Chondroitin sulfate as a regulator of neuronal patterning in the retina.

Highly sulfated proteoglycans are correlated with axon boundaries in the developing central nervous system which suggests that these molecules affect neural pattern formation. In the developing mammalian retina, gradual regression of chondroitin sulfate may help control the onset of ganglion cell differentiation and initial direction of their axons. Changes induced by the removal of chondroitin sulfate from intact retinas in culture confirm the function of chondroitin sulfate in retinal histogenesis.

Reduced tumorigenicity of rat glioma cells in the brain when mediated by hygromycin phosphotransferase.

OBJECT: A variant of C6 glioma cells, C6R-G/H cells express hygromycin phosphotransferase (HPT) and appear to have reduced tumorigenicity in the embryonic brain. The goal of this study was to investigate their reduced capacity to generate tumors in the adult rat brain. METHODS: Cell lines were implanted into rat brains and tumorigenesis was evaluated. After 3 weeks, all rats with C6 cells showed signs of neurological disease, whereas rats with C6R-G/H cells did not and were either killed then or allowed to survive until later. Histological studies were performed to analyze tumor size, malignancy, angiogenesis, and cell proliferation. Cells isolated from rat brain tumors were analyzed for mutation to HPT by testing their sensitivity to hygromycin. CONCLUSIONS: The results indicate that HPT suppresses tumor formation. Three weeks after implantation, only 44% of animals implanted with C6R-G/H cells developed tumors, whereas all animals that received C6 glioma cells developed high-grade gliomas. The C6R-G/H cells filled a 20-fold smaller maximal cross-sectional area than the C6 cells, and exhibited less malignant characteristics, including reduced angiogenesis, mitosis, and cell proliferation. Similar results were obtained in the brain of nude rats, indicating that the immune system did not play a significant role in suppressing tumor growth. The combination of green fluorescent protein (GFP) and HPT was more effective in suppressing tumorigenesis than either plasmid by itself, indicating that the GFP may protect against inactivation of the HPT. Interestingly. hygromycin resistance was lost in tumor cells that were recovered from a group of animals in which C6R-G/H cells formed tumors, confirming the correlation of HPT with reduced tumorigenicity.

Multiple factors govern intraretinal axon guidance: a time-lapse study.

In this study, the multiple factors that govern the unidirectional path of intraretinal axons, as well as the cellular movements prior to and during early axonogenesis, were investigated using time-lapse videomicroscopy. For several hours prior to overt axon elongation, young retinal ganglion cells send out transient minor processes in all directions at the pial surface. Time-lapse analysis of the chondroitin sulfate (CS)-containing matrix that has been suggested to play an important role in regulating this early differentiative event revealed the dynamic, wavelike properties of this extracellular matrix component. As the CS matrix dissipates across the immature ganglion cells, only one minor process, away from the highest concentration of CS peripherally and in the direction of the optic fissure centrally, is retained and becomes the mature axon. Focal concentrations of L1 appear at points of neurite contact with previously established axons, suggesting that this growth-promoting molecule is also involved with establishing the precise, unidirectional outgrowth pattern of retinal ganglion cell axons. NCAM was diffusely distributed on neural elements and on the neuroepithelial endfeet in the central and peripheral retina and, thus, may not be an essential unidirectional axon growth cue. Growth cones mechanically deflected 180 degrees from the optic fissure after the CS wave had receded from the central retina had morphologies and rates of elongation similar to those oriented in the proper direction. Growth cones deflected obliquely toward the ventral retinal periphery entered a territory of increasing CS-containing proteoglycan matrix and neurons with minor processes. As these deflected axons entered more deeply into this region they slowed down and sent out long transient branchlike processes. These observations illustrate the complex organization of the changing cell surface and matrix components within the retina during axonogenesis and axon outgrowth. The results also elucidate the potential importance of a cell state where immature neurons probe their environment via minor processes. These specialized neurites may provide the neuron with a way to sample a full 360 degrees of terrain around them. This method of exploring the environment could afford the cell a mechanism with which to sample, summate, and respond to physical structures as well as simultaneously occurring negative and positive molecular influences that are distributed unequally on either side of the cell body.

Exogenous glycosaminoglycans induce complete inversion of retinal ganglion cell bodies and their axons within the retinal neuroepithelium.

Prior to forming an axon, retinal ganglion cells retain a primitive radial configuration while maintaining ventricular and vitreal endfeet attachments. During their subsequent differentiation, ganglion cells polarize their cell body and axon only along the vitreal surface. When the ventricular surfaces of intact retinas in organ culture were exposed to free chondroitin sulfate (CS) in solution, both the cell body and nerve fiber layers were repolarized to the opposite side of the neuroepithelium. However, the basal lamina remained in its usual position. Thus, the ability to initiate an axon is not restricted to the vitreal endfoot region of differentiating neurons, and in addition, the radial position at which the axon emerges can be mediated by the location and concentration of the extracellular CS milieu.

Unique changes of ganglion cell growth cone behavior following cell adhesion molecule perturbations: a time-lapse study of the living retina.

