Kai Zinn

zinnk@caltech.edu
Ph.D., 1984, Harvard University

See also the Zinn group website:

Summary of Zinn group research as of March 2005.

Our group studies the molecular mechanisms of axon guidance and synaptogenesis. The fruit fly Drosophila melanogaster is our primary experimental system. In the embryo, we examine signaling systems involved in motor and CNS axon guidance. In the larva, we study synaptogenesis and synaptic plasticity in the neuromuscular system. Our approaches combine genetics, molecular biology, electrophysiology, biochemistry, and cell biology.

Motor axon guidance. The Drosophila motor axon network has provided one of the best systems in which to study growth cone pathfinding mechanisms. The network is simple: 32 motoneurons innervate 30 body wall muscle fibers in each abdominal segment. Each motoneuron axon is targeted to a specific muscle fiber, and very few projection errors are made during normal development. Thus, the motor axon network is a genetically hard-wired map, and is an ideal system in which to study how genes control the formation of specific synaptic connections. In much of our work, we have focused on the roles of tyrosine phosphorylation in regulating motor axon guidance decisions. We are now also conducting screens to determine the mechanisms by which cell surface proteins label specific muscle fibers for recognition by motor axon growth cones.

Genetics of receptor tyrosine phosphatases. In the 1990s, we showed that receptor-linked protein tyrosine phosphatases (RPTPs) are selectively expressed on CNS axons and growth cones in the Drosophila embryo, and that these RPTPs regulate motor and CNS axon guidance during embryonic development. RPTPs directly couple cell recognition via their extracellular domains to control of tyrosine phosphorylation via their cytoplasmic enzymatic domains. The extracellular regions of the fly RPTPs all contain immunoglobulin-like (Ig) and/or fibronectin type III (FN3) domains, which are usually involved in recognition of cell-surface or extracellular matrix ligands. Their cytoplasmic regions contain either one or two PTP enzymatic domains. The fly genome encodes six RPTPs (DLAR, DPTP10D, DPTP69D, DPTP99A, DPTP52F, DPTP4E), and we have generated or obtained mutations in all six of the genes encoding these proteins. We have now performed a detailed characterization of the genetic interactions among all six RPTPs. We find that each growth cone guidance decision in the neuromuscular system has a requirement for a unique subset of RPTPs; thus, in a sense, there is an "RPTP code" for each decision. In some cases, the RPTPs work together, so that defects are only observed when two or more are removed. In other cases, however, phenotypes produced by removal of one RPTP are suppressed when a second RPTP is also absent. Our results provide evidence for three types of relationships among the RPTPs: partial redundancy; collaboration; and competition. Our major efforts in this area are now directed toward understanding these relationships at the biochemical level, through definition of upstream (ligands) and downstream (substrates) components of RPTP signaling pathways (see below). We are also continuing to characterize novel Rptp phenotypes. One current project in this area concerns the role of DPTP4E, for which we only recently obtained mutations. Our preliminary results suggest that DPTP4E may regulate glial development. We are also examining how the RPTPs regulate topographical layer formation during development of the mushroom body, a part of the fly brain that is essential for learning and memory.

Searching for RPTP substrates. It is difficult to identify PTP substrates biochemically because PTPs usually do not display strong specificity in vitro. To find substrates, we performed yeast two-hybrid screens with substrate-trap mutant versions of DPTP10D, DPTP69D, DPTP52F, and DPTP99A. These trap proteins form stable complexes with tyrosine-phosphorylated substrates because they bind normally but cannot catalyze dephosphorylation. We introduced a constitutively activated chicken Src tyrosine kinase into yeast together with the PTP trap constructs and the cDNA library, in the hope that it would phosphorylate relevant substrate fusion proteins made from cDNA library plasmids. We identified several classes of clones whose interactions with the substrate-trap RPTPs are dependent on coexpression of the tyrosine kinase, suggesting that they may be substrates. These include a cell-surface receptor and a protein with many SH3 binding motifs. We are currently examining whether these proteins interact with the RPTPs in vivo and if they are required for RPTP signaling.

