Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 1643-1647, March 1990 Developmental Biology Neuronal potentialities of cells in the optic nerve of the chicken embryo are revealed in culture (optic stalk/differentiation/neuroepithelium/neuronal precursor cells/neurofilaments) MARIE-CLAUDE GIESS, PHILIPPE COCHARD*, AND ANNE-MARIE DUPRAT Centre de Biologie du D6veloppement, Centre National de la Recherche Scientifique, Unite de Recherche Associde 675 affilide A l'Institut National de la Sante et de la Recherche Mddicale, Universitd Paul Sabatier, Toulouse, France Communicated by J. B. Gurdon, November 13, 1989 (receivedfor review September 29, 1989) ABSTRACT Neuronal potentialities in neuroepithelial elegant and thorough work of Raff and his colleagues (8-13) cells of the chicken embryonic optic nerve were studied in has led to the characterization of a glial precursor cell in the culture by using neurofilament antibodies as neuronal mark- optic nerve, called an O-2A cell, generating both oligoden- ers. Embryonic day4 and -5 (E4 and ES) optic stalks were drocytes and fibrous, or type 2, astrocytes. This 0-2A explanted in vitro. Within the first few days of culture, numer- progenitor cell is not recognizable in the optic stalk before ous morphologically identifiable neurons extending long neu- day 16 of gestation [embryonic day 16 (E16)] and appears to rites developed. These neurons and their processes were spe- migrate into the optic nerve from external, possibly cerebral, cifically labeled with neurofilament antibodies. Similar results sources (14). From these results, it has been inferred that were obtained by explanting only the medial portion ofE7 optic intrinsic neuroepithelial cells of the optic stalk are unable to stalks away from possibly contaminating cerebral or retinal generate neurons and that their development may be re- tissue. To determine whether neuronal potentialities persisted stricted only to the protoplasmic (type 1) astrocytic pheno- at later embryonic stages, cultures of dissociated optic stalks describes neuronal were established at Ell, E1S, and E18. Neurons labeled with type. On the other hand, a report (15) the various neurofilament antibodies appeared in all cultures of differentiation in vitro from optic stalk cells of the mouse Eli and E15 optic stalks. However, typical neurons could not embryo for a short period between E10 and E11.5 during be recognized in cultures of E18 optic nerves. These results gestation. Thus, cells with neuronal potentialities may occur indicate that cells with neuronal potentialities are present in the in this region ofthe CNS but only at very early developmental embryonic optic nerve from early stages of development and stages. persist until at least E1S. Since the adult optic nerve is devoid We have been studying the question of the occurrence of of nerve cell bodies, our observations are consistent with the cells with neuronal potentialities in the chicken embryonic hypothesis that axons of retinal ganglion cells, which course optic nerve. Our' strategy has been to isolate the optic stalk through the optic stalk, repress neuronal potentialities within from the embryonic environment at various stages of devel- a subpopulation ofprecursor cells during normal development. opment and culture it in vitro. Using morphological and immunocytochemical criteria, we demonstrate that, at least During the development of the vertebrate central nervous between 4 and 15 days of'incubation, neuronal cells develop system (CNS), it remains to be determined how undifferen- from these cultured optic stalks. tiated cells of the primitive CNS anlage are committed to a specific lineage, particularly, how neuronal and glial cell MATERIAL AND METHODS types are segregated. Whether precursor cells of the neural plate or neural tube are capable of giving rise indifferently to Optic Peduncle Disetion and Culture. Eggs (White Leghorn neurons and macroglial cells or are irreversibly committed to chicken) were incubated at 380C for 4-18 days. Developmental a given cell type is a matter of debate, despite various stages were verified using the series ofHamburger and Hamil- attempts to solve this problem (e.g., see refs. 1-7). ton (16). Embryos were placed in sterile Tyrode solution. The A model system that can be used to address this question optic nerve primordium was carefully dissected as follows is the optic nerve. The optic nerve arises from the dienceph- (Fig. 1): a semicircular dorsal incision was made around the alon, which, soon after neural tube closure, bulges bilaterally eyeball and the eyeball was then retracted to expose its to form the optic vesicles. As the optic vesicle extends to posterior surface where the optic nerve emerges from the contact