In the mammalian retina, multiple mechanisms are responsible for guiding retinal ganglion cell axons to the optic fissure. In the present study we have used time-lapse videomicroscopy to show that, within the center of the retinal neuroepithelium, growth cones use a scaffold of previously formed axons as a substrate for guidance. High magnification time-lapse videomicroscopy of normal growth cones in the midretina have shown that they have the ability to alter their shape from long, streamlined forms that hug other axons to more flattened forms that move between axons or neuroepithelial endfeet. In studies on the role of specific cell interactions in these events, Fab fragments against L1 and NCAM, administered either alone or in combination, were found to have dramatic and distinct effects on retinal ganglion cell growth cones. Anti-L1 Fab fragments severely disrupted radial growth cone orientation and rate of outgrowth. The anti-L1-treated growth cones initially stalled for 2 h, then changed direction and, thereafter, resumed an elongation rate twice as fast as in control preparations. By contrast, anti-NCAM Fab did not affect growth cone direction, but caused subsets of growth cones to speed up initially, then to dramatically increase in size, stall, and eventually halt. These results imply that L1 and NCAM play different roles in the promotion and direction of axon growth and, along with repulsive molecules and physical channels, provide essential information for the unidirectional growth of retinal axons into the optic fissure.

The earliest patterns of neuronal differentiation and migration in the mammalian central nervous system.

With the use of four independent cell markers and Brd-U birthdating we have charted the earliest stages of neuronal differentiation and migration in the developing rat central nervous system, including the cortex, spinal cord, and retina. One of the markers, the monoclonal antibody 2G12, labeled a large subpopulation of differentiating cells that uniformly lined the ventricles throughout these CNS regions at unexpectedly early ages. Immunocytochemistry demonstrated that, in cortex, the 2G12 antigen could appear in cells during mitosis. More mature looking 2G12-positive cell types initially had a primitive radial morphology and were axonless. However, in strict spatio-temporal sequences, the most mature looking 2G12-positive cells had the ability to sprout GAP-43-positive axons before or after the cell body left the ventricular surface and before or after detachment of their pial or ventricular endfoot processes. Double label experiments with 2G12 and Brd-U showed that none of these three 2G12-positive cell types incorporated Brd-U after a short pulse. The primitive neuroepithelial shape of the immature neurons was verified with a polyclonal GAP-43 antibody, a type III beta tubulin antibody, and DiI labeling from a distal portion of the axon. In the cortex and retina, the 2G12 marker persisted in cells that had reached prospective neuronal layers. However, in all CNS regions observed, 2G12 immunoreactivity disappeared from the cell body as the axon extended from the young neuron. Based on the smooth progression of changing 2G12-positive cell shapes, but also because of the transient nature of this label, we can only speculate that the 2G12 epitope may be marking a continuum of neuronal cell states throughout the earliest period of differentiation and migration. Thus, our hypothesis suggests that many of the youngest CNS neurons may have a widespread distribution and may begin their differentiation, and even remain axonless for a time, while retaining a neuroepithelial morphology. Once differentiation resumes, a major mode of transformation into mature neurons during the earliest stages of development could occur via translocation of the cell soma into the pial process. Importantly, these markers have verified at later stages, and especially in cortex, that multiple mechanisms exist for neuronal migration in the CNS depending on the region and stage of development.

Fibroblast growth factor receptor function is required for the orderly projection of ganglion cell axons in the developing mammalian retina.

During the early stages of development various cell adhesion molecules (CAMs) and fibroblast growth factor receptors (FGFR) are expressed throughout the retinal neuroepithelium. The ability of retinal ganglion cells to project their axons to the optic fissure depends, in part, on cell-cell interactions mediated by cell adhesion molecules. In the present study we show that the ability of the firstborn rat retinal ganglion cells to extend axons in vitro can be stimulated by NCAM and L1, but not N-cadherin. Both CAM responses can be fully inhibited by antibodies that block neuronal fibroblast growth factor receptor function and by agents that block defined steps in the FGFR signal transduction cascade. When added to living E13.5 rat retinal whole-mount preparations the same agents induced errors in the orderly establishment of young axon patterns in the retinal periphery and caused axons in the retinal center to defasciculate. These results suggest that the activation of the fibroblast growth factor receptor signal cascade not only promotes survival and proliferation of various cell types but can also mediate intraretinal axon guidance.

Generation of a radial-like glial cell line.

Rat C6 glioma is a cell line that has been used extensively as a model of astroglia. Although this cell line retains many of the properties of developing glia, it does not resemble morphologically the specialized form of glia found embryonically, the radial glia. In experiments designed to study a mutant form of receptor protein tyrosine phosphatase beta, we isolated a subclone of C6 called C6-R which, like radial glia, assumes a highly polarized radial-like morphology in culture. C6-R cells and, to a somewhat lesser extent, C6 cells, express cytoskeletal proteins found in developing astroglia including glial fibrillary acidic protein and RC1. As seen with radial glia, cerebellar granule cell bodies and neurites migrated along radial processes of C6-R cells in culture. Morphological analysis of dye-labeled cells injected into the developing forebrain revealed that a large fraction (approximately 60%) of the C6-R cells in the cortex assumed a radial orientation and about half of these (approximately 30%) made contact with the pial surface. In contrast, the parental C6 cells generally formed aggregates and only displayed a radial alignment when associated with blood vessels. These results suggest that we have generated a stable cell line from C6 glioma which has adopted certain key features of radial glia, including the ability to promote neuronal migration in culture and integrate radially in vivo in response to local cues. This cell line may be particularly useful for studying receptors on radial glia that mediate neuronal migration.