Current Research

Our laboratory focuses on two main topics: peripheral nervous system (PNS) development and molecular mechanisms of synaptic vesicle trafficking. Our goal is to define the precise functions of proteins implicated in these processes in flies. In addition, we are involved with the Gene Disruption Project in generating new tools for the Drosophila community.

Molecular mechanisms of PNS development

The fly peripheral nervous system (PNS) is a model system to identify proteins that play a role in nervous system development. To identify novel genes that affect PNS development, we carry out genetic screens. One gene that we isolated — named senseless because in its absence almost all the cells of the PNS are lacking — plays a pivotal role early in PNS development. We are characterizing this gene, which encodes a zinc finger transcription factor expressed in every sensory organ precursor (SOP) of the PNS. In addition, senseless is necessary and sufficient to specify SOPs and initiate full differentiation of most PNS organs.

We have recently shown that senseless functions as a binary switch in proneural clusters. Prepatterning genes activate proneural genes in clusters of cells that become competent to initiate neuronal development. The proneural genes then activate senseless transcription in a portion of the proneural cluster. Expression of senseless can then be subdivided into two domains: a domain with a low level of expression, and a single cell in which a high level of expression is observed. At low levels, Senseless binds a specific DNA sequence to repress proneural genes transcriptionally, thereby quenching the ability of these cells of the proneural cluster to become neural precursors. In the future SOP, however, senseless and the proneural genes synergize, leading to up-regulation of proneural gene expression. This synergism is required for the ectodermal cell to become a neural precursor. Interestingly, this synergism does not depend on DNA binding but rather on the ability of the zinc fingers to interact with proneural proteins and possibly other proteins. We are exploring the mechanism by which senseless synergizes with proneurals to understand how it blocks Notch signaling in the SOP.

More recently we have carried out large-scale genetic mosaic screens to identify new players that control PNS development. We have identified numerous new genes, including a number of genes that affect Notch signaling. These include rumi, which encodes a sugar-modifying enzyme that alters the sugar composition of Notch EGF (epidermal growth factor) repeats and affects all the known functions of Notch. We also discovered that a series of proteins that control actin polymerization affect the formation of an actin structure in SOPs and impair Notch signaling during the specification of the SOPIIb. In addition, we identified a novel protein that is involved in cysteine bridge formation of specific repeats that are only found in Notch proteins. Finally, we are currently mapping a number of other genes that affect neurogenesis or cell fate identity in the cell lineage of the PNS.

The function of presynaptic proteins in synaptic vesicle trafficking

The endocytic molecular scaffold. Numerous proteins have been implicated in vesicle trafficking during endocytosis. The precise function of many of these proteins remains to be determined. We have isolated mutations in seven key players that affect endocytosis: AP180, synaptojanin (synj), endophilin (endo), dynamin-associated protein/intersectin (Dap160), eps15, tweek, and flower. These genes encode proteins that are dramatically enriched in the nervous system and localized to nerve terminals, including the neuromuscular junctions (NMJs). In addition, the presence of some of these proteins in the nervous system is essential, as neural expression of endo, eps15, Dap160, and flower rescues the lethality and phenotypes associated with the loss of the respective genes. This implies that they do not play a major role in tissues other than in the nervous system. The tweek and flower proteins have not previously been identified, though both are conserved from C. elegans to human and affect endocytosis at different stages.

The picture that is emerging is that there are three major forms of endocytosis at fly NMJs: a fast kiss-and-run mode of release at active zones, a slower clathrin-mediated form in periactive zones, and bulk endocytosis of membrane. The presence of at least two modes of retrieval has also been documented in hippocampal neurons but may not occur in all synapses. Our data indicate that synaptojanin (a polyphospoinositide phosphatase) and endophilin (a lysophosphatidic acid transferase) are required at precisely the same step during clathrin uncoating. Mutations in both genes display virtually identical phenotypes in FM 1-43 uptake, transmission electron microscopy (TEM) at NMJs and photoreceptor terminals, and electrophysiological properties of NMJs and photoreceptors. In addition, double mutants (synj; endo) display phenotypes virtually identical to single mutants in flies and worms. Since most synaptic vesicles (SVs) are lost in endo and synj mutants but numerous SVs remain at active zones even after prolonged and intense synaptic activity, and since SVs fail to uptake FM 1-43, we propose that vesicles at active zones mainly use a kiss-and-run mode of release in these mutants. Our data also indicate that synj and endo are not required for kiss-and-run release but are mainly required for clathrin uncoating.

