Identifying Genes that Control Wing

Shape in Flies

Gregory CampbellCentral 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

Wild-type

Wing from wild-type fly

In narrow (nw) mutants the wing is narrow

narrow (nw)

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

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.

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.

Protein Y

Nw protein

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)

• 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

Nw protein

Protein X

Protein X (overactive)

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

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)ED250nw[D]/+

narrow mutant with Dfnarrow mutant

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)ED250nw[D]/+

narrow mutant with Dfnarrow mutant

gen

es

Region deleted in Df(2L)ED250

To identify which gene is responsible for the

phenotype, test more Dfs in 24F-25A 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 24F-25A region

Narrow + no Df

no Df

ED250

ed-dp

BSC51

ED7853

narrower

narrower

narrower

Same

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]/+)

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)

nw[Ddp[olvR]/+

Yes! Reducing Dp results in narrower nw wings

nw[D]dp[ov1]/+

nw[D]/+

Narrow + no Df

Nw

mu

tan

t 10

0% D

pN

w m

uta

nt

50%

Dp

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

Nw protein

Protein X

Protein X (overactive)

• Is Dp protein Y?

Ori

gin

al h

ypo

thes

is

• If it was this simple, then complete absence of Dp protein should result in narrow wings…but

Protein Y

Nw protein

Nw protein

Protein X

Protein X (overactive)

Ori

gin

al h

ypo

thes

is

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)

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/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 LabDepartment of Biological ScienceUniversity of Pittsburgh

Shape changes in nw mutants are similar to those in vertebrate embryos

Frog embryosIncreasing age

Convergent extension

Wings during metamorphosis in pupa Increasing age