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.     

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.     

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.     

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.     

Neuronal pathfinding in developing wings of the moth Manduca sexta

The neural pattern of the moth wing is a simple two-dimensional network nestled between the two epithelial monolayers that form the upper and lower surfaces of the wing. All neural elements within the wing blade are sensory and their axons grow proximally toward the mesothoracic ganglion. The sensory nerves of the wing are intimately associated with the basal lamina of the upper epithelial layer; and the molding of neural pattern is coupled with cues in the basal lamina. The global landscape of the basal lamina can be altered by exchange of epithelial grafts. Axons generally cross control grafts as well as grafts that have been displaced distally. However, axons generally avoid grafts that have been transposed proximally. This asymmetric response of growing axons implies that directional cues in the substratum are also asymmetric along the length of the wing. The asymmetric, graded distribution of extracellular matrix molecules associated with the basal lamina of the wing's upper epithelium could provide the short-range cues that guide sensory axons in a particular direction.     

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.     

Tissue remodeling during maturation of the Drosophila wing

The final step in morphogenesis of the adult fly is wing maturation, a process not well understood at the cellular level due to the impermeable and refractive nature of cuticle synthesized some 30 h prior to eclosion from the pupal case. Advances in GFP technology now make it possible to visualize cells using fluorescence after cuticle synthesis is complete. We find that, between eclosion and wing expansion, the epithelia within the folded wing begin to delaminate from the cuticle and that delamination is complete when the wing has fully expanded. After expansion, epithelial cells lose contact with each other, adherens junctions are disrupted, and nuclei become pycnotic. The cells then change shape, elongate, and migrate from the wing into the thorax. During wing maturation, the Timp gene product, tissue inhibitor of metalloproteinases, and probably other components of an extracellular matrix are expressed that bond the dorsal and ventral cuticular surfaces of the wing following migration of the cells. These steps are dissected using the batone and Timp genes and ectopic expression of alphaPS integrin, inhibitors of Armadillo/beta-catenin nuclear activity and baculovirus caspase inhibitor p35. We conclude that an epithelial-mesenchymal transition is responsible for epithelial delamination and dissolution.     

Programmed cell death at the periphery of the pupal wing of the butterfly,Pieris rapae

The outline of the adult wing of lepidopteran insects (butterflies and moths) emerges as a result of disappearance of a group of cells at the periphery of the pupal wing. Histological observation of the pupal wing of Pieris rapae showed that, just after apolysis of the wing epithelium from the pupal cuticle, there occurs a rapid and localized decrease of the number of cells at the periphery of the wing. This decrease occurs through cell death, which lasts 1–1.5 days at 20°C. Dying cells lose contact with the neighbouring cells and show condensation of chromatin and cytoplasm. They then appear to be phagocytosed by neighbouring epithelial cells or discharged through the basal surface of the epithelium into the lumen within the wing and taken up by phagocytes. Fragmentation of DNA in the nuclei was detected in the dead cells or their debris. These results indicate that programmed cell death in the lepidopteran wing proceeds through a mechanism closely similar to that of apoptosis in the vertebrate.     

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.     

Formation of scale spacing patterns in a moth wing: I. Epithelial feet may mediate cell rearrangement

A new staining procedure reveals outlines of individual, scattered cells within an epithelial monolayer and shows that cells form intimate contacts not only with adjacent cells but also with nonadjacent (and often relatively distant) cells. Cell interactions in the two-dimensional monolayer of the Manduca wing are more complex than originally supposed. Cells extend long basal processes at the time that major changes in epithelial pattern are occurring. The pattern of regularly spaced scale rows in the wing arises from the rearrangement of irregularly distributed scale primordial cells and is probably mediated by short-range and long-range interactions of these epithelial processes.     

Metamorphosis of the ecdysis motor pattern in the hawkmoth, Manduca sexta

Summary The hawkmoth,Manduca sexta, under-goes periodic molts during its growth and metamorphosis. At the end of each molt, the old cuticle is shed by means of a hormonally-activated ecdysis behavior. The pharate adult, however, must not only shed its old cuticle but also dig itself out from its underground pupation chamber. To accomplish this, the adult performs a series of abdominal retractions and extensions; the extensions are coupled with movements of the wing bases. This ecdysis motor pattern is distinct from the slowly progressing, anteriorly-directed, abdominal peristalses expressed by ecdysing larvae and pupae.We have found that the ability to produce the larval-like ecdysis pattern is retained in the adult. Although this behavior is not normally expressed by the adult, larval-like ecdysis could be unmasked when descending neuronal inputs, originating in the pterothoracic ganglion, were removed from the unfused abdominal ganglia. Transformation of the adult-specific ecdysis pattern to the larval-like pattern was accomplished by transecting the connectives between the pterothorax and the abdomen, or by reversibly blocking neuronal activity with a cold-block. A comparative analysis of the ecdysis motor patterns expressed by larvae and by isolated adult abdomens indicates that the two motor patterns are indistinguishable, suggesting that the larval ecdysis motor pattern is retained through metamorphosis. We speculate that its underlying neural circuitry is conserved through development and later modulated to produce the novel ecdysis pattern expressed in the adult stage.Abbreviations A(n) nth abdominal segment - DL dorsal longitudinal - EH eclosion hormone - ISMs intersegmental muscles - MN motoneuron - SEG subesophageal ganglion - T1,T2,T3 prothoracic, mesothoracic, and metathoracic ganglion - TSMs tergosternal muscles - TX thorax     

