The inhibitor kappaB-ortholog Cactus is necessary for normal neuromuscular function in Drosophila melanogaster.

The Drosophila inhibitor-kappaB ortholog Cactus acts as an inhibitor of the Rel-transcription factors Dorsal and Dif. In blastoderm cells and immune competent cells, Cactus inhibits Dorsal and Dif by preventing their nuclear localization. Cactus, Dorsal and Dif are also expressed in somatic muscles, where Cactus and Dorsal, but not Dif, are enriched at the neuromuscular junction. Mutations in dorsal cause neuromuscular defects and mislocalization of Cactus. Here, we investigated whether mutations in cactus affect the neuromuscular system and subcellular localization of Dorsal and Dif. Using locomotion assays, as well as physiological and immunochemical methods, we found that wild type Cactus is necessary for the normal function of the larval neuromuscular system. The phenotype comprises i) altered bouton numbers and impaired neurotransmitter release in the neuromuscular junctions in the abdominal segments, ii) muscular weakness and iii) poor locomotion performance, probably reflecting a general neuromuscular impairment. Interestingly, in cactus mutants the subcellular localization of Dorsal and Dif in muscle is not affected, whereas cactus protein is not detected in the nucleus. This suggests, together with the similarities between the phenotypes induced by cactus and dorsal mutations, that in larval muscles the function of Cactus might be cooperation to the transcriptional activity of Rel proteins more than their cytoplasmic retention. The similarities with inhibitor-kappaB/nuclear factor kappaB interactions and muscle pathology in mammals point to Drosophila as a suitable experimental system to clarify the complex interactions of these proteins in muscle postembryonic development and activity.

Spalt modifies EGFR-mediated induction of chordotonal precursors in the embryonic PNS of Drosophila promoting the development of oenocytes.

Genes of the spalt family encode nuclear zinc finger proteins. In Drosophila melanogaster, they are necessary for the establishment of head/trunk identity, correct tracheal migration and patterning of the wing imaginal disc. Spalt proteins display a predominant pattern of expression in the nervous system, not only in Drosophila but also in species of fish, mouse, frog and human, suggesting an evolutionarily conserved role for these proteins in nervous system development. Here we show that Spalt works as a cell fate switch between two EGFR-induced cell types, the oenocytes and the precursors of the pentascolopodial organ in the embryonic peripheral nervous system. We show that removal of spalt increases the number of scolopodia, as a result of extra secondary recruitment of precursor cells at the expense of the oenocytes. In addition, the absence of spalt causes defects in the normal migration of the pentascolopodial organ. The dual function of spalt in the development of this organ, recruitment of precursors and migration, is reminiscent of its role in tracheal formation and of the role of a spalt homologue, sem-4, in the Caenorhabditis elegans nervous system.

members only encodes a Drosophila nucleoporin required for rel protein import and immune response activation.

Many developmental and physiological responses rely on the selective translocation of transcriptional regulators in and out of the nucleus through the nuclear pores. Here we describe the Drosophila gene members only (mbo) encoding a nucleoporin homologous to the mammalian Nup88. The phenotypes of mbo mutants and mbo expression during development are cell specific, indicating that the nuclear import capacity of cells is differentially regulated. Using inducible assays for nucleocytoplasmic trafficking we show that mRNA export and classic NLS-mediated protein import are unaffected in mbo mutants. Instead, mbo is selectively required for the nuclear import of the yeast transcription factor GAL4 in a subset of the larval tissues. We have identified the first endogenous targets of the mbo nuclear import pathway in the Rel proteins Dorsal and Dif. In mbo mutants the upstream signaling events leading to the degradation of the IkappaB homolog Cactus are functional, but Dorsal and Dif remain cytoplasmic and the larval immune response is not activated in response to infection. Our results demonstrate that distinct nuclear import events require different nucleoporins in vivo and suggest a regulatory role for mbo in signal transduction.

Muscle structure and innervation are affected by loss of Dorsal in the fruit fly, Drosophila melanogaster.

In Drosophila, the Rel-protein Dorsal and its inhibitor, Cactus, act in signal transduction pathways that control the establishment of dorsoventral polarity during embryogenesis and the immune response during postembryonic life. Here we present data indicating that Dorsal is also involved in the control of development and maintenance of innervation in somatic muscles. Dorsal and Cactus are colocalized in all somatic muscles during postembryonic development. In larvae and adults, these proteins are distributed at low levels in the cytoplasm and nuclei and at much higher levels in the postsynaptic component of glutamatergic neuromuscular junctions. Absence of Dorsal, in homozygous dorsal mutant larvae results in muscle misinsertions, duplications, nuclear hypotrophy, disorganization of actin bundles, and altered subcellular distribution of Cactus. Some muscles show very abnormal neuromuscular junctions, and some motor axon terminals are transformed into growth cone-like structures embedded in synaptotagmin-enriched vesicles. The detailed phenotype suggests a role of Dorsal signalling in the maintenance and plasticity of the NMJ.

adrift, a novel bnl-induced Drosophila gene, required for tracheal pathfinding into the CNS.

Neurons and glial cells provide guidance cues for migrating neurons. We show here that migrating epithelial cells also contact specific neurons and glia during their pathfinding, and we describe the first gene required in the process. In wild-type Drosophila embryos, the ganglionic tracheal branch navigates a remarkably complex path along specific neural and glial substrata, switching substrata five times before reaching its ultimate target in the CNS. In adrift mutants, ganglionic branches migrate normally along the intersegmental nerve, but sporadically fail to switch to the segmental nerve and enter the CNS; they wind up meandering along the ventral epidermis instead. adrift encodes a novel nuclear protein with an evolutionarily conserved motif. The gene is required in the trachea and is expressed in the leading cells of migrating ganglionic branches where it is induced by the branchless FGF pathway. We propose that Adrift regulates expression of tracheal genes required for pathfinding on the segmental nerve, and FGF induction of adrift expression in migrating tracheal cells promotes the switch from the intersegmental to the segmental nerve.

Dif and cactus are colocalized in the larval nervous system of Drosophila melanogaster.

The Rel protein Dif is a transcription factor suggested to control part of the immune response in the fruit fly Drosophila melanogaster. In uninfected animals, Dif is normally located in the cytoplasm, most likely in a complex with an IkappaB molecule such as Cactus. Upon infection, Dif is enriched in the nucleus of immunoresponsive tissues such as fat body and blood cells. Rel proteins in mammals not only participate in the control of the immune response, but are also thought to play important roles in the function of the nervous system. Here, we demonstrate that both Dif and Cactus are expressed in the central nervous system (CNS) of Drosophila. Interestingly, Dif and Cactus colocalize in their distribution, suggesting a functional link between these proteins in the CNS. In the larval CNS, both Dif and Cactus are expressed at relatively low levels in most cells and at high levels in the mushroom bodies and in small subsets of neurosecretory cells. The cytoplasmic localization of Dif and Cactus in the CNS cells is not affected by bacterial challenge. Instead, we observed changes in nuclear versus cytoplasmic localization of Cactus (but not Dif) along the dark-light cycle, with a strong nuclear localization in perineurial glia toward the end of the dark period. In the CNS of the prepupa, the intensity of the immunostaining for both Dif and Cactus is higher than in the larva. Interestingly, in fat body of uninfected prepupae, the Dif localization was mainly nuclear, suggesting a function for Dif during the process of pupariation.

Preparation and dissolution rate of gliquidone-PVP K30 solid dispersions.

Solid dispersions of gliquidone in PVP K30 were prepared by the solvent method. These dispersions were characterized using X-ray diffraction. In comparison with the drug alone, the physical mixtures and even more the solid dispersions showed an increase in the dissolution rate. Moreover these solid dispersions were stable during storage.

Genetic control of epithelial tube fusion during Drosophila tracheal development.

During development of tubular networks such as the mammalian vascular system, the kidney and the Drosophila tracheal system, epithelial tubes must fuse to each other to form a continuous network. Little is known of the cellular mechanisms or molecular control of epithelial tube fusion. We describe the cellular dynamics of a tracheal fusion event in Drosophila and identify a gene regulatory hierarchy that controls this extraordinary process. A tracheal cell located at the developing fusion point expresses a sequence of specific markers as it grows out and contacts a similar cell from another tube; the two cells adhere and form an intercellular junction, and they become doughnut-shaped cells with the lumen passing through them. The early fusion marker Fusion-1 is identified as the escargot gene. It lies near the top of the regulatory hierarchy, activating the expression of later fusion markers and repressing genes that promote branching. Ectopic expression of escargot activates the fusion process and suppresses branching throughout the tracheal system, leading to ectopic tracheal connections that resemble certain arteriovenous malformations in humans. This establishes a simple genetic system to study fusion of epithelial tubes.

