Development of the enteric nervous system in chick embryonic duodenum in terms of the distribution of tubulin

An immunohistochemical method that uses anti-tubulin was utilized to observe the development of the enteric nervous system in chick embryonic duodenum. Neural crest cells, and enteric neuroblasts, or enteric ganglia, which derive from neural crest cells were clearly shown as sharp immunoreactive regions of tubulin. The distributions of enteric neuroblasts and enteric ganglia in chick duodena were in agreement with results of previous reports in which different techniques were used. The initial stage at which cells of neural crest origin were present in the duodenal walls (4-day-old embryos) was earlier than the initial stage (about 6-day-old embryos) reported earlier. This was verified by transmission electron microscopy. Also, the tubulin that is a component of the enteric nervous system was shown to be stable at a low temperature. This tubulin-immunostaining method provides a useful histochemical technique with which to study the development of the enteric ganglion and the function of tubulin as a component of the enteric nervous system.     

Divergent patterns of neural development in larval echinoids and asteroids

The development and organization of the nervous systems of echinoderm larvae are incompletely described. We describe the development and organization of the larval nervous systems of Strongylocentrotus purpuratus and Asterina pectinifera using a novel antibody, 1E11, that appears to be neuron specific. In the early pluteus, the antibody reveals all known neural structures: apical ganglion, oral ganglia, lateral ganglia, and an array of neurons and neurites in the ciliary band, the esophagus, and the intestine. The antibody also reveals several novel features, such as neurites that extend to the posterior end of the larva and additional neurons in the apical ganglion. Similarly, in asteroid larvae the antibody binds to all known neural structures and identifies novel features, including large numbers of neurons in the ciliary bands, a network of neurites under the oral epidermis, cell bodies in the esophagus, and a network of neurites in the intestine. The 1E11 antigen is expressed during gastrulation and can be used to trace the ontogenies of the nervous systems. In S. purpuratus, a small number of neuroblasts arise in the oral ectoderm in late gastrulae. The cells are adjacent to the presumptive ciliary bands, where they project neurites with growth cone-like endings that interconnect the neurons. In A. pectinifera, a large number of neuroblasts appear scattered throughout the ectoderm of gastrulae. The cells aggregate in the developing ciliary bands and then project neurites under the oral epidermis. Although there are several shared features of the larval nervous systems of echinoids and asteroids, the patterns of development reveal fundamental differences in neural ontogeny.     

Ultrastructure and immunocytochemistry of the nervous system of the larvae ofLingula anatina andGlottidia sp. (Brachiopoda)

Summary Planktotrophic brachiopod larvae ofGlottidia sp. have been investigated for the occurrence of glyoxylic acid induced fluorescence in catecholamines (CA), and serotonin-like (5-HT) and neuropeptide FMRFamidelike (FMRFamide) immunoreactivity (ir). The location of CA, 5-HT-ir and FMRFamide-ir cells and processes were compared with the location of neurons and nerve processes found by transmission electron microscopy. The apical ganglion contains 5-HT-ir and FMRFamideir cells and processes and CA processes. From the dorsal part of the apical ganglion extend dorsal 5-HT-ir and FMRFamide-ir processes; from the nine pairs of tentacles stage (9. pt) they project to the ventral ganglion. These dorsal lophophore processes follow themusculus lophophoralis and them. brachialis. The 5-HT-ir and some of the FMRFamide-ir processes project along the muscles to the tentacles. From the ventral part of the apical ganglion extend CA, 5-HT-ir and FMRFamide-ir processes which follow the ciliary band of the lophophore and project to the tentacles. An intense band of CA processes was also observed in the lophophore, but the dorsal/ventral location could not be ascertained. The ventral ganglion contains 5-HT-ir and FMRFamide-ir cells which project either caudally on the metasome or rostrally as part of the dorsal lophophore processes. The neuropil of the ventral ganglion contains CA, 5-HT-ir and FMRFamide-ir processes. The nervous system of the planktotrophic brachiopod larvae seems to consist of a ventral lophophore system innervating the ciliary bands and a dorsal lophophore system including the ventral ganglion innervating the body musculature. The latter system develops later in ontogeny and is regarded as a specialization due to the presence of shells and associated musculature. The former system is regarded as homologous with the nervous system of actinotroch larvae (Phoronida) and planktotrophic larvae of the echinoderms.     

Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence

Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.     

Neural factors that stimulate ecdysteroid synthesis by the larval ring gland ofDrosophila melanogaster

Summary The larval ring gland ofDrosophila melanogaster is the source of ecdysteroids responsible for larval-larval and larval-pupal molting. An extract prepared from theDrosophila larval central nervous system, that presumably contains prothoracicotropic hormone, elicits a significant and dose-dependent in vitro increase in ecdysteroid synthesis by ring glands from wandering third instar larvae. The synthesis of all three ecdysteroids previously identified as ring gland products is elevated by more than two-fold in the presence of neural extract. The maximum response occurs within 30 min and can be sustained for at least two hours after a 30 min exposure to neural extract. No non-neural tissue extracts evoke a response and most of the prothoracicotropic activity originates in the ventral ganglion. However, while extract prepared from larval brains elicits only a slight increase in ecdysteroid synthesis, it enhances the activity of a submaximal dose of ventral ganglion extract. This suggests that two or more neural factors, at least one from the brain lobes and another from the ventral ganglion, interact to stimulate ecdysteroid synthesis by the larval ring gland.Abbreviations CNS central nervous system - HPLC high performance liquid chromatography - PTTH prothoracicotropic hormone - RIA radioimmunoassay     

Nervous system development of two crinoid species, the sea lily Metacrinus rotundus and the feather star Oxycomanthus japonicus

Nervous system development in echinoderms has been well documented, especially for sea urchins and starfish. However, that of crinoids, the most basal group of extant echinoderms, has been poorly studied due to difficulties in obtaining their larvae. In this paper, we report nervous system development from two species of crinoids, from hatching to late doliolaria larvae in the sea lily Metacrinus rotundus and from hatching to cystidean stages after settlement in the feather star Oxycomanthus japonicus. The two species showed a similar larval nervous system pattern with an extensive anterior larval ganglion. The ganglion was similar to that in sea urchins which is generally regarded as derived. In contrast with other echinoderm and hemichordate larvae, synaptotagmin antibody 1E11 failed to reveal ciliary band nerve tracts. Basiepithelial nerve cells formed a net-like structure in the M. rotundus doliolaria larvae. In O. japonicus, the larval ganglion was still present 1 day after settlement when the adult nervous system began to appear inside the crown. Stalk nerves originated from the crown and extended down the stalk, but had no connections with the remaining larval ganglion at the base of the stalk. The larval nervous system was not incorporated into the adult nervous system, and the larval ganglion later disappeared. The aboral nerve center, the dominant nervous system in adult crinoids, was formed at the early cystidean stage, considerably earlier than previously suggested. Through comparisons with nervous system development in other ambulacraria, we suggest the possible nervous system development pattern of the echinoderm ancestor and provide new implications on the evolutionary history of echinoderm life cycles.     

Nervous system development of the sea cucumber Stichopus japonicus

The nervous system development of the sea cucumber Stichopus japonicus was investigated to explore the development of the bilateral larval nervous system into the pentaradial adult form typical of echinoderms. The first nerve cells were detected in the apical region of epidermis in the late gastrula. In the auricularia larvae, nerve tracts were seen along the ciliary band. There was a pair of bilateral apical ganglia consisted of serotonergic nerve cells lined along the ciliary bands. During the transition to the doliolaria larvae, the nerve tracts rearranged together with the ciliary bands, but they were not segmented and remained continuous. The doliolaria larvae possessed nerves along the ciliary rings but strongly retained the features of auricularia larvae nerve pattern. The adult nervous system began to develop inside the doliolaria larvae before the larval nervous system disappears. None of the larval nervous system was observed to be incorporated into the adult nervous system with immunohistochemistry. Since S. japonicus are known to possess an ancestral mode of development for echinoderms, these results suggest that the larval nervous system and the adult nervous system were probably formed independently in the last common ancestor of echinoderms.     

