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.     

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

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

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

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

Transformations of epithelial monolayers during wing development of Manduca sexta

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

Mosaic Drosophila wings reveal regional heterogeneity in the guidance of ectopic axons

In most studies of axon guidance in the peripheral tissues of insects, the ability of experimentally perturbed axons to pathfind was examined only along their normal pathways. This means that regions normally devoid of axons have not been sampled for their ability to influence axonal trajectories. To examine this question, we have induced the formation of single sensory neurons in a variety of abnormal locations in the developing wing of Drosophila and have examined the course taken by their axons. The axons of such ectopic neurons have a regionally varying tendency to grow in the normal, proximal direction. This proximal bias approaches 100% for neurons located in the distal part of vein L2 and 70% in distal vein L4 but falls to chance (50%) along vein L5. Thus, neurons forming in ectopic regions of the wing, especially those found near the normal axon pathways (veins L1 and L3), have a high probability of growing axons in the correct direction. We conclude that information relevant to axon outgrowth is not restricted to the normal pathways. Whether this information is intrinsic or extrinsic to the neurons, and why its strength shows such conspicuous regional variation, awaits further study.     

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.     

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.     

Progress in outgrowth culture from rabbit tracheal explants: Balance between proliferation and maintenance of differentiated state in epithelial cells

Summary Primary cultures of rabbit tracheal cells were obtained as outgrowths from explants of tracheal mucosa. A 30% collagen substratum containing serum and minimal essential medium was required for obtaining an outgrowth of epithelial cells keeping their differentiated characteristics. The tracheal epithelial cells obtained near the explant in the first days of culture presented morphologic similarities with normal tracheal epithelium. Cultures contained basal cells and epithelial polarized cells that exhibited apical tight junctions and desmosomes. Ciliated cells stayed functional during all time culture. Their number slightly increased at the beginning of the culture and then stayed constant when the total number of cells increased. Development of the outgrowth was rapid and significant inasmuch as the outgrowth surface reached 30 times that of the explant after less than 8 days. This was linked to cellular proliferation, as demonstrated by the incorporation of bromodeoxyuridine (BrdU) in phase-S nuclei and the revelation of BrdU using an immunofluorescence technique. The epithelial nature of the outgrowth cells and the absence of contamination with fibroblasts were established by positive staining with anti-keratin antibody and by negative staining with anti-vimentin antibody, respectively. This work was supported by DRET and by CIFRE grant awarded to S. R.     

Axon guidance in cultured wing discs and disc fragments of Drosophila

The sensory neurons of the Drosophila wing differentiate during the initial stages of metamorphosis, appearing in the imaginal wing disc as it everts and flattens. These identifiable neurons arise in a stereotyped sequence, and lay down a specific pattern of axon bundles which travel proximally to the CNS. In several locations, the early arising "pioneer" neurons send axons in the direction of more proximal pioneer neurons, later joining with these to form continuous peripheral nerves. It is possible that distal neurons can contact more proximal neurons by random filopodial search, and use this information to guide axonal outgrowth. To test this "guidepost" hypothesis, everting wing discs were raised in vitro to allow surgical manipulation. Neural outgrowth was largely normal in vitro, though growth of the wing was stunted. If such discs were cut into proximodistal fragments before or at the time of initial axonogenesis, neural outgrowth remained normal: distal axons still grew in the direction of the now missing proximal neurons. Thus, proximal neurons are not necessary for the correct guidance of distal neurons in the developing wing.     

The Drosophila ribbon gene encodes a nuclear BTB domain protein that promotes epithelial migration and morphogenesis.

During development of the Drosophila tracheal (respiratory) system, the cell bodies and apical and basal surfaces of the tracheal epithelium normally move in concert as new branches bud and grow out to form tubes. We show that mutations in the Drosophila ribbon (rib) gene disrupt this coupling: the basal surface continues to extend towards its normal targets, but movement and morphogenesis of the tracheal cell bodies and apical surface is severely impaired, resulting in long basal membrane protrusions but little net movement or branch formation. rib mutant tracheal cells are still responsive to the Branchless fibroblast growth factor (FGF) that guides branch outgrowth, and they express apical membrane markers normally. This suggests that the defect lies either in transmission of the FGF signal from the basal surface to the rest of the cell or in the apical cell migration and tubulogenesis machinery. rib encodes a nuclear protein with a BTB/POZ domain and Pipsqueak DNA-binding motif. It is expressed in the developing tracheal system and other morphogenetically active epithelia, many of which are also affected in rib mutants. We propose that Rib is a key regulator of epithelial morphogenesis that promotes migration and morphogenesis of the tracheal cell bodies and apical surface and other morphogenetic movements.     

