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.

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.

Building the central complex of the grasshopper Schistocerca gregaria: axons pioneering the w, x, y, z tracts project onto the primary commissural fascicle of the brain.

The central complex is a major neuropilar structure in the insect brain whose distinctive, modular, neuroarchitecture in the grasshopper is exemplified by a bilateral set of four fibre bundles called the w, x, y and z tracts. These columns represent the stereotypic projection of axons from the pars intercerebralis into commissures of the central complex. Each column is established separately during early embryogenesis in a clonal manner by the progeny of a subset of four identified protocerebral neuroblasts. We report here that dye injected into identified pioneers of the primary brain commissure between 31 and 37% of embryogenesis couples to cells in the pars intercerebralis which we identify as progeny of the W, X, Y, or Z neuroblasts. These progeny are the oldest within each lineage, and also putatively the first to project an axon into the protocerebral commissure. The axons of pioneers from each tract do not fasciculate with one other prior to entry into the commissure, thereby prefiguring the modular w, x, y, z columns of the adult central complex. Within the commissure, pioneer axons from columnar tracts fasciculate with the growth cones of identified pioneers of the existing primary fascicle and do not pioneer a separate fascicle. The results suggest that neurons pioneering a columnar neuroarchitecture within the embryonic central complex utilize the existing primary commissural scaffold to navigate the brain midline.

Embryonic development of a peripheral nervous system: nerve tract associated cells and pioneer neurons in the antenna of the grasshopper Schistocerca gregaria.

The grasshopper antenna is an articulated appendage associated with the deutocerebral segment of the head. In the early embryo, the meristal annuli of the antenna represent segment borders and are also the site of differentiation of pioneer cells which found the dorsal and ventral peripheral nerve tracts to the brain. We report here on another set of cells which appear earlier than the pioneers during development and are later found arrayed along these tracts at the border of epithelium and lumen. These so-called nerve tract associated cells differ morphologically from pioneers in that they are bipolar, have shorter processes, and are not segmentally organized in the antenna. Nerve tract associated cells do not express horseradish peroxidase and so are not classical neurons. They do not express antigens such as repo and annulin which are associated with glia cells in the nervous system. Nerve tract associated cells do, however, express the mesodermal/mesectodermal cell surface marker Mes-3 and putatively derive from the antennal coelom and then migrate to the epithelium/lumen border. Intracellular recordings show that such nerve tract associated cells have resting potentials similar to those of pioneer cells and can be dye coupled to the pioneers. Similar cell types are present in the maxilla, a serially homologous appendage on the head. The nerve tract associated cells are organized into a cellular scaffold which we speculate may be relevant to the navigation of pioneer and sensory axons in the early embryonic antennal nervous system.

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.

Embryonic development of the sensory innervation of the clypeo-labral complex: further support for serially homologous appendages in the locust.

The clypeo-labrum, or upper lip, of insects is intimately involved in feeding behavior and is accordingly endowed with a rich sensory apparatus. In the present study we map the temporal appearance of all major clusters of sensory cells on this structure in the locust during the first half of embryogenesis. The identities of these sensory cell clusters were defined according to the origin of the branching point of their axons from the labral sensory nerve as seen at mid-embryogenesis. The first sensory cells to differentiate from the labral epithelium do so at stereotypic sites beginning at around 32% of embryogenesis. Bilaterally symmetrical clusters of differentiated neurons rapidly appear and pioneering of the labral sensory nerve on each side is performed by a specific cell from each cluster. This cell directs its axon anteriorly towards a bilaterally symmetrical pair of cells, the frontal commissure pioneers, on either side of the developing frontal ganglion. The final trajectory of the sensory nerve within the labrum closely matches the pattern of Repo-expressing glial cells. The majority of the sensory cell clusters differentiate during embryogenesis, but the number of sensory cells in some clusters are modified significantly during postembryonic development. Comparing the innervation pattern of the clypeo-labrum with that of other mouthparts and the leg at mid-embryogenesis, we find a striking similarity in organization which we interpret as support for the homologous appendage hypothesis.

Morphological and molecular data argue for the labrum being non-apical, articulated, and the appendage of the intercalary segment in the locust.

Our analysis of head segmentation in the locust embryo reveals that the labrum is not apical as often interpreted but constitutes the topologically fused appendicular pair of appendages of the third head metamere. Using molecular, immunocytochemical and retrograde axonal staining methods we show that this metamere, the intercalary segment, is innervated by the third brain neuromere-the tritocerebrum. Evidence for the appendicular nature of the labrum is firstly, the presence of an engrailed stripe within its posterior epithelium as is typical of all appendages in the early embryo. Secondly, the labrum is innervated by a segmental nerve originating from the third brain neuromere (the tritocerebrum). Immunocytochemical staining with Lazarillo and horseradish peroxidase antibodies reveal that sensory neurons on the labrum contribute to the segmental (tritocerebral) nerve via the labral nerve in the same way as for the appendages immediately anterior (antenna) and posterior (mandible) on the head. All but one of the adult and embryonic motoneurons innervating the muscles of the labrum have their cell bodies and dendrites located completely within the tritocerebral neuromere and putatively derive from engrailed expressing tritocerebral neuroblasts. Molecular evidence (repo) suggests the labrum is not only appendicular but also articulated, comprising two jointed elements homologous to the coxa and trochanter of the leg.

