Surface Glycoproteins and Diffusible Factors T.M. Jessell, M. Placzek, M. Tessier-Lavigne, P. Bovolenta, Center for Neurobiology, Columbia University, New York, NY 10032 An initial step in the establishment of specific neural connections is the selection of distinct pathways by developing growth cones and axons. To determine mechanisms that contribute to the guidance of axons in mammalian CNS, we are analyzing pathway selection by neurons in the rat spinal cord. We have characterized surface proteins that are restricted to subsets of spinal axons and growth cones. A 135 kDa cell surface glycoprotein, TAG-1, is expressed transiently on subsets of embryonic spinal cord axons and growth cones. TAG-1 is immunochemically distinct from the cell adhesion molecules NCAM and L1 (NILE) and is expressed on commissural neurons over the period of their inital axon extension toward the ventral midline of the spinal cord. TAG-1 and L1 appear to be temporally and spatially segregated to different segments of the same commissural axons. The change in expression of these two glycoproteins occurs as growth cones cross the ventral midline of the spinal cord in contact with a specialized group of epithelial cells, termed the floor plate. Recent in vitro studies have provided evidence that the floor plate may release diffusible factor(s) that contribute to the ventral guidance of commissural neurons. In addition, mouse mutants that lack a floor plate exhibit perturbations in axon projection patterns at the ventral midline of the spinal cord. The floor plate may function as a specialized intermediary target that influences the guidance of commissural neurons by (1) releasing tropic factors, (2) regulating surface axonal glycoprotein expression, and (3) imposing mechanical or adhesive constraints on axonal projections at the ventral midline of the spinal cord. Molecular Control of Phenotypic Decisions in the Sympathoadrenal Lineage Paul H. Patterson Division of Biology, California Institute of Technology, A variety of signals can control the choice of phenotype made by neural crest derivatives in culture. Precursor cells from the embryonic rat sympathetic ganglion or adrenal medulla can be influenced to become noradrenergic or cholinergic neurons, chromaffin, or small, intensely fluorescent (SIF) cells. These cells differ in their classical transmitters, peptides, vesicle ultrastructure, and surface and secreted protein profiles. The precursor cells share some of the properties of chromaffin cells. The choices among these phenotypes can be controlled by NGF, FGF, EGF, two proteins secreted by certain nonneuronal cells, a developmentally regulated protein in rat serum, neuronal surface components, corticosteriods, insulin, and by depolarization and Ca++ influx. The conversion of noradrenergic neurons into fully functional cholinergic neurons also occurs during normal development in vivo. The protein that mediates this conversion in culture has been purified and partially sequenced. Antisera against a synthetic peptide of this sequence can precipitate the native cholinergic factor, and can also block its activity directly. In addition, the synthetic peptide itself can affect the activity of the factor when both are added to cultured neurons. The sequence information is also being used in an effort to clone the gene for the cholinergic factor. REFERENCES Fukada, K., Proc. Natl. Acad. Sci. 82:8795-8799, 1985. Doupe, A.; Patterson, P.; and Landis, S., J. Neurosci. 5:2143-2160, 1985. Wolinsky, E., and Patterson, P., J. Neurosci. 5:1509-1512, 1985. Anderson, D., and Axel, R., Cell 47:1079-1090, 1986. Stemple, D.; Mahanthappa, N.; and Anderson, D., submitted. Development II Activation of Transcription In Vitro From the Rat Somatostatin Promoter by A 43 kD DNA Binding Protein Jack Dixon, Ourania Andrisani, Alex Zhu, and David Pot The somatostatin gene, which encodes a 14 amino acid peptide hormone, is selectively expressed in neuronal and endocrine tissues. The cis-acting promoter element essential for expression of the somatostatin gene in CA-77 cells is located between position -60 to -43 of the promoter (Andrisani et al., Nucl. Acids Res. 15:5715-5728, 1987) and contains the TGACGTCA octanucleotide module. We have identified three sequence-specific DNAprotein complexes (b1-b3) which are formed when HeLa