Proximal tissues and patterned neurite outgrowth at the lumbosacral level of the chick embryo: partial and complete deletion of the somite

The development of patterned axon outgrowth and dorsal root ganglion (DRG) formation was examined after partially or totally removing chick somitic mesoderm. Since the dermamyotome is not essential and a full complement of limb muscles developed, alterations in neural patterns could be ascribed to deletion of sclerotome. When somitic tissue was completely removed, axons extended and DRG formed, but in an unsegmented pattern. Therefore the somite does not elicit outgrowth of axons or migration of DRG precursors, it is not a manditory substratum and it is not required for DRG condensation. These results suggest that posterior sclerotome is relatively inhibitory to invasion, an inhibition that is released when sclerotome is absent. When somites were partially deleted, axonal segmentation was not lost proportionally with the amount of sclerotome removed, suggesting that properties that may vary with sclerotome volume (such as diffusible cues) do not play a primary role. Instead, spinal nerves lost segmentation only when ventral sclerotome was deleted, regardless of whether dorsal sclerotome was or was not removed. This strongly suggests that axonal segmentation is imposed by direct interactions between growth cones and extracellular matrices or surfaces sclerotome cells. While DRG tended to be normally segmented when ventral sclerotome was deleted and to lose segmentation when dorsomedial sclerotome was absent, a coordinate loss of DRG segmentation with sclerotome volume could not be ruled out. However it is clear that axonal and DRG segmentation are independent. Observations on a subset of embryos in which the notochord was displaced relative to the spinal cord suggest that the ventromedial sclerotome surrounding the notochord inhibits axon advance. Posterior and ventromedial sclerotome are hypothesized to act as barriers to axon outgrowth due to some feature of their common cartilaginous development. Specific innervation patterns were also examined. When the notochord was displaced toward the control limb, axons on this side made and corrected projection errors, suggesting that the notochord can influence the precision of axonal pathway selection. In contrast, motor axons that entered the limb on all operated sides innervated muscle with their normal precision despite the absence of the somite and axonal segmentation. Therefore, the somite and the process of spinal nerve segmentation are largely irrelevant to the specificity of motoneuron projection.     

Role of polysialic acid on outgrowth of rat olfactory receptor neurons.

Towards elucidating the role of polysialic acid (PSA) in developing olfactory neuron of the rat, we injected neuraminidase (endo-N) into the olfactory nerve pathway under whole embryo culture, then employed immunohistochemistry to (i) detect expression of highly sialylated neural cell adhesion molecules (NCAM-H) and (ii) identify olfactory neurons via anti-microtubule-associated protein 1B (MAP1B) antibody. Olfactory axonal outgrowth from basal lamina occurred at the 31-somite stage and reached the olfactory bulb primordium at the 42-somite stage, being coincident with the timing and expression of NCAM-H immunoreactivity. Enzymatic removal of PSA by endo-N remarkably affected developmental processes of axonal outgrowth, extension, and pathfinding, i.e. individual axons appeared to have become stuck in the mesenchyme. Results indicate that PSA is critically involved with anti-adhesion cues associated with individual axonal growth during olfactory system development.     

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.     

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.     

Local presentation of L1 and N-cadherin in multicomponent, microscale patterns differentially direct neuron function in vitro

The ability to pattern multiple bioactive cues on a surface is valuable for understanding how neurons interact with their complex extracellular environment. In this report, we introduce a set of methods for creating such surfaces, with the goals of understanding how developing neurons integrate multiple biologically relevant signals and as a tool for studying interactions between multiple neurons. Multiple microcontact printing steps are combined on a single surface to produce an array of polylysine nodes, interconnected by lines of proteins based on the extracellular domains of L1 or N-cadherin. Surprisingly, the N-cadherin protein could also be directly printed onto surfaces while retaining its biological activity. Rat hippocampal neurons selectively attached to the polylysine nodes, differentially extending axonal and dendritic processes along the patterns of L1 and N-cadherin, thus demonstrating control over neuron attachment and outgrowth. Combining these three biomolecules on a single surface revealed a highly complex pattern of protein recognition. Dendrites extended exclusively on N-cadherin patterns, while axons exhibited a very high degree of selectivity on L1 patterns, preferentially at distances greater than 55 mum from the cell body. At shorter distances, axonal processes recognized both L1 and N-cadherin, revealing a new aspect of neuron polarity and axon specification. This onset of L1 selectivity correlated with the establishment of intracellular L1 polarity, suggesting a functional outcome of the process of neuron polarization that has implications in development of neural tissues and creation of in vitro neuron networks.     

THE UPPER CELL SURFACE: ITS INABILITY TO SUPPORT ACTIVE CELL MOVEMENT IN CULTURE

A variety of epithelial cells and fibroblasts fail to move over one another's upper surfaces in culture, resulting in monolayering. The failure of seeded fibroblasts to adhere to and spread on epithelial cell surfaces suggests that monolayering in culture is due to the lack of adhesion of the upper cell surface, at least of epithelial cells. Seeded fibroblasts and postmitotic, rounded fibroblasts likewise fail to spread on the upper surfaces of spread fibroblasts, suggesting that the inability of the upper cell surface to support spreading may be a general phenomenon. Inert particles and cell processes do not adhere directly to the upper cell surface. However, they can initiate adhesions to the surface at a cell's free margin, suggesting a variation of adhesive properties over a cell's surface.     

The fine structure of the light organ of the New Zealand glow-worm Arachnocampa luminosa (Diptera: Mycetophilidae).

