Chick Optic Nerve Research Articles

Sonic hedgehog promotes the migration and proliferation of optic nerve oligodendrocyte precursors

Optic nerve (ON) oligodendrocyte precursors (OPCs) are generated under the influence of the Sonic hedgehog (Shh) in the preoptic area from where they migrate to colonise the entire nerve. The molecular events that control this migration are still poorly understood. Recent studies suggested that Shh is often used by the same cell population to control different processes, including cell proliferation and migration, raising the possibility that Shh could contribute to these aspects of OPC development. In support of this idea, we show here that Shh induces the proliferation of OPCs derived from embryonic mouse ON explants and acts as a chemoattractant for their migration. In ovo injections of hybridomas secreting Shh-specific blocking antibody decreases the number of OPCs present in chick ONs, particularly in the retinal portion of the nerve. Altogether these data indicate that Shh contributes to OPC proliferation and distribution along the ON, in addition to their specification.

Development of macroglial cells in the embryonic chick optic nerve

Macroglia development in the embryonic chick optic nerve was immunohistochemically examined. The astrocytes with glial fibrillary acidic protein immunoreactivity were initially restricted to the retinal end of the optic nerve at stage 40, but had widely dispersed within the optic nerve in an out-side-in manner by stage 44. Oligodendrocytes with myelin basic protein immunoreactivity appeared at stage 38, and were widely distributed at stage 40. Electron microscopic observation confirmed the presence of intermediate filaments in the glial fibers and developing myelin at stages 40–41. The present results suggest that oligodendrocyte precursors undergo terminal differentiation slightly earlier than astrocyte precursors with respect to the expression of marker proteins.

Focal Ventricular Origin and Migration of Oligodendrocyte Precursors into the Chick Optic Nerve

During central nervous system (CNS) development, oligodendrocyte precursors originate in specific locations and subsequently migrate to all regions of the CNS. Here, we demonstrate that the chick optic nerve is populated by oligodendrocyte precursors, which initially appeared in a focal region at the ventral midline of the third ventricle at stage 26–27. Oligodendrocyte precursors migrated into the chiasmal end of the nerve by stage 29 and became uniformly distributed by stage 35. Migrating precursors were restricted to the anterior region of the nerve, closely apposed to axons, and had a bipolar morphology. In contrast to the polysialic acid (PSA)–dependent cooperative streaming migration of olfactory neuronal precursors, the migration of oligodendrocyte precursors along the optic nerve appeared axophilic and unaffected by removal of neural cell adhesion molecule (N-CAM)–associated PSA. These data indicate that during development, defined domains of the ventricular zone give rise to distinct cell types that utilize discrete mechanisms to navigate specific migrational pathways.

Laminin alpha 2 chain (M chain) is found within the pathway of avian and murine retinal projections

Laminin-1 is found at the end-feet of neuroepithelial cells along the outer margin of the optic pathway during early stages of development. Prior to the establishment of most retinal projections in vivo, laminin-1 expression becomes restricted to basement membranes associated with the eye and optic pathway. We report that, in contrast to the alpha 1, beta 1, and gamma 1 chains of laminin-1, laminin alpha 2 chain (formerly laminin M chain) is expressed within the pathway of avian and murine retinal ganglion cell (RGC) growth cones as they extend into the optic nerve, across the optic chiasm and into the brain. Expression of laminin alpha 2 chain is reduced soon after formation of the visual projections but nevertheless maintained at non-basal lamina sites within the adult optic nerve. Laminin alpha 2 chain, in contrast to laminin-1 chains, is also highly expressed in the developing avian tectobulbar pathway. Chick optic nerve derived type-1 astrocytes in culture express laminin alpha 2 chains as extracellular fibrils on their surface. Laminin alpha 2 chain was also detected on the surface of cultured embryonic retinal neurons and developing RGCs. These results suggest that astrocytes and/or RGCS may synthesize laminin alpha 2 chain along the developing optic pathway, and imply that laminin alpha2--in a complex with non-beta1 and non-gamma1 laminin chains-may serve as an adhesive substrate and possibly as a guidance cue for elongating RGC growth cones in vivo.

