Drosophila PS2 and PS3 integrins play distinct roles in retinal photoreceptors–glia interactions

Cellular migration and differentiation are important developmental processes that require dynamic cellular adhesion. Integrins are heterodimeric transmembrane receptors that play key roles in adhesion plasticity. Here, we explore the developing visual system of Drosophila to study the roles of integrin heterodimers in glia development. Our data show that αPS2 is essential for retinal glia migration from the brain into the eye disc and that glial cells have a role in the maintenance of the fenestrated membrane (Laminin‐rich ECM layer) in the disc. Interestingly, the absence of glial cells in the eye disc did not affect the targeting of retinal axons to the optic stalk. In contrast, αPS3 is not required for retinal glia migration, but together with Talin, it functions in glial cells to allow photoreceptor axons to target the optic stalk. Thus, we present evidence that αPS2 and αPS3 integrin have different and specific functions in the development of retinal glia. GLIA 2015;63:1155–1165     

Organization of astrocytes in the visual pathways of the goldfish: An immunohistochemical study

We have used antisera directed against glial cytoskeletal proteins to examine the distribution and organization of astrocytes in the visual pathways of the goldfish. We describe two different types of cells, which may be distinguished by their unique cytoskeletal proteins. Antibodies raised against a 48 Kd optic nerve protein react with stellate astrocytes in the optic nerve but virtually no glial cells in the brain (although blood vessels and the meninges in the brain were stained). The optic nerve astrocytes form a dense meshwork of processes through which the optic fibers pass. The intraorbital and intracranial segments of the nerve are divided into fascicles, each bounded by a glia limitans, which extend across the optic chiasm. Astroglial cells in the brain bind antibodies raised against a 50 Kd brain cytoskeletal protein. These antibodies show a very limited cross-reactivity with optic nerve cells. Brain astrocytes have filiform profiles and most appear to be deployed as radial glia. The glial fabric of the brain, as revealed by these antibodies, is far more loosely woven than that of the optic nerve. There is a sharp boundary between the two types of glial cells, immediately behind the optic chiasm. Glial processes in the optic tracts arise from cells in the preoptic area, whereas those in the optic tectum arise from cells that reside locally. In the optic tract, a glia limitans was often difficult to discern, whereas in the tectum one was always evident and composed of endfeet at the pial extremities of radial glial processes. These findings are discussed both in the context of previous observations by other workers as well as with regard to their possible functional implications.     

Immunological probes reveal spatial and developmental diversity in insect neuroglia

A set of monoclonal antibodies (MAbs) has been generated that recognizes distinct classes of neuroglia in the adult nervous system of the cricket Acheta domesticus corresponding to glial types distinguished by morphological criteria. These include antibodies that bind to the neuroglia of the ganglionic cortex, perineurium, neuropil, and glia associated with the glial lacunar system (interface) and fiber tracts. Another MAb specifically labels components of the neural lamella, a complex extracellular matrix secreted by underlying perineurial cells. Selected adult glial-specific MAbs recognize particular glial antigens expressed during embryonic development of Acheta. Immunohistochemical staining of frozen sections of late- (90-95%) and intermediate- (50-55%) stage whole embryos reveals that the spatial distribution, degree of tissue restriction, or level of expression of some glial determinants changes as development proceeds. Labeling of certain neuroblasts in the embryonic CNS at 50-55% development by an antibody (MAb 3G6) that binds to neuropil glia in the adult CNS implies that at least 1 class of insect glia may be generated by these cells.     

Immunocytochemical localization of a Manduca sexta gamma-aminobutyric acid transporter

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in insect central and peripheral nervous systems. Although much work has focused on the downstream targets of GABA, signal termination at insect GABAergic synapses has received very little attention. One of the major mechanisms of terminating synaptic transmission involves transport of the neurotransmitter molecules into presynaptic neurons or surrounding glia. Here we report the immunolocalization of a GABA transporter in the tobacco hornworm, Manduca sexta (MasGAT), using an affinity-purified antibody developed to the C-terminus. This is the first demonstration of an insect neurotransmitter transporter immunolocalization study. Results showed strong staining in the neuropil regions of embryonic, larval, and pharate adult central nervous system. Expression pattern in the pharate adult brain mostly mimicked that observed for GABA, with staining in parts of the optic and antennal lobes, mushroom body, lateral protocerebrum, and central complex. Certain longitudinal and lateral connectives of ganglia were observed to have immunostained fibers representing axons. These data support the view that GABA is involved in visual and olfactory processing in the insect brain.     

