Antibodies against the T61 antigen inhibit neuronal migration in the chick optic tectum.

Cell migration in the central nervous system depends, in part, on receptors and extracellular matrix molecules that likewise support axonal outgrowth. We have investigated the influence of T61, a monoclonal antibody that has been shown to inhibit growth cone motility in vitro, on neuronal migration in the developing optic tectum. Intraventricular injections of antibody-producing hybridoma cells or ascites fluid were used to determine the action of this antibody in an in vivo environment. To document alterations in tectal layer formation, a combination of cell-nuclei staining and axonal immunolabeling methods was employed. In the presence of T61 antibody, cells normally destined for superficial layers accumulated in the ventricular zone instead, leading to a reduction of the cell-dense layer in the tectal plate. Experiments with 5-bromo-2'-deoxyuridine labeling followed by antibody staining confirmed that the nonmigrating cells remaining in the ventricular zone were postmitotic and had differentiated. The structure of radial glial cells, as judged by staining with a glia-specific antibody and the fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), remained intact in these embryos. Our findings suggest that the T61 epitope is involved in a mechanism underlying axonal extension and neuronal migration, possibly by influencing the motility of the leading process

Embryonic neurons as in vitro inducers of differentiation of nephrogenic mesenchyme

Nephrogenic mesenchyme differentiates into epithelium as a result of morphogenetic tissue interactions. In vivo, the ureter bud is thought to induce tubular differentiation of the mesenchyme. In vitro recombination experiments have shown that various embryonic tissues can act as inducers when put in close proximity to nephrogenic mesenchyme. Induction also occurs across a porous filter. In the present study we show that only a few embryonic tissues are potent inducers in transfilter cultures in which mesenchyme and inducing tissue are separated by a membrane filter. Of the tissues tested, only embryonic spinal cord and brain were effective, whereas the ureter bud did not induce. All tissues tested sent processes through the filter. Weak inducing capacity of embryonic tissues is thus not due to a failure of the cells to make contact with the mesenchyme. To analyze which cell type within the embryonic brain possesses inducing capacity, neurons were selectively removed from primary cultures of chick tectal cells by antibody and complement-mediated cell lysis. These cultures, consisting of glial and undifferentiated cells, were then recombined with nephrogenic mesenchyme. They proved to be ineffective in inducing tubulogenesis, whereas cell populations containing neurons retained their inducing capacity. In transfilter cultures, ingrowth of neuronal processes deep into the mesenchyme, as assayed by anti-neurofilament staining, occurred within the first 24 hr of culture. Thus, it is not the time needed for processes to grow through the filter, but the time needed to grow into the mesenchyme that corresponds to the minimal induction time. These studies suggest that embryonic neurons are the most effective inducers of nephrogenic mesenchyme in vitro. Differentiation may be triggered by neuronal processes that establish cell contacts deep within the mesenchyme. Neurons might be important for nephrogenesis in vivo as well, although we can present no direct evidence to support this idea, since we failed to detect neurons at early stages of kidney development when the first tubules are induced

Avoidance of posterior tectal membranes by temporal retinal axons

Membrane carpets consisting of alternating membrane stripes were prepared from plasma membranes of anterior and posterior chick optic tectum. Axons from retinal explants extend neurites on these carpets. Axons of the nasal retina do not distinguish between the stripes. Axons of the temporal retina prefer to extend neurites on anterior tectal membranes. Treatment of the membrane fragments with high temperature interferes with the pattern of neurite outgrowth from temporal axons. When growing on carpets consisting of treated anterior and posterior tectal membranes, temporal retinal axons no longer distinguish between the stripes. Treatment of posterior membranes alone is sufficient to abolish the preference of temporal axons to extend neurites on anterior tectal membranes. Treatment of the anterior membranes alone has no effect. This result is best explained by a repulsive component in the posterior tectal membranes. Temporal, but not nasal, axons specifically recognize and avoid that component, with the result that they do not extend neurites on posterior tectal membrane stripes. Once the repulsive component is destroyed, temporal axons are able to extend neurites on posterior tectal membranes

Spatial arrangement of radial glia and ingrowing retinal axons in the chick optic tectum during development

