cells during their period of rearrangement within a monolayer and during the transient apposition of the wing's upper and lower monolayers. The differential distribution of protein 2F5 on lateral surfaces and basal processes of scale precursor cells and generalized epithelial cells may account in part for their orderly segregation into alternating rows as well as for the transient interaction of the two wing monolayers.     

Transformations of epithelial monolayers during wing development of Manduca sexta

The two epithelial monolayers of the insect wing undergo striking morphogenetic changes during the course of adult development, but the exact interactions between these monolayers were not evident until the ultrastructure of the cells was carefully examined. The interaction of the dorsal monolayer with the ventral monolayer continually changes as the two initially separate monolayers first lose their pupal basal laminae and then come together along a sharp interface to form microtubule-associated junctions. As blood space between the two monolayers expands 2 days later, new adult basal laminae and cuticle form. Concomitantly the epithelial cells stretch along their apicobasal axes to create a thin cellular M layer halfway between the dorsal and ventral surfaces of the wing that represents the site where connections between the monolayers are maintained at specialized basal junctions. The elongated processes of each monolayer that make up this M layer first fasciculate and then span the space separating the two monolayers, but only at relatively widely-spaced intervals. During later stages of adult development, dense aggregates of microtubules appear in these epithelial processes and presumably contract as cells dramatically shorten along their apicobasal axes during expansion of the wing. Examination of the ultrastructure of the developing adult wing has revealed how certain cellular events can account for the mechanics of cuticle and wing expansion after adult emergence.     

Pattern formation of scale cells in lepidoptera by differential origin-dependent cell adhesion

We present a model for the formation of parallel rows of scale cells in the developing adult wing of moths and butterflies. Precursors of scale cells differentiate throughout each epithelial monolayer and migrate into rows that are roughly parallel to the body axis. Grafting experiments have revealed what appears to be a gradient of adhesivity along the wing. What is more, cell adhesivity character is maintained after grafting. Thus we suggest that it is a cell’s location prior to migration that determines its interactions during migration. We use nonlinear bifurcation analysis to show that differential origin-dependent cell adhesion can result in the stabilization of rows over spots.     

Topographical features of the substratum for growth of pioneering neurons in the Manduca wing disc

The sensory neurons of the Manduca wing form a planar network nestled between the wing's upper and lower monolayers. The pioneering axons of this network grow in a distal-to-proximal direction over the basal surface of the upper epithelial monolayer. The basal surface of this monolayer has been examined ultrastructurally during the period of axonal outgrowth. The cellular terrain traversed by axons shows a graded distribution of epithelial processes, with the number of processes increasing in a proximal direction. Growth cones of axons, therefore, encounter increasing surface areas for contact with their substratum as they move toward the base of the wing. Because a basal lamina is laid down over these epithelial processes after axons have pioneered the neural pathways of the wing, axonal guidance cues apparently lie on surfaces of these basal processes. At branch points of the neural pathway examined in this study, axons avoid pathways in which the basal surfaces of cells in the upper wing monolayer interdigitate with basal surfaces of underlying tracheal cells. This interaction between wing epithelial cells and tracheal epithelial cells could act as a physical barrier to axonal outgrowth.     

Evocation of venation pattern in the wing of a moth,Manduca sexta

The insect wing is formed from an epithelial sheet that folds during development to establish a saclike tissue with an upper and a lower epithelial monolayer. The adult cuticle formed by the upper and lower monolayers has a distinctive pattern of thickened regions called veins. The venation pattern on the lower surface matches that on the upper surface. As demonstrated by transposition of grafts from the upper monolayer, determination of venation pattern occurs prior to pupation in both wing monolayers. However, the pattern is not expressed until later in adult development. Expression of this determined pattern occurs autonomously in most circumstances. One circumstance in which the pattern fails to be expressed is in pieces of the upper monolayer that are isolated from the lower monolayer before adult cuticle deposition and expression of venation pattern. The only evident interaction between the two monolayers of the wing occurs during a 3-day period, 6–8 days after pupation. During this time, the basal laminae segregating upper monolayer from lower monolayer disappear, and the basal ends of cells form desmosomal junctions at the interface between upper and lower monolayer. Transposition as well as isolation of tissue fragments from the upper monolayer suggest that this interaction between the basal surfaces of the two monolayers is a prerequisite for evocation of venation pattern.     