Glia in development, function, and neurodegeneration of the adult insect brain

Glial cells have long been viewed as a passive framework for neurons but in the meanwhile were shown to play a much more active role in brain function and development. Several reviews have described the function of glia in the insect embryo. The focus of this review is the role of glial cells in the development and function of the normal and diseased adult brain. In different insect species, a considerable variety of central nervous system glia has been described indicating adaptation to different functional requirements. In the development of the adult visual and olfactory system, glial cells guide incoming axons acting as intermediate targets. Glia are part of the insect blood-brain barrier, provide nourishment for neurons, and help to regulate the extracellular concentration of ions and neurotransmitters. To fulfill these tasks insect glial cells, like vertebrate glia, interact with each other and with neurons, thus influencing neural activity. The examples presented suggest that crosstalk between all brain cells is necessary not only to develop and maintain the complex insect brain but also to endow it with the capacity to respond and adapt to the changing environment.     

Binding of an antibody against a noncompact myelin protein to presumptive glial cells in the visual system of the crab Ucides cordatus

Glial cells, in both vertebrate and invertebrate nervous systems, provide an essential environment for developmental, supportive, and physiological functions. However, information on glial cells themselves and on glial cell markers, with the exception of those of Drosophila and other insects, is not abundant in invertebrate organisms. A common ultrastructural feature of invertebrate nervous systems is that layers of glial cell cytoplasm-rich processes ensheath axons and neuronal and glial somata. In the present study, we have examined the binding of a monoclonal antibody to 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in the compound eye and optic lobe of the crab Ucides cordatus using both light and electron microscopy. CNPase is a noncompact myelin protein that is a phenotypic marker of oligodendroglial and Schwann cells, is apparently involved in the ensheathment step prior to myelin compaction, and is also expressed by the potentially myelinating olfactory ensheathing glia. CNPase has raised much interest, first by virtue of its unusual enzymatic activity and more recently by its membrane-skeletal features and possible involvement in migration or expansion of membranes. We have found CNPase-like immunoreactivity in most cells of the compound eye basement membrane and both in optic cartridges of the synaptic layer and cells of the outer sublayer of the lamina ganglionaris. The results suggest that in the crab visual system some, but not all, glial cells, including some adaxonal glia, may express the noncompact myelin protein CNPase or a related protein.     

Embryonic development of the Drosophila brain. II. Pattern of glial cells

Glial cells in Drosophila and other insects are organized in an outer layer that envelops the surface of the central and peripheral nervous system (subperineurial glia, peripheral glia), a middle layer associated with neuronal somata in the cortex (cell body glia), and an inner layer surrounding the neuropile (longitudinal glia, midline glia, nerve root glia). In the ventral nerve cord, most glial cells are formed by a relatively small number of neuro-glioblasts; subsequently, glial cell precursors migrate and spread out widely to reach their final destination. By using a glia-specific marker (antibody against the Repo protein) we have reconstructed the pattern of glial cell precursors at successive developmental stages, focusing on the glia of the supraesophageal ganglion and subesophageal ganglion which are not described in previous studies. Digitized images of consecutive optical sections were used to generate 3-D models that show the spatial pattern of glial cell precursors in relationship to the neuropile, brain surface, and peripheral nerves. Similar to their spatial organization in the ventral nerve cord, glial cells of the brain populate the brain nerves and outer surface, cortical cell body layer, and cortex-neuropile interface. Neuropile-associated glial cells arise from a cluster located at the base of the supraesophageal ganglion; from this position, they migrate dorsally along the developing axon tracts and by late embryonic stages form a sheath around all neuropile compartments, including the supraesophageal commissure. Surface and cell body glial cells derive from several discrete foci, notably two large clusters at the deuterocerebrum/protocerebrum boundary and the posterior protocerebrum. From these foci, glial cells then fan out to envelop the surface of the supraesophageal ganglion. J. Comp. Neurol. 402:32–47, 1998. © 1998 Wiley-Liss, Inc.     

