Induction of invagination in insect epithelium: paradigm for embryonic invagination.

The proposal that adhesive disparities between inpocketing populations of cells and surrounding epithelia drive epithelial invagination was tested in grafting experiments with moth pupal wing epithelium. Evidence exists that a cellular adhesiveness gradient spans the proximodistal axis of the wing. Although pupal wing cells normally do not invaginate or evaginate, epithelial folding can be induced after exchange of grafts from opposite ends of the proximodistal axis. The hypothesis that cytoskeletal elements are the primary agents in epithelial invagination should be reevaluated.

Expression of lacunin, a large multidomain extracellular matrix protein, accompanies morphogenesis of epithelial monolayers in Manduca sexta.

Morphogenesis is a complex process operating at several levels of organization--organism, tissues, cells, and molecules. Complex interactions occur between and within these levels. Many of the molecules that mediate these interactions are predictably turning out to be large multidomain proteins. Here we describe one such novel protein associated with remodeling of epithelial monolayers in embryos and developing wings of the moth Manduca sexta. On the basis of its sequence and its expression pattern along lacunae of developing wings, we propose the name lacunin for this extracellular matrix protein that contains nine different types of domains, most of which are present in multiple copies. These include domains of various types: Kunitz proteinase inhibitors, thrombospondin type I, immunoglobulin-like, and several newly defined domains of unknown function (PAL, PLAC, and lagrin domains). This rich patchwork of distinct domains probably exerts multiple effects on a variety of cell behaviors associated with the complex phenomenon of epithelial morphogenesis.

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.

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.

Fine structure of cells specialized for secretion of aggregation pheromone in a nitidulid beetle Carpophilus freemani (coleoptera: Nitidulidae).

The cells that secrete the aggregation pheromone of the male nitidulid beetle Carpophilus freemani are exceptionally large and lie within the body cavity. These secretory cells share many ultrastructural features with cells of other pheromone and defense glands, but they also have several unique features. A deep invagination of the surface of each of these cells acts as the secretory surface for the pheromone. The invaginated surface is highly convoluted and surrounds a narrow cuticular ductule that is connected to the tracheal system. This surface is not covered with microvilli as the comparable surfaces are in other insect secretory cells. Each secretory cell is filled with an abundance of lipid spheres that presumably contain precursors for the pheromone. Examining cells from beetles producing different levels of pheromone showed that sizes of secretory cells are positively correlated with rates of pheromone production. Whereas secretory and ductule cells of other insect glands are usually epidermal cells, these cells of nitidulid beetles represent the first pheromone glands in which oenocytes are believed to have been recruited for pheromone production and tracheal cells have been recruited as ductules for these cells.

Specialization of midgut cells for synthesis of male isoprenoid pheromone components in two scolytid beetles, Dendroctonus jeffreyi and Ips pini.

Endodermal or midgut cells have only recently been recognized as the site of pheromone synthesis in bark beetles. Midgut cells are not only specialized for digestion, but they have also been recruited to form isoprenoid compounds that function as pheromone components in Ips pini and Dendroctonus jeffreyi. Male bark beetle midgut cells are competent to produce isoprenoid pheromones after feeding or stimulation by juvenile hormone (JH) III. Competent midgut cells share many ultrastructural features with cells that do not secrete isoprenoid pheromone, but they are distinguished from these by abundant and highly ordered arrays of smooth endoplasmic reticula. During secretion, both midgut cells that produce pheromone and cells that do not are characterized by the presence of apical extrusions (apocrine secretion) rather than the presence of vesicles that fuse with the apical membrane and undergo exocytosis (eccrine secretion). Pheromone-producing cells of the midgut do not represent a population of cells that are distinct from cells involved in digestion. All, or most, midgut cells of male I. pini and D. jeffreyi can secrete pheromones as well as digestive enzymes.

Morphogenesis in wing imaginal discs: its relationship to changes in the extracellular matrix.

An extracellular matrix (ECM) lies between the upper and lower epithelial layers of the wing imaginal discs of moths. Organization and composition of this extracellular matrix, as revealed by staining with ruthenium red, tannic acid, and alcian blue, changes in concert with levels of hormones in the haemolymph. The ECM of the wing imaginal disc is an environment for cellular movements. Reorganization of the matrix and increase in ecdysteroid level is coupled with the proximal----distal migration of tracheal cells as well as the distal----proximal outgrowth of sensory neurons.