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.     

Development of polyploidy of scale-building cells in the wings of Manduca sexta

The developing wings of butterflies and moths are composed of two epithelial monolayers. Each epithelial sheet is made up of two kinds of cells, diploid cells that make up the epidermal surface and body of the wing, and large polyploid cells that become the scale-building cells whose cytoplasmic projections develop into the scales that will cover the adult wing and bear the pigment pattern. We studied the development of polyploidization of the scale-building cells during the pupal stage of the tobacco hornworm moth, Manduca sexta. The endomitotic divisions of the presumptive scale-building cells and the mitotic divisions of the diploid epithelial cells begin on day 3 of the pupal stage and continue until day 7. We show that scales of different colors and positions on the wing differ in size, and that the size of the scale is proportional to the ploidy of the scale-building cell. Scale-building cells are arranged in irregular rows and within each row there is an alternation of ploidy levels, with the lower ploidy cells giving rise to the underscales and the higher ploidy cells giving rise to the cover scales that carry the color pattern. Along the wing there is a proximo-distal decreasing gradient of average ploidy and scale size. Scale-building cells of high ploidy are surrounded by fewer epidermal cells than those of low ploidy. This inverse relationship is known as Henke's compensation principle, which posits that the number of endomitoses of a pre-polyploid cell and the number of mitotic divisions of its diploid daughter cell add up to a constant. We show that the inverse relationship fits the predictions of the compensation principle and does not fit constraints imposed by packing density, and we discuss mechanisms that could give rise to the inverse relationship.     

Simulation of Cell Patterning Triggered by Cell Death and Differential Adhesion in Drosophila Wing

The Drosophila wing exhibits a well-ordered cell pattern, especially along the posterior margin, where hair cells are arranged in a zigzag pattern in the lateral view. Based on an experimental result observed during metamorphosis of Drosophila, we considered that a pattern of initial cells autonomously develops to the zigzag pattern through cell differentiation, intercellular communication, and cell death (apoptosis) and performed computer simulations of a cell-based model of vertex dynamics for tissues. The model describes the epithelial tissue as a monolayer cell sheet of polyhedral cells. Their vertices move according to equations of motion, minimizing the sum total of the interfacial and elastic energies of cells. The interfacial energy densities between cells are introduced consistently with an ideal zigzag cell pattern, extracted from the experimental result. The apoptosis of cells is modeled by gradually reducing their equilibrium volume to zero and by assuming that the hair cells prohibit neighboring cells from undergoing apoptosis. Based on experimental observations, we also assumed wing elongation along the proximal-distal axis. Starting with an initial cell pattern similar to the micrograph experimentally obtained just before apoptosis, we carried out the simulations according to the model mentioned above and successfully reproduced the ideal zigzag cell pattern. This elucidates a physical mechanism of patterning triggered by cell apoptosis theoretically and exemplifies, to our knowledge, a new framework to study apoptosis-induced patterning. We conclude that the zigzag cell pattern is formed by an autonomous communicative process among the participant cells.     

Scale Arrangement Pattern in a Lepidopteran Wing. 1. Periodic Cellular Pattern in the Pupal Wing of Pieris rapae

In most species of lepidopteran insects, anteroposterior rows formed by scales are arranged at regular intervals in the adult wing; within each row two kinds of scales are alternately arranged. To investigate the cellular basis for the scale arrangement pattern, we examined cell arrangement in the epidermal monolayer of the pupal wing of a small white cabbage butterfly, Pieris rapae , by scanning electron microscopy and light microscopy.
The arrangement of scale precursor cells, closely resembling that of scales in the adult wing, was observed in the wing epidermis of the early pupa. Scale precursor cells are proximodistally elongated and form anteroposterior rows. Within a row two kinds of scale precursor cells are nearly alternately arranged, which is not so precise as the alternation of scales in the adult wing. Individual rows of scale precursor cells are separated by rows of single or double undifferentiated general epidermal cells. Occasionally, arrangement abnormalities occur both in the adult and the pupal wing. The cellular basis for the regular spacing of scale rows is discussed.     

