The Drosophila Compound Eye
The developing fly eye is an ideal system in which to study cellular and molecular processes that mediate development. The eye is elegant in its cellular simplicity and sophisticated in its signaling complexity. Although comprised of just six cell types, their exquisite assembly into a precise array reiterated 800 times requires the activation of multiple complex signaling pathways. These pathways regulate parallel processes in vertebrates, so what we learn about these pathways in the eye can be applied to mammalian systems.

Several advantages to using the fly eye to study epithelial polarity include: the series of events that pattern the eye has been well documented, cell types can be distinguished by morphological features and molecular markers, and ommatidia, the 20-cell unit eyes of the compound eye, are not clonally derived. These features facilitate the assignment of function to specific genes.

Epithelial Polarity
Cells within many epithelial sheets are polarized along an axis perpendicular to the apical-basal axis. In the fly, tissue polarity is manifested in the regular orientation of hairs and bristles on the body surface and in the mirror symmetric arrangement of ommatidia in the dorsal and ventral halves of the eye. In vertebrates, tissue polarity is critical in ordering the stereocilia and in convergent extension movements that take place during gastrulation; mutations in this family of genes have been implicated in deafness and neural tube defects, such as spina bifida.

Appropriate establishment of polarity relies on ubiquitous developmental events, such as establishment, transduction and interpretation of positional information cues, correct cell fate determination, cellular differentiation, and a series of well-choreographed morphogenetic movements which rely upon cytoskeletal remodeling. We’re using the fly eye to link signaling pathways to these events.

 

The functional role of Strabismus

Strabismus (Stbm) is thought to be one component of a well-defined complex of proteins that directs dorsal-ventral polarity in the eye. While it is known that its role in this process is tied to its requirement to specify the fate of one photoreceptor cell, the functional contribution of Stbm to this complex has not been defined. Stbm encodes a protein with a PDZ binding motif and multiple additional conserved domains with no known function. We have taken a two-hybrid approach to identify physical interactors of these domains as a means of determining the functional significance of Stbm. This work may also reveal post-translational modifications of Stbm. Characterization of these interactors is ongoing.

Two cadherins regulate dorsal/ventral signaling in the eye
The tissue polarity complex responds to global positional information, which defines the dorsal and ventral hemispheres of the eye. One aspect of our work has been to dissect the signaling intermediates that convey positional information to the tissue polarity complex. We have shown that two cadherins, Fat and Dachsous, act in this capacity, in part by defining the dorsal/ventral midline in the eye.

stbm requires two tissue polarity genes to regulate polarity
We identified flamingo and prickle, which encode a cadherin and a LIM domain-containing protein, respectively, in a dominant modifier screen designed to identify regulators of stbm. Using a combination of genetic and molecular approaches, we found that Fmi and Pk co-localize with Stbm. The basis of the interaction between pk and stbm appears to be one in which Stbm is required to localize, or perhaps stabilize, Pk. In contrast, Fmi and Stbm are not required to localize one another.

Genetic identification of new regulators of tissue polarity
Little is known about either the downstream targets or regulators of the tissue polarity complex. Since we use a system that enables us to genetically manipulate polarity we can use the stbm phenotype to gain access to additional components of the polarity machinery. To circumvent the limits of screening for modification by loss-of-function phenotypes, we carried out an overexpression screen. Such screens allow the identification of 1) genes that do not display a phenotype as loss-of-function alleles, but do display a phenotype when overexpressed, and 2) genes whose phenotypes are masked by functional redundancy by compensating genes. We screened 3600 P-element lines for genes that can drive inappropriate polarity decisions when ectopically expressed in the fly eye and identified 90 lines that modify Stbm. We are currently characterizing a subset of these lines to identify their roles in setting up polarity. We expect the proteins encoded by these genes will represent a diverse collection of new and unexpected molecules that will uncover the signaling pathways and molecular mechanisms that regulate the cascade of events important for the establishment of epithelial polarity.