Glia associated with central complex lineages in the embryonic brain of the grasshopper Schistocerca gregaria

We have investigated the pattern of glia associated with central complex lineages in the embryonic brain of the grasshopper Schistocerca gregaria. Using the glia-specific marker Repo, we identified glia associated externally with such lineages, termed lineage-extrinsic glia, and glia located internally within the lineages, termed lineage-intrinsic glia. Populations of both glial types increase up to 60 % of embryogenesis, and thereafter decrease. Extrinsic glia change their locations over time, while intrinsic ones are consistently found in the more apical part of a lineage. Apoptosis is not observed for either glial type, suggesting migration is a likely mechanism accounting for changes in glial number. Proliferative glia are present both within and without individual lineages and two glial clusters associated with the lineages, one apically and the other basally, may represent sources of glia.     

Postembryonic development of astrocyte-like glia of the central complex in the grasshopper Schistocerca gregaria

Central complex modules in the postembryonic brain of the grasshopper Schistocerca gregaria are enveloped by Repo-positive/glutamine-synthetase-positive astrocyte-like glia. Such cells constitute Rind-Neuropil Interface glia. We have investigated the postembryonic development of these glia and their anatomical relationship to axons originating from the w, x, y, z tract system of the pars intercerebralis. Based on glutamine synthetase immunolabeling, we have identified four morphological types of cells: bipolar type 1 glia delimit the central body but only innervate its neuropil superficially; monopolar type 2 glia have a more columnar morphology and direct numerous gliopodia into the neuropil where they arborize extensively; monopolar type 3 glia are found predominantly in the region between the noduli and the central body and have a dendritic morphology and their gliopodia project deeply into the central body neuropil where they arborize extensively; multipolar type 4 glia link the central body neuropil with neighboring neuropils of the protocerebrum. These glia occupy type-specific distributions around the central body. Their gliopodia develop late in embryogenesis, elongate and generally become denser during subsequent postembryonic development. Gliopodia from putatively type 3 glia within the central body have been shown to lie closely apposed to individual axons of identified columnar fiber bundles from the w, x, y, z tract system of the central complex. This anatomical association might offer a substrate for neuron/glia interactions mediating postembryonic maturation of the central complex.     

Proliferative cell types in embryonic lineages of the central complex of the grasshopper Schistocerca gregaria

The central complex of the grasshopper Schistocerca gregaria develops to completion during embryogenesis. A major cellular contribution to the central complex is from the w, x, y, z lineages of the pars intercerebralis, each of which comprises over 100 cells, making them by far the largest in the embryonic protocerebrum. Our focus has been to find a cellular mechanism that allows such a large number of cell progeny to be generated within a restricted period of time. Immunohistochemical visualization of the chromosomes of mitotically active cells has revealed an almost identical linear array of proliferative cells present simultaneously in each w, x, y, z lineage at 50% of embryogenesis. This array is maintained relatively unchanged until almost 70% of embryogenesis, after which mitotic activity declines and then ceases. The array is absent from smaller lineages of the protocerebrum not associated with the central complex. The proliferative cells are located apically to the zone of ganglion mother cells and amongst the progeny of the neuroblast. Comparisons of cell morphology, immunoreactivity (horseradish peroxidase, repo, Prospero), location in lineages and spindle orientation have allowed us to distinguish the proliferative cells in an array from neuroblasts, ganglion mother cells, neuronal progeny and glia. Our data are consistent with the proliferative cells being secondary (amplifying) progenitors and originating from a specific subtype of ganglion mother cell. We propose a model of the way that neuroblasts, ganglion mother cells and secondary progenitors together produce the large cell numbers found in central complex lineages.     

