AL-1-induced growth cone collapse of rat cortical neurons is correlated with REK7 expression and rearrangement of the actin cytoskeleton.

Previous experiments identified AL-1 as a glycosylphosphatidylinositol (GPI)-linked ligand for the Eph-related receptor, REK7, and showed that a REK7-IgG fusion protein blocks axon bundling in co-cultures of cortical neurons on astrocytes, suggesting a role for REK7 and AL-1 in axon fasciculation. Subsequent identification of RAGS, the chick homologue of AL-1, as a repellent axon guidance molecule in the developing chick visual system led to speculation that AL-1, expressed on astrocytes, provides a repellent stimulus for cortical axons, inducing them to bundle as an avoidance mechanism. Using a growth cone collapse assay to test this hypothesis, we show that a soluble AL-1-IgG fusion protein is a potent collapsing factor for embryonic rat cortical neurons. The response is strongly correlated with REK7 expression, implicating REK7 as a receptor mediating AL-1-induced collapse. Morphological collapse is preceded by an AL-1-IgG-induced reorganization of the actin cytoskeleton that resembles the effects of cytochalasin D. This suggests a pathway whereby REK7 activation by AL-1 leads to perturbation of the actin cytoskeleton, possibly by an effect on actin polymerization, followed by growth cone collapse. We further show that AL-1-IgG causes collapse of rat hippocampal neurons and rat retinal ganglion cells. These data suggest a role for REK7 and AL-1 in the patterning of axonal connections in the developing cortex, hippocampus and visual system.

Rod photoreceptor neurite sprouting in retinitis pigmentosa.

In animal models for retinitis pigmentosa (RP), rod photoreceptors show abnormal distribution of rhodopsin prior to undergoing cell death. To elucidate the steps in degeneration of human photoreceptors, immunocytochemistry was performed on donor retinas from 15 RP patients and five normal subjects. Rhodopsin immunolabeling in the normal retinas was restricted to the rod outer segments. In the RP retinas, rhodopsin was present in shortened rod outer segments and in the surface membranes of the rod inner segments and somata. In regions of photoreceptor death, the surviving rods had sprouted rhodopsin-positive neurites that were closely associated with gliotic Müller cell processes and extended to the inner limiting membrane. Rods and cones in the RP maculas did not form neurites, but the axons of peripheral cones were abnormally elongated and branched. Double immunofluorescence labeling showed that the rod neurites bypassed the horizontal and rod bipolar cells that are normally postsynaptic to rod axons. To our knowledge, this is the first report of rod neurite sprouting in vivo. We were unable to find neurites on degenerate rods in old rds mice, an animal model for RP. The rod neurites in the human RP retinas resemble the long, branched processes formed by rods cultured on Müller cells or purified N-CAM. Neurite growth by surviving rods in the RP retinas may be a response to neurotrophic factor upregulation, loss of inhibitory factors, or changes in molecules associated with reactive Müller cells. Such changes in the retinal microenvironment may impede functional integration of transplanted photoreceptors. The contributions of the rhodopsin-positive rod neurites and abnormal cone axons to the functional abnormalities observed in RP are unknown.

Cell adhesion molecules regulating neurite growth from amacrine and rod photoreceptor cells.

A great deal is now known about the cell adhesion molecules (CAMs) that are responsible for promoting the growth of ganglion cell axons as they project out of the retina through the optic nerve and finally to distant targets in the brain. However, the CAMs important for regulating axon outgrowth from nonprojection neurons, such as amacrine cells and rods, are not known. Such local circuit neurons extend their neurites rather short distances on cellular surfaces not normally encountered by the ganglion cell axons. To study the mechanisms regulating axon or dendrite growth from local circuit neurons, neurite outgrowth from amacrine cells and rod photoreceptor cells derived from the rat was examined in vitro on immunopurified forms of NCAM, L1, and N-cadherin, three well-characterized adhesive molecules found in the developing retina. Either early (P3) or late (P10) postnatal amacrine cells grew neurites on all three CAMs, but there were significant differences in the percentage of the amacrine cells that responded to each CAM. None of the CAMs supported neurite outgrowth from early postnatal rods, but, surprisingly, NCAM stimulated vigorous neurite extension from rods isolated at postnatal day 10. Postnatal ganglion cells were also examined for comparison and were found not to grow neurites on NCAM, but did grow extensive processes on L1 and N-cadherin. These results show that NCAM, L1, and N-cadherin can promote neurite outgrowth from local circuit neurons, but that the effectiveness of any particular CAM is dependent on the cell type and the developmental period.

