Apical Folds Research Articles

Phylogenomics and deep convergence in cockroach hind-wing morphology

Despite regular advances in Blattodea systematics, several relationships remain controversial or untested in formal phylogenetic reconstructions. This common situation for understudied metazoan groups limits our power to answer questions about phenotypic evolution. In this study, we infer the evolutionary history of Blattodea using newly sampled taxa that improve phylogenetic resolution while also illuminating the evolutionary history of an unusual phenotype—the apically folded hind-wing. Taxa newly sequenced include those with a hind-wing apical fold (Anaplecta pulchella, A. pygmaea, A. sp. cf. malaysensis, Diplopterina parva, Prosoplecta semperi, Anaplectoidea klossi, and Oulopteryx illuminata sp. nov. that we describe herein, including its male genitalia) and other rare taxa (Dipteretrum hamstroemi, Duchailluia togoensis, Lauraesilpha mearetoi, Buboblatta vlasaki). The phylogenetic design utilizes 41 genes over 91 species in total, analyzed in a maximum likelihood and coalescent framework. To quantify the phylogenetic uncertainty of the analysis, support for various topologies is assessed. We find unambiguous support for the surprising position of Neotropical Oulopteryx (Oulopterygidae) as sister to New Caledonian/Australian Tryonicidae. This, and other phylogenetic findings, reveal that the apically folded hind-wing may have arisen nine times in Blattodea. Further investigations are needed, notably with an increased taxonomic sampling, to demonstrate stronger support for the placement of rogue taxa (e.g., Anaplecta) and to investigate the evolutionary correlates of wing evolution.

Laminin-111 mutant studies reveal a hierarchy within laminin-111 genes in their requirement for basal epithelial tissue folding

The process of morphogenesis carefully crafts cells into complex organ structures which allows them to perform their unique functions. During brain development, the neuroepithelial tissue must go through apical and basal folding which is mediated through the instruction of both intrinsic and extrinsic factors. While much is known about apical folding, the mechanisms that regulate basal folding are less understood. Using the highly conserved zebrafish midbrain-hindbrain boundary (MHB) as an epithelial tissue model, we have identified the basement membrane protein laminin-111 as a key extrinsic factor in basal tissue folding. Laminin-111 is a highly conserved, heterotrimeric protein that lines the basal surface of the neuroepithelium. Laminin-111 is comprised of one alpha, one beta, and one gamma chain encoded by the genes lama1, lamb1, and lamc1, respectively. Human mutations in individual laminin-111 genes result in disparate disease phenotypes; therefore, we hypothesized that each laminin gene would have a distinctive role in tissue morphogenesis. Using zebrafish mutants for laminin-111 genes, we found that each laminin chain has a unique impact on basal folding. We found that lamc1 is the most critical gene for MHB morphogenesis, followed by lama1, and finally lamb1a. This hierarchy was discovered via three-dimensional single cell shape analysis, localization of myosin regulatory light chain (MRLC), and with analysis of MHB tissue folding later in development. These findings are essential for development of novel techniques in tissue engineering and to elucidate differences in human diseases due to specific chain mutations.

Old method not old-fashioned: parallelism between wing venation and wing-pad tracheation of cockroaches and a revision of terminology

The Comstock–Needham system, the foundation of main vein homology of insects, is grounded in the comparison between wing venation and wing-pad tracheation, particularly that of Plecoptera and Blattodea. However, the trachea-based approach has come into great dispute in recent years. Meanwhile, the terminology used to describe the wing venation of cockroaches requires a revision according to homology and the practical need. These motivated us to examine the wings of cockroaches, comparing the tracheation of nymphs with the tracheation and venation of adults. We find a correspondence between the tracheation and the main vein venation; therefore the trachea-based approach is effective in cockroaches. It is supported that Neopteran insects have six homologous main veins, namely, Sc, R, M, Cu, Pcu and V. We interpret the wing morphology of cockroaches in detail, including veins, furrows, folds and apical folding. RA and RP in hindwing, ScP, CuA and CuP in tegmen and hindwing, are recognized. Hindwing R + M is the only normal venal fusion in cockroaches. In addition, we propose several descriptive terms: cmv, V[1], V[s], pseudostem and characteristic posterior branch. The venation of 41 species (representing 41 genera), and the wing-pad tracheation of four of them including Diploptera punctata, which has unique apical folding, are illustrated.

