Summary of Zinn group research
Our group is interested in the general problem of how genes control the patterns and functions of synaptic connections in the brain. Our primary experimental system is the fruit fly Drosophila melanogaster. We continue to work on the fly because it has unique advantages for the study of neural development. Although the anatomy of the fly brain does not resemble that of the human brain, about two-thirds of the key genes involved in control of nervous system development in Drosophila have human orthologs or relatives. These include many genes implicated in diseases of the human nervous system. Furthermore, the nervous system is mostly hard-wired by genetics and differs little between individual flies. Many neurons can be individually identified and genetically characterized. This allows investigators to clearly evaluate the contributions of genetics to neural wiring, without the complication of variations in the animal’s experience of the world.
The Drosophila larval neuromuscular system is an ideal arena in which to study axonal pathfinding, synaptic targeting, and synaptic plasticity. 32 motoneurons innervate 30 body wall muscle fibers in each abdominal segment. Each axon is targeted to a specific muscle fiber during embryonic development, and the resulting pattern of Type I neuromuscular junction (NMJ) synapses in the larva is highly stereotyped. NMJ presynaptic terminals 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 is a form of synaptic plasticity, and is controlled by retrograde signals 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 that are homologous to vertebrate AMPA receptors.
In addition to the neuromuscular system, we also study the development of the embryonic and larval central nervous system (CNS), and of the larval and pupal optic lobe.
Receptor tyrosine phosphatases (RPTPs) and their ligands
RPTPs are neural cell surface receptors that control axon pathfinding, synapse formation, and synaptic plasticity. Mammalian RPTPs are also required for the development of many other organs. RPTP ligands and downstream signaling pathways are poorly understood. We have identified 16 cell-surface ligands for four Drosophila RPTPs. We are investigating how interactions between RPTPs and these ligands control events in neural development, and have created an ‘interactome’ defining the extracellular interaction network for these RPTPs. One ligand, Stranded at Second (Sas), is expressed on glia and interacts in trans with neuronal Ptp10D. Binding to Ptp10D downregulates glial Sas signaling. Sas controls the subcellular distribution of glial transcription factors. When Sas is overexpressed in glia, it produces a tumor-like overproliferation. Sas has signaling motifs in its cytoplasmic domain that resemble those in the mammalian amyloid precursor protein, APP, which is implicated in Alzheimer’s disease. We are currently investigating the mechanisms by which Sas signaling alters glial cell biology.
Dprs, DIPs, and synaptic circuit formation
Chris Garcia’s group at Stanford, in collaboration with us, conducted a large-scale in vitro interactome study which showed that a subfamily of 21 immunoglobulin-like (Ig) domain cell-surface proteins, the Dprs, interact selectively with an 11-member Ig domain subfamily, the DIPs. Individual Dpr-DIP interactions can be observed in vivo. We are now investigating whether interactions between individual Dprs and DIPs program the formation of synaptic connections in the nervous system. Each Dpr and DIP is expressed in a small subset of neurons in the embryonic, larval, and adult CNS, and the proteins are localized to synapses. At the larval NMJ, interactions between one Dpr and its DIP partner regulate presynaptic terminal development. The data suggest that this is a transsynaptic interaction between presynaptic Dpr and postsynaptic DIP proteins. In the pupal optic lobe, each Dpr and DIP we have examined is expressed in a unique subset of neurons in the higher-order visual areas: the medulla, lobula, and lobula plate.
Control of synaptic translation
Local synaptic translation is a mechanism that may allow neurons to separately adjust the strengths of individual synapses and maintain synaptic properties over long time periods. We identified the translational repressor Pumilio (Pum) as a regulator of postsynaptic translation at the NMJ. Pum negatively regulates expression of the essential translation factor eIF‑4E and the glutamate receptor subunit GluRIIA, and binds selectively to the 3'UTRs of their mRNAs. Pum also represses expression of its cofactor Nanos (Nos). Although Nos is required for Pum function during early development, Pum and Nos have antagonistic functions at the NMJ. Pum represses translation of GluRIIA, while Nos represses expression of the alternative glutamate receptor subunit GluRIIB. Arthropod, nematode, and vertebrate Pum orthologs contain aggregation-prone regions, and we have found that one of these is a negative regulator of synaptic Pum function. We are currently attempting to determine whether aggregation is a mechanism used for control of Pum activity in vivo.
Systematic generation of monoclonal antibodies (mAbs) against native cell-surface proteins
The study of human proteins and cells, and the development of new therapeutics, would be greatly facilitated by the ability to systematically generate mAbs against large numbers of cell-surface proteins. At present, mAbs are typically made by injecting single purified proteins into mice, then conducting spleen fusions on these mice and screening large numbers of hybridomas for rare lines making antibodies against the protein of interest. This is an expensive and time-consuming process. We have developed a scheme in which we express large numbers of human cell-surface protein extracellular domains on the surfaces of 3T3 cells, then inject live cells into syngeneic mice and screen spleen fusions from these mice for reactivity against all of the expressed surface antigens. Using these methods, we hope to be able to make high-affinity mAbs against many human proteins in a single fusion, without the necessity of purifying any of the antigens. In addition to distributing these mAbs to the community, we hope to systematically make mAbs against collections of cell-surface proteins potentially involved in tumor metastasis and angiogenesis, and thereby identify new targets for therapy.