Patterns of dye coupling involving serotonergic neurons provide insights into the cellular organization of a central complex lineage of the embryonic grasshopper Schistocerca gregaria

All eight neuroblasts from the pars intercerebralis of one protocerebral hemisphere whose progeny contribute fibers to the central complex in the embryonic brain of the grasshopper Schistocerca gregaria generate serotonergic cells at stereotypic locations in their lineages. The pattern of dye coupling involving these neuroblasts and their progeny was investigated during embryogenesis by injecting fluorescent dye intracellularly into the neuroblast and/or its progeny in brain slices. The tissue was then processed for anti-serotonin immunohistochemistry. A representative lineage, that of neuroblast 1-3, was selected for detailed study. Stereotypic patterns of dye coupling were observed between progeny of the lineage throughout embryogenesis. Dye injected into the soma of a serotonergic cell consistently spread to a cluster of between five and eight neighboring non-serotonergic cells, but never to other serotonergic cells. Dye injected into a non-serotonergic cell from such a cluster spread to other non-serotonergic cells of the cluster, and to the immediate serotonergic cell, but never to further serotonergic cells. Serotonergic cells tested from different locations within the lineage repeat this pattern of dye coupling. All dye coupling was blocked on addition of an established gap junctional blocker (n-heptanol) to the bathing medium. The lack of coupling among serotonergic cells in the lineage suggests that each, along with its associated cluster of dye-coupled non-serotonergic cells, represents an independent communicating pathway (labeled line) to the developing central complex neuropil. The serotonergic cell may function as the coordinating element in such a projection system.     

Fascicle switching generates a chiasmal neuroarchitecture in the embryonic central body of the grasshopper Schistocerca gregaria

The central body is a prominent neuropilar structure in the midbrain of the grasshopper and is characterized by a fan-shaped array of fiber columns, which are part of a chiasmal system linking anterior and posterior commissures. These columns are established during embryogenesis and comprise axons from cell clusters in the pars intercerebralis, which project to the central body via the so-called w, x, y, z tracts. Up to mid-embryogenesis the primary axon scaffold in both the brain and ventral nerve cord comprises a simple orthogonal arrangement of commissural and longitudinal fiber pathways. No chiasmata are present and this pattern is maintained during subsequent development of the ventral nerve cord. In the midbrain, individual axons entering the commissural system from each of the w, x, y, z tracts after mid-embryogenesis (55%) are seen to systematically de-fasciculate from an anterior commissure and re-fasciculate with another more posterior commissure en route across the midline, a feature we call "fascicle switching". Since the w, x, y, z tracts are bilaterally symmetrical, fascicle switching generates chiasmata at stereotypic locations across the midbrain. Choice points for leaving and entering fascicles mark the anterior and posterior positions of each future column. As the midbrain neuropil expands, the anterior and posterior groups of commissures condense, so that the chiasmata spanning the widening gap between them become progressively more orthogonally oriented. A columnar neuroarchitecture resembling that of the adult central body is already apparent at 70% of embryogenesis.     

An ontogenetic analysis of locustatachykinin-like expression in the central complex of the grasshopper Schistocerca gregaria

We have investigated the ontogenetic basis of locustatachykinin-like expression in a group of cells located in the pars intercerebralis of the grasshopper midbrain. These cells project fibers to the protocerebral bridge and the central body via a characteristic set of fiber bundles called the w, x, y, z tracts. Lineage analyses associate the immunoreactive cells with one of four neuroblasts (termed W, X, Y, Z) in each protocerebral hemisphere of the early embryo. Locustatachykinin is a ubiquitous myotropic peptide among the insects and its expression in the pars intercerebralis begins at approximately 60-65% of embryogenesis. This coincides with the appearance of the columnar neuroarchitecture characteristic of the central body. The number of immunoreactive cells in a given lineage is initially small, increases significantly in later embryogenesis, and attains the adult situation (about 7% of a lineage) in the first larval instar after hatching. Although each neuroblast generates progeny displaying a spectrum of cell body sizes, there is a clear morphological gradient, which reflects birth order within the lineage. Locustatachykinin expressing cells are located stereotypically at or near the tip of their lineage, which an age profile reveals places them amongst the first born progeny of their respective neuroblasts. Although these neuroblasts begin to generate progeny at approximately 25-27% of embryogenesis, their daughter cells remain quiescent with respect to locustatachykinin expression for over 30% of embryogenesis.     