Fold formation at the compartment boundary of Drosophila wing requires Yki signaling to suppress JNK dependent apoptosis.

Compartment boundaries prevent cell populations of different lineage from intermingling. In many cases, compartment boundaries are associated with morphological folds. However, in the Drosophila wing imaginal disc, fold formation at the anterior/posterior (A/P) compartment boundary is suppressed, probably as a prerequisite for the formation of a flat wing surface. Fold suppression depends on optomotor-blind (omb). Omb mutant animals develop a deep apical fold at the A/P boundary of the larval wing disc and an A/P cleft in the adult wing. A/P fold formation is controlled by different signaling pathways. Jun N-terminal kinase (JNK) and Yorkie (Yki) signaling are activated in cells along the fold and are necessary for the A/P fold to develop. While JNK promotes cell shape changes and cell death, Yki target genes are required to antagonize apoptosis, explaining why both pathways need to be active for the formation of a stable fold.

A somitic contribution to the apical ectodermal ridge is essential for fin formation.

The transition from fins to limbs was an important terrestrial adaptation, but how this crucial evolutionary shift arose developmentally is unknown. Current models focus on the distinct roles of the apical ectodermal ridge (AER) and the signaling molecules that it secretes during limb and fin outgrowth. In contrast to the limb AER, the AER of the fin rapidly transitions into the apical fold and in the process shuts off AER-derived signals that stimulate proliferation of the precursors of the appendicular skeleton. The differing fates of the AER during fish and tetrapod development have led to the speculation that fin-fold formation was one of the evolutionary hurdles to the AER-dependent expansion of the fin mesenchyme required to generate the increased appendicular structure evident within limbs. Consequently, a heterochronic shift in the AER-to-apical-fold transition has been postulated to be crucial for limb evolution. The ability to test this model has been hampered by a lack of understanding of the mechanisms controlling apical fold induction. Here we show that invasion by cells of a newly identified somite-derived lineage into the AER in zebrafish regulates apical fold induction. Ablation of these cells inhibits apical fold formation, prolongs AER activity and increases the amount of fin bud mesenchyme, suggesting that these cells could provide the timing mechanism proposed in Thorogood's clock model of the fin-to-limb transition. We further demonstrate that apical-fold inducing cells are progressively lost during gnathostome evolution;the absence of such cells within the tetrapod limb suggests that their loss may have been a necessary prelude to the attainment of limb-like structures in Devonian sarcopterygian fish.

A New Genus of Odontopygid Millipeds from Tanzania (Diplopoda: Spirostreptida: Odontopygidae)

The new generic taxon Calyptomastix is proposed to accommodate the type species Odontopyge kakandae Kraus, 1958, and, tentatively, Odontopyge dorsalis Carl, 1909, Haplothysanus leviceps Attems, 1909, and Spirostreptus pardalis Gerstäcker, 1873, all from Tanzania. This genus is defined by the broad basal separation of the male genitalia, the elongated basal whorl (torsion) of the gonopod telopodite, and the concealment of the solenomere within apical folds of the telopodite.

Mechanism of pectoral fin outgrowth in zebrafish development

Fins and limbs, which are considered to be homologous paired vertebrate appendages, have obvious morphological differences that arise during development. One major difference in their development is that the AER (apical ectodermal ridge), which organizes fin/limb development, transitions into a different, elongated organizing structure in the fin bud, the AF (apical fold). Although the role of AER in limb development has been clarified in many studies, little is known about the role of AF in fin development. Here, we investigated AF-driven morphogenesis in the pectoral fin of zebrafish. After the AER-AF transition at ∼36 hours post-fertilization, the AF was identifiable distal to the circumferential blood vessel of the fin bud. Moreover, the AF was divisible into two regions: the proximal AF (pAF) and the distal AF (dAF). Removing the AF caused the AER and a new AF to re-form. Interestingly, repeatedly removing the AF led to excessive elongation of the fin mesenchyme, suggesting that prolonged exposure to AER signals results in elongation of mesenchyme region for endoskeleton. Removal of the dAF affected outgrowth of the pAF region, suggesting that dAF signals act on the pAF. We also found that the elongation of the AF was caused by morphological changes in ectodermal cells. Our results suggest that the timing of the AER-AF transition mediates the differences between fins and limbs, and that the acquisition of a mechanism to maintain the AER was a crucial evolutionary step in the development of tetrapod limbs.

