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.     

The midline glial cells are required for regionalization of commissural axons in the embryonic CNS of Drosophila

 The ventral nerve cord of arthropods is characterised by the organisation of major axon tracts in a ladder-like pattern. The individual neuromeres are connected by longitudinal connectives whereas the contra-lateral connections are brought about through segmental commissures. In each neuromere of the embryonic central nervous system (CNS) of Drosophila an anterior and a posterior commissure is found. The development of these commissures requires a set of neurone-glia interactions at the midline. Here we show that both the anterior as well as the posterior commissures are subdivided into three axon-containing regions. Electron microscopy of the ventral nerve cord of mutations affecting CNS midline cells indicates that the midline glial cells are required for this subdivision. In addition the midline glial cells appear required for a crossing of commissural growth cones perpendicular to the longitudinal tracts, since in mutants with defective midline glial cells commissural axons frequently cross the midline at aberrant angles. Received: 6 July 1997 / Accepted: 27 August 1997     

The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio

We have examined neuronal differentiation and the formation of axon tracts in the embryonic forebrain and midbrain of the zebrafish, between 1 and 2 days postfertilisation. Axons were visualised with three techniques; immunocytochemistry (using HNK-1 and antiacetylated tubulin antibodies) and horseradish peroxidase (HRP) labelling in whole-mounted brains, and transmission electron microscopy. Differentiation was monitored by histochemical staining for acetylcholinesterase (AChE). These independent methods demonstrated that a simple grid of tracts and commissures forms the initial axon scaffold of the brain. At 1 day, the olfactory nerve, four commissures, their associated tracts and three other non-commissural tracts are present. By 2 days, these tracts and commissures have all greatly enlarged and, in addition, the optic nerve and tract, and three new commissures and their associated tracts have been added. Small applications of HRP at various sites revealed the origins and projections of some of these earliest axons. Retrogradely labelled cell bodies originated from regions that were also positive for AChE activity. At 1 day, HRP-labelled axons were traced: (1) from the olfactory placode through the olfactory nerve to the dorsal telencephalon; (2) from the telencephalon into the tract of the anterior commissure and also to the postoptic region of the diencephalon; (3) from the hindbrain through the ventral midbrain and diencephalon to the postoptic commissure; (4) from the dorsal diencephalon (in or near the epiphysis) to the tract of the postoptic commissure; (5) from ventral and rostral midbrain through the posterior commissure. Three new projections were demonstrated at 2 days: (1) from the retina through the tract of the postoptic commissure to the tectum; (2) from the telencephalon to the contralateral diencephalon; and (3) from the telencephalon to the ventral flexure. These results show that at 1 day, the zebrafish brain is impressively simple, with a few small, well-separated tracts but by 2 days the brain is already considerably more complex. Most of the additional axons added onto pre-existent tracts rather than pioneered new ones supporting the notion that other axons play a crucial role in the guidance of early central nervous system (CNS) axons.     

Building the central complex of the grasshopper Schistocerca gregaria: temporal topology organizes the neuroarchitecture of the w,x, y,z tracts

The central complex is a brain specific structure involved in multimodal information processing and in coordinating motor behaviour. It possesses a highly organized neuroarchitecture, which is remarkably conserved across insect species. A prominent feature of this neuroarchitecture is the stereotypic projection of axons from clusters of neurons in the pars intercerebralis to the central body via the so-called w, x, y and z tracts. Despite extensive analyses of this neuroarchitecture in adults, little is known about its ontogeny in any insect. In this paper we use the expression pattern of the segment polarity gene engrailed to identify those neuroblasts belonging to the protocerebrum of the early embryonic brain of the grasshopper Schistocerca gregaria. We present a new map for this brain region in which the 95 protocerebral neuroblasts in each hemisphere are organized into seven rows, as they are in the neuromeres of the ventral nerve cord. We then identify a subset of four of these neuroblasts as being the progenitor cells for four clusters of neurons, some of whose axons we show project via discrete tracts (w, x, y, z) into the central complex. These tracts begin to form prior to 39% of embryogenesis. We show further, that the cells from one of these clusters (the Z cluster) are organized according to age, and direct axons topologically according to age into the appropriate z tract. This pattern is repeated in each of the other three clusters, thus establishing a clonally based modular system of fibre tracts consistent with the model proposed for this brain region in the adult.     