Hemocytes contribute to both the formation and breakdown of the basal lamina in developing wings of Manduca sexta

Monoclonal antibodies (MAbs) raised against wing tissues of Manduca sexta recognize epitopes shared by both hemocytes and basal laminae. During the last larval stadium, the basal lamina of moth wing epithelium forms after hemocytes have migrated into the space adjacent to basal surfaces of epithelial cells. As adult development commences, hemocytes participate in phagocytosis of the same basal lamina; and as dissolution of the basal lamina proceeds (day 2-day 5 post-pupation), wing epithelial cells send forth long basal processes and rearrange within the plane of the epithelium. During this period of cell rearrangement, the immunoreactivity of the basal lamina decreases in concert with an increase in immunoreactive vesicles within hemocytes; and at the ultrastructural level, hemocytes have been observed to engulf fragments of basal lamina. The distribution of immunolabel in the developing moth wing suggests that hemocytes contribute not only to the formation of the wing's basal lamina but also to its breakdown. Since basal laminae are probably important determinants of epithelial form and pattern, hemocytes also contribute to the shaping of epithelial populations.     

The wing imaginal disc

The Drosophila wing imaginal disc is a tissue of undifferentiated cells that are precursors of the wing and most of the notum of the adult fly. The wing disc first forms during embryogenesis from a cluster of ∼30 cells located in the second thoracic segment, which invaginate to form a sac-like structure. They undergo extensive proliferation during larval stages to form a mature larval wing disc of ∼35,000 cells. During this time, distinct cell fates are assigned to different regions, and the wing disc develops a complex morphology. Finally, during pupal stages the wing disc undergoes morphogenetic processes and then differentiates to form the adult wing and notum. While the bulk of the wing disc comprises epithelial cells, it also includes neurons and glia, and is associated with tracheal cells and muscle precursor cells. The relative simplicity and accessibility of the wing disc, combined with the wealth of genetic tools available in Drosophila, have combined to make it a premier system for identifying genes and deciphering systems that play crucial roles in animal development. Studies in wing imaginal discs have made key contributions to many areas of biology, including tissue patterning, signal transduction, growth control, regeneration, planar cell polarity, morphogenesis, and tissue mechanics.     

THE EVOLUTIONARY GENETICS AND DEVELOPMENTAL BASIS OF WING PATTERN VARIATION IN THE BUTTERFLY BICYCLUS ANYNANA

We have studied interactions between developmental processes and genetic variation for the eyespot color pattern on the adult dorsal forewing of the nymphalid butterfly, Bicyclus anynana. Truncation selection was applied in both an upward and a downward direction to the size of a single eyespot consisting of rings with wing scales of differing color pigments. High heritabilities resulted in rapid responses to selection yielding divergent lines with very large or very small eyespots. Strong correlated responses occurred in most of the other eyespots on each wing surface. The cells at the center of a presumptive eyespot (the “focus”) act in the early pupal stage to establish the adult wing pattern. The developmental fate of the scale cells within an eyespot is specified by the “signaling” properties of the focus and the “response” thresholds of the epidermis. The individual eyespots can be envisaged as developmental homologues. Grafting experiments performed with the eyespot foci of the selected lines showed that additive genetic variance exists for both the response and, in particular, the signaling components of the developmental system. The results are discussed in the context of how constraints on the evolution of this wing pattern may be related to the developmental organization.     

Tracheole migration in an insect wing

Summary Insect tissues are supplied with oxygen by a system of long and highly branched cuticular tubes known as tracheae and tracheoles. During the growth of with imaginal discs in moths and butterflies, tracheole cells migrate distally from the base of the disc. Tracheoles radiate in a distal direction through the extracellular space sandwiched between the upper and lower epithelial surfaces of the wing.Migration of most cells is assumed to be governed by forces intrinsic to the cell. However, the movement of tracheoles is apparently a passive process whose motive force resides in adjacent epithelial cells. After epithelial cells are exposed to ecdysteroid hormones, these cells extend basal processes that are attracted to oxygen-rich tracheoles. By applying traction to the tracheoles with which they establish intimate contact, epithelial cells may control the pattern of their distribution within wing tissue.     

