Identifying Genes that Control Wing Shape in Flies

Identifying Genes that Control Wing Shape in Flies Gregory Campbell Central Catholic High School Pittsburgh

The fruit fly, Drosophila • Used for over 100 years for genetic studies • Including studies in development how you go from embryo to adult • Many mutants identified that disrupt development in different ways used to identify the gene that is required for normal development • Provided insights into how all animals develop, including humans

Wing from wild-type fly Wild-type In narrow (nw) mutants the wing is narrow (nw)

The Nw protein is required to make the wing wide Wild-type Nw protein nw mutant • How does it do this? • We don’t know. • It is secreted from wing cells - looks like a protein that binds to other proteins Goal: to understand how Nw functions by finding other genes it regulates

Wings during metamorphosis in the pupa Increasing age During metamorphosis in the wildtype pupa, normal wings become narrower when cells move in between each other.

nw mutant wings get narrower in the pupa Increasing age In nw mutants the wings become too narrow when cells move in between each other more than they should.

Hypothesis: • Protein X helps make the wing the right shape by making cells move • If Protein X becomes overactive the wing becomes narrow • Possibility 1: Direct - Narrow binds to Protein X to inhibit its function • Possibility 2: Indirect - Narrow binds to Protein Y to inhibit X Protein X Nw protein Protein X (overactive) Protein Y

Hypothesis: Protein X Nw protein Protein X (overactive) Protein Y • Reducing the amount of X in a nw mutant should make the wing less narrow • If Y exists, reducing Y in a nw mutant may make the wing even more narrow ** Therefore in nw mutants • Mutation in gene x should suppress wing shape - wider • Mutation in gene y should enhance the wing shape - narrower

So how to do you find genes x and y? • Search for mutations that enhance and suppress the nw wing shape Use Deficiencies - tests many mutants at the same time • Dfs = small deletions in a chromosome, e. g. Df(2 L)ED 250 • Each Df may remove 10 -50 genes (heterozygous - 50% reduction in each) • 200 Dfs cover most all of the chromosomes Df(2 L)ED 250 • Find Dfs that change the nw wing shape • Find which gene in the Df is responsible

• Find Dfs that change the nw wing shape • Find which gene in the Df is responsible Studies done before I started: 18 Dfs identified that make a nw mutant wing narrower or wider, e. g. Df(2 L)ED 250 narrow mutant nw[D]/+ narrow mutant with Df nw[D]/Df(2 L)ED 250

Deficiency Df(2 L)ED 250 makes the narrow wing narrower narrow mutant nw[D]/+ narrow mutant with Df nw[D]/Df(2 L)ED 250 genes 24 F-25 A region of the chromosome Region deleted in Df(2 L)ED 250 Removing 50% of one of these makes the nw mutant wing narrower Is this gene y?

To identify which gene is responsible for the phenotype, test more Dfs in 24 F-25 A region

Method • Cross nw mutant to different Dfs • Pick out nw/Df flies • Pull wings off and mount on microscope slides • Take pictures • Measure length and width of 10 wings for each genotype • Compare l: w of nw/Df to nw/+ • Use statistical test (t-test) to find if difference is likely to be real (p-value below 5%)

Results for 24 F-25 A region no Df ED 250 narrower ed-dp narrower BSC 51 narrower ED 7853 Narrow + no Df Same

Which gene? Blue = interacts (makes wing narrower) Red = no effect (same as nw[D]/+) Region containing gene interacting with nw • What is known about these genes? • Do mutations in any affect wing shape on their own? Yes - dumpy (dp)

dp mutants have defective wings dp[olv 1]/+ (50% Dp protein) dp[olv 1]/ dp[olv 1] (no Dp protein) • Why dp mutant wings look like this is not understood. • Dp protein is very large and is found on the outside of cells. • Do dp mutants change the shape of nw mutant wings like the Dfs?

Nw mutant 100% Dp Yes! Reducing Dp results in narrower nw wings nw[D]/+ Nw mutant 50% Dp nw[D] dp[ov 1]/+ nw[D dp[olv. R]/+ Narrow + no Df

Conclusion Original hypothesis • Reducing Dp levels causes a nw mutant wing to become even narrower - suggest that Dp may function with Nw to stop the wing becoming too narrow Protein X Nw protein Protein X (overactive) • Is Dp protein Y? Protein Y

Original hypothesis Protein X Nw protein Protein X (overactive) Protein Y • Predicts: removing Y completely (even with full Nw) should result in narrow wings • If it was this simple, then complete absence of Dp protein should result in narrow wings…but dp[olv 1]/ dp[olv 1 (no Dp protein) So my original hypothesis is too simple

Future • Analyse other 17 Dfs - Identify other genes that interact with narrow • Work out why dp mutants have funky wings and why it affects nw wing shape • Dp protein is outside cells, does Nw protein bind to it?

References (1) http: //www. sdbonline. org/fly/aimorph/wing. htm (Genes involved in tissue and organ development) (2) http: //flybase. org/reports/FBgn 0002974. html (Gene report on narrow) (3) http: //fly. bio. indiana. edu/Browse/df/dftop. htm (Use to find Dfs at the Bloomington stock center) (4) http: //flybase. org/static_pages/cytosearch/ cytosearch 15. html (Use to work out which genes are present in a Df) (5) Morgan, T. H. , Bridges, C. B. , Sturtevant, A. H. (1925). The genetics of Drosophila melanogaster. Biblphia Genet. 2: 262 pp. (original report of narrow mutant) Research done: Dr. Gerard Campbell’s Lab Department of Biological Science University of Pittsburgh

Shape changes in nw mutants are to those in vertebrate embryo Inc Frog embryos Convergent extension Wings during metamorphosis in pupa Increasi