Development of midline cell types and commissural axon tracts requires Fgfr1 in the cerebrum

The adult cerebral hemispheres are connected to each other by specialized midline cell types and by three axonal tracts: the corpus callosum, the hippocampal commissure, and the anterior commissure. Many steps are required for these tracts to form, including early patterning and later axon pathfinding steps. Here, the requirement for FGF signaling in forming midline cell types and commissural axon tracts of the cerebral hemispheres is examined. Fgfr1, but not Fgfr3, is found to be essential for establishing all three commissural tracts. In an Fgfr1 mutant, commissural neurons are present and initially project their axons, but these fail to cross the midline that separates the hemispheres. Moreover, midline patterning defects are observed in the mutant. These defects include the loss of the septum and three specialized glial cell types, the indusium griseum glia, midline zipper glia, and glial wedge. Our findings demonstrate that FGF signaling is required for generating telencephalic midline structures, in particular septal and glial cell types and all three cerebral commissures. In addition, analysis of the Fgfr1 heterozygous mutant, in which midline patterning is normal but commissural defects still occur, suggests that at least two distinct FGF-dependent mechanisms underlie the formation of the cerebral commissures.     

DCC plays a role in navigation of forebrain axons across the ventral midbrain commissure in embryonic xenopus

In the developing vertebrate brain, growing axons establish a scaffold of axon tracts connected across the midline via commissures. We have previously identified a population of telencephalic neurons that express NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM that is involved in axon guidance in the forebrain. These axons arise from the presumptive telencephalic nucleus, course caudally along the principal longitudinal tract of the forebrain, cross the ventral midline in the midbrain, and then project to the contralateral side of the brain. In the present study we have investigated mechanisms controlling the growth of these axons across the ventral midline of the midbrain. The axon guidance receptor DCC is expressed by the NOC-2 population of axons both within the longitudinal tract and within the ventral midbrain commissure. Disruption of DCC-dependent interactions, both in vitro and in vivo, inhibited the NOC-2 axons from crossing the ventral midbrain. Instead, these axons grew along aberrant trajectories away from the midline, suggesting that DCC-dependent interactions are important for overcoming inhibitory mechanisms within the midbrain of the embryonic vertebrate brain. Thus, coordinated responsiveness of forebrain axons to both chemostimulatory and chemorepulsive cues appears to determine whether they cross the ventral midline in the midbrain.     

Commissure formation in the embryonic CNS of Drosophila.

In the ventral nerve cord of Drosophila most axons are organized in a simple, ladder-like pattern. Two segmental commissures connect the hemisegments along the mediolateral and two longitudinal connectives connect individual neuromeres along the anterior-posterior axis. Cells located at the midline of the developing CNS first guide commissural growth cones toward and across the midline. In later stages, midline glial cells are required to separate anterior and posterior commissures into distinct axon bundles. To unravel the genes underlying the formation of axon pattern in the embryonic ventral nerve cord, we conducted a saturating ethylmethane sulfonate mutagenesis, screening for mutations which disrupt this process. Subsequent genetic and phenotypic analyses support a sequential model of axon pattern formation in the embryonic ventral nerve cord. Specification of midline cell lineages is brought about by the action of segment polarity genes. Five genes are necessary for the establishment of the commissures. In addition to commissureless, the netrin genes, and the netrin receptor encoded by the frazzled gene, two gene functions are required for the initial formation of commissural tracts. Over 20 genes appear to be required for correct development of the midline glial cells which are necessary for the formation of distinct segmental commissures.     

Interhemispheric connections through the pallial commissures in the brain of Podarcis hispanica and Gallotia stehlinii (Reptilia,Lacertidae)

The cells-of-origin and the mode and site of termination of the interhemispheric connections passing through the anterior and posterior pallial commissures in the telencephalon of two lizards (Podarcis hispanica and Gallotia stehlinii) were investigated by studying the anterograde and retrograde transport of unilaterally injected horseradish peroxidase. The commissural projections arise mainly from pyramidal cells in the medial, dorsomedial, and dorsal cortices (medial subfield). Additionally some non-pyramidal neurons in the medial and dorsal cortices contribute to the commissural system. Medial cortex neurons project to the contralateral anterior septum through the anterior pallial commissure. The dorsomedial cortex projects contralaterally via the anterior pallial commissure to the dorsolateral septum and to the medial, dorsomedial, and dorsal cortices. The projection to the medial cortex terminates in two bands at the inner and outer border, respectively, of the cell layer; the projection to the dorsomedial and dorsal cortex ends in a zone in layer 1 which previously has been described to be Timm-negative, and in a diffuse band in the inner half of layer 3. The medial subfield of the dorsal cortex projects through the anterior pallial commissure to the dorsomedial and dorsal cortices with a similar pattern of termination to that found for the dorsomedial cortex. The posterior pallial commissure contains only the projections from the ventral cortex to its contralateral counterpart and to the ventral part of the caudal medial cortex. The similarities found between this commissural system and the mammalian hippocampal interhemispheric connections are discussed.     