Developmental changes of the antenna and its neurons in the silkworm,Bombyx mori,with special regard to larval-pupal transformation

The larval antenna of Bombyx mori has 13 sensilla and about 52 sensory neurons in its distal portion. The axons form two nerve cords which unite in the cranial hemocoel to supply the brain as the olfactory nerve. The antennal imaginal disc, which is a thick pseudostratified epithelium continuous with the antennal epidermis, thickens markedly during the 5th instar by rapid cell proliferation. At the prepupal stage cell proliferation ceases and the disc everts to form a large pupal antenna. Simultaneously, an extensive cell rearrangement occurs in the antennal epidermis and the disc tissue becomes much thinner because of the abrupt expansion of antennal surface area. The two larval nerve cords thin down markedly by degeneration of axons, but they do not disintegrate totally even after the onset of pupation. The epidermis of the larval antenna forms the distal portion of the pupal antenna, while the imaginal disc forms the more basal portion. Development to the adult antenna occurs almost immediately after the onset of pupation; many adult neurons appear in the simple epidermis facing toward the thick outer side of the newly formed pupal cuticle. By 12 hours after the onset of pupation, these neurons align themselves in many transverse rows which are the first sign of the adult antennal configuration. Addition of these neuronal axons to the once-thinned nerve cords causes resumed thickening of the cords during the first 24 hours and thereafter. Differentiation of adult sensilla begins in the next 24 hours and is almost completed at the third day of pupation, which requires a total of 10 days.     

In vivo and solid state 13C nuclear magnetic resonance studies of tyrosine metabolism during insect cuticle formation

1. Tyrosine metabolism during pupation can be followed in living Heliothis virescens larvae using nuclear magnetic resonance spectroscopy. 2. Loss of 13C signals from a label at the C-3 position of tyrosine during pupation indicates uptake of tyrosine into solid cuticle. 3. Solids 13C NMR spectroscopy of cuticle formed by insects injected with 3-13C labelled tyrosine indicates that little change in chemical shift occurs during cuticle hardening, indicating that the side chain is probably not involved in protein cross-linking.     

Dpp/BMP transport mechanism is required for wing venation in the sawfly Athalia rosae

The pattern of wing venation varies considerably among different groups of insects and has been used as a means of species-specific identification. However, little is known about how wing venation is established and diversified among insects. The decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signaling pathway plays a critical role in wing vein formation during the pupal stages in Drosophila melanogaster. A key mechanism is BMP transport from the longitudinal veins (LVs) to the posterior crossvein (PCV) by the BMP-binding proteins, short gastrulation (Sog) and twisted gastrulation2/crossveinless (Tsg2/Cv). To investigate whether the BMP transport mechanism is utilized to specify insect wing vein patterns in other than Drosophila, we used the sawfly Athalia rosae as a model, which has distinct venation patterns in the fore- and hindwings. Here, we show that Ar-dpp is ubiquitously expressed in both the fore- and hindwings, but is required for localized BMP signaling that reflects distinct wing vein patterns between the fore- and hindwings. By isolating Ar-tsg/cv in the sawfly, we found that Ar-Tsg/Cv is also required for BMP signaling in wing vein formation and retains the ability to transport Dpp. These data suggest that the BMP transport system is widely used to redistribute Dpp to specify wing venation and may be a basal mechanism underlying diversified wing vein patterns among insects.     

Trichomonas vaginalis: Ultrastructural Bases of the Cytopathic Effect

ABSTRACT. The in vitro cytopathic effect of Trichomonas vaginalis on epithelial cells was explored through the interaction of trophozoites of the virulent strain GT-10 with MDCK monolayers. The interaction was analyzed through electrophysiology, video microscopy, and transmission and scanning electron microscopy. Electrical measurements revealed that living parasites produced severe damage to the cell monolayers within 30 min, manifested as a rapid decrease in transepithelial resistance. Microscopic observations demonstrated that when placed in contact with epithelial cells, trichomonas formed clumps through interdigitations and transient plasma membrane junctions between adjacent parasites. Also, attached trophozoites adopted an ameboid shape. The in vitro cytopathic action of T. vaginalis on MDCK cells was initially evident by modifications of the plasma membrane, resulting in opening of tight junctions, membrane blebbing, and monolayer disruption. After 15 min of interaction the damage was focal, concentrating at sites where parasite clumps adhered to the monolayer. At 30 min practically all MDCK cells were dead, whether or not trichomonas were attached to them. These events were followed by detachment of lysed cells and complete disruption of the monolayer at 60 min. Electron microscopy demonstrated a peculiar form of adhesion that appears to be specific for trichomonas, in which the basal surface of T. vaginalis formed slender channels through which microvilli and cytoplasmic fragments of epithelial cells were internalized. The same sequence of lytic events was found with the less virulent GT-3 strain. However, the time course of cytolysis with GT-3 parasites was much slower, and lysis was limited to areas of attachment of T. vaginalis.     