cell or CA-77 cell extracts interact with the -70 to -29 region of the promoter (Andrisani et al., Mol. Cell. Biol., in press). These three sequence-specific complexes form identical DNA/protein contacts with the somatostatin TGACGTCA module as shown by methylation interference analyses. The activities forming complexes b1-b3 have been fractionated by DEAE-Sepharose chromatography and the major b2 forming activity has been purified to homogeneity by sequence-specific DNA affinity chromatography. The affinity purified material gives a single band on silver stained SDS gels and has an apparent molecular weight of 43 kD. In order to evaluate the role of the protein in the transcription of the rat somatostatin gene, we have developed an in vitro HeLa or CA-77 cell free system from which the somatostatin promoter directs accurate transcription. We show that when the extract is depleted of the TGACGTCA binding factor(s) by the affinity procedure, the somatostatin promoter is inactive. The transcriptional activity is restored by addition of the purified 43 kD protein. We conclude that this 43 kD DNA-binding protein is a somatostatin gene transacting factor. Genes That Affect the Development of Specific Serotonergic and Gabaergic Motorneurons of the Nematode Caenorhabditis Elegans H. Robert Horvitz, Chand Desai, Gian Garriga, and Steven McIntire Department of Biology, MIT, Cambridge, MA 02139 What are the genetic and the molecular mechanisms responsible for the development of specific types of nerve cells? To approach this problem, we have identified genes necessary for the development of two classes of motorneurons in the nematode Caenorhabditis elegans. Mutations that define 15 genes perturb the functioning of the GABAergic DD and VD motorneurons, which innervate the body muscles and control locomotion. Some of these genes are necessary for DD and VD process outgrowth, whereas others are necessary for normal levels of GABA expression. Mutations that define 34 genes perturb the functioning of the serotonergic HSN motorneurons, which innervate the vulval muscles and drive egg laying. Some of these genes are involved in the determination of HSN cell fates, whereas others must function for the expression of specific HSN characteristics, including cell migration, nerve process outgrowh, and neurotransmitter expression. By analyzing these genes both genetically and molecularly, we hope to elucidate how each functions in the development of a particular class of neuron. Mutations Affecting Neurogenesis and Y.N. Jan, K. Blochlinger, R. Bodmer, H. Vaessin, E. Giniger Howard Hughes Med. Inst. and the Depts. of Physiology and Biochemistry, UCSF, San Francisco, CA 94143 We are interested in early events of neural development. To study these events at the molecular level, we took a genetic approach and attempted to identify relevant genes. We used the embryonic peripheral nervous system (PNS) as our assay system, and examined lethal mutations and chromosomal deletions for possible defects. After screening approximately 40 percent of Drosophila genome, we found more than 20 genes which, when deleted or mutated, give rise to abnormal PNS. Currently, we are studying at the molecular level several genes that we believe play important roles in neurogenesis or specifying neuronal identity. Two of the genes will be described. (1) 1(3)183. Mutations of this gene affect one class of sensory organ (the chordotonal organ) preferentially, although other cells are also affected. We have cloned the gene. The sequence of this gene product revealed that it is homologous to cyclin (C. Lehrer and P. O'Farrell independently found this gene by using cross homology to sea urchin cyclin). Cyclin has been suspected to play an important role in controlling the cell cycle. The identification of 1(3)183 as the structural gene for Drosophila cyclin should allow us to test this notion and to study the role of the cell cycle in neurogenesis. (2) cut. In the absence of cut gene activity, external sensory (es) organs are transformed into chordotonal organs (ch). Recently, we found that the cut locus codes for a large protein with a homeodomain. This protein is expressed in nuclei of cells in es organs but not in cells within ch organs. These observations suggest that this cut product acts as a nuclear regulatory protein. |