Kai Zinn

Receptor Tyrosine Phosphatases Are Required for Motor Axon Guidance in the Drosophila Embryo

The receptor tyrosine phosphatases DPTP69D and DPTP99A are expressed on motor axons in Drosophila embryos. In mutant embryos lacking DPTP69D protein, motor neuron growth cones stop growing before reaching their muscle targets, or follow incorrect pathways that bypass these muscles. Mutant embryos lacking DPTP99A are indistinguishable from wild type. Motor axon defects in dptp69D dptp99A double mutant embryos, however, are much more severe than in embryos lacking only DPTP69D. Our results demonstrate that DPTP69D and DPTP99A are required for motor axon guidance and that they have partially redundant functions during development of the neuro-muscular system.

Three receptor-linked protein-tyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo

We describe the isolation of seven different protein-tyrosine phosphatase (PTPase) cDNAs from Drosophila embryos, three of which are primarily expressed in the central nervous system (CNS). The CNS-specific PTPases include the previously sequenced DLAR, as well as two novel PTPases (denoted DPTP10D and DPTP99A), which have extracellular domains consisting of multiple fibronectin type III repeats. Each of the Drosophila sequences is most closely related to a different human PTPase. The three PTPase mRNAs are expressed in different patterns of cells in the ventral nerve cord, and all three proteins are restricted to axons. DLAR and DPTP99A are apparently expressed on most or all axons, while DPTP10D is primarily localized to the anterior commissure and its junctions with the longitudinal tracts.

Profilin and the Abl Tyrosine Kinase Are Required for Motor Axon Outgrowth in the Drosophila Embryo

The ability of neuronal growth cones to be guided by extracellular cues requires intimate communication between signal transduction systems and the dynamic actin-based cytoskeleton at the leading edge. Profilin, a small, actin-binding protein, has been proposed to be a regulator of the cell motility machinery at leading edge membranes. However, its requirement in the developing nervous system has been unknown. Profilin associates with members of the Enabled family of proteins, suggesting that Profilin might link Abl function to the cytoskeleton. Here, genetic analysis in Drosophila is used to demonstrate that mutations in Profilin (chickadee) and Abl (abl) display an identical growth cone arrest phenotype for axons of intersegmental nerve b (ISNb). Moreover, the phenotype of a double mutant suggests that these components function together to control axonal outgrowth.

A gain-of-function screen for genes controlling motor axon guidance and synaptogenesis in Drosophila.

BACKGROUND The neuromuscular system of the Drosophila larva contains a small number of identified motor neurons that make genetically defined synaptic connections with muscle fibers. We drove high-level expression of genes in these motor neurons by crossing 2293 GAL4-driven EP element lines with known insertion site sequences to lines containing a pan-neuronal GAL4 source and UAS-green fluorescent protein elements. This allowed visualization of every synapse in the neuromuscular system in live larvae. RESULTS We identified 114 EPs that generate axon guidance and/or synaptogenesis phenotypes in F1 EP x driver larvae. Analysis of genomic regions adjacent to these EPs defined 76 genes that exhibit neuromuscular gain-of-function phenotypes. Forty-one of these (known genes) have published mutant alleles; the other 35 (new genes) have not yet been characterized genetically. To assess the roles of the known genes, we surveyed published data on their phenotypes and expression patterns. We also examined loss-of-function mutants ourselves, identifying new guidance and synaptogenesis phenotypes for eight genes. At least three quarters of the known genes are important for nervous system development and/or function in wild-type flies. CONCLUSIONS Known genes, new genes, and a set of previously analyzed genes with phenotypes in the Adh region display similar patterns of homology to sequences in other species and have equivalent EST representations. We infer from these results that most new genes will also have nervous system loss-of-function phenotypes. The proteins encoded by the 76 identified genes include GTPase regulators, vesicle trafficking proteins, kinases, and RNA binding proteins.

Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function.

The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP) is caused by mutations in the SPG4 (spastin) gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ) phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, we observed the opposite phenotype: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that we observe in spastin mutants may be relevant to an understanding of human Spastins role in maintenance of axon terminals in the spinal cord.