Glial cells in insect ganglia.

Glial cells associated with elements of central neuropils in several insect species were studied with conventional light and electron microscopical techniques, the Golgi procedure, and a combination of the latter with electron microscopy. Different types of cells located in the layer of cells covering the neuropil were found to send complex arborizations into synaptic neuropils. These arborizations grow in clusters that seem to represent discrete compartments circumscribing groups of synaptic terminals. The thinnest glial processes are found deep in the neuropil and consist of compact membrane leaflets lacking cell organelles and with reduced amounts of cytoplasmic matrix. Some of these glial processes also invest neuropil tracheoles in a manner reminiscent of the association between astrocyte end-feet and blood capillaries in the central nervous system of mammals. Other glial cells reside completely in the neuropil, where they enwrap fiber bundles in a similar manner to oligodendrocytes in the central nervous system of mammals.

Electron microscopic analysis of Drosophila midline glia during embryogenesis and larval development using beta-galactosidase expression as endogenous cell marker.

To thoroughly study developmental problems it is often desirable to identify specific cells at the resolution of the electron microscope (TEM). Specific antibodies, and immunogold and other antibody labelling techniques can be successfully used with the TEM. But for these techniques to be successful there must be substantial adjustments for each antibody and tissue analyzed. To develop a more generally applicable labelling method we took advantage of the enhancer trap technique in Drosophila. Enhancer trap fly strains show cell- and/or tissue-specific beta-galactosidase expression which can be visualized by a simple X-gal staining procedure. To combine the power of the enhancer trap approach with electron microscopy, we have improved the fixation and staining conditions, which allow detection of X-gal crystals (by TEM) and thus provide precise information on ultrastructural morphology. We have tested our technique using the well-known midline glial cells and examined these cells between late embryonic and pupal developmental stages. The four embryonic midline glial cells found in each neuromere reside ventrally and dorsally to the midline of the neuropile and are closely associated with unpaired neurons, major commissures, and other types of glial cells. During larval and pupal life dramatic cell growth and endomitotic nuclear replication occur in midline glial cells. By the end of larval life, the giant midline glial cells fragment to give rise to a variable number of small midline glial cells. Here we show that the combination of transmission electron microscopy with cytochemical detection of beta-galactosidase expression represents a promising and valuable tool for the study of the morphology and development of specific cell types.

Glial cells phagocytose neuronal debris during the metamorphosis of the central nervous system in Drosophila melanogaster.

Using electron microscopy we demonstrate that degenerating neurons and cellular debris resulting from neuronal reorganization are phagocytosed by glial cells in the brain and nerve cord of the fruitfly Drosophila melanogaster during the first few hours following pupariation. At this stage several classes of glial cells appear to be engaged in intense phagocytosis. In the cell body rind, neuronal cell bodies are engulfed and phagocytosed by the same glial cells that enwrap healthy neurons in this region. In the neuropil, cellular debris in tracts and synaptic centres resulting from metamorphic re-differentiation of larval neurons is phagocytosed by neuropil-associated glial cells. Phagocytic glial cells are hypertrophied, produce large amounts of lysosome-like bodies and contain a large number of mitochondria, condensed chromatin bodies, membranes and other remains from neuronal degeneration in phagosomes.

Evidence that locustatachykinin I is involved in release of adipokinetic hormone from locust corpora cardiaca.

The glandular cells of the corpus cardiacum of the locust Locusta migratoria, known to synthesize and release adipokinetic hormones (AKH), are contacted by axons immunoreactive to an antiserum raised against the locust neuropeptide locustatachykinin I (LomTK I). Electron-microscopical immunocytochemistry reveals LomTK immunoreactive axon terminals, containing granular vesicles, in close contact with the glandular cells cells. Release of AKH I from isolated corpora cardiaca of the locust has been monitored in an in vitro system where the amount of AKH I released into the incubation saline is determined by reversed phase high performance liquid chromatography with fluorometric detection. We could show that LomTK I induces release of AKH from corpora cardiaca in a dose-dependent manner when tested in a range of 10-200 microM. This is thus the first clear demonstration of a substance inducing release of AKH, correlated with the presence of the substance in fibers innervating the AKH-synthesizing glandular cells, in the insect corpora cardiaca.

