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.     

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.     

Short-lived, transitory cell-cell interactions foster migration-dependent aggregation

During embryonic development, motile cells aggregate into cohesive groups, which give rise to tissues and organs. The role of cell migration in regulating aggregation is unclear. The current paradigm for aggregation is based on an equilibrium model of differential cell adhesivity to neighboring cells versus the underlying substratum. In many biological contexts, however, dynamics is critical. Here, we provide evidence that multicellular aggregation dynamics involves both local adhesive interactions and transport by cell migration. Using time-lapse video microscopy, we quantified the duration of cell-cell contacts among migrating cells that collided and adhered to another cell. This lifetime of cell-cell interactions exhibited a monotonic decreasing dependence on substratum adhesivity. Parallel quantitative measurements of cell migration speed revealed that across the tested range of adhesive substrata, the mean time needed for cells to migrate and encounter another cell was greater than the mean adhesion lifetime, suggesting that aggregation dynamics may depend on cell motility instead of the local differential adhesivity of cells. Consistent with this hypothesis, aggregate size exhibited a biphasic dependence on substratum adhesivity, matching the trend we observed for cell migration speed. Our findings suggest a new role for cell motility, alongside differential adhesion, in regulating developmental aggregation events and motivate new design principles for tuning aggregation dynamics in tissue engineering applications.     

Marrow-derived stem cell motility in 3D synthetic scaffold is governed by geometry along with adhesivity and stiffness

Design of 3D scaffolds that can facilitate proper survival, proliferation, and differentiation of progenitor cells is a challenge for clinical applications involving large connective tissue defects. Cell migration within such scaffolds is a critical process governing tissue integration. Here, we examine effects of scaffold pore diameter, in concert with matrix stiffness and adhesivity, as independently tunable parameters that govern marrow‐derived stem cell motility. We adopted an “inverse opal” processing technique to create synthetic scaffolds by crosslinking poly(ethylene glycol) at different densities (controlling matrix elastic moduli or stiffness) and small doses of a heterobifunctional monomer (controlling matrix adhesivity) around templating beads of different radii. As pore diameter was varied from 7 to 17 µm (i.e., from significantly smaller than the spherical cell diameter to approximately cell diameter), it displayed a profound effect on migration of these stem cells—including the degree to which motility was sensitive to changes in matrix stiffness and adhesivity. Surprisingly, the highest probability for substantive cell movement through pores was observed for an intermediate pore diameter, rather than the largest pore diameter, which exceeded cell diameter. The relationships between migration speed, displacement, and total path length were found to depend strongly on pore diameter. We attribute this dependence to convolution of pore diameter and void chamber diameter, yielding different geometric environments experienced by the cells within. Bioeng. 2011; 108:1181–1193. © 2010 Wiley Periodicals, Inc.     

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.     

Morphometric analysis of nymphalid butterfly wings: number,size and arrangement of scales,and their implications for tissue‐size determination

Animal body size and tissue size depend on genetic and environmental factors, but the precise mechanisms of how tissue size is determined in proportion to body size remain unknown. Here we focused on wings from three nymphalid butterflies, Junonia orithya (Linnaeus), Vanessa cardui (Linnaeus) and Danaus chrysippus (Linnaeus) (Lepidoptera: Nymphalidae), to examine the contributions of the number and size of scales to macroscopic structures, represented by wing compartments, and to investigate the positional dependence of scale size, density and arrangement. The whole wing area and wing compartment area exhibited a high correlation in all three species. Similarly, the wing compartment area and the blue or orange area showed a high correlation in three species, indicating isometric relationships among wings of different sizes. However, only in J. orithya, the blue area was highly correlated with the number of constituent scales and, to a lesser extent, with scale size. In contrast, reasonable correlations were obtained between the blue or orange area and the number of rows in all three species. These results suggest that variations of the background area accompany changes in the number of scales through changes in the number of rows. In a background region of the compartment, scale size gradually decreased and scale density increased from the proximal to the distal side in all three species. Our findings suggest that butterfly wing tissue size is determined primarily by the number of scale cells and secondarily by the size change of scale cells before or during the period of row arrangement.     

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.     

The mechanism of axon growth: what we have learned from the cell adhesion molecule L1

Cell adhesion molecules (CAMs) are not just an inert glue that mediates static cell-cell and cell-extracellular matrix (ECM) adhesion; instead, their adhesivity is dynamically controlled to enable a cell to migrate through complex environmental situations. Furthermore, cell migration requires distinct levels of CAM adhesivity in various subcellular regions. Recent studies on L1, a CAM in the immunoglobulin superfamily, demonstrate that cell adhesion can be spatially regulated by the polarized internalization and recycling of CAMs. This article examines the molecular mechanism of axon growth, with a particular focus on the role of L1 trafficking in the polarized adhesion and migration of neuronal growth cones.     