The swollen distal tips of the Malpighian tubules of the glow-worm Arachnocampa luminosa constitute the light organ. The ventral and lateral surfaces are covered by a tracheal ‘reflector’ and the nervous supply to the light organ comes from the ganglion in the penultimate segment. Fine nerve terminals, axons, and glial cells can be seen in close proximity to the basal surface of the cells of the light organ. The epithelial cells of the light organ are large, the cytoplasm dense, homogeneous and acidophilic. The cytoplasm gives a strong positive reaction for protein. The cytoplasm contains a high density of free ribosomes, patches of dense material, smooth endoplasmic reticulum, glycogen and scattered microtubules. Mitochondria are numerous; they are large, randomly distributed and packed with fine cristae. These cells lack the features characteristic of Malpighian tubule epithelial cells; infolding of the apical and basal cell surfaces is reduced and the cytoplasm contains few organelles. These cells do not contain secretory or photocyte granules and the grainy cell matrix is thought to be the luciferin substrate. Oxygen is supplied via the tracheal layer (which may have secondary reflecting properties) and light production controlled by neurosecretory excitation either directly via synapses, or by hormones. There are no other reports of Malpighian tubules of insects producing light and the fine structure of these cells is distinct. Thus, the swollen distal tips of the Malpighian tubules of the glow-worm undoubtedly constitute a unique luminescent organ.     

Regulation of SC1/DM-GRASP during the migration of motor neurons in the chick embryo brain stem

The hindbrain of the chick embryo contains three classes of motor neurons: somatic, visceral, and branchial motor. During development, somata of neurons in the last two classes undergo a laterally directed migration within the neuroepithelium; somata translocate towards the nerve exit points, through which motor axons are beginning to extend into the periphery. All classes of motor neuron are immunopositive for the SC1/DM-GRASP cell surface glycoprotein. We have examined the relationship between patterns of motor neuron migration, axon outgrowth, and expression of the SC1/DM-GRASP mRNA and protein, using anterograde or retrograde axonal tracing, immunohistochemistry, and in situ hybridization. We find that as motor neurons migrate laterally, SC1/DM-GRASP is down-regulated, both on neuronal somata and axonal surfaces. Within individual motor nuclei, these lateral, more mature neurons are found to possess longer axons than the young, medial cells of the population. Labelling of sensory or motor axons growing into the second branchial arch also shows that motor axons reach the muscle plate first, and that SC1/DM-GRASP is expressed on the muscle at the time growth cones arrive. 1994 John Wiley & Sons, Inc.     

The spread of herpes simplex virus type 1 from trigeminal neurons to the murine cornea: an immunoelectron microscopy study

An animal model has been developed to clarify the mechanism for spread of herpes simplex virus (HSV) from neuron to epithelial cells in herpetic epithelial keratitis. HSV was introduced into the murine trigeminal ganglion via stereotaxic guided injection. After 2 to 5 days, the animals were euthanized. Ganglia and corneas were prepared for light and electron microscopic immunocytochemistry with antisera to HSV. At 2 days, labeled axons were identified in the stromal layer. At 3 days, we could detect immunoreactive profiles of trigeminal ganglion cell axons that contained many vesicular structures. By 3 and 4 days, the infection had spread to all layers of epithelium, and the center of a region of infected epithelium appeared thinned. At 5 day, fewer basal cells appeared infected, although infection persisted in superficial cells where it had expanded laterally. Mature HSV was found in the extracellular space surrounding wing and squamous cells. Viral antigen was expressed in small pits along the apical surfaces of wing and squamous cells but not at the basal surface of these cells or on basal cells. This polarized expression of viral antigen resulted in the spread of HSV to superficial cells and limited lateral spread to neighboring basal cells. The pathogenesis of HSV infection in these mice may serve as a model of the human recurrent epithelial disease in the progression of focal sites of infection and transfer from basal to superficial cells.     

Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat

The molecular pathways for fluid transport in pulmonary, oral,and nasal tissues are still unresolved. Here we use immunocytochemistry and immunoelectron microscopy to define the sites of expression of fouraquaporins in the respiratory tract and glandular epithelia, where theyreside in distinct, nonoverlapping sites. Aquaporin-1 (AQP1) is presentin apical and basolateral membranes of bronchial, tracheal, andnasopharyngeal vascular endothelium and fibroblasts. AQP5 is localizedto the apical plasma membrane of type I pneumocytes and the apicalplasma membranes of secretory epithelium in upper airway and salivaryglands. In contrast, AQP3 is present in basal cells of tracheal andnasopharyngeal epithelium and is abundant in basolateral membranes ofsurface epithelial cells of nasal conchus. AQP4 resides in basolateralmembranes of columnar cells of bronchial, tracheal, and nasopharyngealepithelium; in nasal conchus AQP4 is restricted to basolateralmembranes of a subset of intra- and subepithelial glands. These sitesof expression suggest that transalveolar water movement, modulation ofairway surface liquid, air humidification, and generation ofnasopharyngeal secretions involve a coordinated network of aquaporinwater channels.