Identification of RPTP ligands. The ligands recognized by RPTPs in vivo have not been identified in any system. In order to understand how RPTPs regulate axon guidance, it is essential to know when and where they engage ligands, and how ligand binding affects enzymatic activity and/or localization. Our current approach to identifying ligands is based on our observation that fusion proteins in which the extracellular domains of RPTPs are joined to human placental alkaline phosphatase (AP) can be used to stain live Drosophila embryos. Each of the six fusion proteins (DLAR-AP, DPTP69D-AP, DPTP10D-AP, DPTP99A-AP, DPTP4E-AP, DPTP52F-AP) binds in a specific manner. Each fusion protein stains a subset of CNS axons and also binds to other cell types in the periphery. We are now screening a ‘deficiency (Df) kit’ of ~225 fly lines, each of which lacks a specific region of the genome, by staining homozygous Df embryos from each line with each of the fusion proteins. This method may be able to identify the genomic regions encoding the RPTP ligands. Using this screen, we have already found a Df that contains a gene encoding a ligand that binds to DLAR-AP, and have identified this ligand as Syndecan (Sdc). Sdc is a heparan sulfate proteoglycan (HSPG). Our results show that DLAR binds to the glycosaminoglycan side chains of Sdc with nanomolar affinity, and that Sdc is required for DLAR-mediated axon guidance. We can generate motor axon guidance errors by overexpressing DLAR on neurons, and find that the same errors are generated by ectopically expressing Sdc on muscles. This Sdc gain-of-function (GOF) phenotype is suppressed by loss-of-function (LOF) mutations in the Lar gene, indicating that DLAR is epistatic to (downstream of) Sdc. This result shows that muscle Sdc can function as a ligand for DLAR on neuronal growth cones, and suggests that binding to Sdc increases DLAR's signaling activity.

Targeting of motor axons to specific muscle fibers. Despite the advances in characterizing molecules that regulate motor axon pathfinding, we still understand little about how specific muscle fibers are recognized as targets for synapse formation by these axons. Many mutations affect pathfinding decisions, leading to aberrant wiring of the neuromuscular system, but no single LOF mutations are known that block recognition of specific muscle targets. These results are most easily explained by invoking genetic redundancy in target labeling. If each muscle fiber were defined by a combination of several cell surface labels, removing one of the labels might not have a major effect on targeting of axons to that fiber. This would explain why targeting molecules have not been identified in conventional loss-of-function (LOF) genetic screens. Studies of gain-of-function (GOF) phenotypes by the Keshishian, Chiba, and Nose groups are consistent with the redundancy hypothesis. For example, the homophilic cell adhesion molecule Fasciclin III (Fas III) is expressed on only two muscle fibers, 6 and 7, and on the growth cone of the RP3 neuron that innervates these two fibers. Fas III appears to be a functional target label, because when it is ectopically expressed on other muscle fibers near 6 and 7, the RP3 neuron makes abnormal synapses on these Fas III-expressing fibers. However, when Fas III is removed by a LOF mutation, there is no effect on targeting of RP3 to 6 and 7. These results imply that Fas III can be used for muscle targeting, but that targeting of 6 and 7 can still proceed in its absence, presumably because these fibers are also labeled by other surface molecules that can be detected by the RP3 growth cone when Fas III is not present. Similar observations have been made for another cell adhesion molecule, Capricious. These findings suggest that cell-surface proteins that label specific targets in the motor axon system might be identifiable by a GOF genetic screen in which candidate labels are ectopically expressed on all muscle fibers. If these proteins are functional labels, their misexpression might produce alterations in target recognition, as observed in the Fas III experiments described above. By identifying all the genes encoded in the Drosophila genome that can confer GOF phenotypes in which targeting of specific muscle fibers is altered, we will acquire the tools to understand the mechanisms involved in target recognition in this system. This type of screen should allow us to overcome the redundancy problem. For example, suppose one could identify three different cell-surface proteins that are normally expressed on a specific muscle fiber, but whose misexpression on other muscle fibers produces targeting errors. One might then predict that removing all three of these proteins by making a triple LOF mutant (through conventional or RNAi techniques) would now prevent targeting of this muscle fiber. Through these kinds of experiments, we could begin to understand the combinatorial code for muscle targeting. Insights into the motor axon targeting code would be likely to facilitate an understanding of targeting in other neuronal systems (e.g. the antennal lobe, optic lobe, and mushroom body), since candidate target labels are usually expressed by a variety of neuronal and non-neuronal cell types. To conduct this GOF screen, we first created a database of all cell-surface and secreted (CSS) proteins in Drosophila that are likely to be involved in specific cell-cell interactions. The database was generated by defining all fly genes encoding proteins that contain domains known to be present in CSS proteins in other eukaryotes. It currently contains 1005 genes. To drive expression of these genes in muscles, we used the ‘EP’ system, in which a P element containing a block of UAS sequences that are responsive to the yeast transcription factor GAL4 is jumped around the genome. Like other P elements, EPs usually land upstream of genes. If a line bearing an EP upstream of a gene is crossed to a ‘driver’ line expressing GAL4 in all muscle fibers, the gene will now be expressed at high levels in muscles in the resulting progeny embryos and larvae. To find EP-like elements upstream of the CSS genes, we searched through about 40,000 different insertions, including the original EP set generated by Pernille Rorth, the EY insertions lines generated in the Bellen lab, the GS lines developed in Japan, and the GE lines developed by GenExel, Inc. We were able to identify insertions that can confer expression of about 300 of the 1005 CSS genes in our database, representing members of all cell-surface protein families. To screen for genes encoding potential targeting molecules, we are crossing each of these insertions to a muscle GAL4 driver and visualizing motor axons and neuromuscular junction synapses in the resulting F1 progeny larvae by immunostaining. We have already identified a number of genes that cause specific mistargeting phenotypes when they are expressed on muscles.