Postembryonic development of corazonin-containing neurons and neurosecretory cells in the blowfly, Phormia terraenovae.

An antiserum against the cockroach cardioactive peptide corazonin was used to investigate the distribution of immunoreactive neurons and neurosecretory cells in the nervous system of the blowfly, Phormia terraenovae, during postembryonic development. A small number of corazonin-immunoreactive neurons was found at larval, pupal, and adult stages. At all postembryonic stages two cell groups were found in the protocerebrum of the brain: 1) two lateral cell clusters and 2) two median cells. In the larva eight bilateral cell pairs were found in thoracic and abdominal neuromeres of the fused ventral ganglion. The lateral brain neurons are located in the lateral neurosecretory cell group and extend axons with branches in several components of the retrocerebral neuroendocrine complex, in the stomatogastric nervous system of larvae and adults, and additionally in muscles of the alimentary canal in the adult. The most prominent element of these peripheral processes is a large plexus of varicose fibers located in the wall of the aorta, the main site for the release of neurohormones produced in the brain of blowflies. The presence of corazonin-immunoreactive material in the aortic plexus suggests that this peptide functions as a neurohormone. During metamorphosis, the immunoreactive neurons found in the thoracic-abdominal ganglion of the larva disappear, and in the brain new immunoreactive neurons are added to those that persist from larval stages. The bulk of the corazonin-immunoreactive material extracted from adult brains and corpora cardiaca-aorta complexes was found to co-elute with synthetic corazonin in reversed-phase high-performance liquid chromatography as monitored with enzyme-linked immunosorbent assay.

Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I.

An antiserum against the cockroach neuropeptide leucokinin I (LKI) was used to study peptidergic neurons and their innervation patterns in larvae and adults of three species of higher dipteran insects, the flies Drosophila melanogaster, Calliphora vomitoria, and Phormia terraenovae, as well as larvae of a primitive dipteran insect, the crane fly Phalacrocera replicata. In the larvae of the higher dipteran flies, the antiserum revealed three pairs of cells in the brain, three pairs of ventro-medial cells in the subesophageal ganglion, and seven pairs of ventro-lateral cells in the abdominal ganglia. Each of these 14 abdominal leucokinin-immunoreactive (LKIR) neurons innervates a single muscle of the abdominal body wall (muscle 8), which is known to degenerate shortly after adult emergence. Conventional electron microscopy demonstrates that this muscle is innervated by at least one axon containing clear vesicles and two axons containing dense-cored vesicles. Electron-microscopical immunocytochemistry shows that the LKIR axon is one of these two axons with dense-cored vesicles and that it forms terminals on the sarcolemma of its target muscle. The abdominal LKIR neurons appear to survive metamorphosis. In the adult fly, the efferent abdominal LKIR neurons innervate the spiracles, the heart, and neurohemal regions of the abdominal wall. In the crane fly larva, dorso-medial and ventrolateral LKIR cell bodies are located in both thoracic and abdominal ganglia of the ventral nerve cord. As in the larvae of the other flies, the abdominal ventrolateral LKIR neurons form efferent axons. However, in the crane fly larva there are two pairs of efferent LKIR neurons in each of the abdominal ganglia and their peripheral targets include neurohemal regions of the dorsal transverse nerves. An additional difference is that in the crane fly, a caudal pair of LKIR axons originating from the penultimate pair of dorso-median LKIR cells in the terminal ganglion innervate the hind-gut.

Neurons in the cockroach nervous system reacting with antisera to the neuropeptide leucokinin I.