Adult rat retinal glia in vitro: effects of in vivo crush-activation on glia proliferation and permissiveness for regenerating retinal ganglion cell axons

The effects of optic nerve crush on adult rat retinal glia activation were studied in vitro. In adult rats the optic nerves were crushed and the corresponding retinae were explained 5 to 7 days later and cultured in vitro. The glial response of retinae with precrushed optic nerves was compared to the glial response of retinae without prior optic nerve crush. As a consequence of crush-axotomy more glial cells migrated out from retinal explants and covered significantly larger areas of the substratum than glia from noncrushed retinae. Migration of immunohistochemically distinguishable Vimentin-positive Müller cells and glial fibrillary acidic protein-positive astrocytes could be observed in both types of cultures. Astrocytes as well as Müller cells incorporated bromodeoxyuridine after explantation. In noncrushed retinal explants Thy 1.1-immunopositive flat cells were much more frequent and the relative proportion of glial cells was much lower than in crush-activated cultures. In a second set of experiments the ability of adult rat retinal glia to support retinal ganglion cell regeneration was examined. Normal retinal explants (without optic nerve crush) which usually do not substantially regenerate axons were cultured on retinal glia from normal and crush-activated explants. Both glia preparations supported axon growth from retinal explants after 3 days in vitro. Neuritic growth was significantly better when retinal explants from normal adult rats were cultured on crush-activated retinal glia as compared to glia derived from noncrushed retinae. It is concluded that activated adult rat retinal glia, unlike adult glia found in other brain regions, support adult rat retinal ganglion cell regeneration in vitro.     

R- and B-cadherin expression defines subpopulations of glial cells involved in axonal guidance in the optic nerve head of the chicken

Glial cells play a crucial role in the organization and function of the nervous system. Cell-cell adhesion receptors of the cadherin family have been shown to participate in distinct morphogenetic processes throughout the development of the CNS, but little is known about glial expression of cadherins. Applying immunofluorescence and confocal laser scanning microscopy, we investigated R- and B-cadherin expression in relation to the glial cell differentiation in the optic nerve head and pecten oculi of developing chicken. Throughout embryonic development, R- and B-cadherin were expressed in distinct cell populations, which differentiated into distinct subtypes of glial cells. R-cadherin was located in the glia limitans perivascularis et superficialis of the optic nerve and in cells bordering the optic nerve head, where it comes in contact with the retina. B-cadherin was located in the glia limitans perivascularis et superficialis of the pecten oculi and in a subset of cells at the retinal border. R-cadherin-expressing cells differentiated unequivocally into a glial fibrillary acidic protein (GFAP)-positive but glutamine synthetase (GS)-negative phenotype, whereas B-cadherin-expressing glia developed into a GS-positive but GFAP-negative phenotype. In addition, the B-cadherin-positive population developed into a highly pigmented cell type, which was consistently associated with pecten-type capillaries. By contrast, the R-cadherin-positive glia remained unpigmented and surrounded normal brain-phenotype capillaries. These data suggest that glial cells, like neurons, may use the expression of different cadherins to segregate and differentiate into distinct subtypes, which goes hand in hand with their involvement in special functions and morphogenetic processes. To address this issue, we selectively lysed both glial subtypes in developing embryos by microinjection of R- and B-cadherin antibodies with complement. First evidence is presented for R-cadherin-positive glial cells as crucial to the organization of the optic nerve and axonal guidance at its lateral margin. B-cadherin-positive cells are involved in the axonal guidance at the pecteneal margin, avoiding the ingrowth of axons into the pecten.     

Progenitor cells and glioma formation.

The gliomas are a collection of tumors that arise within the central nervous system and have characteristics similar to astrocytes, oligodendrocytes, or their precursors. Whether or not the glial characteristics of these tumors mean that they arise from the differentiated glia that they resemble or their precursors has been debated. Even under normal circumstances the cells within the central nervous system of an adult can trans-differentiate to other cell types. In addition, mutations found in gliomas further destabilize the differentiation status of these cells making a determination of what cell type gives rise to a given tumor histology difficult. Lineage tracing studies in animals can be used to correlate some specific cell characteristics with the histology of gliomas that arise from these cells. From these experiments it appears that undifferentiated cells are more sensitive to the oncogenic effects of certain signaling abnormalities than are differentiated cells, but that with the appropriate genetic abnormalities differentiated astrocytes can act as the cell-of-origin for gliomas. These data imply that small molecules that promote differentiation may be a rational component of glioma therapy in combination with other drugs aimed at specific molecular signaling targets.     