Primary commissure pioneer neurons in the brain of the grasshopper Schistocerca gregaria: development, ultrastructure, and neuropeptide expression.

The bilaterally paired primary commissure pioneer neurons in the median domain of the grasshopper brain are large, descending interneurons that uniquely express the TERM-1 antigen, even in the adult. After pioneering the primary interhemispheric brain commissure, these neurons extend TERM-1-immunoreactive collaterals into most parts of the brain except the mushroom bodies. In this report, the authors show that the TERM-1 antigen is located in the cell body cytoplasm of these neurons and not on the membranes. Screening with antisera to insect neuropeptides reveals that an antiserum recognizing peptides of the leucokinin family labels the cell body cytoplasm of the primary commissure neurons. Leucokinin-related peptides are known to modulate motility of visceral muscle, play a role in diuresis, and are likely to be neuromodulators in the insect nervous system. The primary commissure neurons differ ultrastructurally from median neurosecretory cells in that their cell body cytoplasm is more extensive, contains high numbers of mitochondria and extensive endoplasmic reticulum, but does not contain neurosecretory granules. In the adult, the cell somata are enveloped by multiple glia membranes and associated trophospongia. According to these ultrastructural characteristics, the primary commissure pioneers are not classical neurosecretory cells.

Lazarillo expression reveals a subset of neurons contributing to the primary axon scaffold of the embryonic brain of the grasshopper Schistocerca gregaria.

The authors studied the contribution of seven clusters of Lazarillo-expressing cells to the primary axon scaffold of the brain in the grasshopper Schistocerca gregaria from 26% to 43% of embryogenesis. Each cluster, which was numbered according to when Lazarillo expression first appeared, was uniquely identifiable on the basis of its stereotypic position in the brain and the number of Lazarillo-expressing cells it contained. At no time during embryogenesis was Lazarillo expression found in brain neuroblasts: It was found only in progeny. For ease of analysis, axogenesis was followed in a cell cluster that contained only a single Lazarillo-expressing cell (the lateral cell) in the dorsal median domain of the brain midline. Bromodeoxyuridine incorporation revealed the presence of only a single midline precursor cell in this region during embryogenesis. Intracellular injection of Lucifer yellow into the lateral cell at various ages showed that there was no dye coupling to the midline precursor or to the nearby term-1-expressing primary commissure pioneers. The lateral cell is not related lineally to these cells and most likely differentiates directly from the neuroectoderm of the brain midline. Lazarillo expression appears at the onset of axogenesis as the lateral cell projects an axon laterally toward the next Lazarillo-expressing cell cluster. The cells of this target cluster direct axons into separate brain regions, thereby establishing an orthogonally organized scaffold that the lateral cell axon follows as it navigates away from the brain midline. The primary axon scaffold of the brain results from a stepwise interlinking of discrete brain regions, as exemplified by axons from neighboring Lazarillo-expressing cell clusters.

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.

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.

Neurogenesis in the median domain of the embryonic brain of the grasshopper Schistocerca gregaria.

Embryonic development in the median domain of the brain of the grasshopper Schistocerca gregaria was investigated with immunohistochemical, histological, and intracellular dye injection techniques. The early head midline is divisible into a dorsal median domain and a ventral median domain based on the orientation of cell somata in each region. At 25% of embryogenesis, a single large midline precursor differentiates in the dorsal median domain and produces a lineage of six neuronal progeny before degenerating. No further precursors arise. In addition, the primary commissure pioneers and a pair of lateral neurons differentiate directly from the ectoderm in this region. Lucifer yellow dye injected into the midline precursor stains only this cell and its progeny. Similarly, there is no dye coupling from the primary commissure pioneers to the midline lineage or to neuroblasts of the brain hemispheres. Neurogenesis in the dorsal median domain therefore proceeds separately within each subset of cells, and is not related to development in the brain hemispheres. Beginning at 42% of embryogenesis, the primary commissure pioneers undergo a morphological transformation and concomittantly express the Term-1 antigen. Expression continues throughout embryogenesis and into the adult, where the midline primary commissure pioneer cells are the only ones labeled by Term-1 in the entire brain. The cellular organization of the dorsal median domain therefore remains remarkably conserved throughout embryogenesis, even as the brain undergoes extensive morphological transformation.

Building a brain: developmental insights in insects.

Understanding the cellular, molecular and genetic mechanisms involved in building the brain remains one of the most challenging problems of neurobiology. In this article, we review recent work on the developmental mechanisms that generate the embryonic brain in insects. We compare some of the early developmental events that occur in the insect brain with those that operate during brain development in vertebrates and find that numerous parallels are present at both the cellular and the molecular levels. Thus, the roles of glial cells in prefiguring axon pathways, the function of pioneer neurons in establishing axon pathways, and the formation of a primary axon scaffolding are features of embryonic brain development in both insects and vertebrates. Moreover, at the molecular genetic level homologous regulatory genes control morphogenesis, regionalization and patterning during embryonic brain development in both insects and vertebrates. This indicates that there might be universal mechanisms for brain development, and that knowledge gained from Drosophila and other insects is relevant to our understanding of brain development in other more complex organisms, including man.