Antisera were raised against the myotropic neuropeptide leucokinin I, originally isolated from head extracts of the cockroach Leucophaea maderae. Processes of leucokinin I immunoreactive (LKIR) neurons were distributed throughout the nervous system, but immunoreactive cell bodies were not found in all neuromeres. In the brain, about 160 LKIR cell bodies were distributed in the protocerebrum and optic lobes (no LKIR cell bodies were found in the deuto- and tritocerebrum). In the ventral ganglia, LKIR cell bodies were seen distributed as follows: eight (weakly immunoreactive) in the subesophageal ganglion; about six larger and bilateral clusters of 5 smaller in each of the three thoracic ganglia, and in each of the abdominal ganglia, two pairs of strongly immunoreactive cell bodies were resolved. Many of the LKIR neurons could be described in detail. In the optic lobes, immunoreactive neurons innervate the medulla and accessory medulla. In the brain, three pairs of bilateral LKIR neurons supply branches to distinct sets of nonglomerular neuropil, and two pairs of descending neurons connect the posterior protocerebrum to the antennal lobes and all the ventral ganglia. Other brain neurons innervate the central body, tritocerebrum, and nonglomerular neuropil in protocerebrum. LKIR neurons of the median and lateral neurosecretory cell groups send axons to the corpora cardiaca, frontal ganglion, and tritocerebrum. In the muscle layer of the foregut (crop), bi- and multipolar LKIR neurons with axons running to the retrocerebral complex were resolved. The LKIR neurons in the abdominal ganglia form efferent axons supplying the lateral cardiac nerves, spiracles, and the segmental perivisceral organs. The distribution of immunoreactivity indicates roles for leucokinins as neuromodulators or neurotransmitters in central interneurons arborizing in different portions of the brain, visual system, and ventral ganglia. Also, a function in circuits regulating feeding can be presumed. Furthermore, a role in regulation of heart and possibly respiration can be suggested, and probably leucokinins are released from corpora cardiaca as neurohormones. Leucokinins were isolated by their myotropic action on the Leucophaea hindgut, but no innervation of this portion of the gut could be demonstrated. The distribution of leucokinin immunoreactivity was compared to immunolabeling with antisera against vertebrate tachykinins and lysine vasopressin.

Postembryonic development of leucokinin I-immunoreactive neurons innervating a neurohemal organ in the turnip moth Agrotis segetum.

In the abdominal ganglia of the turnip moth Agrotis segetum, an antibody against the cockroach neuropeptide leucokinin I recognizes neurons with varicose fibers and terminals innervating the perisympathetic neurohemal organs. In the larva, the abdominal perisympathetic organs consist of a segmental series of discrete neurohemal swellings on the dorsal unpaired nerve and the transverse nerves originating at its bifurcation. These neurohemal structures are innervated by varicose terminals of leucokinin I-immunoreactive (LKIR) fibers originating from neuronal cell bodies located in the preceding segment. In the adult, the abdominal segmental neurohemal units are more or less fused into a plexus that extends over almost the whole abdominal nerve cord. The adult plexus consists of peripheral nerve branches and superficial nerve fibers beneath the basal lamina of the neural sheath of the nerve cord. During metamorphosis, the LKIR fibers closely follow the restructuration of the perisympathetic organs. In both larvae and adults the LKIR fibers in the neurohemal structures originate from the same cell bodies, which are distributed as ventrolateral bilateral pairs in all abdominal ganglia. The transformation of the series of separated and relatively simple larval neurohemal organs into the larger, continuous and more complex adult neurohemal areas occurs during the first of the two weeks of pupal life. The efferent abdominal LKIR neurons of the moth Agrotis segetum thus belong to the class of larval neurons which persist into adult life with substantial peripheral reorganization occurring during metamorphosis.

Dual peptidergic innervation of the blowfly hindgut: a light- and electron microscopic study of FMRFamide and proctolin immunoreactive fibers.

1. The innervation of the hindgut, rectal valve, rectum and rectal papillae of the adult blowfly, Calliphora erythrocephala, was studied by means of light and electron microscopic immunocytochemistry, using antibodies against the neuropeptides proctolin and FMRFamide. 2. Branches from the abdominal nerves reaching the posterior portion of the gut were found to contain mostly neurosecretory type axons and to innervate the muscle coat of all hindgut structures studied. 3. Some of the axons found in these nerve branches innervating the gut display proctolin- others FMRFamide-like immunoreactivity. Both types of peptidergic axons were found to have abundant terminals in the muscle coat of the hindgut, rectum and rectal valve and in the medulla of the rectal papillae. 4. It is clear that two separate peptidergic systems derived from the abdominal ganglion are supplying the hindgut structures, and, possibly, they use proctolin- and FMRFamide-like peptides respectively as their transmitters or modulators.

