Biomedical subjects
Publications and source records attributed to H Keshishian.
During embryogenesis, Drosophila melanogaster generates a set of muscles and motoneurons that are restructured during pupal metamorphosis to give rise to an adult motor system. The study of neuromuscular development in embryos and pupae has recently revealed important insights into the cellular and molecular mechanisms controlling both muscle patterning and the establishment of synaptic connections.
This article reveals a novel aspect in the regulation of synaptic connectivity in Drosophila. Reducing neural activity genetically or pharmacologically disrupts the normally precise embryonic and larval neuromuscular connections. In third instar larvae with mutations that affect sodium channel function or expression such as no action potential, temperature-induced paralysis E, or seizure1, foreign neuromuscular synapses, arising from inappropriate nerve sources, are observed on muscle fibers throughout the abdominal body wall. Their frequencies increase as neural activity is further reduced in double mutant combinations. These foreign connections are first observed during late embryogenesis as filopodial-like contacts, but critical period analysis suggests that neural activity must be reduced during both late embryogenesis and the first larval instar to promote the differentiation of these embryonic contacts into foreign motor synapses. In addition, the loss of electrical activity in the motoneuron, as opposed to the loss of postsynaptic potentials in the muscle fibers, appears to be responsible for these changes in connectivity. Our experiments suggest that neural activity may function during development by preventing inappropriate connections and thereby maintaining the precise connectivity achieved during nerve outgrowth and target selection.
Synaptogenesis can be analyzed in a simple array of motoneurons and muscle fibers of the embryos and larvae of Drosophila melanogaster. Each abdominal hemisegment contains a stereotypic array of 30 muscle fibers. During middle to late embryogenesis, motoneurons exit the central nervous system to make precise synaptic connections with specific muscle fibers. Target recognition has been tested using both genetic and microsurgical manipulations, which indicate that motoneurons actively recognize specific muscle fibers. The molecular basis of target recognition has been examined by screens for mutations that disrupt both guidance events and correct innervation. In addition, the motoneurons and muscle fibers both express an array of putative cell adhesion molecules whose functions may contribute to normal connectivity. Postsynaptic specializations, including glutamate receptor distribution, depend on innervation and neural activity. The neuromuscular system is not "hardwired," as motoneurons are capable of altering both their branch arborizations and connectivity in response to local denervation and blockade of synaptic function. Collectively, these studies show that the Drosophila motor innervation is a powerful model system for testing at the cellular and molecular level the mechanisms that govern synaptic development.
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The neuromuscular connections of Drosophila are ideally suited for studying synaptic function and development. Hypotheses about cell recognition can be tested in a simple array of pre- and postsynaptic elements. Drosophila muscle fibers are multiply innervated by individually identifiable motoneurons. The neurons express several synaptic cotransmitters, including glutamate, proctolin, and octopamine, and are specialized by their synaptic morphology, neurotransmitters, and connectivity. During larval development the initial motoneuron endings grow extensively over the surface of the muscle fibers, and differentiate synaptic boutons of characteristic morphology. While considerable growth occurs postembryonically, the initial wiring of motoneurons to muscle fibers is accomplished during mid-to-late embryogenesis (stages 15-17). Efferent growth cones sample multiple muscle fibers with rapidly moving filopodia. Upon reaching their target muscle fibers, the growth cones rapidly differentiate into synaptic contacts whose morphology prefigures that of the larval junction. Mismatch experiments show that growth cones recognize specific muscle fibers, and can do so when the surrounding musculature is radically altered. However, when denied their normal targets, motoneurons can establish functional synapses on alternate muscle fibers. Blocking synaptic activity with either injected toxins or ion channel mutants does not derange synaptogenesis, but may influence the number of motor ending processes. The molecular mechanisms governing cellular recognition during synaptogenesis remain to be identified. However, several cell surface glycoproteins known to mediate cellular adhesion events in vitro are expressed by the developing synapses. Furthermore, enhancer detector lines have identified genes with expression restricted to small subsets of muscle fibers and/or motoneurons during the period of synaptogenesis. These observations suggest that in Drosophila a mechanism of target chemoaffinity may be involved in the genesis of stereotypic synaptic wiring.
How a neuron finds its synaptic target is one of the key questions of developmental neurobiology. It is a problem that must, at least in part, be explained in molecular terms. In light of this, several groups have recently examined the synapses that are made between individual motoneurons and muscle fibers in the embryos and larvae of the fruit fly Drosophila melanogaster. The work combines the traditions of small system neurobiology, which is founded on the analysis of singly identified cells, with those of developmental genetics. An important insight emerging from the work is that many of the familiar features of vertebrate synaptogenesis occur in Drosophila, where a rich array of genetic and molecular methods may be readily applied.
