Leslie C. Timpe

frazzled Encodes a Drosophila Member of the DCC Immunoglobulin Subfamily and Is Required for CNS and Motor Axon Guidance

We have identified a Drosophila member of the deleted in colorectal cancer (DCC) gene family. The frazzled gene encodes transmembrane proteins that contain four immunoglobulin C2 type domains, six fibronectin type III repeats, and a cytoplasmic domain of 278 amino acids. Like vertebrate members of the DCC family, Frazzled is expressed on axons in the embryonic central nervous system and on motor axons in the periphery. Frazzled is also expressed on epidermis and gut epithelium. Null mutants in frazzled are defective in axon guidance in the central nervous system and in motor axon guidance and targeting in the periphery. The phenotypes strongly resemble those of a deletion of the two Drosophila Netrin genes. We have rescued the frazzled CNS and motor axon defects by expressing Frazzled specifically in neurons; expression in target tissues does not rescue the phenotype. These data, together with vertebrate studies showing binding of DCC to netrin, suggest that Frazzled may function in vivo as a receptor or component of a receptor mediating Netrin-dependent axon guidance.

Four cDNA clones from the Shaker locus of Drosophila induce kinetically distinct A-type potassium currents in Xenopus oocytes

The Shaker gene encodes the A-channel of larval and pupal muscle, or one or more of its subunits. Alternative splicing produces messages for several different proteins; two mRNA species have previously been shown to induce the expression of A-currents in Xenopus oocytes. Two additional mRNAs have now been tested and found to produce A-currents in oocytes. The four currents differ in kinetics of inactivation, indicating that the Shaker products may contribute to kinetic diversity in A-channels of the fly and that sequences in both the amino- and carboxy-terminal regions are important for inactivation.

Molecular Studies of Voltage-gated Potassium Channels

The cloning and characterization of the voltage-activated Shaker potassium channel gene in Drosophila have led to the identification of structural elements involved in potassium channel gating. As found for the voltage-activated sodium channel, the S4 segment, located in the conserved core of the protein, plays a central role in voltage-dependent activation. Potassium channels appear to be formed by the assembly of several polypeptides into multisubunit channels. This is directly analogous to the proposed folding of the four internally homologous pseudosubunits of sodium and calcium channels. The amino- and carboxy-terminal regions of Shaker channels are specialized for, and appear to interact in, inactivation gating. This interaction probably includes interaction between subunits, as may be said for the role in inactivation gating of the junction between the carboxyl terminus of the third domain and amino terminus of the fourth domain of sodium channel (Vassilev et al. 1988). The capacity for coassembly in potassium channels extends not only to the alternatively spliced products of the same gene, but also to the products of different genes. Heteromultimeric channels that are formed in this way have kinetic and pharmacological properties that differ from homomultimers of their constituents and, as such, broaden the functional diversity of channels that can be produced by any given number of compatible potassium channel genes.

Cyclic nucleotide-gated cation channel expression in embryonic chick brain

Cyclic nucleotide-gated cation channels mediate sensory transduction in vertebrate photoreceptors and olfactory epithelium. These channels are also present in some non-sensory cells, but little is known of their physiological roles outside sensory systems. Using in situ hybridization we found that cyclic nucleotide channel mRNA is expressed specifically in the embryonic chicken forebrain, thalamus, optic tectum, basal midbrain and hindbrain, as well as in the branchial arches, limb buds and skin. Cyclic nucleotide gated channels may thus contribute to development or to cellular differentiation in the brain and in other tissues.

Application of drosophila molecular genetics in the study of neural function — studies of the shaker locus for a potassium channel

Abstract One general approach to studying molecules that are important for neuronal function or development, even if the gene products are not defined biochemically, is to make use of classical and molecular genetics; these tools have been well developed for organisms such as Caenorhabditis elegans and Drosophila melanogaster . The cytogenetics made possible by the polytene chromosomes in Drosophila further facilitates gene cloning. If one can identify genes that are important for the nervous system, one should be able to clone them and then study these genes and their products in molecular terms.

Chapter 4 Studies of Shaker Mutations Affecting a K+ Channel in Drosophila

Publisher Summary This chapter reviews the evidence that the Shaker locus in Drosophila is the site for the structural gene of a K+ channel. It discusses the strategies to be used for the molecular cloning of the Shaker locus. The recently isolated hybrid dysgenesis-induced Shaker mutants, which can be useful in the initial cloning and subsequent analysis of DNA from the Shaker locus, are described. The molecular studies of K+ channels are important because these channels play important roles in the control of neuronal activity and synaptic efficacy. The genetics of Drosophila and mutations of the Shaker locus offer an alternative approach for cloning K+ channels in the absence of high-affinity toxins or antibodies against K+ channels. In addition to providing a starting point for cloning, dysgenesis-induced Shaker mutants can also supply abundant new mutations that are considered useful in later molecular analysis. Genetic analyses using electrophysiological assays including voltage clamping provide strong evidence that the Shaker locus contains the structural gene for a K+ channel, the A channel.

A Family of Potassium Channels from the Shaker Locus of Drosophila

Potassium channels are found in nerve, muscle, epithelial, endocrine, exocrine and immune cells. Despite their importance in the control of excitation and ion transport, biochemical understanding of the channels has grown slowly because there are no high abundance sources of these channels and because high affinity ligands for them have only recently been found (Carbone et al., 1982; Miller et al, 19854; Seagar et al., 1986; Sternsfeld et al., 1987). An alternative route to studying these molecules has been provided by the fruit fly, Drosophila melanoqaster. Many neurological mutations of this organism have been found that define loci that may encode important neuronal proteins (Ganetzky and Wu, 1986). One of these, the Shaker locus, appeared likely from electrophysiological and genetic studies (Jan et al., 1977; Salkoff and Wyman, 1983; Timpe and Jan, 1987) to contain the structural gene for a type of potassium channel, called the A channel, that opens in response to depolarization and inactivates thereafter. The gene was mapped to band 16F on the X-chromosome (Tanouye et al., 1981). An entry point to cloning the region was provided by a cDNA, cloned for independent reasons (Wollfner, 1980); the cDNA is not related to Shaker but hybridizes to the same band on the X-chromosome.