Multipotent neuroblasts generate a biochemical neuroarchitecture in the central complex of the grasshopper Schistocerca gregaria

We have examined the developmental expression of the neuromodulators locustatachykinin, leucokinin-1, allatostatin and serotonin in a subset of lineages (Y, Z) of the central complex in the brain of the grasshopper Schistocerca gregaria. First, we show that all these neuromodulators are expressed in the same lineages during embryogenesis. The neuroblasts generating these lineages are therefore biochemically multipotent. Second, the neurons expressing the different neuromodulators are found clustered at stereotypic locations in their respective lineages. Locustatachykinin and leucokinin-1 map to the apical region of the lineage, allatostatin medially and serotonin to the base of the lineage. Since the location in these lineages translates into their birth order, we have been able ontogenetically to analyse their biochemical expression patterns. The age-profile within a lineage reveals that locustatachykinin- and leucokinin-1-expressing neurons are born first, then allatostatin neurons and finally serotoninergic neurons. Co-expression has been tested for serotonin with locustatachykin, leucokinin-1 or allatostatin and is negative but is positive for locustatachykinin and leucokinin-1, consistent with the stereotypic location of cells in the lineages. The delay between the birth of a neuron and the expression of its neuromodulator is stereotypic for each substance. Combined with a known birth date, this delay translates into a developmental expression pattern for the central complex itself.     

Astrocyte-like glia associated with the embryonic development of the central complex in the grasshopper <Emphasis Type="Italic">Schistocerca gregaria</Emphasis>

In this study we employed the expression of the astrocyte-specific enzyme glutamine synthetase, in addition to the glia-specific marker Repo, to characterize glia cell types associated with the embryonic development of the central complex in the grasshopper Schistocerca gregaria. Double labeling experiments reveal that all glutamine synthetase-positive cells associated with the central complex are also Repo-positive and horseradish peroxidase-negative, confirming they are glia. Early in embryogenesis, prior to development of the central complex, glia form a continuous population extending from the pars intercerebralis into the region of the commissural fascicles. Subsequently, these glia redisperse to envelop each of the modules of the central complex. No glial somata are found within the central complex neuropils themselves. Since glutamine synthetase is expressed cortically in glia, it allows their processes as well as their soma locations to be visualized. Single cell reconstructions reveal one population of glia as directing extensive ensheathing processes around central complex neuropils such as the central body, while another population projects columnar-like arborizations within the central body. Such arborizations are only seen in central complex modules after their neuroarchitecture has been established suggesting that the glial arborizations project onto a prior scaffold of neurons or tracheae.     

Embryonic expression of muscle-specific antigens in the grasshopper Schistocerca gregaria

Monoclonal antibodies (MAbs) are used to investigate molecules that are expressed during embryonic muscle differentiation and that may be involved in muscle pioneer and muscle attachment site formation. MAb F2A5 immunoreactivity appears in all muscle pioneers as soon as they extend processes, and continues in all muscle precursors. MAb 4H1 immunoreactivity is strongly expressed only after mesodermal cells have fused with the muscle pioneers; then it is concentrated at their growth-cone-like ends near developing attachment sites. During later embryonic development, MAb F2A5 and MAb 4H1 immunoreactivity become associated with the myofibrillar network. Biochemical experiments indicate that MAb 4H1 recognises a 47 kDa antigen, and MAb F2A5 recognises an 80 kDa antigen.     