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

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

09-P097 Apical fold morphogenesis in zebrafish fin; differences from tetrapod limb development

09-P097 Apical fold morphogenesis in zebrafish fin; differences from tetrapod limb development

Optomotor-blind suppresses instability at the A/P compartment boundary of the Drosophila wing

Formation and function of the A/P compartment boundary of the Drosophila wing have been studied intensely. The boundary prevents mingling of A and P cells, is characterized by an expression discontinuity of several genes like engrailed, Cubitus interruptus, hedgehog and decapentaplegic and is essential for patterning the wing. Compared with segmental or compartmental boundaries in several other systems which generally manifest as folds or clefts, the wing A/P boundary is morphologically inconspicuous in both the larval and adult stage. We show here that the Drosophila wing A/P boundary, too, is susceptible to fold and cleft formation and that these processes are suppressed by the T-box transcription factor Optomotor-blind (Omb). Using a targeted deletion encompassing the omb wing enhancer [Sivasankaran, R., Vigano, M.A., Muller, B., Affolter, M., Basler, K., 2000. Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker. EMBO J. 19, 6162–6172] and omb-RNAi to strongly and specifically reduce Omb, we show that omb is required in P cells to prevent aberrant apical fold formation at the A/P boundary of the larval wing disc and cleft formation in the adult wing blade. In the larval A/P fold, DE-cadherin-based adherens junctions appeared intact but the apical microtubule web was strongly reduced.

Laminin α5 is essential for the formation of the zebrafish fins

The vertebrate fin fold, the presumptive evolutionary antecedent of the paired fins, consists of two layers of epidermal cells extending dorsally and ventrally over the trunk and tail of the embryo, facilitating swimming during the embryonic and larval stages. Development of the fin fold requires dramatic changes in cell shape and adhesion during early development, but the proteins involved in this process are completely unknown. In a screen of mutants defective in fin fold morphogenesis, we identified a mutant with a severe fin fold defect, which also displays malformed pectoral fins. We find that the cause of the defect is a non-sense mutation in the zebrafish lama5 gene that truncates laminin α5 before the C-terminal laminin LG domains, thereby preventing laminin α5 from interacting with its cell surface receptors. Laminin is mislocalized in this mutant, as are the membrane-associated proteins, actin and β-catenin, that normally form foci within the fin fold. Ultrastructural analysis revealed severe morphological abnormalities and defects in cell–cell adhesion within the epidermis of the developing fin fold at 36 hpf, resulting in an epidermal sheet that can not extend away from the body. Examining the pectoral fins, we find that the lama5 mutant is the first zebrafish mutant identified in which the pectoral fins fail to make the transition from an apical epidermal ridge to an apical fold, a transformation that is essential for pectoral fin morphogenesis. We propose that laminin α5, which is concentrated at the distal ends of the fins, organizes the distal cells of the fin fold and pectoral fins in order to promote the morphogenesis of the epidermis. The lama5 mutant provides novel insight into the role of laminins in the zebrafish epidermis, and the molecular mechanisms driving fin formation in vertebrates.

Dackel acts in the ectoderm of the zebrafish pectoral fin bud to maintain AER signaling.