the lateral ectoderm, the part of the neuroepithelium retina. Muscle and connective tissues were dissected away to connecting the optic vesicle to the diencephalon narrows and expose the entire length of optic stalk until it penetrates the forms the optic stalk, presumptive territory of the optic brain. Two transverse sections performed in the middle por- nerve. Thus, the optic stalk is merely a neuroepithelial tion of the nerve-i.e., at some distance from the retina and extension of the diencephalon. However, the adult optic diencephalon-allowed us to remove the optic stalk without nerve, although totally devoid of nerve cell bodies, contains contamination by other CNS tissue (Fig. 1 Inset). axons ofretinal ganglion cells surrounded by several glial cell At stages ranging from 4 to 7 days of incubation, optic types, including fibrous astrocytes, protoplasmic astrocytes, stalks were cultured as explants. At later stages, optic stalks and oligodendrocytes. Therefore, the question arises whether were dissociated at 370C in 0.04% collagenase (Boehringer) in optic stalk neuroepithelial cells are committed from the onset Ca2l- and Mg2"-free Tyrode solution. After 20 min, the to glial lineages or, at least for some time, have the potential explants were washed with Tyrode solution and gently trit- to develop along the neuronal lineages. urated with a blunt Pasteur pipette. The cell suspension was Until now, developmental potentialities ofoptic nerve cells centrifuged, washed in complete medium, and plated in have been documented mostly in the perinatal rat. The Abbreviations: CNS, central nervous system; NF, neurofilaments; The publication costs of this article were defrayed in part by page charge FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine payment. This article must therefore be hereby marked "advertisement" isothiocyanate; E, embryonic day. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 1643 Downloaded by guest on September 24, 2021 1644 Developmental Biology: Giess et al. Proc. Natl. Acad. Sci. USA 87 (1990) tional de la Recherche Scientifique, Strasbourg, France), used at 1:100 dilution. The specificity of all antibodies was verified on Western blots containing chicken nervous system intermediate fila- ments, separated on polyacrylamide gels (18) and transferred to nitrocellulose (19). Results for the three anti-NF mono- clonal antibodies are shown in Fig. 2. Immunofluorescence Staining. Cultures were fixed at var- ious stages (usually 5, 7, or 15 days after plating) with 3.5% (vol/vol) formaldehyde in phosphate-buffered saline (Pi/ NaCl = 0.01 M sodium phosphate, pH 7.4/0.9% NaCl). After extensive rinsing in P,/NaCl, coverslips were preincubated for 15 min with Pi/NaCl containing 2% (wt/vol) lyophilized skimmed milk and 0.25% Triton X-100. All antibodies were diluted in P1/NaCl/milk. Incubation with specific antibodies was at room temperature for 30 min. After three 5-min rinses in P1/NaCl/milk, coverslips were incubated with the appro- priate fluorescein isothiocyanate (FITC)- or tetramethyl- rhodamine isothiocyanate (TRITC)-conjugated anti-immuno- globulin antibody, under the same conditions. The conju- gated reagents and their working dilutions were as follows: goat anti-mouse immunoglobulin/FITC (Nordic, Tilburg, FIG. 1. Schematic drawing of the head of an E7 chicken embryo immu- summarizing the dissection procedure of the embryonic optic nerve. The Netherlands) at a 1:100 dilution; goat anti-mouse The optic stalk (OS), well-individualized at this stage, is exposed noglobulin/TRITC (Nordic) at a 1:20 dilution; goat anti- from a dorsal view. CG, ciliary ganglion; E, eyeball. (Inset) Enlarge- rabbit IgG/FITC (Nordic) at a 1:40 dilution; and goat anti- ment of the dissection field. Two transverse sections (double arrows) rabbit IgG/TRITC (Immunotech, Marseille, France) at a 1:50 allow the medial part of the optic stalk to be removed. dilution. In double-labeling experiments, cells were incubated se- 4-well plastic dishes (Nunc) onto 16-mm glass coverslips quentially with the specific antibodies, washed, and stained, coated with rat-tail collagen (Sigma). again sequentially, with the appropriate FITC- or TRITC- The culture medium was Dulbecco's modified Eagle's conjugated antibodies. None of these fluorescent antisera medium supplemented with 10% (vol/vol) fetal calf serum cross-reacted with inappropriate immunoglobulins. (Flow Laboratories) and gentamicin (Sigma) (50 gg/ml). Control coverslips, for which the primary antibody or, for Cultures were maintained at 370C in a humidified atmosphere double-labeling experiments, either the primary or secondary of'5% C02/95% air. Cultures were fed every 3 days. antibody was omitted, were always included. Antibodies. Antibodies