Programmed cell death in the wing of Orgyia leucostigma (Lepidoptera: Lymantriidae)

Programmed cell death is an integral and ubiquitous phenomenon of development that is responsible for the reduction of wing size in female moths of Orgyia leucostigma (Lymantriidae). Throughout larval and pupal life, cells of the wing epithelium proliferate and interact to form normal imaginal discs and pupal wings in both sexes. But at the onset of adult development, most cells in female O. leucostigma wings degenerate over a brief, 2-day period. Lysosomes and autophagic vacuoles appear in cells of the wing epithelium shortly after it retracts from the pupal cuticle. Hemocytes actively participate in removing the resulting cellular debris. By contrast, epithelial cells in wings of developing adult males of O. leucostigma do not undergo massive cell death. Wing epithelium of female pupae transferred to male pupal hosts behaves autonomously in this foreign environment. By pupation, cells of the female wing apparently are committed to self-destruct even in a male pupal environment. Normal interactions among epithelial cells within the plane of a wing monolayer as well as between the upper and lower monolayers of the wing are disrupted in female O. leucostigma by massive cell degeneration. Despite this disruption, the remaining cells of the wing contribute to the formation of a diminutive, but reasonably proportioned, adult wing with scales and veins.     

Mutations in the polycomb group gene polyhomeotic lead to epithelial instability in both the ovary and wing imaginal disc in Drosophila

Background

Most human cancers originate from epithelial tissues and cell polarity and adhesion defects can lead to metastasis. The Polycomb-Group of chromatin factors were first characterized in Drosophila as repressors of homeotic genes during development, while studies in mammals indicate a conserved role in body plan organization, as well as an implication in other processes such as stem cell maintenance, cell proliferation, and tumorigenesis. We have analyzed the function of the Drosophila Polycomb-Group gene polyhomeotic in epithelial cells of two different organs, the ovary and the wing imaginal disc.

Results

Clonal analysis of loss and gain of function of polyhomeotic resulted in segregation between mutant and wild-type cells in both the follicular and wing imaginal disc epithelia, without excessive cell proliferation. Both basal and apical expulsion of mutant cells was observed, the former characterized by specific reorganization of cell adhesion and polarity proteins, the latter by complete cytoplasmic diffusion of these proteins. Among several candidate target genes tested, only the homeotic gene Abdominal-B was a target of PH in both ovarian and wing disc cells. Although overexpression of Abdominal-B was sufficient to cause cell segregation in the wing disc, epistatic analysis indicated that the presence of Abdominal-B is not necessary for expulsion of polyhomeotic mutant epithelial cells suggesting that additional POLYHOMEOTIC targets are implicated in this phenomenon.

Conclusion

Our results indicate that polyhomeotic mutations have a direct effect on epithelial integrity that can be uncoupled from overproliferation. We show that cells in an epithelium expressing different levels of POLYHOMEOTIC sort out indicating differential adhesive properties between the cell populations. Interestingly, we found distinct modalities between apical and basal expulsion of ph mutant cells and further studies of this phenomenon should allow parallels to be made with the modified adhesive and polarity properties of different types of epithelial tumors.     

Spatial patterns of correlated scale size and scale color in relation to color pattern elements in butterfly wings

Complex butterfly wing color patterns are coordinated throughout a wing by unknown mechanisms that provide undifferentiated immature scale cells with positional information for scale color. Because there is a reasonable level of correspondence between the color pattern element and scale size at least in Junonia orithya and Junonia oenone, a single morphogenic signal may contain positional information for both color and size. However, this color–size relationship has not been demonstrated in other species of the family Nymphalidae. Here, we investigated the distribution patterns of scale size in relation to color pattern elements on the hindwings of the peacock pansy butterfly Junonia almana, together with other nymphalid butterflies, Vanessa indica and Danaus chrysippus. In these species, we observed a general decrease in scale size from the basal to the distal areas, although the size gradient was small in D. chrysippus. Scales of dark color in color pattern elements, including eyespot black rings, parafocal elements, and submarginal bands, were larger than those of their surroundings. Within an eyespot, the largest scales were found at the focal white area, although there were exceptional cases. Similarly, ectopic eyespots that were induced by physical damage on the J. almana background area had larger scales than in the surrounding area. These results are consistent with the previous finding that scale color and size coordinate to form color pattern elements. We propose a ploidy hypothesis to explain the color–size relationship in which the putative morphogenic signal induces the polyploidization (genome amplification) of immature scale cells and that the degrees of ploidy (gene dosage) determine scale color and scale size simultaneously in butterfly wings.     