Genes controlling synaptogenesis in the larval neuromuscular system. Motor growth cones reach their muscle targets during late embryogenesis and then mature into presynaptic terminals that are functional by the time of hatching. The pattern of Type I neuromuscular junction (NMJ) synapses in the larva is simple and highly stereotyped, with boutons restricted to specific locations on each muscle fiber. These synapses continue to expand and change as the larva grows, because their strengths must be matched to the sizes of the muscle fibers they drive. This growth represents a form of synaptic plasticity, because it is controlled by feedback from the muscle to the neuron. Studies of NMJ synapses in flies are relevant to an understanding of synaptic plasticity in the mammalian brain, because the fly NMJ is a glutamatergic synapse, organized into boutons, that uses ionotropic glutamate receptors homologous to vertebrate AMPA receptors. To identify genes involved in synaptogenesis in larvae, we devised and executed a gain-of-function (GOF) screen of live third instar larvae (pdf available on website). Our screen identified 41 known genes (those with published mutant alleles) and 35 new genes for which high-level neuronal expression produces axonal/synaptic phenotypes. The products encoded by the 76 genes identified in our screen include kinases, protein and lipid phosphatases, GTPases, guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), ATPases, cell-surface receptors, RNA-binding proteins, transcriptional regulators, and a variety of other molecules likely to be involved in protein trafficking, modification, and degradation.

Control of synaptic local translation. Our screen has led us into the area of control of synaptic protein translation, and this has become one of our major focuses. Local translation at synapses has been studied in Aplysia, mammalian, and arthropod systems. It has attracted interest because it is a mechanism that allows neurons to separately adjust the strengths of individual synapses. In the screen, we identified pumilio (pum), which encodes an RNA-binding protein that shuts down translation of specific mRNAs by binding to their 3' untranslated regions. Translational repression by Pum controls posterior patterning during embryonic development. In a recent Neuron paper (pdf available on website), we showed that Pum is an important mediator of synaptic growth and plasticity at the NMJ. Pum is localized to the postsynaptic side of the NMJ in third instar larvae, and is also expressed in larval neurons. Neuronal Pum regulates synaptic growth. In its absence, NMJ boutons are larger and fewer in number, while Pum overexpression increases bouton number and decreases bouton size. Postsynaptic Pum negatively regulates expression of the essential translation factor eIF-4E (the cap-binding protein) at the NMJ, and Pum binds selectively to the 3’UTR of eIF-4E mRNA. These data suggest that Pum is a direct regulator of local eIF-4E translation, and that eIF-4E (which is normally limiting for translation) in turn switches on translation of other synaptic mRNAs. These mRNAs probably include that encoding the GluRIIa glutamate receptor, which is also upregulated in pum mutants. These results, together with genetic epistasis studies, suggest that postsynaptic Pum modulates synaptic function via direct control of local synaptic translation. We are now examining a number of other RNA-binding proteins that may regulate postsynaptic translation at the NMJ. These include FMRP (Fragile X mental retardation protein ortholog), Orb (CPEB ortholog), and Nanos (Pum's partner during early development). We are also studying whether controlled aggregation of Pum regulates its ability to repress translation.

Synaptic microtubule dynamics. Another gene identified in our GOF screens is spastin, which is orthologous to a human disease gene. 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 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 Spastin’s role in maintenance of axon terminals in the spinal cord (pdf of paper available on website). We have now begun to characterize the other members of the small family of AAA ATPases to which Spastin belongs. Our preliminary results indicate that CG1193, a protein closely related to Katanin, is also a regulator of synaptic microtubule dynamics.

Other current projects. Blue cheese (bchs), another gene identified in our screen, encodes a a huge protein that is closely related to the human protein whose loss causes Chediak-Higashi syndrome, a lethal disease affecting lysosomes and related organelles. In flies, we have found that loss of Bchs protein causes a specific pair of motor neurons to die during larval development. We are currently trying to determine the mechanisms involved in this specific neuronal death. We have also begun a systematic analysis of the LOF phenotypes conferred by the other interesting new genes identified in the screen. We are doing this by generating transgenic RNAi lines to knock out expression of each of these genes.

Selected Publications for Kai Zinn