Müller cells are a preferred substrate for in vitro neurite extension by rod photoreceptor cells.

To define the factors important in photoreceptor cell morphogenesis, we have examined the ability of rods to extend neurites in vitro. Retinas from neonatal rats were dissociated and plated onto substrate-bound extracellular matrix (ECM) components or cell monolayers. When rods, identified with monoclonal antibodies to opsin, were in contact exclusively with purified ECM (e.g., laminin, fibronectin, type I collagen, or Matrigel), neurite outgrowth was extremely limited. By contrast, rods extended long neurites on Müller cells. Retinal or brain astrocytes, endothelial cells, 3T3 fibroblasts, or other retinal neurons were less supportive of rod process outgrowth. These data demonstrate regional specificity in the promotion of neurite outgrowth by glia and suggest that not all neurons within the retina require the same morphogenic factors.

Optic nerve tissue sections derived from myelin-deficient mutant mice as culture substrata for fibroblast adhesion and spreading.

The substrate properties were compared between normal and myelin-deficient central nervous system (CNS) tissues by an in vitro assay of cell attachment and spreading. Fibroblasts (3T3) were plated onto culture substrata consisting of optic nerve tissue sections cut from normal or two myelin-deficient mutant mice, Shiverer and Quaking. Optic nerve sections from either of the mutant animals supported more 3T3 fibroblast spreading and adhesion than sections derived from animals with normal myelin. These results demonstrate that CNS myelin influences the ability of cells to attach and spread and that it is the actual presence of myelin which is inhibitory rather than the presence of optic nerve axons or oligodendrocytes.

Age of differentiation determines rat retinal germinal cell phenotype: induction of differentiation by dissociation.

We are interested in the mechanisms that control cell phenotype during the development of the CNS. Since different neuronal types arise at different times during neurogenesis in the retina, we predicted that the factors that determine cell type must be developmentally regulated as well. To test this hypothesis, we induced retinal germinal cells to differentiate at different ages by dissociating the retina into single cells and culturing them on a variety of substrates. Prior to dissociation, the S-phase germinal cells were labeled with 3H-thymidine so that their fate could be specifically followed. We found that our culture conditions promoted the differentiation of the majority of the germinal cells and that these cells differentiated into different neuronal types depending on the age of the animal from which the retina had been taken; embryonic day 14 germinal cells differentiated primarily into ganglion cells, and never produced rods, while germinal cells from postnatal day 1 retina differentiated into rods, but not ganglion cells. These results are consistent with the hypothesis that temporally regulated factors determine cell phenotype during the development of the retina.

Early development of photoreceptors in the ventral retina of the zebrafish embryo.

The first photoreceptor outer segments in the retina of the zebrafish Brachydanio rerio appear in the embryo 2.5 days after fertilization, as revealed by scanning and transmission electron microscopy. These outer segments arise in a small region ventral to the exit of the optic nerve. Ultrastructural features of the developing photoreceptor cells, especially those that distinguish the rods and cones, are described. By 3 days after fertilization, the time of hatching, photoreceptor outer segments are widespread in the retina. However, at this time and in the young larva the early developing ventral region remains distinctive because of its conspicuous population of rods.

Colloidal gold fluorescent microspheres: a new retrograde marker visualized by light and electron microscopy.

A new retrograde tracer, rhodamine latex microspheres, permits labeled neurons to be visualized with fluorescence light microscopy. However, their use has been limited to the light microscope. We now have developed colloidal gold fluorescent microspheres which identify retrogradely labeled neurons first by fluorescence microscopy and then by electron microscopy. This new fluorescent/EM tracer will find widespread use in the field of neuroscience to elucidate the ultrastructural integrity of neuronal networks.