UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles.

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.

Molecular gradients along the proximal-distal axis of embryonic insect legs: possible guidance cues of pioneer axon growth.

It has been proposed that gradients of environmental cues direct the proximal growth of pioneer axons in embryonic insect legs. Hybridoma techniques have been used to produce 3 monoclonal antibodies (mAbs) that bind to components associated with the basal lamina/extracellular matrix that are non-uniformly distributed along the proximal-distal axis of cockroach legs at the time of pioneer axon growth. Two of these mAbs, PROD-1 and PROD-2, label the proximal parts of the leg more intensely than the distal ends. The other mAb, DIP-1, has the reverse pattern of binding with the distal parts of the leg labeled more intensely. The graded distribution of these antigens only occurs just prior to and during the growth period of the Ti1 pioneer axons. Western blot analyses and immunoprecipitations have identified the protein antigens recognized by these mAbs. The spatial and temporal distributions of these molecules in the legs and the CNS make them good candidates for environmental guidance cues of pioneer axon growth.

An axon growth associated antigen is also an early marker of neuronal determination.

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.

Developmental stage-specific antigens in the nervous system of the cockroach.

A specific effort was made to obtain monoclonal antibodies that bind to macromolecules that play a role in the development of the nervous system. It was considered that good candidates for such molecules were those that were only transiently present in the embryonic nervous system. Hybridomas were prepared from spleen cells taken from mice that had been immunized with nerve cords from cockroach embryos at the 43-50% stage of development. The hybridoma supernatants were screened for antibody binding to frozen sections of both embryonic and adult thoracic ganglia. Cell lines that produced monoclonal antibodies that transiently bound to the embryonic nervous system were saved and cloned. These developmental stage-specific monoclonal antibodies either did not bind to the adult nervous system or bound to it with a pattern very different from that in the embryonic nervous system. The developmental stage-specific antigens detected by these monoclonal antibodies were organized into four categories based on the part of the embryonic nervous system in which they were transiently localized. These include binding to the cell bodies of all neurons, cell bodies of subsets of neurons or neuroblasts, subsets of axons, and the neuropile. Preliminary biochemical characterization of the antigens showed that many of these antibodies were recognizing carbohydrate epitopes. Functions for these antigens, most of which are components of the cell surface, are tentatively proposed.