Postnatal development of photoreceptor-specific proteins in mice with hereditary retinal degeneration. An immunocytochemical study.

The postnatal development of immunoreactivity for photoreceptor-specific markers was studied in mice carrying the genes rd (retinal degeneration) and rds (retinal degeneration slow) in different combinations. Antibodies raised against three specific photoreceptor proteins (opsin, alpha-transducin and S-antigen) were applied on retinae from mice with the following allelic combinations at the rd and rds loci: +/+, +/+ (control); rd/rd, +/+; rds/rds, +/+; rds/+, +/+; and rd/rd, rds/rds. Immunoreactivity for each antibody appeared simultaneously in normal and mutants. Thereafter, the immunoreactivity patterns in the mutants diverged from the normal phenotype. Except for a dramatic loss of photoreceptor cells in the mutants, the main divergence from the normal development consisted of a progressive loss of the intracellular immunoreactivity compartmentalization for each protein. As degeneration progressed, the remaining photoreceptors became homogeneously labelled; one this labelling pattern was acquired, it was maintained during subsequent stages of development. It is proposed that this pattern, common for all phenotypes studied, may be due to the loss of structural and biochemical polarity of the photoreceptor cells undergoing degeneration, and that this may be an important primary or secondary aspect of the disease process.

Postnatal development of photoreceptor proteins in mutant mice and Abyssinian cats with retinal degeneration.

Using immunocytochemical techniques, development of opsin, transducin alpha and S-antigen in photoreceptor cells of the mice homozygous or heterozygous for the rd or rds genes has been found to be similar to that of control animals during the first postnatal week. Even though the absolute amounts of these proteins are low (eg. opsin in the rds retina) or decrease in the postnatal period (as in the rd retina), we can demonstrate their persistence during the entire degeneration process. In fact, the content of the proteins in the photoreceptor perikarya actually appear to be higher after postnatal day 11 in all mutants studied. Thus, one of the major manifestations of the mutant retinae is a loss of polarity of the photoreceptor cells at the time of ROS degeneration without a loss in the ability to synthesize these important proteins of the visual cycle. This correlates well with the apparent defect in IRBP secretion and its intracellular accumulation in mutant photoreceptor cells as previously observed (van Veen et al. 1986). In the Abyssinian cat model for progressive retinal atrophy, the development an cellular distribution of all the proteins studied are similar in affected and control retinae until the beginning of stage 2 of the disease. At this time, outer segments begin to degenerate and immunoreactivity increases in the photoreceptor perikarya. The IRBP content of the retina declines markedly at stage 2, preceding extensive loss of photoreceptors.

Serotonin and gastrin/cholecystokinin-like immunoreactive neurons in the larval retrocerebral complex of the blowfly Calliphora erythrocephala.

The presence and distribution of neurons immunoreactive against antibodies to serotonin (5-HT) and gastrin/cholecystokinin (gastrin/CCK) has been studied in the larval retrocerebral complex of the blowfly Calliphora erythrocephala, a composite structure which consists of the corpus cardiacum, the corpus allatum, the thoracic gland and a portion of the cephalic aorta. Immunoreactive material was found in all these elements except in the corpus allatum. Six to eight cell bodies in the corpus cardiacum and four to eight cell bodies in the thoracic gland were 5-HT immunoreactive (5-HTi). These 5-HTi cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the cephalic aorta, corpus cardiacum and ventral part of the thoracic gland. Six to eight cell bodies in the corpus cardiacum and four to six cell bodies in the thoracic gland reacted with antibodies against gastrin/CCK. These cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the corpus cardiacum and the cephalic aorta in a pattern resembling that of the 5-HTi fibers. Additional gastrin/CCK-like immunoreactive fibers were shown to come from the central nervous system via the two nervi corporis cardiaci. An electron-microscopical analysis was performed to analyze further the morphological features revealed by the light-microscopic immunocytochemical technique. This confirmed the existence of neurosecretory-like terminals among the gland cells of the thoracic glands and the existence of neurohemal sites in several regions of the larval retrocerebral complex. Some functional aspects of the retrocerebral complex are discussed on the basis of the presented data.