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

2. 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

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

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

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

6. 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.

7. Protein Y Nw protein Protein X 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 (overactive)

8. 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 Hypothesis: Protein Y Nw protein Protein X Protein X (overactive)

9. Df(2L)ED250 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(2L)ED250 Each Df may remove 10-50 genes (heterozygous - 50% reduction in each) 200 Dfs cover most all of the chromosomes Find Dfs that change the nw wing shape Find which gene in the Df is responsible

10. Studies done before I started: 18 Dfs identified that make a nw mutant wing narrower or wider, e.g. Df(2L)ED250 Find Dfs that change the nw wing shape Find which gene in the Df is responsible nw[D]/Df(2L)ED250 nw[D]/+ narrow mutant with Df narrow mutant

11. Removing 50% of one of these makes the nw mutant wing narrower Is this gene y? 24F-25A region of the chromosome Deficiency Df(2L)ED250 makes the narrow wing narrower nw[D]/Df(2L)ED250 nw[D]/+ narrow mutant with Df narrow mutant genes Region deleted in Df(2L)ED250

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

13. 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%)

14. Results for 24F-25A region Narrow + no Df no Df ED250 ed-dp BSC51 ED7853 narrower Same

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

16. dp mutants have defective wings dp[olv1]/ dp[olv1] dp[olv1]/+ (50% 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? (no Dp protein)

17. nw[D dp[olvR]/+ Yes! Reducing Dp results in narrower nw wings nw[D] dp[ov1]/+ nw[D]/+ Narrow + no Df Nw mutant 100% Dp Nw mutant 50% Dp

18. Conclusion 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 Y Nw protein Protein X Protein X (overactive) Is Dp protein Y? Original hypothesis

19. If it was this simple, then complete absence of Dp protein should result in narrow wings…but Protein Y Nw protein Protein X Protein X (overactive) Original hypothesis So my original hypothesis is too simple Predicts: removing Y completely (even with full Nw) should result in narrow wings dp[olv1]/ dp[olv1 (no Dp protein)

20. 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?

21. References (1) http://www.sdbonline.org/fly/aimorph/wing.htm (Genes involved in tissue and organ development) (2) http://flybase.org/reports/FBgn0002974.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/ cytosearch15.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: 262pp. (original report of narrow mutant) Research done: Dr. Gerard Campbell’s Lab Department of Biological Science University of Pittsburgh

22. Shape changes in nw mutants are similar to those in vertebrate embryos Frog embryos Increasing age Convergent extension Wings during metamorphosis in pupa Increasing age Too much convergent extension?