Ebony protein in the Drosophila nervous system: optic neuropile expression in glial cells

The Drosophila ebony mutation (Bridges and Morgan, [1923] Publs Carnegie Inst Wash 327:50) reveals a pleiotropic phenotype with cuticular and behavioral defects. To understand Ebony function in the nervous system, particularly in transmission of the visual signal, it is essential to know the cell type and temporal characteristics of its expression throughout development. Therefore, we raised an antiserum against an Ebony peptide to detect the protein in whole-mount and slice preparations of Drosophila. Attention was focused on ebony expression in the adult optic neuropiles of the fly. Colocalization of Ebony with neuronal or glial cell markers in frozen sections showed non-neuronal expression of ebony in the lamina and medulla neuropiles. Furthermore, colocalization with glial cell markers demonstrated glial expression of ebony in epithelial glia of the lamina and neuropile glia of the distal medulla. This finding was confirmed for the lamina epithelial glia by electron microscopic examination of immunolabeling by using the diaminobenzidine method. These glia have in common that they match the two sites of histamine release from the compound eye's photoreceptors. Possible ways in which the biochemical activity of Ebony might function with respect to histamine release are considered.     

Developmental dynamics of peripheral glia in Drosophila melanogaster

To study the roles of peripheral glia in nervous system development, a thorough characterization of wild type glial development must first be performed. We present a developmental profile of peripheral glia in Drosophila melanogaster that includes glial genesis, developmental morphology, the establishment of transient cellular contacts, migration patterns, and the extent of nerve wrapping in the embryonic and larval stages. In early embryonic development, immature peripheral glia that are born in the CNS seem to be intermediate targets for neurites that are migrating into the periphery. During migration to the PNS, peripheral glia follow the routes of pioneer neurons. The glia preferentially adhere to sensory axonal projections, extending cytoplasmic processes along them such that by the end of embryogenesis peripheral glial coverage of the sensory system is complete. In contrast, significant lengths of motor branch termini are unsheathed in the mature embryo. During larval stages however, peripheral glia further extend and elaborate their cytoplasmic processes until they often reach to the neuromuscular junction. Throughout the embryonic and larval developmental stages, we have also observed a number of similarities of peripheral glia to vertebrate Schwann cells and astrocytes. Peripheral glia seem to have dynamic and diverse roles and their similarities to vertebrate glia suggest that Drosophila may serve as a powerful tool for analysis of glial roles in PNS development in the future.     

Early development of the optic chiasm in the gray short-tailed opossum,Monodelphis domestica

We have studied the early development of the uncrssed retinofugal projection in the gray short-tailed opossum. Axons that form the adult uncrossed retinofugal projection arise from the temporal crescent of the retina and reach the optic chiasm on postnatal day 7. The sites at which the uncrossed fibres segregate from the crossed fibres and the pattern of this segregation are very different from those seen in eutherian mammals. In the opossum, the uncrossed fibres segregate from the crossed fibres within the juxtachiasmatic part of the optic nerve before they have encountered either the fibres of the other eye or midline structures of the ventral diencephalon. The uncrossed fibres turn perpendicular to the axis of the nerve and grow dorsoventrally through the crossed projection to gather as a discrete bundle at the ventral edge of the nerve. The abrupt divergence of the uncrossed fibres occurs at a border between two glial cell types: the interfascicular glia that characterise the main part of the optic nerve and the radial glia of the juxtachiasmatic part of the nerve. At the ventral part of the nerve, the bundle of uncrossed fibres turns caudally across the axis of the nerve and enters the ipsilateral optic tract. When retinofugal fibres encounter the border between the interfascicular and radial glia, a very specific axonal reorganisation occurs in marsupials, and this is strikingly different from the axonal reorganisation that occurs at the same site in eutherians, where essentially all retinofugal fibres reorganise, not just the uncrossed component. We believe this to be an important example of an identified cellular element that has quite distinct axon-guidance properties in different species. © 1994 Wiley-Liss, Inc.     

Gfap‐positive radial glial cells are an essential progenitor population for later‐born neurons and glia in the zebrafish spinal cord

Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial‐derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later‐born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter‐driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap‐negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood–brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170–1189     

Optic Disc and Optic Nerve of the Blind Cape Mole-Rat (Georychus capensis): A Proposed Model for Naturally Occurring Reactive Gliosis

Recent studies of the visual system of animal species that live in a subterranean environment show not only regressive but also progressive morphological features. In this regard the aim of the present investigation is to describe the structural organisation of the eye and optic nerve of the adult Cape mole-rat, with special emphasis on both glial cell population and myelination. The main results are: (a) astrocytes show identical features to those occurring in reactive gliosis; (b) optic fibers vary greatly in diameter; (c) very small axons are myelinated and are often surrounded by a thicker sheath than larger optic fibers; (d) a large onion bulb-like structure composed of optic fibers, glia, and ganglion cells is found within the choriocapillary layer. These results suggest that the Cape mole-rat and probably other subterranean rodents may serve as a model to study spontaneous gliosis as well as mechanisms involved in myelination and degenerative processes.     

Morphological diversity and development of glia in Drosophila

Insect glia represents a conspicuous and diverse population of cells and plays a role in controlling neuronal progenitor proliferation, axonal growth, neuronal differentiation and maintenance, and neuronal function. Genetic studies in Drosophila have elucidated many aspects of glial structure, function, and development. Just as in vertebrates, it appears as if different classes of glial cells are specialized for different functions. On the basis of topology and cell shape, glial cells of the central nervous system fall into three classes (Fig. 1A-C): (i) surface glia that extend sheath-like processes to wrap around the entire brain; (ii) cortex glia (also called cell body-associated glia) that encapsulate neuronal somata and neuroblasts which form the outer layer (cortex) of the central nervous system; (iii) neuropile glia that are located at the interface between the cortex and the neuropile, the central domain of the nervous system formed by the highly branched neuronal processes and their synaptic contacts. Surface glia is further subdivided into an outer, perineurial layer, and an inner, subperineurial layer. Likewise, neuropile glia comprises a class of cells that remain at the surface of the neuropile (ensheathing glia), and a second class that forms profuse lamellar processes around nerve fibers within the neuropile (astrocyte-like or reticular glia). Glia also surrounds the peripheral nerves and sensory organs; here, one also recognizes perineurial and subperineurial glia, and a third type called "wrapping glia" that most likely corresponds to the ensheathing glia of the central nervous system. Much more experimental work is needed to determine how fundamental these differences between classes of glial cells are, or how and when during development they are specified. To aid in this work the following review will briefly summarize our knowledge of the classes of glial cells encountered in the Drosophila nervous system, and then survey their development from the embryo to adult.     

Neural organization of afferent pathways from the stomatopod compound eye

Crustaceans and insects share many similarities of brain organization suggesting that their common ancestor possessed some components of those shared features. Stomatopods (mantis shrimps) are basal eumalacostracan crustaceans famous for their elaborate visual system, the most complex of which possesses 12 types of color photoreceptors and the ability to detect both linearly and circularly polarized light. Here, using a palette of histological methods we describe neurons and their neuropils most immediately associated with the stomatopod retina. We first provide a general overview of the major neuropil structures in the eyestalks lateral protocerebrum, with respect to the optical pathways originating from the six rows of specialized ommatidia in the stomatopod's eye, termed the midband. We then focus on the structure and neuronal types of the lamina, the first optic neuropil in the stomatopod visual system. Using Golgi impregnations to resolve single neurons we identify cells in different parts of the lamina corresponding to the three different regions of the stomatopod eye (midband and the upper and lower eye halves). While the optic cartridges relating to the spectral and polarization sensitive midband ommatidia show some specializations not found in the lamina serving the upper and lower eye halves, the general morphology of the midband lamina reflects cell types elsewhere in the lamina and cell types described for other species of Eumalacostraca.     

Drosophila as a developmental paradigm of regressive brain evolution: proof of principle in the visual system

Evolutionary developmental biology focuses heavily on the constructive evolution of body plan components, but there are many instances such as parasitism, cave adaptation, or postembryonic growth rate optimization where evolutionary regression is of adaptive value. This is particularly true in the nervous system because of its massive energy costs. However, comparatively little effort has thus far been made to understand the evolutionary developmental trajectories of adaptive nervous system reduction. This review focuses on the organization and evolution of the Drosophila larval brain, which represents an exceptional example of miniaturization, most dramatically in the visual system. It is specifically discussed how the dependency of outer optic lobe development on retinal innervation can be assumed to have facilitated a first evolutionary phase of larval visual system reduction. Afferent input-contingent development of neu- ral compartments very likely plays a widespread role in adaptive brain evolution. Understanding the complete deconstruction of the larval optic neuropiles in Drosophila awaits expanded comparative analysis but has the promise to inform about further developmental trajectories and mechanisms underlying regressive evolution of the brain.