Neuroanatomical tracing of retinal axons and axonal terminals with the fluorescent dye, DiI, was combined with immunohistochemical characterization of radial glial cells in the developing chick retinotectal system. Emphasis was placed on the mode of the tectal innervation by individual retinal axons and on the distribution and fate of the tectal radial glial cells and their spatial relation to retinal axons. It was obvious from fluorescent images obtained from anterogradely filled axons that these axons deserted the superficial stratum opticum (SO) to penetrate the stratum griseum et fibrosum superficiale (SGFS) by making right-angled turns within the SO. Frequently, axons which had invaded the SGFS were bifurcated and had a superficial branch which remained within the SO. Terminal axonal arborization occurred at various depths within the SGFS. Characterization of the tectal glial cells and their radial fibers by means of the anti-filament antibody, R5, and post-mortem staining with the fluorescent dye, DiI, revealed the following. (a) At least from day E8 to P1, tectal glial fibers traversed all tectal layers from the periventricular location of their somata to the superficial interface between SO and pia mater. In this interface they enlarged and formed characteristic endfeet. (b) Glial endfeet covered the whole tectal surface. They showed at early ages anterior-posterior differences having a higher density in the posterior tectum. These differences disappeared at embryonic day E13. (c) After innervation, glial endfeet of the anterior tectal third were arranged in rows parallel to the retinal fibers within the SO. This arrangement was not observed in eyeless embryos. (d) Radial glial fibers could be stained with R5 from day E8 to late embryonic stages throughout their entire length. (e) At the first posthatching days, only the segments of the radial glial fibers restricted to the thickness of the SO were R5-positive, although the fibers still traversed throughout the depth of the tectum. The results are discussed in context to the genesis of the retinotectal projection

Antibodies to cell surface ganglioside GD3 perturb inductive epithelial-mesenchymal interactions

Most epithelial sheets emerge during embryogenesis by a branching and growth of the epithelium. The surrounding mesenchyme is crucial for this process. We report that branching morphogenesis and the formation of a new epithelium from the mesenchyme in the embryonic kidney can be blocked by a monoclonal antibody reacting with a surface glycolipid, disialoganglioside GD3. In contrast, a more than 10-fold excess of antibodies to adhesive glycoproteins (N-CAM, L-CAM, fibronectin) fails to inhibit morphogenesis. Although the anti-GD3 antibody affected epithelial development, the disialoganglioside GD3 was expressed not in the epithelium, but in the mesenchyme surrounding the developing epithelia. The data raise the intriguing possibility that the anti-GD3 antibody inhibits epithelial development by interfering with epithelial-mesenchymal interactions

Antiproliferative and cytotoxic properties of bevacizumab on different ocular cells

AIM: To evaluate the antiproliferative and cytotoxic properties of bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), on human retinal pigment epithelium (ARPE19) cells, rat retinal ganglion cells (RGC5), and pig choroidal endothelial cells (CEC). METHODS: Monolayer cultures of ARPE19, RGC5, and CEC were used. Bevacizumab (0.008–2.5 mg/ml), diluted in culture medium, was added to cells that were growing on cell culture dishes. Cellular proliferative activity was monitored by 5′‐bromo‐2′‐deoxyuridine (BrdU) incorporation into cellular DNA and the morphology assessed microscopically. For cytotoxicity assays ARPE19, RGC5, and CEC cells were grown to confluence and then cultured in a serum depleted medium to ensure a static milieu. The MTT test was performed after 1 day. The “Live/Dead” viability/cytotoxicity assay was performed and analysed by fluorescence microscopy after 6, 12, 18, 24, 30, 36, and 48 hours of incubation. Expression of VEGF, VEGF receptors (VEGFR1 and VEGFR2) and von Willebrand factor was analysed by immunohistochemistry. RESULTS: No cytotoxicity of bevacizumab on RGC5, CEC, and ARPE19 cells could be observed after 1 day. However, after 2 days at a bevacizumab concentration of 2.5 mg/ml a moderate decrease in ARPE19 cell numbers and cell viability was observed. Bevacizumab caused a dose dependent suppression of DNA synthesis in CEC as a result of a moderate antiproliferative activity (maximum reduction 36.8%). No relevant antiproliferative effect of bevacizumab on RGC5 and ARPE19 cells could be observed when used at a concentration of 0.8 mg/ml or lower. CEC and ARPE 19 cells stained positively for VEGF, VEGFR1, and VEGFR2. More than 95% of the CEC were positive for von Willebrand factor. CONCLUSIONS: These experimental findings support the safety of intravitreal bevacizumab when used at the currently applied concentration of about 0.25 mg/ml. Bevacizumab exerts a moderate growth inhibition on CEC when used in concentrations of at least 0.025 mg/ml. However, at higher doses (2.5 mg/ml) bevacizumab may be harmful to the retinal pigment epithelium