The mechanism of axon growth

Cell adhesion molecules (CAMs) are not just an inert glue that mediates static cell-cell and cell-extracellular matrix (ECM) adhesion; instead, their adhesivity is dynamically controlled to enable a cell to migrate through complex environmental situations. Furthermore, cell migration requires distinct levels of CAM adhesivity in various subcellular regions. Recent studies on L1, a CAM in the immunoglobulin superfamily, demonstrate that cell adhesion can be spatially regulated by the polarized internalization and recycling of CAMs. This article examines the molecular mechanism of axon growth, with a particular focus on the role of L1 trafficking in the polarized adhesion and migration of neuronal growth cones.     

Intercellular adhesivity and pupal morphogenesis inDrosophila melanogaster

Summary Leg and wing imaginal discs of mature larvae ofDrosophila melanogaster when treated with 0.1% trypsin for 5–10 min underwent a change in shape that closely resembled normal pupal morphogenesis. Simultaneously, the cells of the disc epithelium changed in shape from tall columnar to cuboidal. Colcemid eliminated microtubules but was without effect on the shape of the imaginal discs or their cells. Tryptic digestion reduced non-junctional intercellular adhesivity but septate desmosomes and gap junctions remained intact.It is proposed that the structure of imaginal discs permits the packaging of the anlagen of the adult integument so that they can change shape and replace the larval structures in a brief period. Apparently most of the definitive form of the pupal leg is built into the disc and becomes visible within a few minutes as intercellular adhesivity is changed.     

Cell shape and epithelial patterning in the Drosophila embryonic epidermis

The development of denticle rows on the ventral Drosophila embryo is a valuable system for studying the genetic control of epithelial patterning. During late embryogenesis, the apical surfaces of denticle-producing cells acquire a distinctive rectangular morphology with long anteroposterior boundaries, along which the denticles form, and short ventrolateral boundaries that stain strongly for adherens junction proteins. We observe that ventrolateral denticle cell boundaries are also convoluted, suggesting that the strong adherens staining results, at least in part, from the additional membrane in these regions. Embryos mutant for the Planar Cell Polarity (PCP) Effector gene multiple wing hairs (mwh), or expressing dominant negative form of the small GTPase Rac1, have cells present between the normal denticle cell rows. These 'Interloper Cells' do not have convoluted ventrolateral boundaries with strong adherens protein staining, but have normal denticle placement, suggesting that adherens protein localization is not critical for denticle cell PCP. Based on these and other observations, we propose that denticle cell morphology arises from an epithelial stretch without junction remodeling. A crude mechanical model suggests that this mechanism can generate both the straight anteroposterior boundaries and the compacted ventrolateral boundaries typical of denticle cells. We discuss the significance of cell adhesion for denticle cell morphogenesis, especially given the established role for Rac1 in cell adhesion.     

Tissue-specific laminin expression facilitates integrin-dependent association of the embryonic wing disc with the trachea in Drosophila

The interaction of heterologous tissues involves cell adhesion mediated by the extracellular matrix and its receptor integrins. The Drosophila wing disc is an ectodermal invagination that contacts specific tracheal branches at the basolateral cell surface. We show that an alpha subunit of laminin, encoded by wing blister (wb), is essential for the establishment of the interaction between the wing and trachea. During embryogenesis, wing disc cells present Wb at their basolateral surface and extend posteriorly, expanding their association to more posteriorly located tracheal branches. These migratory processes are impaired in the absence of the trachea, Wb, or integrins. Time-lapse and transmission electron microscopy analyses suggest that Wb facilitates integrin-dependent contact over a large surface and controls the cellular behavior of the wing cells, including their exploratory filopodial activity. Our data identify Wb laminin as an extracellular matrix ligand that is essential for integrin-dependent cellular migration in Drosophila.     

Nudel and FAK as Antagonizing Strength Modulators of Nascent Adhesions through Paxillin

Adhesion and detachment are coordinated critical steps during cell migration. Conceptually, efficient migration requires both effective stabilization of membrane protrusions at the leading edge via nascent adhesions and their successful persistence during retraction of the trailing side via disruption of focal adhesions. As nascent adhesions are much smaller in size than focal adhesions, they are expected to exhibit a stronger adhesivity in order to achieve the coordination between cell front and back. Here, we show that Nudel knockdown by interference RNA (RNAi) resulted in cell edge shrinkage due to poor adhesions of membrane protrusions. Nudel bound to paxillin, a scaffold protein of focal contacts, and colocalized with it in areas of active membrane protrusions, presumably at nascent adhesions. The Nudel-paxillin interaction was disrupted by focal adhesion kinase (FAK) in a paxillin-binding–dependent manner. Forced localization of Nudel in all focal contacts by fusing it to paxillin markedly strengthened their adhesivity, whereas overexpression of structurally activated FAK or any paxillin-binding FAK mutant lacking the N-terminal autoinhibitory domain caused cell edge shrinkage. These results suggest a novel mechanism for selective reinforcement of nascent adhesions via interplays of Nudel and FAK with paxillin to facilitate cell migration.     