Classical embryological studies have implied the existence of an apical ectodermal maintenance factor (AEMF) that sustains signaling from the apical ectodermal ridge (AER) during vertebrate limb development. Recent evidence suggests that AEMF activity is composed of different signals involving both a sonic hedgehog (Shh) signal and a fibroblast growth factor 10 (Fgf10) signal from the mesenchyme. In this study we show that the product of the dackel (dak) gene is one of the components that acts in the epidermis of the zebrafish pectoral fin bud to maintain signaling from the apical fold, which is homologous to the AER of tetrapods. dak acts synergistically with Shh to induce fgf4 and fgf8 expression but independently of Shh in promoting apical fold morphogenesis. The failure of dak mutant fin buds to progress from the initial fin induction phase to the autonomous outgrowth phase causes loss of both AER and Shh activity, and subsequently results in a proximodistal truncation of the fin, similar to the result obtained by ridge ablation experiments in the chicken. Further analysis of the dak mutant phenotype indicates that the activity of the transcription factor engrailed 1 (En1) in the ventral non-ridge ectoderm also depends on a maintenance signal probably provided by the ridge. This result uncovers a new interaction between the AER and the dorsoventral organizer in the zebrafish pectoral fin bud.

Na+/K+-ATPase activity and immunocytochemical labeling in podobranchial filament and lamina of the freshwater crayfish Astacus leptodactylus Eschscholtz: evidence for the existence of sodium transport in the filaments

In the crayfish Astacus leptodactylus, the podobranch, bearing respiratory and ion-transporting filaments, is attached to the lamina. The Na+/K+-ATPase activity is higher in filaments than in lamina. Using a mouse monoclonal antibody directed against the α subunit of Na+/K+-ATPase, and indirect immunofluorescence microscopy, we can observe that the immunoreactivity of the antibody is different for each kind of structure. The lamina presents an ion-transporting type epithelium, which is thick and presents a developed apical infolding system but no developed basolateral infolding system. The immunoreactivity of the antibody in the lamina is very weak. Respiratory filaments present a thin epithelium with a few small apical folds and scarce mitochondria. The immunoreactivity of the antibody in the respiratory filaments is very weak. In contrast, the ion-transporting filament epithelium is thick, presents a short apical infolding system, a well-developed basal infolding system, and numerous mitochondria. The immunoreactivity of the antibody in the ion-transporting filaments is strong. Ion-transporting filaments, which have a cuticle permeable to cations, seem particularly implicated in Na+regulation, by way of the sodium pump.

Ultrastructural study of the proboscis endothelium of Riseriellus occultus (Nemertea, Heteronemertea)

We have examined with transmission electron microscopythe epithelial layer exposed to the rhynchocoel fluidof the proboscis in the heteronemertine Riseriellus occultus. This epithelium is organized asa monociliated, pseudostratified myoepitheliumconsisting of two cell types: apically situatedmonociliated supportive cells and subapical myocyteslacking cilia. The low supportive cells form acontinuous adluminal sheet and reach with numerouscytoplasmic processes into the extracellular matrix;these cells are characterized by numerous, irregularlyshaped, apical folds projecting into the rhynchocoelfluid, delimiting broad extracellular spaces. Theauthors suppose that both apical and basal folds couldaccommodate stretching of the endothelium when theproboscis is everted. The apical folds of thesupportive cells increase the interface of these withthe rhynchocoel fluid; this feature, together with thepresence of pinocytotic vesicles in such cells,suggest that they could be involved in the exchange ofsubstances between the rhynchocoel fluid and theproboscis. The myocytes are scattered singly withinthe monociliated pseudostratified myoepithelium. Theyare situated between the supportive cells and thesubjacent extracellular matrix. Basement membraneseparating both cells types is lacking. Myofibrillarparts protrude basally from the myocyte somata. Themyofibrillar parts lie in direct apposition to theextracellular matrix, and are oriented circular to thelongitudinal axis of the proboscis. We consider themyocytes to be intra-epithelial, myoepithelial cells.

