identifying genes that control wing shape in flies gregory campbell central catholic high school...
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Identifying Genes that Control Wing
Shape in Flies
Gregory CampbellCentral Catholic High School
Pittsburgh
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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
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Wild-type
Wing from wild-type fly
In narrow (nw) mutants the wing is narrow
narrow (nw)
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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
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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.
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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.
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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)
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• 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)
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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
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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
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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
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To identify which gene is responsible for the
phenotype, test more Dfs in 24F-25A region
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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%)
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Results for 24F-25A region
Narrow + no Df
no Df
ED250
ed-dp
BSC51
ED7853
narrower
narrower
narrower
Same
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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]/+)
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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)
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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
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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
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• 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)
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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?
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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
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Shape changes in nw mutants are similar to those in vertebrate embryos
Frog embryosIncreasing age
Convergent extension
Wings during metamorphosis in pupa Increasing age