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In the developing wing of Drosophila a set of early differentiating neurons pioneer the axon courses observed in the adult. The possibility that these first cells are indispensable for establishing the normal neural pathways has been tested. The differentiation of particular neurons was suppressed by inducing cell clones homozygous for two scute deficiencies, mutations that inhibit the differentiation of sensilla and their associated neurons. From the analysis of the nerve patterns in wings lacking specific sensilla, it has been demonstrated that none of the identified neurons are essential for guiding other axons along the correct path. However, the possibility remains that the presence of certain cells may increase the probability of establishing the normal pattern of peripheral nerves.

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 role of the notochord on the early development of ventral horn neuroblasts was investigated in chick embryos by implanting an additional notochord fragment near the right side of the thoracic neural tube. When the implant was located directly lateral to the neural tube, an enlargement of the right half of the neural tube and of the area of neuroblasts occurred, and axons were found to pass through the outer membrane of the neural tube over a broad dorsoventral trajectory. When the notochord was located ventrolaterally a population of neuroblasts including their efferent axons was found at a more dorsal location. It is concluded that a notochordal implant is able to influence the differentiation of neuroblasts.

Computerized image analysis was used to determine the normal axonal count and axon diameter distribution in 12 normal human eyes. Mean axon count per nerve was 969,279 +/- 239,740 and mean axon diameter was 0.72 +/- 0.07 micron. Multiple linear regression disclosed 4909 axons lost yearly (P = 0.08). Statistical analysis did not show a relationship between axon diameter and age or time to fixation. The inferotemporal sector of the nerve had the highest fiber density (P = 0.02). The superonasal nerve had higher mean diameters (P = 0.02). This study may provide a baseline for future pathologic studies.

The sensory neurons of the Drosophila wing differentiate during the initial stages of metamorphosis, appearing in the imaginal wing disc as it everts and flattens. These identifiable neurons arise in a stereotyped sequence, and lay down a specific pattern of axon bundles which travel proximally to the CNS. In several locations, the early arising "pioneer" neurons send axons in the direction of more proximal pioneer neurons, later joining with these to form continuous peripheral nerves. It is possible that distal neurons can contact more proximal neurons by random filopodial search, and use this information to guide axonal outgrowth. To test this "guidepost" hypothesis, everting wing discs were raised in vitro to allow surgical manipulation. Neural outgrowth was largely normal in vitro, though growth of the wing was stunted. If such discs were cut into proximodistal fragments before or at the time of initial axonogenesis, neural outgrowth remained normal: distal axons still grew in the direction of the now missing proximal neurons. Thus, proximal neurons are not necessary for the correct guidance of distal neurons in the developing wing.

We have previously described the generation of a monoclonal antibody (DSS-3) that binds to all neurons in cockroach embryos at 50% development and to only a small subset of interneurons in the adult nervous system. This developmental stage-specific antigen was observed to reappear in all axotomized adult neurons that were undergoing axonal regeneration. In the present study the time course of the appearance of this growth-associated antigen during embryonic development was determined. Unexpectedly, the antigen was observed to be present in embryonic neurons long before axon growth. In addition, all cells in the CNS neuronal lineage (neuroblasts, ganglion mother cells, and neurons) bind the antibody as soon as they can be morphologically identified. However, the antigen is also transiently present in all neuroepithelial cells at a stage prior to the morphological differentiation of some of them to neuroblasts. Analogous patterns of DSS-3 binding to cells involved in the development of sensory neurons and leg pioneer neurons are observed. The DSS-3 antigen is therefore a very early marker for the capacity of ectodermal epithelial cells to develop along a neuronal lineage.