EVOLUTION OF THE HIND WING IN COLEOPTERA

AbstractA survey is made of the major features of the venation, articulation, and folding in the hind wings of Coleoptera. The documentation is based upon examination of 108 Coleoptera families and 200 specimens, and shown in 101 published figures. Wing veins and articular sclerites are homologized with elements of the neopteran wing groundplan, resulting in wing vein terminology that differs substantially from that generally used by coleopterists. We tabulate the differences between currently used venational nomenclature and the all-pterygote homologous symbols. The use of the neopteran groundplan, combined with the knowledge of the way in which veins evolved, provides many strong characters linked to the early evolutionary radiation of Coleoptera. The order originated with the development of the apical folding of the hind wings under the elytra executed by the radial and medial loop. The loops, which are very complex venational structures, further diversified in four distinctly different ways which mark the highest (suborder) taxa. The remaining venation and the wing articulation have changed with the loops, which formed additional synapomorphies and autapomorphies at the suborder, superfamily, and sometimes even family and tribe levels. Relationships among the four currently recognized suborders of Coleoptera are reexamined using hind wing characters. The number of wing-related apomorphies are 16 in Coleoptera, seven in Archostemata + Adephaga–Myxophaga, four in Adephaga–Myxophaga, seven in Myxophaga, nine in Archostemata, and five in Polyphaga. The following phylogenetic scheme is suggested: Polyphaga [Archostemata (Adephaga + Myxophaga)]. Venational evidence is given to define two major lineages (the hydrophiloid and the eucinetoid) within the suborder Polyphaga. The unique apical wing folding mechanism of beetles is described. Derived types of wing folding are discussed, based mainly on a survey of recent literature. A sister group relationship between Coleoptera and Strepsiptera is supported by hind wing evidence.

Absorption of phosphorus from the spermatophore through the cuticle of the bursa copulatrix of the butterfly, Calpodes ethlius

In lepidoptera the spermatophore is deposited in a bursa copulatrix, which is lined by a cuticle through which solubilized components of the spermatophore must be absorbed by the underlying epithelium. This study follows the route taken by the phosphorus which, with calcium, forms a significant portion of the spermatophore of Calpodes ethlius in the form of homogeneous, amorphous concretions. Lead nitrate was used in the primary fixative to precipitate the phosphorus in order to follow the pathway taken by it through the cuticle and bursal epithelium. The epicuticle-free pits, which cover the bursal cuticle, are identified as the points of entry, and the epicuticular filaments as major pathways for transport of phosphorus and presumably soluble molecules. Lead phosphate deposits also occurred in the epithelium extracellularly between the apical microvilli, lateral membranes and basal folds, in calcium granules and in large compound structures which appear to be made up, at least in part, of calcium granules. The confinement of the phosphorus to extracellular compartments of the epithelium suggests that its function is to be found elsewhere.

Ultrastructural defects in the apical neural folds in mutant embryos with spina bifida.

Ultrastructural pathology in the apical neural folds was analyzed by means of tannic acid (TA) and ruthenium red (RR) cytochemistry in abnormal (vl/vl) mutant mouse embryos ranging in age from 17-35 somites. At lumbosacral levels of the spinal cord where closure fails to occur, as well as at more cranial levels where closure occurs but results in dorsal midline abnormalities, normal deposition of TA-positive and RR-positive material occurred in the space that develops between the overlying surface ectoderm (SE) and neuroepithelium (NE). However, in lumbosacral regions, pleomorphic excrescences projected abnormally from the apices of the transitional zone cells between SE and NE cells of the open neural folds. These abnormal projections consisted of enlarged cytoplasmic blebs, as well as entire cells. The cells were not necrotic nor did they show evidence of incipient degeneration. However, it is possible that they represent aberrant putative neural crest cells, as indicated by their location in the transitional zone and by the filopodia and lamellipodia projecting from their luminal surfaces.

Studies on the mechanisms of neurulation in the chick: morphometric analysis of force distribution within the neuroepithelium during neural tube formation.