Expression of a Cell Adhesion Molecule, Neuropilin, in the Developing Chick Nervous System

Neuropilin (previously known as the A5 protein) is a membrane protein identified in Xenopus and is presumed to be involved in the target recognition of the optic nerve fibers. We have isolated cDNAs encoding the chick homologue of neuropilin, using the Xenopus neuropilin cDNA as a hybridization probe. The predicted amino acid sequence of chick neuropilin is 75% identical to that of the Xenopus homologue. A cell aggregation assay showed that fibroblasts transfected with the chick neuropilin cDNA acquired cell adhesiveness. This adhesion is mediated by a heterophilic interaction between neuropilin and protease-sensitive molecules on fibroblasts. The expression of chick neuropilin is restricted to certain neuronal circuits and is dynamically regulated during development, as is the Xenopus homologue. However, their expression patterns differed significantly in the visual systems between the two species: In the chick optic tectum, the localization of neuropilin is confined to layers d and e of SGFS, two of the six layers receiving the retinal input; the chick optic nerve fibers do not express neuropilin; in the chick retina, amacrine cells transiently express neuropilin. Cultured neurons of the dorsal root ganglia express chick neuropilin on their neurites including growth cones. These results suggest that neuropilin functions as a cell adhesion molecule during the formation of certain neuronal circuits in vivo.

Cells from the early chick optic nerve generate neurons but not oligodendrocytes in vitro

We have recently described neuronal potentialities in neuroepithelial cells of the embryonic chicken optic nerve (Giess et al., Proc. Natl. Acad. Sci. USA, 87 (1990), 1643–1647) To further investigate the developmental repertoire of optic nerve cells, oligodendroglial development was studied in cultures of optic nerve explanted at various developmental stages. Oligodendrocyte differentiation was analyzed using antibodies directed against galactocerebrosides (Gal-C) and against sulfatides. Optic nerves removed at embryonic days 5 and 6 (E5-E6) never gave rise in culture to differentiated oligodendrocytes, even after 3 weeks in vitro. In contrast, in cultures of optic nerves removed from E7 or older embryos, cells expressing both oligodendrocyte markers were rapidly and invariably observed. Absence of oligodendrocytes before E7 was not due to culture conditions being inadequate to support the differentiation of early precursors along this pathway, since neuroepithelial cells from E2 and E4 trunk neural tube cultivated in the same conditions expressed Gal-C after respectively 16 and 10 days. These results demonstrate that the optic nerve territory is initially devoid of oligodendrocyte potentialities. Whether oligodendrocyte precursors that, around E7, populate the optic nerve are induced by a specific developmental signal occurring at this stage or migrate from outside the optic nerve remains to be determined.

Ganglion cells in the juvenile chick retina and their ability to regenerate axons in vitro

Ganglion cells in the chicken retina fail to regenerate their axons upon mechanical injury. In order to determine whether this failure to regenerate axons is intrinsic to the neurons or is mediated by the environment, we asked whether ganglion cells possess an ability to regrow their injured axons in the absence of their natural environment, namely in vitro. Since the retina contains morphologically different types of ganglion cells, it became desirable to investigate whether all types of ganglion cells contribute to regeneration of axons. Ganglion cells were labelled post-mortem with the fluorescent dye DiI and described morphologically. Morphometric parameters like the sizes of their perikarya, their dendrites, and the patterns of dendritic ramification and stratification were considered for grouping cells. Although a strong classification of the cells could not be achieved because of the high diversity among this population of neurons, the chick retinal ganglion cells could be separated into seven regular groups which have their somata located within the ganglion cell layer and into one group with the somata located within the inner nuclear layer (displaced ganglion cells). The experimental procedure for regeneration combines crush injury of the chick optic nerve in situ with explantation of retinal pieces 1 week later for organ cultures in a serum-free medium. Under these conditions, the ganglion cells extended axons 1 day after explantation on polylysine/laminin. The densities of ganglion cells contributing to the axonal regrowth reached up to 1447 cells mm −2 (mean 1028 cells mm −2; s.d. 237). This density correspons to 13% of the ganglion cell density in the normal retina, averaged across the total retina area. Although the dendrites of some cells whose axons had regenerated were altered in comparison with the normal ganglion cells, all morphological types of ganglion cells including those with displaced cell bodies contributed propotionally to the regrowth of axons from the explants.

