The role of liquid facets-Related in Drosophila development

The goal of my graduate research is to find the role of a Drosophila gene, liquid facets-Related, encoding an ENTH (Epsin N-term homology) domain protein using developing Drosophila eye as a model system. The ENTH domain is a well-conserved globular domain with affinity to phosphoinositides, and found in endocytic Epsins and Golgi Epsins. With the ENTH domain and peptide motifs, such as clathrin binding motifs and other protein binding motifs, endocytic Epsins and Golgi Epsins are localized to the plasma membrane and the Golgi membrane, respectively. The main function of Epsins is to facilitate clathrin-dependent vesicle formation. An interesting finding from endocytic Epsin research using Drosophila is that this seemingly generic factor has in fact a specific role in the Notch signaling pathway by mediating ligand endocytosis which is crucial for receptor activation in the adjacent cell. The role of Golgi Epsin, on the other hand, has not been understood in a multi-cellular context. A former graduate student in our lab, Erin Overstreet, generated loss-of-function mutants of liquid facets-Related and found that this gene is essential for viability and important for cell growth and patterning in Drosophila eye development. Her finding suggests that liquid facets-Related has a specific role in development. She also found that the ENTH domain is dispensable for the function of liquid facets-Related. This is an interesting result because studies using other model organisms show that ENTH domain directly recognizes the cargos, suggestive of essential function it in Golgi Epsin. Therefore, I aimed to figure out what is the function of liquid facets-Related in a multi-cellular context using the Drosophila eye as a model system. To address this, I further characterized the mutant phenotype, screened for dominant modifiers of the hypomorphic eye phenotype, and performed structure/function assays that helped me to generate specific hypotheses and then I tested them. My graduate research contributed to understanding the role of liquid facets-Related by providing the in vivo function, identifying genetic interactions, and specifying the domain necessary and sufficient for its function. First of all, characterization of the mutant phenotype indicated that liquid facets-Related is crucial for proliferation, suppression of apoptosis, insulin receptor-independent cell growth, and progression of the morphogenetic furrow at the D/V midline in the developing Drosophila eye. Secondly, from a forward genetic screen, I found Delta, neuralized, polychaetoid (ZO-1), string (cdc25), and altered disjunction (Mps1) as dominant enhancers of the hypomorphic eye phenotype, suggestive of the role of liquid facets-Related in the Notch signaling pathway and cell cycle regulation. I also found that wingless and armadillo dominantly enhance the hypomorphic phenotype of liquid facets-Related, which suggests that liquid facets-Related has a role in the Wingless signaling pathway. Indeed, the expression of a transcriptional target of the Wingless signal, dachsous, is reduced in liquid facets-Related null cells. Baso-lateral levels of E-cadherin and Armadillo are increased in the liquid facets-Related null cells, which is consistent with the fact that E-cadherin antagonizes the Wingless signal activity. Finally, an unexpected result from structure/function analysis is that exon 6 of the liquid facet-Related gene is necessary and sufficient to rescue all visible morphological defects of null mutants. This is interesting because exon 6 is conserved in Golgi Epsin gene only in several insects but not in most other species including yeast, nematode, mouse, and human. In fact, exon 6 is a homolog of a recently studied gene known as tel2. Evidence suggests that liquid facets-Related is Drosophila tel2. As the function of Tel2 is not clearly understood, this study may contribute to better understand the essential role of Tel2 in Drosophila and other model systems.