Embryonic development of the pars intercerebralis/central complex of the grasshopper.

We have studied the embryonic development of the pars intercerebralis/central complex in the brain of the grasshopper using immunocytochemical and histochemical techniques. Expression of the cell-surface antigen lachesin reveals that the neuroblasts of the pars intercerebralis first differentiate from the neuroectoderm at around 26% of embryogenesis. Differentiation of medial and lateral neuroblasts occurs first. By the 28% stage a more or less uniform sheet of 20 neuroblasts has formed. As a result of both cell proliferation and cell translocation, the pars intercerebralis proliferative cluster in each hemisphere expands so that at 30% the most medial neuroblasts lie apposed at the midline. We followed the further development of the pars intercerebralis of each brain hemisphere using bromo-deoxy-uridine incorporation and osmium-ethyl-gallate staining. Within the pars intercerebralis itself, the neuroblasts redistribute into discrete subsets. The neuroblasts of each subset generate clusters of progeny which extend in a stereotypic, subset-specific direction in the brain. We have used this feature to identify one subset of four neuroblasts as being the likely progenitor cells for four clusters of embryonic neurons (W, X, Y, Z) which develop at around 55% of embryogenesis. We show that these progeny project axons via four discrete fascicles (w, x, y, z) into the embryonic central complex. At the single cell level, Golgi impregnation reveals that the axons from these neighbouring cell clusters remain discrete, and those from the same cluster tightly fasciculated, as they project into the central complex, consistent with a modular organization for this brain region.

Morphogenetic reorganization of the brain during embryogenesis in the grasshopper.

We have studied the morphogenetic reorganization that occurs in the grasshopper brain during embryogenesis. We find that morphogenetic movements occur at three organizational levels during brain development. First, the entire developing brain changes its orientation with respect to the segmental chain of ventral ganglia. A 90 degrees shift in the attitude of the brain neuraxis occurs during embryogenesis due to a gradual upward movement of the cerebral structures in the head. Second, the clusters of proliferating neuroblasts and progeny that generate the neuroarchitecture of the mature brain move relative to one another and to nonneural structures such as the stomodeum. This is especially pronounced for the pars intercerebralis and for the tritocerebrum, as shown by annulin and engrailed immunoreactivity. Third, individual neuroblasts within a given proliferative cluster undergo positional reorganization during embryogenesis. Identified neuroblasts of the tritocerebrum and the pars intercerebralis are displaced within the brain. We conclude that the transformation of the simple sheet-like structure of the early embryonic brain into the highly differentiated structure of the mature brain involves a series of morphogenetic movements that occur in virtually all parts of the brain.

Axogenesis in the embryonic brain of the grasshopper Schistocerca gregaria: an identified cell analysis of early brain development.

Axogenesis in the embryonic brain was studied at the single cell level in the grasshopper Schistocerca gregaria. A small set of individually identifiable pioneer neurons establishes a primary axon scaffold during early embryogenesis. At the beginning of scaffold formation, pioneering axons navigate along and between glial borders that surround clusters of proliferating neuroblasts. In each brain hemisphere, an axonal outgrowth cascade involving a series of pioneer neurons establishes a pathway from the optic ganglia to the brain midline. At the midline the primary preoral commissural interconnection in the embryonic brain is pioneered by a pair of midline-derived pioneer neurons. A second preoral commissural connection is pioneered by two pairs of pars intercerebralis pioneer neurons. Descending tracts are pioneered by the progeny of identified neuroblasts in the pars intercerebralis, deutocerebrum and tritocerebrum; the postoral tritocerebral commissure is pioneered by a pair of tritocerebral neurons. All of the pioneering brain neurons express the cell adhesion molecule fasciclin I during initial axon outgrowth and fasciculation. Once established, the primary axon scaffold of the brain is used for fasciculation by subsequently differentiating neurons and, by the 40% stage of embryogenesis, axonal projections that characterize the mature brain become evident. The single cell analysis of grasshopper brain development presented here sets the stage for manipulative cell biological experiments and provides the basis for comparative molecular genetic studies of embryonic brain development in Drosophila.

Organization of the commissural fibers in the adult brain of the locust.

The brain (supraoesophageal ganglion) is the most complex of the segmental ganglia composing the nerve cord of the locusts Schistocerca gregaria and Locusta migratoria. In this paper, we describe the ground plan of the commissures crossing the midline of the brain and propose a nomenclature with the aim of making a complex neuropil more understandable at the level of individual neurons. For developmental and comparative reasons the neuroarchitecture of the brain is related to the neural axis, not to the body axis. We have identified 73 commissural fiber bundles belonging to the adult brain, and these are named according to their location (ventral, dorsal, anterior, posterior, medial) with respect to the central complex as reference point. Reconstructions of identified neurons from intracellular stainings, cobalt backfills, or immunohistochemical studies demonstrate the various configurations in which fibers cross the brain.