Studies of the developing chick retina using monoclonal antibody 8A2 that recognizes a novel set of gangliosides

A monoclonal antibody, Mab 8A2, that recognizes a novel set of gangliosides was produced by immunizing a mouse with Embryonic Day 14 chick optic nerve. Immunohistochemical studies of the developing chick retina revealed a complex pattern of Mab 8A2 immunoreactivity. Initially, staining is concentrated in the optic fiber layer in the central retina. Later in development, the most intense staining is seen at the periphery of the retina and 8A2 immunoreactivity appears in other retina layers. In the adult retina, 8A2 immunoreactivity is lost from the optic fiber layer but persists in the inner plexiform layer, inner nuclear layer, and outer plexiform layer. Cell culture experiments showed intense staining of neurites from retinal ganglion cells but no staining of Muller cells. Biochemical characterization of the epitope recognized by Mab 8A2 suggests that it includes a 9- O-acetyl group that is present on five different gangliosides. The 8A2 immunoreactive gangliosides are distinct from and have slower mobilities on thin-layer chromatographs than those recognized by Mab D1.1 which recognizes 9- O-acetyl GD3.

Proliferation of glial precursors during the early development of the chick optic nerve.

The mitotic patterns and cytoarchitecture of the optic stalk were studied in the chick embryo during the period of formation of the optic cup until the elimination of the stalk lumen. Cell proliferation in the superficial regions of the stalk ventral wall is described. Superficial cell proliferation, whose beginning coincides with penetration of the earliest optic fibers, gives rise to an early glioblast plate located internally with respect to the marginal lamina of ganglion cell axon fascicles. The early glioblasts are transformed into marginal glioblasts, which undergo radial mitosis. Radially oriented division seems to favour glioblast penetration towards more internal zones of the stalk. Thus the marginal glioblasts are transformed into inner glioblasts, which continue to proliferate. Cell death in the ventral wall of the optic stalk is in close topographical relation with extracellular spaces which loosen up the consistency of the stalk tissue, favouring invasion of the ventral stalk by optic fibers and the addition of new glioblasts by proliferation of preexisting cells.

Evidence for self-absorption of terminals by developing axons of retinal ganglion cells in the chick

The appearance of membrane-bound degenerative organelles in chick optic nerve axons was studied at the electron microscopic level. A semiquantitative analysis revealed a sharp increase in the number of axons containing accumulations of such organelles during the second day after hatching. In dark-reared chicks this increase was retarded, suggesting the presence of a light-influenced event in the early post-hatch period.

Developmental changes of chick cerebellar cortex revealed by monoclonal antibodies

Immunohistochemistry studies of the embryonic and newly hatched chick cerebellum were performed with 13 monoclonal antibodies (MAbs) raised against the embryonic chick optic nerve and a MAb which binds to cell nuclei. Neural MAbs differentially stained Purkinje cells, the external granular layer, molecular layer, internal granular layer, climbing fibers, basket cell axons, Bergmann glia and Ramón y Cajal's ansiform fibers. At the different developmental stages each component responded to MAbs differently. For example, staining of Purkinje cells with MAbs 23C10, 82E10 and 94C2 appeared on day 11 of incubation and disappeared sequentially after day 18. These results reveal molecular heterogeneity not only in cerebellar neurons but also at various developmental stages.

Monoclonal antibodies selective for the inner portion of the chick retina

Five monoclonal antibodies were selected by immunizing mice with embryonic chick optic nerve and screening the resultant hybridoma antibodies by immunofluorescence on sections of the retina. The optic nerve was chosen as the immunizing tissue since it is presumably enriched in ganglion cell-specific antigens. These antibodies bind to antigens which are largely localized to the inner part of the retina. Two of the antibodies, RET1 and RET2, show highly selective binding to the optic fiber layer. Another, RET3, stains the optic fiber layer, ganglion cell layer, and inner plexiform layer. Antibody RET4 stains the optic fiber layer, ganglion cell layer, inner plexiform layer, and the inner one-half of the inner nuclear layer; this antibody binds to an antigen on or associated with the plasma membrane. Antibody RET5 stains the retinal ganglion cell layer faintly and the inner and outer plexiform layers strongly. The potential utility of these antibodies for future studies on retinal development is discussed.