Peripodial cells regulate proliferation and patterning of Drosophila imaginal discs

Cells employ a diverse array of signaling mechanisms to establish spatial patterns during development. Nowhere is this better understood than in Drosophila, where the limbs and eyes arise from discrete epithelial sacs called imaginal discs. Molecular-genetic analyses of pattern formation have generally treated discs as single epithelial sheets. Anatomically, however, discs comprise a columnar cell monolayer covered by a squamous epithelium known as the peripodial membrane. Here we demonstrate that during development, peripodial cells signal to disc columnar cells via microtubule-based apical extensions. Ablation and targeted gene misexpression experiments demonstrate that peripodial cell signaling contributes to growth control and pattern formation in the eye and wing primordia. These findings challenge the traditional view of discs as monolayers and provide foundational evidence for peripodial cell function in Drosophila appendage development.     

Comparative morphology and evolutionary aspects of the reflective under wing scale-pattern in Fritillary butterflies (Nymphalidae: Argynnini)

The wing scale ultrastructure of the reflective under wing pattern found in many Argynnini butterflies are examined for all recognised genera and subgenera and compared to that of some basal Heliconiini. A true reflective pattern probably evolved once within the Argynnini. But the phylogenetic information in these structures is limited due to a high degree of homoplasy in the scale ultrastructure related to the reflective patterns. The degree of specialisation is also homoplastic. The general morphological modification responsible for the reflective patterns seems to be a “closing” of the large windows, which generally occupy most of the inter-ridge space on the abwing surface in the scales of higher Lepidoptera. The fact that the Argynnis niobe morphs with a silvery pattern have scales with ‘closed windows’ whereas the Argynnis niobe morphs without a silvery pattern have typical non-reflective scales with very large windows supports this conclusion.The degrees of modification of the scales, including whether both cover and ground scales or only cover scales are modified, are to some extent correlated to the degree of reflectiveness in the wing pattern. Boloria eunomia has, as the only species, more modified ground scales than cover scales.     

Involvement of Lgl and Mahjong/VprBP in Cell Competition

During the initial stages of carcinogenesis, transformation events occur in a single cell within an epithelial monolayer. However, it remains unknown what happens at the interface between normal and transformed epithelial cells during this process. In Drosophila, it has been recently shown that normal and transformed cells compete with each other for survival in an epithelial tissue; however the molecular mechanisms whereby “loser cells” undergo apoptosis are not clearly understood. Lgl (lethal giant larvae) is a tumor suppressor protein and plays a crucial role in oncogenesis in flies and mammals. Here we have examined the involvement of Lgl in cell competition and shown that a novel Lgl-binding protein is involved in Lgl-mediated cell competition. Using biochemical immunoprecipitation methods, we first identified Mahjong as a novel binding partner of Lgl in both flies and mammals. In Drosophila, Mahjong is an essential gene, but zygotic mahjong mutants (mahj ^−/−) do not have obvious patterning defects during embryonic or larval development. However, mahj ^−/− cells undergo apoptosis when surrounded by wild-type cells in the wing disc epithelium. Importantly, comparable phenomena also occur in Mahjong-knockdown mammalian cells; Mahjong-knockdown Madin-Darby canine kidney epithelial cells undergo apoptosis, only when surrounded by non-transformed cells. Similarly, apoptosis of lgl ^−/− cells is induced when they are surrounded by wild-type cells in Drosophila wing discs. Phosphorylation of the c-Jun N-terminal kinase (JNK) is increased in mahj ^−/− or lgl ^−/− mutant cells, and expression of Puckered (Puc), an inhibitor of the JNK pathway, suppresses apoptosis of these mutant cells surrounded by wild-type cells, suggesting that the JNK pathway is involved in mahj- or lgl-mediated cell competition. Finally, we have shown that overexpression of Mahj in lgl ^−/− cells strongly suppresses JNK activation and blocks apoptosis of lgl ^−/− cells in the wild-type wing disc epithelium. These data indicate that Mahjong interacts with Lgl biochemically and genetically and that Mahjong and Lgl function in the same pathway to regulate cellular competitiveness. As far as we are aware, this is the first report that cell competition can occur in a mammalian cell culture system.