Ontogeny of identified cells from the median domain in the embryonic brain of the grasshopper Schistocerca gregaria

In this paper, we propose an ontogeny for previously identified cells from the median domain in the midline of the embryonic brain of the grasshopper Schistocerca gregaria. The so-called lateral cells (LCs) are characteristically located laterally within the median domain at its border with the protocerebral hemispheres. The LC occurs singly and can be identified in the early embryo on the basis of their expression of the cell surface lipocalin Lazarillo. Using immunocytochemical, dye injection, electron microscopical and histological methods, we show that these LC are neurons and derive as postmitotic cells directly from the epithelium of the median domain. Further, they and the other identified cells of the median domain such as the protocerebral commissure pioneers (PCP), co-express the Mes-3 antigen, consistent with a derivation from the mesectodermal germ layer of the embryo. Subsequent to axogenesis, electron microscopy reveals that these Mes-3-expressing LC fasciculate with the co-expressing PCPs within the developing protocerebral commissure. We present a model for the origin of all these cells based on histological data and bromodeoxyuridine incorporation. The model suggests a delamination of cells from the mesectoderm followed by a migration to their ultimate sites within the median domain.     

Gliogenesis in the embryonic brain of the grasshopper Schistocerca gregaria with particular focus on the protocerebrum prior to mid-embryogenesis

I investigate the pattern of gliogenesis in the brain of the grasshopper Schistocerca gregaria prior to mid-embryogenesis, with particular focus on the protocerebrum. Using the glia-specific marker Repo and the neuron-specific marker HRP, I identify three types of glia with respect to their respective positions in the brain: surface glia form the outmost cell layer ensheathing the brain; cortex glia are intermingled with neuronal somata forming the brain cortex; and neuropil glia are associated with brain neuropils. The ontogeny of each glial type has also been studied. At 24 % of embryogenesis, a few glia are observed in each hemisphere of the proto-, deuto- and tritocerebrum. In each protocerebral hemisphere, such glia form a cluster that expands rapidly during later development. Closer examination reveals proliferative glia in such clusters at ages spanning from 24 to 36 % of embryogenesis, indicating that glial proliferation may account for the expansion of the clusters. Data derived from 33–39 % of embryogenesis suggest that, in the protocerebrum, each type of glia is likely to be generated by its respective progenitor-forming clusters. Moreover, the glial cluster located at the anterior end of the brain can give rise to both surface glia and cortex glia that populate the protocerebrum via subsequent migration. Proliferation is observed for all three glial types, indicating a possible source for the glia.     

Germ line development in the grasshopper Schistocerca gregaria: vasa as a marker

Vasa is a widely conserved germline marker, both in vertebrates and invertebrates. We identify a vasa orthologue, Sgvasa, and use it to study germline development in the grasshopper Schistocerca gregaria, a species in which no germ plasm has been identified. In adults, Sgvasa is specifically expressed in the ovary and testis. It is expressed at high levels during early oogenesis, but no detectable vasa RNA and little Vasa protein are present in mature unlaid eggs. None appears to be localized to any defined region of the egg cortex, suggesting that germline specification may not depend on maternal germ plasm expressing vasa. Vasa protein is expressed in most cleavage energids as they reach the egg surface and persists at high levels in most cells aggregating to form the embryonic primordium. However, after gastrulation, Vasa protein persists only in extraembryonic membranes and in cells at the outer margin of the late heart-stage embryo. In the embryo, it then become restricted to cells at the dorsal margin of the forming abdomen. In older embryos, these Vasa-positive cells move toward the midline; Vasa protein accumulates asymmetrically in their cytoplasm, a pattern closely resembling that of germ cells in late embryonic gonads. Thus, we suggest that the Vasa-stained cells in the abdominal margin are germ cells, as proposed by Nelson (1934), and not cardioblasts, as has been proposed by others.     

