Transcript PJAS Gregory 2010-11d - Central Catholic High School

Does cell
polarization
influence the shape
of fly wings?
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
Question: how do organs and
appendages achieve their correct size
and shape?
….why is the fly wing this shape?
Mutants have been identified with
wings with different shapes
Wing from wild-type fly
Wild-type
In narrow (nw) mutants the
wing is narrow
narrow
(nw)
Goal: to understand why the narrow
mutant wings are narrow…or..what
does the wild-type Narrow protein do
to make the wings the correct shape
Nw wings are narrow because of
too much cell movement during
metamorphosis
Increasing age
This type of cell movement, when cells
move in between each other, is known
as Convergent Extension (CE)
CE also occurs in vertebrates
embryos to make them elongate
Increasing age
Frog embryos
PCP genes control CE
in vertebrates
PCP = planar cell polarity
PCP genes identified first in flies
- control hair orientation
Wild-type
PCP
mutant
All hairs
point
towards the
tip
But not in
PCP
mutants
Each cell is polarized
PCP proteins are polarized
at the edges of each cell some on one side some
on the other
tip of
wing
Frizzled
Prickle
Van Gough
Dishevelled
Starry Night
On both sides
If you remove one protein
none of the others is polarized
PCP proteins are needed for CE cell
movements in vertebrate embryos.
Are they needed for the extra cell
movement in narrow mutants?
Nw protein
PCP induced cell
movement
Hypothesis: the normal function of Narrow
protein is to prevent too much CE cell
movements
..so that in narrow mutants where there is no
Nw protein, too much CE takes place and
the wings become narrow
..so Nw protein inhibits the ability of PCP
proteins to make cells move
Experimental approach
Reduce the level of PCP proteins in nw
mutants to 50%
(if you reduce it to 100% the flies usually die
before they become adults)
Nw protein
PCP induced cell
movement
Expectation
If the hypothesis is correct this should result in
the wings becoming less narrow
If we could knock out all Nw and all PCP the
wings may look like wild-type
Method
• Cross nw mutant to different PCP
mutants
• Pick out nw/PCP 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/PCP to nw/+
• Use statistical test (t-test) to find if
difference is likely to be real (p-value
below 5%)
Results
Narrow mutant, no PCP mutant
(nwD/+)
Narrow mutant, prickle mutant
(nwD pk/+ )
Some PCP mutants clearly make
the nw wing less narrow
1.85
Starry night
Dishevelled
frizzled
Van Gough
prickle
No PCP mutant
Wild-type no nw
Length:width
Results
2.40
2.35
2.30
2.25
2.20
2.15
2.10
2.05
2.00
1.95
1.90
*p<0.05
Genotype (over nwD)
What do these results mean?
Frizzled
Prickle
Van Gough
Dishevelled
Starry Night
On both sides
Hypothesis: reducing PCP genes will result in wings
becoming less narrow
Results:
Prickle, Frizzled and Dishevelled experiments
support hypothesis
Van Gough: does not support, unclear why?
Starry night: has opposite effect than expected! It is
not polarized to one side so this may produce this
outcome although we don’t understand why.
Conclusion
• Reducing levels of some PCP proteins does prevent
the wing elongating in narrow wings
• Supports hypothesis that PCP-induced Convergent
Extension cell movements are required to achieve
the narrow wing
• And so in wild-type wings, Narrow protein functions
to prevent these movements making the wing its
normal shape
• However, it is unclear why reducing one PCP
protein, Van Gough, does not produce the same
result and why reducing another, Starry Night, has
the opposite outcome.
Nw protein
PCP induced cell
movement
Future
Knock down PCP protein levels
more than 50% - more complicated
genetic techniques (RNAi).
Prediction - narrow wings will look
even more like wild-type
Investigate how PCP makes cells
move: what does Nw protein do to
stop CE cell movement.
Biochemistry - find proteins that
bind to Nw - test PCP proteins for
binding to Nw?
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