New Model for Studying the Migration of Immune Cells into Intestinal Epithelial Cell Monolayers

A novel cell culture system was constructed to analyze the direct interaction between intestinal epithelial cells and immune cells. Human intestinal epithelial Caco-2 cells were monolayer-cultured on the under side of a permeable membrane (12 μm pore size) in a Millicell insert. Integrated monolayers of Caco-2 cells had formed after 12 days of culture. Human monocyte/macrophage-like THP-1 cells were then added to the upper chamber of the insert, and their migration into the Caco-2 cell monolayers was observed by confocal laser scanning microscopy, after staining the cells with specific antibodies. When MCP-1, a β-chemokine, was added to the apical side of the monolayer, a greater number of THP-1 cells migrated into the Caco-2 cell monolayers. This cell culture system will be useful for studying the behavior of macrophages in the intestinal epithelial cell monolayers at the initial stage of an intestinal immune reaction.     

现存蜉蝣翅基纵脉走向及愈合模式(昆虫纲：蜉蝣目)

有翅昆虫翅基纵脉的走向及愈合模式在系统发育重建中占有重要地位。然而,现存蜉蝣翅基纵脉的走向及愈合状况在大部分种类变化极大,无法推测其原始状况,只在极少数种类保留有部分可见残迹。中国拟短丝蜉Siphluriscus chinensis的翅基保留有独立的亚前缘脉弓、部分中脉M和肘脉Cu主干以及前中脉MA及径分脉Rs的走向痕迹。据此并结合红斑蜉Ephemera rufomaculata 和大网脉蜉Chromarcys magnifica翅基的相关特征,本文提出了蜉蝣目主要纵脉基部走向及愈合的基本模式,其要点有：中脉主干在基部与径脉主干独自发出后先接近或愈合后又分离、它们各自分成两支后的前中脉及径分脉又先愈合再分离、肘脉始终独立。这种中脉与径脉先接近或愈合后分离的模式非常接近新翅类的情况而与蜻蜓很不相同(在蜻蜓,中脉与肘脉在基部愈合) 。亚前缘脉弓的作用相信是加强了因翅基骨板发达而相互远离的纵脉间的连结作用。这个假说也可以来解释蜻蜓复杂脉相的形成原因。     

Stability of polarity in the epidermis of a beetle, Tenebrio molitor L.

Cell polarity in the insect epidermis may be coupled to the orientation of anisometric cuticle components. Rotation of squares of sternite epidermis in the larva results in a corresponding rotation in the highly ordered orientation of cuticle fibers in the adult “crossply” cuticle. The patterns of fiber orientation resulting from graft rotation can be explained by the presence of an axial gradient of positional information.The polarity of the rotated tissue is, however, not fixed. Interaction between the polarity of the graft and host tissue may result in a partial shift of graft polarity toward the axial polarity of the host tissue. This interaction is apparently restricted to a limited period of the cell cycle: cell division. In Tenebrio, the sternite epidermis proliferates only once during metamorphosis, 140–90 hr before pupation. Rotational grafts performed before, during, and after this period present a graded series of “relaxation” patterns in fiber orientation in the graft area. Maximal graft repolarization coincides with maximal cell division on the sternite. The epidermal gradient, or cell response to the gradient, appears to be nonlinear along the segment.If no cell division intervenes between graft rotation and fiber deposition, graft polarity remains stable. This stability necessitates a “memory” component in the epidermis. It is suggested that periodic assessment of tissue polarity occurs concomitant with a particular process of cell division.     

Cellular Interactions in Morphogenesis of the Thyroid Gland

The existence of an epithelio-mesodermal interaction in theembryonic chick thyroid has been tested by reaggregation of8- and 16-day epithelial monolayers with capsule, mesentery,heart ventricle, and perichondrial cells. Establishment of thehistological anil cytological pattern of the thyroid occurredonly in the presence of capsule. The loss of endoplasmic reticulumwhich occurs in monolayer culture was shown not to be a resultof spreading, since organ cultures of immediately reaggregatedcells undergo the same degenerative changes. Dissociation itselfalso cannot be the explanation for the loss since membrane destructionand recovery occur at different times in raft cultures and inchorio-allantoic grafts. The similarity of the results afterimmediate reaggregation and in combinations of epithelial cellswith capsule after monolayer culture suggests that the roleof the mesodermal component is to support the specialized cytoplasmicstructure and function of the epithelial cell type.