A Cross-eyed View of Development

Polarization is essential in all multi-cellular organisms. For example, the epithelium of the gut polarizes to form secretory and absorptive cells on the apical surface, and structural and supportive cells on the basal surface. The individual cells of a tissue must know the correct ways in which to orient themselves in order to form a functional tissue. At the Tanya Wolff lab, I studied the proteins that establish polarization of the compound eye of Drosophila melanogaster, commonly referred to as the fruit fly.

Because of various advantages, Drosophila is a popular laboratory system. It reproduces quickly and requires little maintenance. It is also attractive because of the recent completion of its genomic sequence. Moreover, its compound eyes, each consisting of about 800 unit eyes, or ommatidia, make Drosophila a particularly appealing organism in which to study polarity. A closer inspection of a cross-section of the compound eye under the microscope reveals a ubiquitous organization of photoreceptor rhabdomeres in the shape of trapezoids. Each trapezoid of rhabdomeres corresponds to the interior of each ommatidium. On the dorsal half of the compound eye, the trapezoids point dorsally, while on the ventral half of the compound eye, the trapezoids point ventrally. The prevalent organization of the ommatidia photoreceptors indicates the presence of polarity and serves as an assay by which we can determine whether or not the correct eye development occurs.

Prior to working at Washington University, Tanya Wolff linked proper polarization of the eye to a gene she named strabismus. Mutant strabismus flies lack the "smooth" eye lattice that wild type flies possess. The trapezoids of ommatidial receptors point in various directions, effectively making the fly cross-eyed. Hence, the name "strabismus," which is the medical term for cross-eyed. My projects over the summer involved working with the strabismus gene as well as working with the fat gene, another gene that is required for setting up polarity in the eye.

Very little is known about the Strabismus protein with the exception of the PDZ domain binding motif found at the C-terminus. Jake Guinto, a fellow lab worker, took advantage of the PDZ domain binding motif by building DNA constructs that contained both a portion of the C-terminal end of strabismus and the green fluorescent protein (GFP) marker, both of which he placed downstream of a GMR promoter, which causes expression in the fly eye. His intent is to insert the construct into the DNA of a fly embryo using a P-element. When the embryo develops into a fly, he will take samples of the eyes and place them under a certain wavelength of light to check for the green fluorescent protein. The location of the dye should signal the location of the attached PDZ binding domains of Strabismus protein. My particular assignment in this experiment was to determine if the constructs he had made were the correct sequence. I made minipreps of the plasmid in which the construct was being built. Then, I digested the plasmid, ran it on a gel, and sequenced it. A control construct was also made by changing two of four bases in the PDZ domain binding motif.

Besides learning where the strabismus gene is expressed, learning the expression locations of the fat gene was another of my projects. I worked with Amy Schrecengost, another lab worker, to make progress towards this goal. I performed a PCR reaction on a specific portion of the genomic DNA and transcribed the product with DIG-labeled urasil. I then hybridized the RNA probe to the corresponding mRNA molecules. The hybridization was done on 3rd instar larval eye discs; the eye discs are developing precursors of adult eyes, and the localization of fat in the eye discs gives insights into the development and polarization process of the eyes.

Polarity occurs in many tissues and is often required for proper development. Learning about polarization using the fly eye gives clues to the process of polarization in other organisms. strabismus, for instance, is a conserved gene through evolution; it has mammalian homologues. Although I did not have the opportunity to see the construct project through to completion, I did view the results of the fat probe in situ hybridization. The labeled probe was treated with anti-DIG, and then was subjected to an enzymatic reaction which dyed the anti-DIG labels. fat mRNA seemed to be prevalent throughout the eye disc, but was especially concentrated in the furrow and folds. The data I collected is just another piece of the puzzle in our quest for understanding of the polarization process.