Retinal Axon Target Selection in Drosophila Is Regulated by a Receptor Protein Tyrosine Phosphatase

Different Drosophila photoreceptors (R cells) connect to neurons in different optic lobe layers. R1-R6 axons project to the lamina; R7 and R8 axons project to separate layers of the medulla. We show a receptor tyrosine phosphatase, PTP69D, is required for lamina target specificity. In Ptp69D mutants, R1-R6 project through the lamina, terminating in the medulla. Genetic mosaics, transgene rescue, and immunolocalization indicate PTP69D functions in R1-R6 growth cones. PTP69D overexpression in R7 and R8 does not respecify their connections, suggesting PTP69D acts in combination with other factors to determine target specificity. Structure-function analysis indicates the extracellular fibronectin type III domains and intracellular phosphatase activity are required for targeting. We propose PTP69D promotes R1-R6 targeting in response to extracellular signals by dephosphorylating substrate(s) in R1-R6 growth cones.

Slit Branches Out: A Secreted Protein Mediates Both Attractive and Repulsive Axon Guidance

Slit is a large, modular extracellular matrix protein containing four arrays of leucine-rich repeat (LRR) sequences, followed by a string of epidermal growth factor (EGF)–like repeats (Rothberg et al. 1990). slit mutations were first identified in the famous Nusslein-Volhard/Wieschaus patterning screen because they affect external midline structures in the Drosophila embryo ( Nusslein-Volhard et al. 1984). Drosophila and C. elegans have a single slit gene, while humans and rats have three ( 5, 6, 11, 2 and 10). Slit is expressed by midline glia in the fly embryo; and in slit mutants these glia are ventrally displaced and the ladder-like axon scaffold of the central nervous system (CNS) collapses down to a single tract at the midline ( Figure 1B). Mutations that delete all midline glia produce similar phenotypes, so Slit was thought to be primarily involved in the control of midline cell fates. The collapse of the axon ladder was assumed to be a secondary consequence of these cell fate changes. A series of recent papers in Cell and Neuron ( 2, 9, 10, 17 and 12) and a paper in press in Development ( Battye et al. 1999), however, now show that Slit’s major functions are likely to be in the direct control of axon guidance decisions. Remarkably, Slit has been shown to have at least two distinct guidance activities, discovered through complementary genetic and biochemical approaches.

The Leucokinin Pathway and Its Neurons Regulate Meal Size in Drosophila

BACKGROUND Total food intake is a function of meal size and meal frequency, and adjustments to these parameters allow animals to maintain a stable energy balance in changing environmental conditions. The physiological mechanisms that regulate meal size have been studied in blowflies but have not been previously examined in Drosophila. RESULTS Here we show that mutations in the leucokinin neuropeptide (leuc) and leucokinin receptor (lkr) genes cause phenotypes in which Drosophila adults have an increase in meal size and a compensatory reduction in meal frequency. Because mutant flies take larger but fewer meals, their caloric intake is the same as that of wild-type flies. The expression patterns of the leuc and lkr genes identify small groups of brain neurons that regulate this behavior. Leuc-containing presynaptic terminals are found close to Lkr neurons in the brain and ventral ganglia, suggesting that they deliver Leuc peptide to these neurons. Lkr neurons innervate the foregut. Flies in which Leuc or Lkr neurons are ablated have defects identical to those of leucokinin pathway mutants. CONCLUSIONS Our data suggest that the increase in meal size in leuc and lkr mutants is due to a meal termination defect, perhaps arising from impaired communication of gut distension signals to the brain. Leucokinin and the leucokinin receptor are homologous to vertebrate tachykinin and its receptor, and injection of tachykinins reduces food consumption. Our results suggest that the roles of the tachykinin system in regulating food intake might be evolutionarily conserved between insects and vertebrates.

engrailed controls glial/neuronal cell fate decisions at the midline of the central nervous system

The molecular mechanisms involved in glial/neuronal fate decisions during embryonic development are largely unknown. Here we show that the segment-polarity gene engrailed, which encodes a homeodomain protein, controls these decisions within an insect CNS lineage. The grasshopper median neuroblast (MNB) generates both neurons and midline glia in distinct temporal phases. engrailed expression in MNB progeny can be inhibited by injection of antisense oligodeoxynucleotides into the MNB nucleus. This produces a phenotype in which the midline glia do not develop and extra midline neurons are generated. In the absence of engrailed function, midline glial precursors are apparently converted into neuronal precursors. Thus, engrailed is required for execution of the decision between the glial and neuronal fates.

A Screen of Cell-Surface Molecules Identifies Leucine-Rich Repeat Proteins as Key Mediators of Synaptic Target Selection

In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, we screened 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, we showed that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron.