Transcript Genetics of flies Greg Sci Proj 2010-78 over

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
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
Nw protein
Protein X
(overactive)
Protein Y
Hypothesis:
Protein X
Nw protein
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(2L)ED250
• Each Df may remove 10-50 genes
(heterozygous - 50% reduction in each)
• 200 Dfs cover most all of the
chromosomes
Df(2L)ED250
• 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(2L)ED250
narrow mutant
nw[D]/+
narrow mutant with Df
nw[D]/Df(2L)ED250
Deficiency Df(2L)ED250 makes
the narrow wing narrower
narrow mutant
nw[D]/+
narrow mutant with Df
nw[D]/Df(2L)ED250
genes
24F-25A region of the chromosome
Region deleted
in Df(2L)ED250
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 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
no Df
ED250
narrower
ed-dp
narrower
BSC51
narrower
ED7853
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[olv1]/+
(50% Dp
protein)
dp[olv1]/ dp[olv1]
(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[ov1]/+
nw[D
dp[olvR]/+
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
Nw protein
Protein X
(overactive)
• Is Dp protein Y?
Protein Y
Original hypothesis
Protein X
Nw protein
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[olv1]/ dp[olv1
(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/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
Shape changes in nw mutants are
to those in vertebrate embryo
Inc
Frog embryos
Convergent
extension
Wings during metamorphosis in pupa
Increasi