Drosophila cortex and neuropile glia influence secondary axon tract growth, pathfinding, and fasciculation in the developing larval brain

Glial cells play important roles in the developing brain during axon fasciculation, growth cone guidance, and neuron survival. In the Drosophila brain, three main classes of glia have been identified including surface, cortex, and neuropile glia. While surface glia ensheaths the brain and is involved in the formation of the blood-brain-barrier and the control of neuroblast proliferation, the range of functions for cortex and neuropile glia is less well understood. In this study, we use the nirvana2-GAL4 driver to visualize the association of cortex and neuropile glia with axon tracts formed by different brain lineages and selectively eliminate these glial populations via induced apoptosis. The larval central brain consists of approximately 100 lineages. Each lineage forms a cohesive axon bundle, the secondary axon tract (SAT). While entering and traversing the brain neuropile, SATs interact in a characteristic way with glial cells. Some SATs are completely invested with glial processes; others show no particular association with glia, and most fall somewhere in between these extremes. Our results demonstrate that the elimination of glia results in abnormalities in SAT fasciculation and trajectory. The most prevalent phenotype is truncation or misguidance of axon tracts, or abnormal fasciculation of tracts that normally form separate pathways. Importantly, the degree of glial association with a given lineage is positively correlated with the severity of the phenotype resulting from glial ablation. Previous studies have focused on the embryonic nerve cord or adult-specific compartments to establish the role of glia. Our study provides, for the first time, an analysis of glial function in the brain during axon formation and growth in larval development.     

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.     

Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube

Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-l-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube.     

Identification of motifs that are conserved in 12 Drosophila species and regulate midline glia vs. neuron expression

Functional complexity of the central nervous system (CNS) is reflected by the large number and diversity of genes expressed in its many different cell types. Understanding the control of gene expression within cells of the CNS will help reveal how various neurons and glia develop and function. Midline cells of Drosophila differentiate into glial cells and several types of neurons and also serve as a signaling center for surrounding tissues. Here, we examine regulation of the midline gene, wrapper, required for both neuron–glia interactions and viability of midline glia. We identify a region upstream of wrapper required for midline expression that is highly conserved (87%) between 12 Drosophila species. Site-directed mutagenesis identifies four motifs necessary for midline glial expression: (1) a Single-minded/Tango binding site, (2) a motif resembling a pointed binding site, (3) a motif resembling a Sox binding site, and (4) a novel motif. An additional highly conserved 27 bp are required to restrict expression to midline glia and exclude it from midline neurons. These results suggest short, highly conserved genomic sequences flanking Drosophila midline genes are indicative of functional regulatory regions and that small changes within these sequences can alter the expression pattern of a gene.     

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.     

Distinct mechanisms triggering glial differentiation in Drosophila thoracic and abdominal neuroblasts 6-4

Neurons and glia are produced in stereotyped patterns after neuroblast cell division during development of the Drosophila central nervous system. The first cell division of thoracic neuroblast 6-4 (NB6-4T) is asymmetric, giving rise to a glial precursor cell and a neuronal precursor cell. In contrast, abdominal NB6-4 (NB6-4A) divides symmetrically to produce two glial cells. To understand the relationship between cell division and glia-neuron cell fate determination, we examined and compared the effects of known cell division mutations on the NB6-4T and NB6-4A lineages. Based on observation of expression of glial fate determination and early glial differentiation genes, the onset of glial differentiation occurred in NB6-4A but not in NB6-4T when both cell cycle progression and cytokinesis were genetically arrested. On the other hand, glial differentiation started in both lineages when cytokinesis was blocked with intact cell cycle progression. These results showed that NB6-4T, but not NB6-4A, requires cell cycle progression for acquisition of glial fate, suggesting that distinct mechanisms trigger glial differentiation in the different lineages.     

Lineage of radial glia in the chicken optic tectum.

In many parts of the central nervous system, the elongated processes of radial glial cells are believed to guide immature neurons from the ventricular zone to their sites of differentiation. To study the clonal relationships of radial glia to other neural cell types, we used a recombinant retrovirus to label precursor cells in the chick optic tectum with a heritable marker, the E. coli lacZ gene. The progeny of the infected cells were detected at later stages of development with a histochemical stain for the lacZ gene product. Radial glia were identified in a substantial fraction of clones, and these were studied further. Our main results are the following. (a) Clones containing radial glia frequently contained neurons and/or astrocytes, but usually not other radial glia. Thus, radial glia derive from a multipotential progenitor rather than from a committed radial glial precursor. (b) Production of radial glia continues until at least embryonic day (E) 8, after the peak of neuronal birth is over (approximately E5) and after radial migration of immature neurons has begun (E6-7). Radial glial and neuronal lineages do not appear to diverge during this interval, and radial glia are among the last cells that their progenitors produce. (c) As they migrate, many cells are closely apposed to the apical process of their sibling radial glia. Thus, radial glia may frequently guide the migration of their clonal relatives. (d) The population of labelled radial glia declines between E15 and E19-20 (just before hatching), concurrent with a sharp increase in the number of labelled astrocytes. This result suggests that some tectal radial glia transform into astrocytes, as occurs in mammalian cerebral cortex, although others persist after hatching. To reconcile the observations that many radial glia are present early, that radial glia are among the last offspring of a multipotential stem cell, and that most clones contain only a single radial glial cell, we suggest that the stem cell is, or becomes, a radial glial cell.     