Rearrangement of epithelial cell types in an insect wing monolayer is accompanied by differential expression of a cell surface protein.

The distribution of adhesive molecules on surfaces of cells represents covert information for specifying positions of cells within a tissue. In insect wing epithelia where cells are arranged in two monolayers separated by an extracellular space, these adhesive molecules are found on basal and lateral surfaces of cells. Protein 3B11 is one surface protein whose expression changes in concert with movement and alignment of cells in wing monolayers of Manduca as well as with migration of tracheoles between the two monolayers of the wing. As epithelial cells segregate into periodic, transverse rows of alternating cell types (scale cells and generalized epithelial cells), the expression of 3B11 changes from a uniform distribution throughout the epithelial monolayer to a distribution correlated with a cell's final position and phenotype. Initially protein 3B11 is uniformly expressed on nonadherent surfaces of cells, but with the inception of cell rearrangement, differential expression of 3B11 on basolateral surfaces of cells--both adherent and nonadherent surfaces--becomes a function of epithelial cell type. At the completion of the cell movements associated with segregation of cell types, 3B11 is once again uniformly expressed throughout the wing epithelium. Also, as the upper and lower epithelial monolayers interact at their basal surfaces during adult development, 3B11 is expressed at the interface between the two epithelial monolayers and presumably functions in the nonspecific interaction between these monolayers. Examining the expression patterns of this protein as well as other adhesion molecules in wing epithelia should reveal general rules about either the simplicity or the complexity of the molecular prepatterns that orchestrate overt tissue patterns in epithelial monolayers.

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.

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.

Expression of a surface epitope on cells that link branches in the tracheal network of Manduca sexta.

A monoclonal antibody (MAb 2F5) to a cell surface epitope labels a small subpopulation of tracheal epithelial cells in each thoracic and abdominal segment of Manduca. These cells (nodes) represent the sites within the tracheal network at which invaginating tracheal tubes join during embryonic establishment of the tracheal network. Tracheal nodes are also the sites at which tracheal cuticle fractures during each molt. Since tracheal cuticle is shed through each spiracle, a tracheal node lies between each pair of contralateral spiracles within a segment (commissural node) and between each pair of adjacent, ipsilateral spiracles (lateral longitudinal node). MAb 2F5 first labels presumptive nodal cells of tracheal epithelium immediately prior to the linking of epithelial tubes from successive and opposite spiracles. One cell at the tip of each invaginating tracheal branch labels with MAb 2F5. The highly localized expression of the cell surface epitope recognized by MAb 2F5 may be instrumental in the orderly coupling of tracheal branches during embryonic development. On the basis of immunolabeling of Western blots and tissues, MAb 2F5 is believed to recognize Manduca fasciclin II, a member of a class of molecules involved in cell adhesion/recognition.

Polarity and gradients in lepidopteran wing epidermis. I. Changes in graft polarity, form, and cell density accompanying transpositions and reorientations.

Lepidopteran wing epidermis has certain advantages for studying the spatial organization of cell populations: ease of accessibility and manipulation, large size, an essentially two-dimensional structure, and direct expression by the scale cells of their polarity. Grafting experiments reveal that polarity and density of graft cells as well as overall graft form are functions of a graft's source and its transplantation site; graft polarity is also determined by the orientation of the graft. The results are consistent with the existence of at least one morphogenetic gradient along the proximo-distal axis of the wing's upper epidermal layer. Various gradient models that might explain the experimental observations are considered.

Polarity and gradients in lepidopteran wing epidermis. II. The differential adhesiveness model: gradient of a non-diffusible cell surface parameter.

For explaining the Manduca wing gradient (Nardi & Kafatos, 1976) a model which postulates a proximo-distal gradient in cellular adhesiveness is considered. The model is based on Steinberg's (1963) differential adhesiveness hypothesis. Rosette formation in certain transposed and/or reoriented grafts can be adequately explained by this model. Several predictions, formulated by using the concept of surface free energy as a thermodynamic measure of adhesiveness, have been tested and proven correct. (1) Transposed grafts tend to assume circular forms, which are configurations of minimum free energy. (2) Because of the pressure difference expected across the interface of two cell populations with different surface free energies, cell densities increase in both distally and proximally transposed grafts. As a corollary to this rule, final size of a graft is a function of its distance from the original position. (3) Histological sections of host-graft boundaries suggest minimal cell contact at the interface. In proximal grafts placed in distal regions, cell density is far lower near the host-graft interface, as compared to the high interior density; the peripheries of distal grafts do not show this effect. (4) Juxtaposition of three different wing regions in all possible arrangements yields the expected two-dimensional configurations. (5) Differences in adhesiveness can be demonstrated by allowing two different wing grafts to interact in an essentially neutral environment (i.e. at a leg or antenna site). as the distance between two given graft regions increases, the extent of their final contact decreases. When applied to other insect systems, the model not only offers an alternative interpretation for results currently explained by diffusible substance models, but also accounts for certain features that were unexplained by other models.