Pyrokinin/PBAN-like peptides in the central nervous system of Drosophila melanogaster

The pyrokinin/pheromone biosynthesis activating neuropeptide (PBAN) family of peptides found in insects is characterized by a 5-amino-acid C-terminal sequence, FXPRLamide. The pentapeptide is the active core required for diverse physiological functions, including stimulation of pheromone biosynthesis in female moths, stimulation of muscle contraction, induction of embryonic diapause in Bombyx mori, and stimulation of melanization in some larval moths. Recently, this family of peptides has been implicated in accelerating the formation of the puparium in a dipteran. Using bioassay and immunocytochemical techniques, we demonstrate the presence of pyrokinin/PBAN-like peptides in the central nervous system of Drosophila melanogaster. Pheromonotropic activity was shown in the moths Helicoverpa zeaand Helicoverpa armigera by using dissected larval nervous systems and adult heads and bodies of D. melanogaster. Polyclonal antisera against the C-terminal ending of PBAN revealed the location of cell bodies and axons in the central nervous systems of larval and adult flies. Immunoreactive material was detected in at least three groups of neurons in the subesophageal ganglion of 3rd instar larvae, pupae, and adults. The ring gland of both larvae and adults contained immunoreactivity. Adult brain-subesophageal ganglion complex possessed additional neurons. The fused ventral ganglia of both larvae and adults contained three pairs of neurons that sent their axons to a neurohemal organ connected to the abdominal nervous system. These results indicate that the D. melanogasternervous system contains pyrokinin/PBAN-like peptides and that these peptides could be released into the hemolymph.     

Development of the nervous system in the brittle star Amphipholis kochii

There are several studies of neural development in various echinoderms, but few on ophiuroids, which develop indirectly via the production of pluteus larvae, as do echinoids. To determine the extent of similarity of neuroanatomy and neural development in the ophiuroids with other echinoderm larvae, we investigated the development of the nervous system in the brittle star Amphipholis kochii (Echinodermata: Ophiuroidea) by immunohistochemistry. Immunoreactive cells first appeared bilaterally in the animal pole at the late gastrula stage, and there was little migration of the neural precursors during A. kochii ontogeny, as is also the case in echinoids and holothuroids. On the other hand, neural specification in the presumptive ciliary band near the base of the arms does occur in ophiuroid larvae and is a feature they share with echinoids and ophiuroids. The ophiopluteus larval nervous system is similar to that of auricularia larvae on the whole, including the lack of a fine network of neurites in the epidermis and the presence of neural connections across the oral epidermis. Ophioplutei possess a pair of bilateral apical organs that differ from those of echinoid echinoplutei in terms of relative position. They also possess coiled cilia, which may possess a sensory function, but in the same location as the serotonergic apical ganglia. These coiled cilia are thought to be a derived structure in pluteus-like larvae. Our results suggest that the neural specification in the animal plate in ophiuroids, holothuroids, and echinoids is a plesiomorphic feature of the Ambulacraria, whereas neural specification at the base of the larval arms may be a more derived state restricted to pluteus-like larvae.     