Another picture is emerging for Dap160 and eps15, which have been shown to bind to each other, as well as numerous other endocytic proteins. Our data suggest that Dap160 and eps15 are required as scaffolding proteins to localize dynamin properly and to control actin dynamics. In their absence, endocytosis is only mildly impaired. A severe defect in endocytosis is observed, however, when the mutants are shifted to the restrictive temperature (34°C). In the absence of Dap160, numerous collared pits are observed at active zones and periactive zones. We also observe numerous irregularly sized vesicles in NMJ synapses and highly variable miniature excitatory junction potentials, similar to what we observed in AP180 mutants, suggesting a defect in clathrin caging. Dap160 and eps15 appear to act at the same step in endocytosis, and double eps15; dap160 mutants exhibit many of the same features as eps15 or dap160 single mutants. Their presence seems important to ensure proper localization of dynamin.

Novel players in presynaptic vesicle trafficking. To identify mutations in novel genes as well as genes that have previously been implicated in SV trafficking, we used the eyeless-Flp/FRT system to carry out four forward F1 chemical mutagenesis screens. This system allows us to generate flies that have eyes that are homozygous mutant in an otherwise heterozygous fly. We opted to carry out five sequential assays to isolate mutations that affect synaptic transmission or synapse development. First, we only tested flies with apparent normal eye morphology (300,000 flies) for their ability to phototax (retained 10,000). Second, we screened for loss of ON and OFF responses in electroretinograms (ERGs) (retained 650). Third, flies with aberrant ERGs were used to establish stocks and retested (retained 450). Fourth, brains of adult flies were immunohistochemically stained to determine if photoreceptor synapses appear normal (450). We then carried out TEM in the lamina to determine ultrastructural defects of photoreceptor synapses. We identified 62 genes with two or more alleles that affect neurotransmitter release or synapse formation. To map these genes, we developed a novel high-throughput mapping strategy. This strategy is based on the availability of P element insertions every 16 kB in the genome. Using this approach, we identified the molecular lesions in numerous genes, a vast improvement to any other mapping strategy, including deficiency mapping. We identified sec15, a homolog of the yeast exocyst component SEC15, which was originally isolated in a yeast secretion screen. We have shown that Sec15 plays a highly specific role delivering cell adhesion molecules as well as the Delta protein in some cells. Loss of Sec15 causes a specific targeting defect during synaptic partner selection, as well as cell fate specification defects in the PNS.

We have characterized other mutants that play a role in synaptic vesicle fusion, including the V₀ ATPase a1 100-kDa subunit. Some mutations in this gene severely reduce SV fusion but do not affect vesicle acidification, implying that SNAREs (soluble NFS-attachment protein receptors) are not sufficient to promote fusion and that other proteins are required in the process. Most recently we have identified mutations in hip14 (Huntingtin interacting protein 14). Hip14 is a palmitoyl transferase, and we have uncovered a new substrate for this protein that plays a pivotal role in the control of synaptic transmission.

Genome-wide disruption/tagging of Drosophila genes

To permit rapid generation of targeted mutations for functional analysis of most Drosophila genes, we are attempting to create a transposable element insertion in every gene. In collaboration with the laboratories of Allan Spradling (HHMI, Carnegie Institution of Washington), Roger Hoskins (Lawrence Berkeley National Laboratory), and Gerald Rubin (HHMI, Janelia Farm Research Campus), we have created insertions in more than 50 percent of all Drosophila genes. We have now switched from P elements to Minos transposable elements and have developed new Minos-based elements that greatly enhance the usefulness of the insertion stocks. All the stocks that we create are deposited in the Bloomington Drosophila Stock Center and are available to the fly community.

We have also developed a new transgenesis platform for Drosophila, named P[acman]. This vector allows us to clone and manipulate very large pieces of DNA and to integrate them at specific genomic sites in the fly genome via ΦC31-mediated integration. We have recently generated two genomic libraries encompassing DNA fragments that average 20 or 80 kb, and we soon will make these libraries available to the community. These libraries will allow unprecedented manipulations of more than 99 percent of the fly genes.