Changes in the shape of neuroepithelial cells, particularly apical constriction, are generally thought to play a major role in generating the driving forces for neural tube formation. Our previous study [Nagele and Lee (1987) J. Exp. Zool., 241:197-205] has shown that, in the developing midbrain region of stage 8+ chick embryos, neuroepithelial cells showing the greatest degree of apical constriction are concentrated at sites of enhanced bending of the neuroepithelium (i.e., the floor and midlateral walls of neural tube), suggesting that driving forces resulting from apical constriction are concentrated at these sites during closure of the neural tube. In the present study, we have used morphometric methods to 1) measure regional variations in the degree of apical constriction and apical surface folding at selected regions along the anteroposterior axis of stage 8+ chick embryos, which closely resemble the various ontogenetic phases of neural tube formation, and 2) investigate how forces resulting from apical constriction are distributed within the neuroepithelium during transformation of the neural plate into a neural tube. Results show that, during neural tube formation, driving forces resulting from apical constriction are not distributed uniformly throughout the neuroepithelium but rather are concentrated sequentially at three distinct locations: 1) the floor (during transformation of the neural plate to a V-shaped neuroepithelium), 2) the midlateral walls (during transformation of the V-shaped neuroepithelium into a C-shaped neuroepithelium), and 3) the upper walls (during the transformation of the C-shaped neuroepithelium into a closed neural tube).

Ultrastructure of osmoregulatory organs in larvae of the brackish-water mosquito, Culiseta inornata (Williston).

The ultrastructure of the Malpighian tubules, ileum, rectum, anal canal, and anal papillae of larvae of the mosquito Culiseta inornata was examined. The Malpighian tubules, rectum, and anal papillae have many of the ultrastructural features characteristic of ion transport tissues, i.e., elaboration of the basal and apical membranes and a close association of these membranes with mitochondria. The Malpighian tubules possess two cell types, primary and stellate. The larval rectum of C. inornata is composed of a single segment containing a homogenous population of cells. In this respect, the larval rectum of C. inornata is distinct from that of saline-water species of Aedes. The cells in the larval rectum of C. inornata, however, closely resemble those of one cell type, the anterior rectal cells, of the saline-water mosquito Aedes campestris with regard to cell and nuclear size, the percentage of the cell occupied by apical folds, and mitochondrial density and distribution. No similarities can be found between the rectum of C. inornata and the posterior segment of the saline-water Aedes, which functions as a salt gland. On this basis, we have postulated that the rectum of C. inornata does not function as a site of hyperosmotic fluid secretion. The ultrastructure of the anal papillae of C. inornata is consistent with a role in ion transport. The significance of these findings to comparative aspects of osmoregulatory strategies in mosquito larvae is discussed.

An analysis of in vivo cell migration during teleost fin morphogenesis.

In the teleost embryo the pectoral fin bud initially displays an apical ectodermal ridge along its entire distal margin. The ridge subsequently becomes transformed into an apical fold as the distal ectodermal epithelium grows and folds to enclose an extracellular space between the apposed basal surfaces of the epithelium. Collagen fibrils up to 2 micron in diameter, termed 'actinotrichia', are deposited along the proximo-distal axis in two (dorsal and ventral) arrays. The actinotrichia are aligned parallel to one another with a regular spacing along the greater part of their length. Mesenchymal cells migrating distally from the base of the fin bud encounter the dorsal and ventral arrays of actinotrichia and move between them apparently using the fibrils as a substratum. The entire structure is transparent and, using the killifish Aphyosemion scheeli, we have investigated the migration of the mesenchymal cells between 135 and 220 h of development, using Nomarski interference contrast microscopy and time-lapse video recording. The number of cellular processes per cell increased significantly during the period of observation. These processes could be graded according to their diameters. Processes of diameter greater than 2 micron were not usually aligned along actinotrichia and arose at any aspect of the cell body. In contrast, processes with diameters less than 2 micron appeared to be confined to the distal aspects of the migrating cells and showed an increasing tendency to become aligned as development progressed. Time-lapse video recordings revealed that such aligned processes move faster (mean speed 17.98 (+/- 2.25) micron/h) than non-aligned processes (mean speed 4.66 (+/- 0.67) micron/h). Whole cell translocation was generally slower than rates of process movement: the lowest mean value (1.52(+/- 0.36) micron/h) was recorded between 135 and 160 h of development rising to a maximum mean rate (4.72(+/- 0.42) micron/h) between 195 and 220 h; the period of the fastest rate of cell translocation correlated with maximum process alignment along actinotrichia. Thin 1 micron plastic sections revealed that, generally, aligned processes were in close association with the surface of the actinotrichial fibrils and not the spaces between them.