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.     

Dual function of polysialic acid during zebrafish central nervous system development.

Polysialic acid (PSA), a carbohydrate epitope attached to the neural cell adhesion molecule, serves as a modulator of axonal interactions during vertebrate nervous system development. We have used PSA-specific antibodies and whole-mount immunocytochemistry to describe the spatiotemporal expression pattern of PSA during zebrafish central nervous system development. PSA is transiently expressed on all cell bodies and, except for the posterior commissure, it is not found on axons. Floorplate cells in the spinal cord and hindbrain strongly express PSA throughout development. Enzymatic removal of PSA leads to a defasciculated growth pattern of the posterior commissure and also affects distinct subsets of commissural axons in the hindbrain, which fail to cross the midline. Whereas the disordered growth pattern of hindbrain commissures produced by PSA-removal could be mimicked by injections of soluble PSA, the growth of axons in the posterior commissure was unaffected by such treatment. These results suggest that there are distinct mechanisms for PSA action during axon growth and pathfinding in the developing zebrafish CNS.     

GABA-like immunoreactivity in the suboesophageal ganglion of the locust Schistocerca gregaria

Summary Neurones in the suboesophageal ganglion of the locust Schistocerca gregaria were stained with an antiserum raised against gamma amino butyric acid (GABA). This ganglion consists of the fused mandibular, maxillary and labial neuromeres. Immunoreactive cell bodies of similar size and distribution occur in the lateral, ventral and middorsal regions of all three neuromeres. Approximately 200 cell bodies stain in both the mandibular and maxillary neuromeres and 270 in the labial neuromere. A few distinctly larger cells occur in the ventral groups and one large pair occurs in the lateral group of the maxillary neuromere. Dorsal commissures DCIV and DCV are composed mainly of stained fibres, while DCI–DCIII are largely unstained. A ventral commissure also stains in the maxillary neuromere. All longitudinal tracts contain both stained and unstained fibres. Many processes within the neuropil are also immunoreactive. A stained axon is found in the posterior tritocerebral commissure which enters the anterior dorsal region of the mandibular neuromere. The salivary branch of the 7th nerve contains one stained axon and two axons stain in nerve 8 which innervates neck muscles.     

PKCγ免疫反应定位小鼠皮质脊髓束的实验研究

目的探讨显示皮质脊髓束在成年小鼠脑和脊髓中定位分布的简便有效方法。方法运用蛋白激酶Cγ（PKCγ）免疫组织化学染色法,观察成年ICR小鼠脑和脊髓中皮质脊髓束的定位和分布情况。结果PKCγ免疫阳性产物分布于大脑运动皮层第V层锥体细胞胞体和轴突中,锥体细胞的阳性纤维经内囊、中脑大脑脚底、脑桥基底部、下行至延髓锥体中。在延髓下段,PKCγ阳性纤维经锥体交叉后进入对侧脊髓灰质后联合背侧,形成背侧皮质脊髓束,在脊髓白质的后索腹侧深层下行,至骶髓3-4节段以下逐渐消失。在整个脊髓前索和外侧索中未见有PKCγ阳性纤维。结论PKCγ特异地表达于脊髓后索皮质脊髓束中,提示PKCγ免疫组织化学法是一种显示和观察皮质脊髓束精确定位的有效方法。     

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.     

Embryonic development of the insect central complex: insights from lineages in the grasshopper and Drosophila

The neurons of the insect brain derive from neuroblasts which delaminate from the neuroectoderm at stereotypic locations during early embryogenesis. In both grasshopper and Drosophila, each developing neuroblast acquires an intrinsic capacity for neuronal proliferation in a cell autonomous manner and generates a specific lineage of neural progeny which is nearly invariant and unique. Maps revealing numbers and distributions of brain neuroblasts now exist for various species, and in both grasshopper and Drosophila four putatively homologous neuroblasts have been identified whose progeny direct axons to the protocerebral bridge and then to the central body via an equivalent set of tracts. Lineage analysis in the grasshopper nervous system reveals that the progeny of a neuroblast maintain their topological position within the lineage throughout embryogenesis. We have taken advantage of this to study the pioneering of the so-called w, x, y, z tracts, to show how fascicle switching generates central body neuroarchitecture, and to evaluate the roles of so-called intermediate progenitors as well as programmed cell death in shaping lineage structure. The novel form of neurogenesis involving intermediate progenitors has been demonstrated in grasshopper, Drosophila and mammalian cortical development and may represent a general strategy for increasing brain size and complexity. An analysis of gap junctional communication involving serotonergic cells reveals an intrinsic cellular organization which may relate to the presence of such transient progenitors in central complex lineages.     

