Thymidine-labeling and PCNA-staining experiments indicate that in the absence of NT-3, a higher proportion of proliferating cells is observed compared with age-matched controls

Thymidine-labeling and PCNA-staining experiments indicate that in the absence of NT-3, a higher proportion of proliferating cells is observed compared with age-matched controls. E-3810 == NT-3 influences early retinaldevelopment == At E6, fewer retinal ganglion cells are seen in the retina of anti-NT-3-treated embryos, and fewer axons are counted in the optic nerve. Seven different cell types are generated within a short period of time, and cell lineage analyses indicate that the embryonic retina contains multipotent progenitor cells (Turner and Cepko, 1987;Holt et al., 1988). In addition,in vitroexperiments with dissociated cells suggest that the retina generates intrinsic signals that contribute to the determination of cell fate and cell survival (Anchan et al., 1991; Althuser and Cepko, 1992;Watanabe and Raff, 1992; also seeKelley et al., 1994and references therein). Hitherto, few such signals have been identified and shown to be physiologically relevant during normal development. In this study, we examine the role of neurotrophin-3 (NT-3) in the development of the chick retina. Like other members of the neurotrophin gene family, NT-3 plays an essential role in the development of the nervous system. Deletion of the gene coding for NT-3 (Ernfors et al., 1994;Farias et al., 1994) or the NT-3 receptor trkC (Klein et al., 1994), or the administration of antibodies blocking the activity of NT-3 (Gaese et al., 1994), results in neuronal losses in peripheral E-3810 ganglia. So far, however, there are no clear indications that NT-3, or indeed any other neurotrophins, also controls neuronal numbers in the CNS during normal development. Both NT-3 and trkC are expressed in many structures in the developing CNS (Tessarollo et al., 1993;Kahane and Kalcheim, 1994), and previous studies have demonstrated the presence of high-affinity NT-3 receptors on dissociated retinal cells as early as the fourth day of development (Rodrguez-Tbar et al., 1993). In addition, NT-3 also promotesin vitrothe neuronal differentiation of thymidine-labeled precursor cells (De la Rosa et al., 1994). These results prompted us to test the role of NT-3in vivoduring chick retinal development, using a monoclonal antibody specifically blocking the biological activity of NT-3. == MATERIALS AND METHODS E-3810 == == == == Antibodies == The monoclonal antibodies 12 and 27/21 used here specifically block the biological activities of NT-3 and NGF, respectively (Gaese et al., 1994). Both belong to the mouse IgG1 subclass, and levels of 1220 g/gm wet weight were measured in the tectum of treated embryos using an enzyme immunoassay (Gaese et al., 1994). In the retina, the levels were 8.1 2.1 g/gm at embryo day 6 (E6), 9.1 2.1 g/gm at E9, and 10.5 5 g/gm at E11. The monoclonal antibody G4 labels a glycoprotein located on neurites of a subset of differentiated neurons (Rathjen et al., 1987). It stains 1012% of differentiated neurons in dissociated cultures from the E9 chick retina (data not shown), including the retinal ganglion cells Rabbit Polyclonal to ZNF682 and a population of neurons in the inner nuclear layer. The monoclonal antibody against chick Thy-1 specifically recognizes retinal ganglion cells (Cohen et al., 1986; Sheppard, 1991). == Chick embryos == Fertilized eggs from White-Leghorn hens were obtained from a local supplier and incubated at 38.5C in 70% humidity atmosphere, and the embryos were staged according toHamburger and Hamilton (1951). Hybridoma cells secreting anti-NT-3 and anti-NGF antibodies were grownin ovoaccording to the procedure described previously for quail embryos (Rohrer et al., 1988). Eggs were incubated for 3 d (stages 1415 of development), and a suspension of 2 106hybridoma cells in 50 l PBS was applied onto the chorioallantois membrane. == Cell culture == Retinae from E8 or E9 embryos were dissected free from pigmented epithelium and dissociated as described previously (Rodrguez-Tbar et al., 1989). Briefly, retinae were placed in 1 ml Ca2+, Mg2+-free PBS containing 3 mg/ml bovine serum albumin and treated with 1 mg/ml trypsin (Worthington, Freehold, NJ) for 15 min at 37C. Digestion was stopped by adding 2 mg/ml soybean trypsin inhibitor (Sigma, St. Louis, MO). Cells were dissociated by passing 510 times through a wide-bore Pasteur pipette, suspended in culture medium, and plated onto 10 mm round glass coverslips in 4-well dishes (Greiner, Frickenhausen, Germany). The coverslips were coated previously with polyornithine (Sigma) and laminin (Gibco, Paisley, Scotland), according toCollins E-3810 (1978). The initial density of the cultures was between 48,700 and 54,100 cells/cm2. Cells were cultured in 50% DMEM/50%.