Influence of increased concentration of Ca2+, La3+, PVP and urea on primary adhesivity of embryonic brain cells. Ultrastructural pattern of adhering cells

With the help of a previously described experimental arrangement the influence of increased external concentration of Ca2+, La3+, PVP and urea was tested on the initial stages of brain cell adhesivity and its kinetics. Urea, an inhibitor of hydrogen bonds, significantly inhibited the adhesivity of the treated cells. PVP significantly increased cell adhesivity. The adhesivity was enhanced and speeded up by increased concentrations of Ca2+ and La3+. It is evident that the membrane surface potential, zeta potential and formation of H+ bonds and bridges are highly important for cellular adhesivity. EM control of freshly dissociated cells disclosed that a part of the cells had been damaged. According to the ultrastructural organisation, the surface membrane is damaged to a small or greater extent. Intercellular contacts were formed in vitro either between non damaged surfaces of membranes, or between fragments of membranes, or contacts were mediated by membrane debris. Because cellular debris disappeared during rotation from single adhesive complexes, it is probable, that disrupted membranes are used for restoration of membranes, or serve as a metabolic substrates, or are catabolized.     

JUNCTIONAL PORES AND CELL WALL DEPRESSIONS OF HORMOSCILLA PRINGSHEIMII (CYANOPHYCEAE)1

Sassoon's isolate identified as Borzia trilocularis (but recently renamed Hormoscilla pringsheimii Anagnostidis and Komárek (1988) was studied with transmission electron microscopy because of its unusual combination of longitudinal wall features, described here in development for the first time. Junctional pores (linear rows of circumferential L-II layer pores near crosswalls) developed into multiple, parallel series, unlike the pores in many other species, which form only single rows. In dividing cells, two single pore rows appear opposite crosswalls after crosswall initiation, but an additional parallel row is usually added to each row by completion of fission. Elongating cells reveal 3–6 parallel and uniformly spaced pore rows developing on each side of crosswall pairs; these rows may end up toward the center of the cell wall after cell elongation. Pores are 18–24 nm wide with a center-to-center and row-to-row distance of 24–36 nm and occur in an especially thick L-II area. The second group of pit-like pores of longitudinal cell walls are 50–135 nm-wide depressions and have a center-to-center distance of 100–1000 nm. These depressions arise when the L-II layer fails to form and appear next to a row-pair of junctional pores soon after fission. Most depressions form single rows, but when they form several rows they may cover much of the surface of the cell. The L-III and L-IV wall layers line these L-II layer cavities; the outer surface of the L-IV layer around and within the depressions is covered with fibrous mucilage. Given their diversity, pores and depressions of longitudinal walls deserve further attention from functional and taxonomic points of view.     

A model for the kinetics of homotypic cellular aggregation under static conditions.

We present the formulation and testing of a mathematical model for the kinetics of homotypic cellular aggregation. The model considers cellular aggregation under no-flow conditions as a two-step process. Individual cells and cell aggregates 1) move on the tissue culture surface and 2) collide with other cells (or aggregates). These collisions lead to the formation of intercellular bonds. The aggregation kinetics are described by a system of coupled, nonlinear ordinary differential equations, and the collision frequency kernel is derived by extending Smoluchowski's colloidal flocculation theory to cell migration and aggregation on a two-dimensional surface. Our results indicate that aggregation rates strongly depend upon the motility of cells and cell aggregates, the frequency of cell-cell collisions, and the strength of intercellular bonds. Model predictions agree well with data from homotypic lymphocyte aggregation experiments using Jurkat cells activated by 33B6, an antibody to the beta 1 integrin. Since cell migration speeds and all the other model parameters can be independently measured, the aggregation model provides a quantitative methodology by which we can accurately evaluate the adhesivity and aggregation behavior of cells.     

Transformation-induced alterations in fibroblast adhesion: masking by trypsin treatment.

As assessed by initial rates of cell aggregation, adhesivity of EDTA-suspended cells is increased by SV 40 transformation of mouse 3T3 and human WI 38 fibroblasts. Preparation of cell suspensions by trypsin treatment decreases the aggregation rate and abolishes the difference between transformed and untransformed cells. These findings suggest that increased cellular adhesivity accompanies the surface alterations induced by viral transformation and that trypsinization masks these changes. Thus, use of freshly trypsinized cells in quantitative assessments of cellular adhesivity may yield results which do not reflect the actual cell properties.     

Changes in cell adhesivity and cytoskeleton‐related proteins during imatinib‐induced apoptosis of leukemic JURL‐MK1 cells

The fusion protein Bcr–Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr–Abl protein used in front‐line CML therapy, on the adhesivity of JURL‐MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL‐MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr–Abl inhibition and secondary changes related to the apoptosis. Imatinib treatment caused a transient increase in JURL‐MK1 cell adhesivity to fibronectin, possibly due to the switch off of Bcr–Abl activity. Subsequently, we observed a number of changes including a decrease in cell adhesivity, F‐actin decomposition, reduction of integrin β1, CD44, and paxillin expression levels and a marked increase in cofilin phophorylation at Ser3. These events were generally related to the proceeding apoptosis but they differed in their sensitivity to the individual caspase inhibitors. J. Cell. Biochem. 111: 1413–1425, 2010. © 2010 Wiley‐Liss, Inc.