Time-lapse imaging reveals stereotypical patterns of Drosophila midline glial migration

The Drosophila CNS midline glia (MG) are multifunctional cells that ensheath and provide trophic support to commissural axons, and direct embryonic development by employing a variety of signaling molecules. These glia consist of two functionally distinct populations: the anterior MG (AMG) and posterior MG (PMG). Only the AMG ensheath axon commissures, whereas the function of the non-ensheathing PMG is unknown. The Drosophila MG have proven to be an excellent system for studying glial proliferation, cell fate, apoptosis, and axon-glial interactions. However, insight into how AMG migrate and acquire their specific positions within the axon-glial scaffold has been lacking. In this paper, we use time-lapse imaging, single-cell analysis, and embryo staining to comprehensively describe the proliferation, migration, and apoptosis of the Drosophila MG. We identified 3 groups of MG that differed in the trajectories of their initial inward migration: AMG that migrate inward and to the anterior before undergoing apoptosis, AMG that migrate inward and to the posterior to ensheath commissural axons, and PMG that migrate inward and to the anterior to contact the commissural axons before undergoing apoptosis. In a second phase of their migration, the surviving AMG stereotypically migrated posteriorly to specific positions surrounding the commissures, and their final position was correlated with their location prior to migration. Most noteworthy are AMG that migrated between the commissures from a ventral to a dorsal position. Single-cell analysis indicated that individual AMG possessed wide-ranging and elaborate membrane extensions that partially ensheathed both commissures. These results provide a strong foundation for future genetic experiments to identify mutants affecting MG development, particularly in guidance cues that may direct migration. Drosophila MG are homologous in structure and function to the glial-like cells that populate the vertebrate CNS floorplate, and study of Drosophila MG will provide useful insights into floorplate development and function.     

In the Absence of Frazzled Over-Expression of Abelson Tyrosine Kinase Disrupts Commissure Formation and Causes Axons to Leave the Embryonic CNS

Background

In the Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra) and the Abelson (Abl) cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abl''s role in attractive signaling, we over-expressed Abl in Fra mutants anticipating rescue of commissures.

Methodology/Principal Findings

The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P-motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS.

Conclusion/Significance

Taken together, these data suggest that Fra activity is required to correctly regulate Abl-dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border.     

Ventral midline cells are required for the local control of commissural axon guidance in the mouse spinal cord.

Specialized cells at the midline of the central nervous system have been implicated in controlling axon projections in both invertebrates and vertebrates. To address the requirement for ventral midline cells in providing cues to commissural axons in mice, we have analyzed Gli2 mouse mutants, which lack specifically the floor plate and immediately adjacent interneurons. We show that a Dbx1 enhancer drives tau-lacZ expression in a subpopulation of commissural axons and, using a reporter line generated from this construct, as well as DiI tracing, we find that commissural axons projected to the ventral midline in Gli2(-/-) embryos. Netrin1 mRNA expression was detected in Gli2(-/-) embryos and, although much weaker than in wild-type embryos, was found in a dorsally decreasing gradient. This result demonstrates that while the floor plate can serve as a source of long-range cues for C-axons in vitro, it is not required in vivo for the guidance of commissural axons to the ventral midline in the mouse spinal cord. After reaching the ventral midline, most commissural axons remained clustered in Gli2(-/-) embryos, although some were able to extend longitudinally. Interestingly, some of the longitudinally projecting axons in Gli2(-/-) embryos extended caudally and others rostrally at the ventral midline, in contrast to normal embryos in which virtually all commissural axons turn rostrally after crossing the midline. This finding indicates a critical role for ventral midline cells in regulating the rostral polarity choice made by commissural axons after they cross the midline. In addition, we provide evidence that interactions between commissural axons and floor plate cells are required to modulate the localization of Nr-CAM and TAG-1 proteins on axons at the midline. Finally, we show that the floor plate is not required for the early trajectory of motoneurons or axons of the posterior commissure, whose projections are directed away from the ventral midline in both WT and Gli2(-/-) embryos, although they are less well organized in Gli2(-/-)mutants.     

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.