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.

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.

The tetraspanin superfamily in insects.

We describe four members of the tetraspanin/TM4SF superfamily of proteins that were identified in expressed sequence tag projects on the antennae of Manduca sexta moths and Apis mellifera honey bees. The three moth genes are expressed in the sensillar epithelium of male antennae, and some are expressed in female antennae, haemocytes, wing scale cell primordia and/or embryonic tissues. These proteins are probably involved in diverse cellular processes, much like their vertebrate homologues. A phylogenetic analysis of all known tetraspanins, including thirty-seven members of the superfamily revealed by the Drosophila melanogaster genome and twenty in the nematode Caenorhabditis elegans genome, reveals some phylum-specific gene amplification, in particular a contiguous array of eighteen genes in the D. melanogaster genome.

The extracellular matrix protein lacunin is expressed by a subset of hemocytes involved in basal lamina morphogenesis.

The extracellular matrix protein of Manduca sexta known as lacunin is localized to basal laminae and granular cells of the hemolymph. Circulating granular cells increase in both number and size as pupal basal laminae break down after the initiation of adult development. Basal laminae of wing epithelia break down as ecdysteroid levels rise during adult development, and lacunin immunoreactivity concurrently passes from basal laminae to endocytic vacuoles of granular cells. Granular cells not only endocytose lacunin protein that they salvage from the remnants of the old basal laminae but they also express transcripts for lacunin. As new adult basal laminae form several days later, the number of circulating granular cells decreases as lacunin immunoreactivity appears in the new basal laminae.

Neuroglian is expressed on cells destined to form the prothoracic glands of Manduca embryos as they segregate from surrounding cells and rearrange during morphogenesis.

A cell surface protein (3B11) is differentially expressed in the embryonic labial segment of Manduca as two circular monolayers of epithelial cells invaginate and segregate from surrounding epithelial cells. The cells that invaginate and preferentially express 3B11 represent the presumptive prothoracic glands. These cells continue to express protein 3B11 as they rearrange to form first a three-dimensional aggregate and later anastomosing filaments of cells. In the differentiated prothoracic gland, expression of 3B11 is restricted to sites of cell-cell contact. Cloning and sequencing of the cDNA for protein 3B11 revealed that this protein is the Manduca counterpart of Drosophila neuroglian and mouse L1. These surface proteins are known to function as adhesion/recognition molecules during development. Manduca neuroglian shares 58 and 31% identity respectively with the Drosophila and mouse proteins and has a cytoplasmic domain of over 100 amino acids.

Diversity of odourant binding proteins revealed by an expressed sequence tag project on male Manduca sexta moth antennae.

A small expressed sequence tag (EST) project generating 506 ESTs from 375 cDNAs was undertaken on the antennae of male Manduca sexta moths in an effort to discover olfactory receptor proteins. We encountered several clones that encode apparent transmembrane proteins; however, none is a clear candidate for an olfactory receptor. Instead we found a greater diversity of odourant binding proteins (OBPs) than previously known in moth antennae, raising the number known for M. sexta from three to seven. Together with evidence of seventeen members of the family from the Drosophila melanogaster genome project, our results suggest that insects may have many tens of OBPs expressed in subsets of the chemosensory sensilla on their antennae. These results support a model for insect olfaction in which OBPs selectively transport and present odourants to transmembrane olfactory receptors. We also found five members of a family of shorter proteins, named sensory appendage proteins (SAPs), that might also be involved in odourant transport. This small EST project also revealed several candidate odourant degrading enzymes including three P450 cytochromes, a glutathione S-transferase and a uridine diphosphate (UDP) glucosyltransferase. Several first insect homologues of proteins known from vertebrates, the nematode Caenorhabditis elegans, yeast and bacteria were encountered, and most have now also been detected by the large D. melanogaster EST project. Only thriteen entirely novel proteins were encountered, some of which are likely to be cuticle proteins.