In Drosophila embryos, each motoneuron is accurately matched to one or more singly identifiable muscle fibers. In this article we altered the number and pattern of the embryonic muscle fibers using genetic, heat shock, and laser ablation methods to test whether motoneuron growth cones are able to recognize specific targets. The choices made by two motoneurons were assayed using both intracellular dye fills and immunocytochemistry. The motoneurons RP1 and RP3 have nearly identical central and peripheral axonal trajectories. However, RP3 innervates the two most ventral longitudinal muscle fibers, 7 and 6, while RP1 grows past these fibers to innervate only muscle fiber 13. In rhomboid mutants muscle fiber 7 does not develop. Despite the loss of one of its targets, RP3 faithfully innervated the remaining muscle fiber 6 in over 80% of the observed cases. Furthermore, neuron RP1 accurately innervated muscle fiber 13, although it traversed one fiber fewer to reach it. Laser ablation of muscle fiber 7 confirmed the target choices shown by the motoneurons. In numb mutants, multiple muscle fibers, including 7, 13, and 12, fail to develop. This allowed us to test whether fibers distal to the target are involved in muscle fiber recognition, possibly by halting the growth cone advance. In mutant embryos, RP3 innervated muscle fiber 6 at the same frequency regardless of the absence of the distal muscle fiber 13. By contrast, RP1, which had lost its target entirely, frequently failed to innervate any muscle fiber during the period examined. Finally, muscle fiber 13 can be duplicated in wild-type embryos by means of a brief heat pulse during myogenesis. Presented with two targets, RP1 innervated both fibers in each case examined, while RP3 synapsed with muscle fibers 7 and 6 normally. Neuron-specific antibodies revealed that the embryonic growth cone choices were not transient, but persisted into the larval neuromuscular projections. These results indicate that each motoneuron growth cone has a primary target preference, which is retained even when the numbers of the muscle fibers, and therefore their relative positions, are altered. We therefore suggest that synaptic recognition by Drosophila motoneuron growth cones relies on unique features of the individual muscle fibers.
The Drosophila embryonic and larval body wall consists of a simple array of segmental muscle fibers, innervated in a precise manner by identified neurons. During development motoneurons were forced to find alternate targets following the selective deletion of a single muscle fiber, the pleural internal oblique #5. We used backfills, intracellular dyefills, and immunocytochemistry in embryos and larvae to characterize the normal motoneurons to the fiber. Deleting the fiber using either a genetic or laser lesioning method yielded essentially the same result. In nearly half the cases examined, an ectopically placed neuromuscular projection was found on either of two neighboring muscle fibers, with one favored eight times more than the other. The ectopic projection derived from the nerve branch that normally supplied the deleted muscle fiber 5. Motoneuronal endings on undeleted muscle fibers elsewhere in the body wall had normal morphology. The ectopically placed motor terminals accumulated glutamate in normally sized synaptic boutons, beneath which transmitter sensitivity was localized. The number of boutons and branches at the ectopic endings did not differ significantly from those on intact muscle fiber 5s. Also, the native motoneurons did not alter their arborization sizes in response to a supernumerary ectopically placed contact. While the orientation of the individual ectopically placed branches was variable, the ectopic endings occupied a predictable site on the surrogate muscle fibers. The results suggest that Drosophila motoneurons can project to body wall destinations in the absence of their muscle fiber targets, and that alternate muscle fibers are selected by their proximity. The muscle fibers will support apparently stable and functional supernumerary motor endings on ectopic sites, and these inputs do not significantly influence the behavior of the native motoneurons. The data suggest that Drosophila motoneurons may behave autonomously when making synapses, and that competition does not play a major role in the matching of motoneuron to muscle fiber.
Somatic and germline cells interact during oogenesis to establish the pattern axes of the Drosophila eggshell and embryo. The role of the oocyte nucleus in pattern formation was tested with the use of laser ablation. Ablation in stage 6 to 9 egg chambers caused partial or complete ventralization of the eggshell, phenotypes similar to those of eggs produced by gurken or torpedo females. Accumulation of vasa protein at the posterior pole of treated oocytes was also disrupted. Thus the oocyte nucleus is required as late as stage 9 for dorsoventral patterning within the follicle cells and for polar plasm assembly in the oocyte.
Each muscle fiber in the segmented body wall of Drosophila larvae is innervated by anatomically stereotyped neuromuscular junctions. These synapses arise through the selective choices of motoneuronal growth cones at their peripheral targets. Using digital optical microscopy of staged intracellular dye fills, we have singly identified embryonic motoneurons and have examined individual growth cones when they contact and differentiate at the target cells. There is a precise connectivity between motoneuron and muscle fiber, which is the direct consequence of growth cone behavior. We have also found that Drosophila muscle fibers possess molecularly heterogeneous cell surfaces that may be involved in growth cone recognition of appropriate targets. Fasciclin III, a homophilic adhesion molecule, is coexpressed by several of the efferent growth cones and in a site-specific fashion by the target muscle fiber's membrane. The fasciclin III expression is transient, corresponding to the period in embryogenesis when the first neuromuscular contacts are made. Upon encountering the target cell surface, the growth cones can sprout stereotypically arrayed filopodial processes, orient along the anterior-posterior axis, and turn in predictable directions. Subsequently, terminal branches are established in a nonrandom order. These phenomena were found to occur in two motoneurons that innervate adjacent muscle fiber targets, and may be general features of neuromuscular synaptogenesis in Drosophila.
A molecular mechanism for selective axonal adhesion is a central question of neural development. Cell adhesion molecules have been identified, but it has been difficult to ascribe functions for these proteins in vivo. Here we show that the neuronal membrane glycoprotein fasciclin I has a role in the adhesion of sister axons during the development of the grasshopper limb bud. To do this we used a new technique, chromophore-assisted laser inactivation (CALI), which causes the precisely timed thermal denaturation of specific proteins by laser light targeted through a dye-labelled antibody, without any other observable damage to living cells. This can be achieved by relaxation of the laser-excited dye which releases heat to denature the bound protein; the rapid dissipation of heat with distance insulates unbound proteins from damage. CALI is a molecular analogue of cellular laser ablation and provides an unprecedented level of spatial and temporal resolution. Using dye-labelled antibodies that recognize fasciclin I, CALI disrupts fasciculation of the pioneer neurons without affecting their growth or guidance.
Although the neuropeptide proctolin has important functions in many arthropods, it is reported to be absent in Lepidoptera. Its possible occurrence in these insects was reinvestigated by bioassays of HPLC fractions and immunocytochemistry. A proctolin-like substance was recovered from the frontal and subesophageal ganglia of Lymantria dispar. This substance has the same chromatographic retention time as proctolin; enzymatic degradation indicates that it is a peptide; it is bound by proctolin antisera; and thus it is indistinguishable from authentic proctolin. A small subpopulation of proctolin-like immunoreactive (PLI) neurons was stained in the larval CNS of L. dispar, Manduca sexta, Trichoplusia ni, Galleria mellonella, and Vanessa cardui. Most prominent of these cells are median neurosecretory neurons in the brain, paired neurons in the frontal ganglion, two clusters of neurons in the subesophageal ganglion, paired lateral neurons in the thoracic ganglia, and dorsomedial neurons in the abdominal ganglia. Also, varicose PLI axons are found in the corpora cardiaca and perivisceral organs. In L. dispar, PLI cells also were found in the corpora cardiaca. The results of this study indicate that proctolin is of general occurrence in the Lepidoptera, that it has an important role in the stomatogastric nervous system, and that it may be released as a local neurohormone from various neurohemal organs.
The outgrowth of peripheral nerves and the development of muscle fiber-specific neuromuscular junctions were examined in Drosophila embryos using immunocytochemistry and computer-enhanced digital optical microscopy. We find that the pioneering of the peripheral nerves and the formation of the neuromuscular junctions occur through a precisely orchestrated sequence of stereotyped axonal trajectories, mediated by the selective growth cone choices of pioneer motoneurons. We have also examined the establishment of the embryonic muscle fibers and, using intracellular dye fills, have identified cells that are putative muscle pioneers. The muscle fibers of the bodywall have completed their morphogenesis prior to the initiation of synaptic contacts, and owing to the timing of neurite outgrowth from the CNS, synaptogenesis is synchronous at muscle fibers throughout the bodywall. At each muscle fiber the innervating axons make their initial contacts on a characteristic surface domain of the target cell's membrane. Through stereotyped growth cone-mediated trajectories the motoneurons actively establish the basic anatomical features of the mature neuromuscular junction, including the stereotyped, muscle fiber-specific branch anatomy. These events occur without significant process pruning or apparent synapse elimination. Our results suggest that the basic elements of the mature neuromuscular innervation, including the details of the ending trajectory on the target cell's surface, are formed by the precise navigation and presumed recognition by the motoneuron growth cones of muscle membrane surface features.
