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.     

Developmental expression of neuromodulators in the central complex of the grasshopper Schistocerca gregaria

The central complex is a major integrative region within the insect brain with demonstrated roles in spatial orientation, the regulation of locomotor behavior, and sound production. In the hemimetabolous grasshopper, the central complex comprises the protocerebral bridge, central body (CB), ellipsoid body, noduli, and accessory lobes, and this modular organization develops entirely during embryogenesis. From a biochemical perspective, a range of neuroactive substances has been demonstrated in these modules of the adult central complex, but little is known about their developmental expression. In this study, we use matrix‐assisted laser desorption/ionization‐imaging mass spectrometry on single brain slices to confirm the presence of several peptide families (tachykinin, allatostatin, periviscerokinin/pyrokinin, FLRFamide, and neuropeptide F) in the adult central complex and then use immunohistochemistry and histology to examine their developmental expression, together with that of the indolamin serotonin, and the endogenous messenger nitric oxide (NO; via its synthesizing enzyme). We find that each neuromodulator is expressed according to a unique, stereotypic, pattern within the various modules making up the central complex. Neuropeptides such as tachykinin (55%) and allatostatin (65%), and the NO‐synthesizing enzyme diaphorase (70%), are expressed earlier during embryonic development than the biogenic amine serotonin (80%), whereas periviscerokinin‐like peptides and FLRFamide‐like peptides begin to be expressed only postembryonically. Within the CB, these neuroactive substances are present in tangential projection neurons before they appear in columnar neurons. There is also no colocalization of serotonin‐positive and peptide‐positive projections up to the third larval instar during development, consistent with the clear dorsoventral layering of the neuropil we observe. Our results provide the first neurochemical fingerprint of the developing central complex in an hemimetabolous insect. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Cell death shapes embryonic lineages of the central complex in the grasshopper Schistocerca gregaria

We have investigated cell death in identified lineages of the central complex in the embryonic brain of the grasshopper Schistocerca gregaria. Progeny from these lineages lie in the pars intercerebralis and direct projections to the protocerebral bridge and then the central body via the w, x, y, z tracts. Osmium‐ethyl gallate staining reveals pycnotic cells exclusively in cortical regions, and concentrated specifically within the lineages of the W, X, Y, Z neuroblasts. Minimal cell death occurs in a sporadic, nonpatterned manner, in other protocerebral regions. Immunohistochemistry reveals pycnotic cells express the enzyme cleaved Caspase‐3 in their cytoplasm and are therefore undergoing programmed cell death (apoptosis). The number of pycnotic bodies in lineages of the pars intercerebralis varies with age: small numbers are present in the Y, Z lineages early in embryogenesis (42%), the number peaks at 67–80%, and then declines and disappears late in embryogenesis. Cell death may encompass up to 20% of a lineage at mid‐embryogenesis. Peak cell death occurs shortly after maximum neurogenesis in the Y, Z lineages, and is maintained after neurogenesis has ceased in these lineages. Cell death within a lineage is patterned. Apoptosis is more pronounced among older cells and almost absent among younger cells. This suggests that specific subsets of progeny will be culled from these lineages, and we speculate about the effect of apoptosis on the biochemical profile of such lineages. J. Morphol. 271:949–959, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

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.     

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.     

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.     

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.     

Building the antennal lobe: engrailed expression reveals a contribution from protocerebral neuroblasts in the grasshopper Schistocerca gregaria

The expression pattern of the engrailed protein was studied in neuroblasts which delaminate at the border of the protocerebrum and antennal lobe of the deutocerebrum in the early embryonic brain of the grasshopper. The antennal lobe is a complex structure comprising both glomerular and non-glomerular components, a cellular organization which distinguishes it from the striate-like neuropil comprising the remainder of the deutocerebrum. Early in embryogenesis engrailed expression in the protocerebrum is restricted to a compact block of neuroblasts located at its interface with the antennal lobe. Subsequently engrailed expression in these cells disappears in a stepwise manner from anterior to posterior so that by 37% of embryogenesis only a single row of three engrailed positive neuroblasts and their progeny remains. Contemporaneously engrailed expression reappears in a group of more anterior progeny deriving from neuroblasts which are no longer immunoreactive. The three remaining engrailed positive neuroblasts then become separated from their non-immunoreactive neighbours by an invagination of the perineurium called the lateral cleft and come to lie completely within the developing antennal lobe. These cells then direct columns of immunoreactive progeny centrifugally towards the centre of the lobe. Such a protocerebral contribution to the antennal lobe suggests that the evolution and ontogeny of this brain region need to be reconsidered.     

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.     

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.     

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.     

A cellular network of dye-coupled glia associated with the embryonic central complex in the grasshopper Schistocerca gregaria

The central complex of the grasshopper (Schistocerca gregaria) brain comprises a modular set of neuropils, which develops after mid-embryogenesis and is functional on hatching. Early in embryogenesis, Repo-positive glia cells are found intermingled among the commissures of the midbrain, but then redistribute as central complex modules become established and, by the end of embryogenesis, envelop all midbrain neuropils. The predominant glia associated with the central body during embryogenesis are glutamine synthetase-/Repo-positive astrocyte-like glia, which direct extensive processes (gliopodia) into and around midbrain neuropils. We used intracellular dye injection in brain slices to ascertain whether such glia are dye-coupled into a communicating cellular network during embryogenesis. Intracellular staining of individual cells located at any one of four sites around the central body revealed a population of dye-coupled cells whose number and spatial distribution were stereotypic for each site and comparable at both 70 and 100% of embryogenesis. Subsequent immunolabeling confirmed these dye-coupled cells to be astrocyte-like glia. The addition of n-heptanol to the bathing saline prevented all dye coupling, consistent with gap junctions linking the glia surrounding the central body. Since dye coupling also occurred in the absence of direct intersomal contacts, it might additionally involve the extensive array of gliopodia, which develop after glia are arrayed around the central body. Collating the data from all injection sites suggests that the developing central body is surrounded by a network of dye-coupled glia, which we speculate may function as a positioning system for the developing neuropils of the central complex.     

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