Embryonic development of the sensory innervation of the antenna of the grasshopper Schistocerca gregaria

The establishment of the sensory nervous system of the antenna of the grasshopper Schistocerca gregaria was examined using immunocytochemical methods and in the light of the appendicular and articulated nature of this structure. The former is demonstrated first by the expression pattern of the segment polarity gene engrailed in the head neuromere innervating the antenna, the deutocerebrum. Engrailed expression is present in identified deutocerebral neuroblasts and, as elsewhere in the body, is continuous with cells of the posterior epithelium of the associated appendage, in this case the antenna. Second, early expression of the glial homeobox gene reversed polarity (repo) in the antenna is by a stereotypic pair of cells at the antenna base, a pattern we show is repeated metamerically for each thoracic appendage of the embryo. Subsequently, three regions of Repo expression (A1, A2, A3) are seen within the antenna, and may represent a preliminary form of articulation. Bromodeoxyuridine incorporation reveals that these regions are sites of intense cell differentiation. Neuron-specific horseradish peroxidase and Lazarillo expression confirm that the pioneers of the ventral and dorsal tracts of the antennal sensory nervous system are amongst these differentiating cells. Sets of pioneers appear simultaneously in several bands and project confluent axons towards the antennal base. We conclude that the sensory nervous system of the antenna is not pioneered from the tip of the antenna alone, but in a stepwise manner by cells from several zones. The early sensory nervous systems of antenna, maxilla and leg therefore follow a similar developmental program consistent with their serially homologous nature.     

Homologous patterns in the embryonic development of the peripheral nervous system in the grasshopper Schistocerca gregaria and the fly Drosophila melanogaster.

To determine the generality of developmental mechanisms involved in the construction of the insect nervous system, the embryonic development of the peripheral nervous system in the grasshopper Schistocerca gregaria was characterized at the level of identified neurons and nerve branches and then compared to that previously described from the fly Drosophila melanogaster. For this, immunocytochemistry using a neuron-specific antibody was carried out on staged grasshopper embryos. Our results show that initially a simple peripheral nerve scaffolding is established in each segment of the animal. This scaffolding consists of a pair of intersegmental nerves that are formed by identified afferent and efferent pioneer neurons and a pair of segmental nerves that are formed by afferent pioneers situated in limb buds. Subsequently, identified sets of sensory neurons differentiate in a stereotyped spatiotemporal pattern in dorsal, lateral and ventral clusters in each segment and project their axons onto these nerves. Although segment-specific differences exist, serial homologs of the developing nerves and sensory neurons can be identified. A comparison of these results with those obtained from Drosophila shows that virtually the same pattern of peripheral nerves and sensory structures is formed in both species. This indicates that the construction of the peripheral nervous system in extremely divergent modern insects relies on conserved developmental mechanisms that evolved in ancestral insects over 300 million years ago.     

A single cell analysis of engrailed expression in the early embryonic brain of the grasshopper Schistocerca gregaria: ontogeny and identity of the secondary headspot cells

The expression pattern of the segment polarity gene engrailed was studied at the single cell level in the protocerebrum of the early embryonic brain of the grasshopper Schistocerca gregaria, the neuromere containing the secondary headspot cells. The engrailed protein is first expressed in the protocerebrum at about 22% of embryogenesis by a group of identified neuroblasts bordering the antennal lobe. The number of immunoreactive neuroblasts increases up to 26% of embryogenesis and then rapidly declines so that by 30% only the three most posterior remain immunoreactive. These three neuroblasts become incorporated into the developing antennal lobe of the deutocerebrum. Subsequently, there is a progressive re-expression of the engrailed protein in the protocerebrum by the so-called six secondary headspot cells. These are the first born sibling progeny of three identified protocerebral neuroblasts which themselves expressed the engrailed protein prior to generating their lineages, and so represents a reacquisition of engrailed expression within identified clones. The secondary headspot cells are neurons which direct axonal processes into the developing optic tract and so contribute to the primary axon scaffold of the brain. From our analysis of their ontogeny, we conclude that the secondary headspot cells do not represent a segmental border in the brain.     