The evolution of the serotonergic nervous system

The pattern of development of the serotonergic nervous system is described from the larvae of ctenophores, platyhelminths, nemerteans, entoprocts, ectoprocts (bryozoans), molluscs, polychaetes, brachiopods, phoronids, echinoderms, enteropneusts and lampreys. The larval brain (apical ganglion) of spiralian protostomes (except nermerteans) generally has three serotonergic neurons and the lateral pair always innervates the ciliary band of the prototroch. In contrast, brachiopods, phoronids, echinoderms and enteropneusts have numerous serotonergic neurons in the apical ganglion from which the ciliary band is innervated. This pattern of development is much like the pattern seen in lamprey embryos and larvae, which leads the author to conclude that the serotonergic raphe system found in vertebrates originated in the larval brain of deuterostome invertebrates. Further, the neural tube of chordates appears to be derived, at least in part, from the ciliary band of deuterostome invertebrate larvae. The evidence shows no sign of a shift in the dorsal ventral orientation within the line leading to the chordates.     

Peculiarities of ultrastructural organization of the metathoracic ganglion in the locustLocusta migratoria larva

In electron microscopic study of structural organization of the thoracic ganglion of the locust larva of the 1st age (1–2 days after hatching), the data on the structure of motoneurons of the 1st nerve, basal and motor neuropil of the larva were obtained. The effector elements of the larval locust CNS are formed rather early and have the structural plan similar to that in adult insects. However, in the larval motoneurons innervating the flight muscles (longitudinal dorsal muscles, wing depressors) the clearly seen features of immaturity of these nervous elements are revealed. Study of the larval ganglion neuropil has shown that the basal neuropil is morphologically formed sufficiently completely as early as in larvae of the first days after hatching. There are shown longitudinal contacts between axons of the ventral neuropil zone, the presence of axons forming theen-passant contacts as well as the synapses with a heterogeneous set of vesicles in the presynaptic area. The presence of the great number of granular vesicles in the basal neuropil of the locust larva may indicate an important role of catecholamines in the early development of the nervous system in the locust larva.     

Neuronal form in the central nervous system of the tadpole larva of the ascidian Ciona intestinalis.

The dorsal tubular central nervous system (CNS) of the ascidian tadpole larva is a diagnostic feature by which the chordate affinities of this group, as a whole, are recognized. We have used two methods to identify larval neurons of Ciona intestinalis. The first is serial electron microscopy (EM), as part of a dedicated study of the visceral ganglion (1), and the second is the transient transfection of neural plate progeny with green fluorescent protein (GFP) (2), to visualize the soma and its neurites of individual neurons in whole-mounted larvae of C. intestinalis. Our observations reveal that ascidian larval neurons are simple inform, with a single axonal neurite arising from a soma that is either monopolar or has only very few, relatively simple neurites arising from it, as part of a presumed dendritic arbor. Somata in the visceral ganglion giving rise to axons descending in the caudal nerve cord are presumed to be those of motor neurons.     

Isolation of an early neural maker gene abundantly expressed in the nervous system of the ascidian, Halocynthia roretzi

Ascidian tadpole larvae possess a primitive nervous system, which is a prospective prototype of the chordate nervous system. It is composed of relatively few cells but sufficient for complex larval behavior. Here we report on HrETR-1, a gene zygotically expressed in a large proportion of the developing neural cells of the ascidian, Halocynthia roretzi. HrETR-1 is an early neural marker which can be used for analyzing neural differentiation. HrETR-1 expression intensified in most neural cells of genes isolated to date, in both central and peripheral nervous systems including palps as early as the 110-cell stage. Using this gene as a probe, we characterized neural cells in the nervous system as well as confirming their origins. Also, we recognized three types of peripheral epidermal neurons which presumably correlate to the larval neurons previously reported for another ascidian. Among these, five bilateral neurons located in the anterior region of the trunk appeared to be derived from a8.26 blastomeres.     