Cdc42 and Gsk3 modulate the dynamics of radial glial growth, inter-radial glial interactions and polarity in the developing cerebral cortex

Polarized radial glia are crucial to the formation of the cerebral cortex. They serve as neural progenitors and as guides for neuronal placement in the developing cerebral cortex. The maintenance of polarized morphology is essential for radial glial functions, but the extent to which the polarized radial glial scaffold is static or dynamic during corticogenesis remains an open question. The developmental dynamics of radial glial morphology, inter-radial glial interactions during corticogenesis, and the role of the cell polarity complexes in these activities remain undefined. Here, using real-time imaging of cohorts of mouse radial glia cells, we show that the radial glial scaffold, upon which the cortex is constructed, is highly dynamic. Radial glial cells within the scaffold constantly interact with one another. These interactions are mediated by growth cone-like endfeet and filopodia-like protrusions. Polarized expression of the cell polarity regulator Cdc42 in radial glia regulates glial endfeet activities and inter-radial glial interactions. Furthermore, appropriate regulation of Gsk3 activity is required to maintain the overall polarity of the radial glia scaffold. These findings reveal dynamism and interactions among radial glia that appear to be crucial contributors to the formation of the cerebral cortex. Related cell polarity determinants (Cdc42, Gsk3) differentially influence radial glial activities within the evolving radial glia scaffold to coordinate the formation of cerebral cortex.     

Developmental regulation of glial cell phagocytic function during Drosophila embryogenesis

The proper removal of superfluous neurons through apoptosis and subsequent phagocytosis is essential for normal development of the central nervous system (CNS). During Drosophila embryogenesis, a large number of apoptotic neurons are efficiently engulfed and degraded by phagocytic glia. Here we demonstrate that glial proficiency to phagocytose relies on expression of phagocytic receptors for apoptotic cells, SIMU and DRPR. Moreover, we reveal that the phagocytic ability of embryonic glia is established as part of a developmental program responsible for glial cell fate determination and is not triggered by apoptosis per se. Explicitly, we provide evidence for a critical role of the major regulators of glial identity, gcm and repo, in controlling glial phagocytic function through regulation of SIMU and DRPR specific expression. Taken together, our study uncovers molecular mechanisms essential for establishment of embryonic glia as primary phagocytes during CNS development.     

Glial cells in the developing and adult olfactory lobe of the moth Manduca sexta

The antennal lobe of the moth contains several classes of glial cells that are likely to play functional roles in both the developing and mature lobe. In this study, confocal and electron microscopy were used to examine in detail the morphology of two classes of glial cells, those associated with olfactory receptor axons as they course to their targets in the lobe and those that form borders around the synaptic neuropil of the olfactory glomeruli. The former, the nerve-layer glia, have long processes with multiple expansions that enwrap axon fascicles; the latter, the neuropil glia, constitute two subgroups: complex glia with large cell bodies and branching, vellate arbors; and simple glia, with multiple, mostly unbranched processes with many lamellate expansions along their lengths. The processes of complex glia appear to be closely associated with axon fascicles as they enter the glomeruli, while those of the simple glia surround the glomeruli as part of a multi-lamellar glial envelope, their processes rarely invading the synaptic neuropil of the body of the glomerulus. The full morphological development of antennal-lobe glial cells requires more than two-thirds of metamorphic development. During this period, cells that began as cuboidal or spindle-shaped cells that were extensively dye-coupled to one another gradually assume their adult form and, at least under nonstimulated conditions, greatly reduce their coupling. These changes are only weakly dependent on the presence of olfactory receptor axons. Glial processes are somewhat shorter and less branched in the absence of these axons, but basic structure and degree of dye-coupling are unchanged.     

A requirement for Notch in the genesis of a subset of glial cells in the Drosophila embryonic central nervous system which arise through asymmetric divisions

In the Drosophila central nervous system (CNS) glial cells are known to be generated from glioblasts, which produce exclusively glia or neuroglioblasts that bifurcate to produce both neuronal and glial sublineages. We show that the genesis of a subset of glial cells, the subperineurial glia (SPGs), involves a new mechanism and requires Notch. We demonstrate that the SPGs share direct sibling relationships with neurones and are the products of asymmetric divisions. This mechanism of specifying glial cell fates within the CNS is novel and provides further insight into regulatory interactions leading to glial cell fate determination. Furthermore, we show that Notch signalling positively regulates glial cells missing (gcm) expression in the context of SPG development.     

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2⁺ neural progenitor, A2B5⁺ astrocyte/ glial progenitor, NG2⁺ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration.