The Role of how in Leg Imaginal
Disc Morphogenesis in Drosophila
melanogaster
Nicole Whyte
Craig Woodard
July 23, 2004
Background and Significance
The processes that lead to the development of
animal body parts include complex steps that
should be executed accurately to ensure the
proper formation.
These developmental processes are controlled by
the interaction of the genes present in the
organism. Genes regulate growth and development
by hormonal signaling, including steroid hormones,
in simple organisms such as insects to more
intricate organisms such as humans.
Introduction
Drosophila melanogaster are interesting organisms
to study.
ßFTZ-F1 vs. how Gene
Examination of leg development in how mutant
fruit flies
Drosophila melanogaster
Pupariation
(Entry into
Metamorphosis)
Morphogenesis of
Adult Body Parts
Destruction of
Larval body Parts
by Programmed
Cell Death
Leg Morphogenesis in Drosophila
melanogaster
Structures such as wings and legs in the fruit fly
originate from the imaginal disc sacs formed
during embryogenesis.
Imaginal discs are small packets of cells in the
larva which undergo morphogenesis during the
metamophosis of the insect.
The discs are covered by a single layer of cells
called the peripodial epithelium.
Third Instar Larva
Leg Disc Eversion
Adult
Cell Shape Changes During Leg Disc
Elongation
a
b
Courtesy of Condic et al. 1991. Development 111:23-33
Normal Leg Development
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how Gene
The how gene found in Drosophila melanogaster
encodes a KH RNA binding protein, and is involved
in processes such as muscle development.
There are different how mutant alleles, some
stronger than others.
Example: howe44 and howstruthio are strong mutant
alleles that results in embryonic death of the
flies, while the howr17 is a weak allele. Some of
these flies survive to adulthood.
how Mutants Show Defects in
Leg Development
how Mutant Leg Development
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Experiment
What causes the short and crooked leg phenotype
seen in how mutants?
The experiment included the characterization of leg
imaginal disc:
length and shape
cell shape changes
comparison of mutation in each leg disc
Materials and Method
The animals were kept on standard
cornmeal/molasses/yeast medium at 250C or 180C.
For the experiment, three strains of animals were
used:
howe44/TM6B,Hu,Tb,e
w; +; howr17/TM6B,Hu,Tb,e
Humeral (Hu)
Tubby (Tb), Ebony (e)
Canton S wild type (CS)
Materials and Methods Cont.
Experimental animals:
howe44/TM6B,Hu,Tb,e
F1:
X
w;+;howr17/TM6B,Hu,Tb,e
howe44/ howr17
Control animals:
howe44/TM6B,Hu,Tb,e X
F1:
howe44/ +
CS (+/+)
Materials and Methods Cont.
Zero hour APF prepupae (white, not
tubby) animals were collected and
maintained at 250C for 6 hours.
The leg imaginal discs were dissected out
from the animals in PBS Buffer solution
at room temperature and fixed in 4%
formaldehyde for 18-24 hours at 40C.
Then permeabilized for 1-2 hours with
0.5% Triton X-100 (w/v in PBS) at room
temeperature.
Materials and Methods Cont.
The leg imaginal discs were stained with the
fluorescent AlexaFluor 488 0.6 µM phalloidin
solution for four hours at room temperature.
Then rinsed in 0.5% Triton X-100 (w/v in PBS) for
2 hours at room temperature, with 3 changes.
The leg imaginal discs were mounted in
VectashieldTM and viewed with a Zeiss LSM 510
Meta laser scanning confocal microscope and
Nikon SMZ 1500 Stereoscope.
Cell Shape in Leg Disc
control
Stubble Mutant
Results
Cell Shape in Mutant Animal
howe44/ howr17
Phenotypic Differences in
6 hr APF Leg Disc
Wild type
howe44/ howr17 mutant
Peripodial Epithelium in Mutant
6 hr APF Leg Disc
howe44/ howr17
Discussion
Normal cell shape change occurs in mutants
The mutation affects each leg differently
The peripodial epithelium is still present around
the leg disc which might contribute to the short
and bent legs observed
Future Work
Determine why the leg discs fail to orient properly
Examine younger animals to see if there are any
defects that explains the observed phenotype
Determine what goes wrong in the peripodial
epithelium in the how mutants
Acknowledgment
Dr. Craig Woodard
Tina Fortier
The Vibrant Fly Lab Team
The National Science Foundation
And All the People who have inspired my Scientific
Career