Guidance and topographic stabilization of nasal chick retinal axons on target-derived components in vitro.

We studied mechanisms underlying the generation of topographic order within the developing chick retinotectal connection by combining the recently introduced stripe assay with a novel membrane protein fractionation technique. Our experiments show a preference of temporal and nasal retinal fibers for growing on cell membranes prepared from their proper target area. In addition, membrane preparations from posterior tectum were found to prolong substantially the survival of nasal neurites in vitro. We conclude that tropic as well as trophic interactions contribute to the generation of topographic maps during embryogenesis, in our case to the homing of nasal axons within the posterior tectum.

Axonal pathfinding in organ-cultured embryonic avian retinae.

Eye cups from stage 14-28 (E2 to E5) chick and quail embryos consisting of neural retina, lens, and vitreous body were cultured for 1 or 2 days. These eyes expanded by proliferation of the retinal cells and the surface areas of the retinae increased several-fold. The area covered by ganglion cells and axons also expanded in vitro. [3H]Thymidine labeling showed extensive proliferation of the neuroepithelial cells including the formation of new ganglion cells. Culturing eyes from embryos before stage 17 results, as in vivo, in the generation of the first ganglion cells of the retina, but unlike in the in vivo situation, the outgrowing axons always formed a random fiber net in the central portion of the retina. A defined axonal pattern identical to the in vivo developed only in specimens from embryos of stage 17 and older. Some aberrant axons, however, were also observed at the retinal periphery in specimens from embryos of more advanced stages (20-24), but only during the second day of culturing. Axons in retinae from embryos of stages 23 to 26 heading toward the optic fissure often crossed the fissure and, in contrast to the situation in vivo, invaded the opposite retinal side. These axons of wrong polarity followed the pathways of axons growing centripetally but in reverse direction. This suggests that the polarity of growing nerve fibers and their course are determined by different factors. Culturing the eyes of embryos from stages 20 to 25 in the presence of antibodies showed that the antibodies penetrated the entire retina with 6 hr. Neither anti-N-CAM nor the T-61 antibody--both recognizing membrane proteins of retinal cells--affected the growth of the eyes in vitro. The development of the axonal pattern in vitro was not affected by incubation with N-CAM-antibodies at concentrations up to 500 micron/ml, whereas the T-61 antibody which is known to block neurite extention in vitro (S. Henke-Fahle, W. Reckhaus, and R. Babiel (l984). "Developmental Neuroscience: Physiological, Pharmacological, and Clinical Aspects," pp. 393-398. Elsevier, Amsterdam/New York) showed inhibition of axonal growth in retina cultures at 50 micron/ml. These results indicate that the eye cultures can be used as a test system for antibodies against antigens which could be involved in axon extension and neurite pathfinding in situ.

The formation of the axonal pattern in the embryonic avian retina.

Both the polarity of the axonal growth and the formation of the optic fiber pattern early in retinal morphogenesis were studied in silver stained whole mounts of embryonic chick, quail, and pigeon retinae. The surface area of the retina and of the optic fiber layer increases in size exponentially, the optic fiber layer expanding faster than the retina. The optic fiber layer covers the retinal surface at E5 in quail and at E6 in chick and pigeon. In all species studied, the retinal fiber layer does not expand homogeneously with the optic nerve head as the center. Instead, the retinal fiber layer enlarges with polarities in the dorsal to ventral and nasal to temporal direction. The very first axon bearing ganglion cells appear at stage 16 in the dorsal and central portion of the retina and grow ventrally to merge at the optic disk. From stage 23 on, the optic fiber layer expands faster in the temporal than in the nasal side. Measurements on the initial polarization of young axonal processes show that the axonal growth is directed toward the optic fissure and the optic nerve head. This growth polarization is found at the onset of growth cone formation and in axons far from the nearest ganglion cells or ganglion cell axons. Therefore axon-axon interaction cannot be involved in the initial axon orientation early in retinal morphogenesis.

Axon growth in embryonic chick and quail retinal whole mounts in vitro.

Whole retinae of 4- to 10-day-old chick and quail embryos were spread on membrane filters and kept in culture for up to 4 days. Axon growth during culture was demonstrated by silver staining, anterograde labeling of fibers with RITC, time-lapse recording, and SEM. Fiber growth was observed in specimens from chick embryos up to 7 days old, with a growth maximum at E6 and from quail embryos up to E6 with the maximum at E5. Newly growing axons followed the optic fiber pattern already existing and, like axons in vivo, grew predominantly toward the optic fissure. Directional and orientational adaptation of newly growing axons to the preexisting fibers increased with the donor age. Retinae from donors up to E5 in chick and up to E4 in quail showed a high proportion of axons which crossed the optic fissure during the culture period and invaded the opposite retinal fiber layer. These fibers showed a correct radial orientation while growing in the opposite direction to normal. Likewise, in cultures from these young donors some fibers grew out initially in the diametrically opposite direction to normal toward the tissue periphery. Since all of the wrongly directed axons grew at the same rate as normal and adapted correctly to the already formed axon pattern, this suggests independent signals for the direction and orientation of growing fibers. Treatment of mounted retinae with collagenase or trypsin removed the vitreal retinal surface, leaving the existing axon pattern intact. Subsequently, new axons grew profusely in culture, but lost both their orientational and directional characteristics.