Localization of a SALMFamide Neuropeptide in the Larval Nervous System of the Sand Dollar Dendraster excentricus

Using immunocytochemical methods we describe the localization of serotonin and the SALMFamide peptide, S1 (GFNSALMFamide), during embryonic and larval development of the echinoid Dendraster excentricus. Anti-SI immunoreactivity first appears in the apical ganglion in late gastrulae at the same time as anti-serotonin immunoreactivity. Initially, anti-S1 immunoreactivity is restricted to fibres of the neuropile, but in later feeding stages, cell bodies are also immunoreactive. Anti-S1 immunoreactivity appears as 2–4 cells in the oral ganglion of early prism stage larvae, whereas anti-serotonin immunoreactivity does not occur in the oral ganglion until the 8-arm stage. Anti-S1 immunoreactivity also occurs in diffuse fibres in the oesophagus and in a single fibre encircling the pyloric sphincter of the gut. A reticular network associated with the apical surface of the epithelial cells of the vestibule of the adult rudiment was anti-S1 immunoreactive. In double-labelling experiments, anti-serotonin and anti-S1 immunoreactivity co-localize in the neuropile of the apical ganglion. The distribution of S1, in association with putative sensory cells in the apical and oral ganglia and with muscles of the oesophagus and gut, suggests S1 may have diverse functions in the larval nervous system. The distribution of anti-S1 immunoreactivity in echinoid embryos and larvae supports the proposal that SALMFamide-like peptides are widely shared in echinoderms and potentially have a fundamental role in neural function.     

Ontogeny of the holothurian larval nervous system: evolution of larval forms

Echinoderm larvae share numerous features of neuroanatomy. However, there are substantial differences in specific aspects of neural structure and ontogeny between the dipleurula-like larvae of asteroids and the pluteus larvae of echinoids. To help identify apomorphic features, we have examined the ontogeny of the dipleurula-like auricularia larva of the sea cucumber, Holothuria atra. Neural precursors arise in the apical ectoderm of gastrulae and appear to originate in bilateral clusters of cells. The cells differentiate without extensive migration, and they align with the developing ciliary bands and begin neurogenesis. Neurites project along the ciliary bands and do not appear to extend beneath either the oral or aboral epidermis. Apical serotonergic cells are associated with the preoral loops of the ciliary bands and do not form a substantial commissure. Paired, tripartite connectives form on either side of the larval mouth that connect the pre-oral, post-oral, and lateral ciliary bands. Holothurian larvae share with hemichordates and bipinnariae a similar organization of the apical organ, suggesting that the more highly structured apical organ of the pluteus is a derived feature. However, the auricularia larva shares with the pluteus larva of echinoids several features of neural ontogeny. Both have a bilateral origin of neural precursors in ectoderm adjacent to presumptive ciliary bands, and the presumptive neurons move only a few cell diameters before undergoing neurogenesis. The development of the holothurian nervous systems suggests that the extensive migration of neural precursors in asteroids is a derived feature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distribution of growth-blocking peptide in the insect central nervous tissue

Parasitization of the armyworm Pseudaletia separata by the endoparasitic wasp Cotesia kariyai inhibits larval growth and delays pupation, conditions necessary for proper maturation of the parasite larvae. Parasitization is correlated with an elevated level of a 25-amino-acid hormone-like peptide, growth-blocking peptide (GBP, ENFSGGCVAGYMRTPDGRCKPTFYQ). Injection of synthetic GBP into nonparasitized larvae dose dependently mimics the effects of parasitization by delaying the larval development. Here we studied the relationship between parasitization and both the production and distribution of GBP in central nervous tissues. We found that parasitization is correlated with an elevated expression of GBP mRNA, and increased concentrations of both proGBP and GBP in the host insect brain and subesophageal ganglion. The increase in proGBP precedes that of the mature GBP by about 12 h. In situ hybridization analysis using sections of parasitized and nonparasitized larval brains showed strong expression of GBP mRNA in perineural cells and/or class I neuroglia in the rind of both larval brains. The expression in parasitized larval brain-subesophageal ganglion is approximately two- to threefold higher than that in nonparasitized larvae. The presence of GBP in insect neural tissue, and its role in inhibiting growth, suggest an involvement in the regulation of neurosecretory cells.