The distribution and morphology of glutamatergic synapses on Drosophila bodywall muscle fibers were examined at the single-synapse level using immunocytochemistry and electrophysiology. We find that glutamate-immunoreactive motor endings innervate the entire larval bodywall musculature, with each muscle fiber receiving at least one glutamatergic ending. The innervation is initiated at stereotyped locations on each muscle fiber from where moderately branched varicose nerve processes project over the internally facing muscle surface. Individual muscle fibers have distinct stereotypic patterns of nerve endings that occupy characteristic regions on the cell surface. The muscle-specific branching pattern of motor endings is reiterated by segmentally homologous fibers. Two morphological types of innervating nerve processes can be distinguished by their bouton size distributions: (1) Type I processes, which have localized branching and a broad size distribution of relatively large varicosities ranging up to 8 microns (mean diameter, 3.1 +/- 1.6 microns; +/- SD, n = 521), and (2) thinner Type II processes, which have a narrower distribution of small varicosities with a mean diameter of only 1.4 +/- 0.6 microns (+/- SD, n = 214). Immunoelectron microscopy with peroxidase-labeled second antibody demonstrates that the varicosities are surrounded by a subsynaptic reticulum, that they contain immunoreactive vesicles of about 30-50 nm, and thus probably represent synaptic release sites. By iontophoretic application of glutamate we mapped the responsive sites on the muscle surface and found an excellent correspondence between transmitter sensitivity and the patterns of endings as described by immunocytochemistry. In contrast to our finding of numerous glutamate iontophoresis-sensitive sites, we did not detect any aspartate-responsive muscles. These data provide strong new evidence for glutamate being an endogenous transmitter at the Drosophila larval neuromuscular junction.
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The cellular localization of the peptide neurotransmitter proctolin was determined for larvae of the fruitfly Drosophila melanogaster. Proctolin was recovered from the CNS, hindgut, and segmental bodywall using reverse-phase HPLC, and characterized by bioassay, immunoassay, and enzymatic analysis. A small, stereotyped population of proctolin-immunoreactive neurons was found in the larval CNS. Several of the identified neurons may be efferents. In the periphery, proctolin-immunoreactive neuromuscular endings were identified on both visceral and skeletal muscle fibers. On the hindgut, the neuropeptide is associated with endings on intrinsic circular muscle fibers. We propose that the hindgut muscle fibers are innervated by central neurons homologous to previously described proctolinergic efferents of grasshoppers. The segmental bodywall innervation consists of a pattern of segment-specific junctions on several singly identifiable muscle fibers. While it is generally accepted that Drosophila muscle fibers are innervated by glutamatergic motoneurons, our data indicate that a specialized subset of muscle fibers are also innervated by peptidergic efferents.
We have examined the developmental acquisition and regional expression of the neurotransmitter proctolin in neurons of the grasshopper embryo, using both assay and immunocytochemistry. There are four stages in the acquisition and accumulation of proctolin in the embryo. The first stage (50 to 60% embryogenesis) consists of a slow accumulation of proctolin prior to the onset of central nervous system (CNS) staining. During the second stage proctolin levels increase at a 5-fold faster rate. During this stage, which ends at 70% of development, a stable pattern of staining is established in the ganglia. The third stage (70 to 95% embryogenesis) consists of a rapid accumulation of peripheral proctolin levels that occurs principally in motor endings, without a significant increase in the CNS. This stage also correlates with the onset of peripheral staining. During the fourth stage, over the final 5% of embryogenesis, there is a 20% loss of proctolin in the periphery. We have attempted to relate these changes to developmental events occurring in identified clusters of neurons, by examining the development of neuromuscular connections made by embryonic terminal ganglion motoneurons to the intrinsic muscles of the hindgut. Of the 30 hindgut motoneurons, only six anterior medial (AM) efferents are proctolin immunoreactive in the embryo. Their axons establish a dense proctolin-staining arborization over the intrinsic muscles. The peptide accumulates in the endings as staining appears in the terminal ganglion. Hindgut proctolin levels increase substantially and then decline in a manner similar to that found for total peripheral proctolin. By the end of embryogenesis the endings will release proctolin, in a calcium-dependent manner, when depolarized. The six proctolin-staining AM neurons constitute a simple model system for studying the determination and expression of neurotransmitters in identified embryonic cells.
Proctolin is a peptide neurotransmitter, discovered in the visceral muscles of the cockroach. The peptide has recently been identified in the nervous systems of many invertebrates. Using reverse phase high pressure liquid chromatography together with a sensitive bioassay, we analyzed the distribution of proctolin in the central nervous system of the grasshopper Schistocerca nitens. We also used immunocytochemistry to identify about 70 diverse proctolin-staining neurons in the postembryonic ventral nerve cord. Proctolin immunoreactivity is rare, being found in only about 0.6% of the ganglionic neurons. Among the cells we stained for proctolin were thoracic skeletal motoneurons, a cluster of efferents to the intrinsic muscles of the hindgut, and both inter- and intraganglionic thoracic interneurons, as well as several interspecies homologues to proctolin-staining neurons in the cockroach. The map of proctolin expression within the central nervous system is not extensively altered during postembryonic life. It is the product of embryonic development, which is the subject of the following paper (Keshishian, H., and M. O'Shea (1985) J. Neurosci. 5: 1005-1015).