Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes

Abstract

Using a large consortium of undergraduate students in an organized program at the University of California, Los Angeles (UCLA), we have undertaken a functional genomic screen in the Drosophila eye. In addition to the educational value of discovery-based learning, this article presents the first comprehensive genomewide analysis of essential genes involved in eye development. The data reveal the surprising result that the X chromosome has almost twice the frequency of essential genes involved in eye development as that found on the autosomes.

Figure 1.—

FLP/FRT system and crossing scheme. (A) Chromosomes bearing a lethal mutation (P[w⁺]) can be made homozygous through FLP-mediated mitotic recombination at FRT sites in the chromosome. The resulting daughter cells are of three potential genetic lineages: homozygous mutant, homozygous wild type, or heterozygous. The students identified FRT recombinant flies using the mini-white (w⁺) gene, a pigmentation marker located in the transposon, by observing mosaic eyes. (B) By using the eye-specific enhancer eyeless (ey) to drive the expression of FLP, the students were able to create homozygous mutant tissue of lethal mutations specifically in the eye in the third generation. These recombinants were then crossed to a stock bearing a Minute (w⁺M) or cell-lethal mutation on its FRT chromosome over a balancer chromosome, which generates a balanced stock of FRT recombinant flies and siblings that have eyes that are mostly homozygous mutant due to the Minute mutation. The scheme shown is specific for the 3L chromosome arm; however, other chromosome arms use this same core scheme. For all of the crossing schemes used in our project, please see our website at http://www.BruinFly.ucla.edu.

Figure 2.—

Examples of eye phenotypes identified in the screen. All images show mosaic eyes with orange, homozygous mutant tissue (arrowheads) and red, heterozygous tissue. The right column is a scanning electron micrograph of the eye shown on the left. (A) An eye with a mutation in a gene that leads to no eye defects; note the perfect symmetrical arrangement of the repeating ommatidia. (B) A rough phenotype results when the mutant gene disrupts the regular pattern of the ommatidia. (C) The cell-lethal phenotype is assigned when the homozygous mutant (orange) tissue is lost or reduced, and the eye is smaller in size. (D) When the mutation leads to a lack of lens secretion, the eye takes on a glossy phenotype.

Figure 3.—

Distribution of unique genes in this study. Genes are mapped by their FlyBase annotated transcriptional start site on the three chromosomes (X, 2, and 3). Green lines indicate transposon insertions that give a wild-type phenotype, while the red lines indicate a mutant phenotype. No centromeric (blue oval) genes were used because they are too close to be recombined with the FRT or are proximal to the FRT and thus inappropriate for mitotic recombination.

Figure 4.—

Clustering analysis of genes on the X chromosome. The scattered dots near the top of the plot show the gene positions. A small amount of random y-axis jitter was added so that dots close to each other can be seen distinctly. Black represents all genes, green represents genes that give a wild-type phenotype, while red represents mutant phenotype. A 2.5-Mbp window (1.25 Mbp inclusive on each side) was centered on each gene. A typical window encompassed ∼10–25 genes; the average number of genes per window was ∼20.1. The negative base 10 log of the hypergeometric P-value for enrichment in a subset (no. of genes in a window that have a phenotype)/(no. of genes in a window) > (no. of genes on X chromosome having a phenotype)/(no. of total genes on X chromosome examined) was computed for each window; higher values indicate more significant enrichment and a P = 0.05 level corresponds to −log₁₀(0.05) = ∼1.301. No corrections were made for multiple testing as without them there was no significantly enriched window (represented by the lines at the bottom). The curves in the middle of the plot show estimated density as a visual aid; they are 1-Mbp-bandwidth Gaussian kernel-smoothed versions of the gene positions.

Figure 5.—

Educational results of introductory URCFG students. (A) Results from standard course evaluations of 223 introductory students when asked about their interest in the subject (research) before and after the course. (B) Eighty-eight students, consisting of students from all four years of our program, voluntarily participated in the SURE II survey (Lopatto 2004) in 2006. The survey has a number of questions measuring gains of knowledge in areas related to scientific research. A five-point scale was used to measure the level of gain: no gain or very small gain, small gain, moderate gain, large gain, and very large gain. All students reported moderate gains or greater from the URCFG program and their average benefit for each subject is plotted on the graph vs. the results from 532 students from across the nation at multiple universities and colleges that had participated in a summer research program in 2006. The full-subject descriptions for the x-axis are as follows: clarification of a career path, skill in the interpretation of results, understanding of the research process in your field, ability to integrate theory and practice, ability to analyze data and other information, understanding science, learning ethical conduct in your field, skill in science writing, self-confidence, understanding of how scientists think, and learning to work independently. The mean scores are graphed ± 95% confidence intervals. T-tests were performed for each question to determine significant differences; P-values are as follows: *<0.05, **<0.01, and ***<0.001.