A role for Fringe in segment morphogenesis but not segment formation in the grasshopper, Schistocerca gregaria

Studies of somitogenesis in vertebrates have identified a number of genes that are regulated by a periodic oscillator that patterns the pre-somitic mesoderm. One of these genes, hairy, is homologous to a Drosophila segmentation gene that also shows periodic spatial expression. This, and the periodic expression of a zebrafish homologue of hairy during somitogenesis, has suggested that insect segmentation and vertebrate somitogenesis may use similar molecular mechanisms and possibly share a common origin. In chicks and mice expression of the lunatic fringe gene also oscillates in the presomitic mesoderm. Fringe encodes an extracellular protein that regulates Notch signalling. This, and the finding that mutations in Notch or its ligands disrupt somite patterning, suggests that Notch signalling plays an important role in vertebrate somitogenesis. Although Notch signalling is not known to play a role in the formation of segments in Drosophila, we reasoned that it might do so in other insects such as the grasshopper, where segment boundaries form between cells, not between syncytial nuclei as they do in Drosophila. Here we report the cloning of a single fringe gene from the grasshopper Schistocerca. We show that it is not detectably expressed in the forming trunk segments of the embryo until after segment boundaries have formed. We conclude that fringe is not part of the mechanism that makes segments in Schistocerca. Thereafter it is expressed in a pattern which shows that it is a downstream target of the segmentation machinery and suggests that it may play a role in segment morphogenesis. Like its Drosophila counterpart, Schistocerca fringe is also expressed in the eye, in rings in the legs, and during oogenesis, in follicle cells. Received: 14 October 1999 / Accepted: 18 January 2000     

Synaptic input from serial chordotonal organs onto segmentally homologous interneurons in the grasshopper Schistocerca gregaria

We investigated the synaptic inputs from the serially homologous pleural, tympanal and wing-hinge chordotonal organs onto a set of identified homologous interneurons (714, 539, 529) in the ventral nerve cord of the grasshopper Schistocerca gregaria. Cobalt backfills show that afferents from all chordotonal organs project into stereotypic tracts in the central nervous system in which intracellular staining reveals the interneurons to have dendritic arborizations. Neuron 714 was found to receive excitatory bilateral synaptic input from all the serial chordotonal organs tested, from the second thoracic segment down to the seventh abdominal segment. Neuron 531, by contrast, only receives input from the chordotonal afferents on the first abdominal segment; those on the axon side are excitatory, while those on the soma side are inhibitory. The pattern of chordotonal input onto neuron 529 is similar to that seen for neuron 714, with the exception that neuron 529 receives no input from the forewing chordotonal organs. The pattern of afferent connectivities onto neurons 714, 531 and 529 differs with respect to those afferents which synapse directly or indirectly with the respective neuron. The synaptic inputs demonstrate a segmental specialization in the chordotonal system and thereby offer an insight into information processing in a modular sensory system.     

Evidence that the primary brain commissure is pioneered by neurons with a peripheral-like ontogeny in the grasshopper Schistocerca gregaria

The commissures represent a major neuroarchitectural feature of the central nervous system of insects and vertebrates alike. The adult brain of the grasshopper comprises 72 such commissures, the first of which is established in the protocerebral midbrain by three sets of pioneer cells at around 30% of embryogenesis. These pioneers have been individually identified via cellular, molecular and intracellular dye injection techniques. Their ontogenies, however, remain unclear. The progenitor cells of the protocerebral midbrain are shown via Annulin immunocytochemistry to be compartmentalized, belonging either to the protocerebral hemispheres or the so-called median domain. Serial reconstructions based on bromodeoxyuridine incorporation confirm that their lineages do not intermingle. Dye injection into progenitor cells and progeny confirms this compartmentalization, and reveals that none of the pioneers are associated with a lineage of cells deriving from a protocerebral neuroblast or midline precursor. Immunocytochemical data as well as dye injection into identified pioneers over several developmental stages indicate that they differentiate directly from epithelial cells, but not from classical progenitor cells. That the commissural pioneers of the protocerebrum represent modified epithelial cells involves a different ontogeny to that described for pioneers in the ventral nerve cord, but parallels that of pioneer neurons of the peripheral nervous system.