Michelle Glaid
BIOL 371W-01
February 14, 2010
Cell development and death in Drosophila melanogaster
Introduction
When studying the development of tissues, it is interesting to note that regulated cell
death, or apoptosis, is present. Apoptosis is used by the organism to make sure that cells with
fates are able to grow, and that cells without fates are removed (Wolff & Ready, 1991). The use
of apoptosis can be seen during the development of the eye disc in Drosophila melanogaster.
When the eye disc first develops, the cells are undifferentiated. Then, a morphogenetic furrow
passes across the eye, causing some cells to begin to develop into photoreceptor cells (Escudero
& Freeman, 2007). Other cells that have not yet differentiated go into cell cycle arrest and
neither divide nor die. A second signal sweeps across the eye disc and causes a wave of mitosis,
which causes the remaining photoreceptor cells and cone cells to be recruited (Wolff & Ready,
1991). Cells that are not recruited undergo apoptosis so that they do not inhibit further
development of the eye or lenses. See Figure 1.
Figure 1. A depiction of the development of the eye disc and the recruitment or death of cells.
After the morphogenetic furrow passes, some photoreceptor cells are recruited. The remaining
photoreceptor cells are recruited by lozenge after the second wave of mitosis (in red), and any
extra cells undergo apoptosis. Picture courtesy of Siddall, et al., 2003.
During cell cycle arrest, the gene lozenge is activated and prevents cells without fates
from undergoing apoptosis too soon. Cells without fates that are told to not divide naturally
undergo apoptosis, and if this occurred, there would not be enough cells to form the remaining
photoreceptor and cone cells. Drosophila strains with mutated expression of the lozenge gene
would therefore have altered amounts and locations of apoptosis. The experimental strain used
was strain 2387, with the genotype lzk. The genotyle lzk contains an insertion of a transposable
element between exons three and four of the gene (Behan, et al., 2005). As a result, the lozenge
gene tends to be overexpressed. The phenotype is slightly varied from the control phenotype.
The control stock used was strain 5, which has normal development of the eye disc.
Presence of apoptosis in eye disc tissue was detected using the TUNEL apoptosis
detection kit, which acts by adding a biotin marker on the ends of cut DNA. Cells undergoing
apoptosis would have a greater number of cut DNA ends than cells not undergoing apoptosis.
This is because during apoptosis, the cell digests its proteins, organelles, and DNA so that it does
not disrupt neighboring cells. After the biotin marker was added, avidin was allowed to bind to
the biotin in the eye disc tissue. Avidin has a strong affinity for biotin and would bind strongly
to it. The avidin also had a fluorescent protein attached to it so that the location of the avidin,
biotin, and cells undergoing apoptosis could be seen. A positive control was created to ensure
that the biotin could attach to the ends of cut DNA. A DNase enzyme was added to the eye disc
tissue and allowed to cut the DNA. The biotin marker and avidin were added in the same way as
the experimental group.
It was predicted that because lozenge was overexpressed in strain 2387 with the mutation
lzk, there would be a decreased amount of cell death in the eye disc as opposed to the wildtype
strain 5.
Methods
D. melanogaster larvae were obtained from the strains 2387 and 5. The larvae were in
the third instar stage. The cephalic complex was obtained containing the brain and developing
eye discs. The tissue was fixed with 4% paraformaldehyde and then permeablized with 0.2%
Triton X-100. After being rinsed in 1x phosphate buffer saline, the tissue was added to 100 µL
Terminal deoxynucleotidyl Transferase buffer for 5 minutes, and then 100 µL TdT end-labeling
cocktail for 60 minutes at 37°C. This cocktail contained biotinylated deoxyuridine triphosphate,
which attached to ends of DNA. The tissue was then washed with a termination buffer several
times to stop the reaction and the tissue was incubated in 100 µL blocking solution at 4°C for
117 hours.
The tissue was then brought to room temperature and incubated in 100 µL avidin-FITC
for 45 minutes at 37°. The tissue was then washed with PBS and the primary monoclonal
antibodies rabbit anti-phospho-histone H3 and mouse anti-ELAV were added. The avidin-FITC
bound to the biotin at the ends of the DNA strands and appeared green under an ultraviolet light.
The anti-phospho-histone H3 attaches to histone H3 that is found in cells undergoing mitosis.
The anti-ELAV attaches to the ELAV protein that is found in the nuclei of nerve cells. It serves
as a marker for the location of nerve cells.
After allowing the primary antibodies to incubate with the fly tissue for 18 hours, the
tissue was washed with PBS and 100 µL of the secondary antibodies were added. The
polyclonal secondary antibodies were goat anti-rabbit CY5 and goat anti-mouse CY3. The goat
anti-rabbit CY5 bound to the primary antibody rabbit anti-phospho-histone H3 and had a
fluorescent tag that showed under an infrared light. The goat anti-mouse CY3 bound to the
primary antibody mouse anti-ELAV and showed red because of the fluorescent tag. The
secondary antibodies were allowed to incubate with the fly tissue for 28 hours at 4°C.
The tissue was then dissected further so that just the eye discs were placed on slides with
Prolong gold. The Prolong gold served as a mounting medium. The eye discs were then
observed under a phase contrast microscope under varying magnifications and wavelengths of
light. The tissue was also observed under a confocal microscope so that the infrared
fluorescence could be seen.
Results
The eye discs were observed with a confocal microscope under varying magnification.
The tissue appeared clear under normal white light, as seen in Figure 2. The smallest divisions
in the scale are 0.1 mm.
Figure 2. D. melanogaster strain 2387 under
white light. The eye disc is located at the center
of the picture. The scale is at 0.5 mm, with the
smaller divisions at 0.1 mm.
The avidin-FITC dye appeared green under an ultraviolet light and shows the location of
cells undergoing apoptosis. The positive control demonstrated that cells undergoing apoptosis
were visible, but it was much more difficult to see cells undergoing apoptosis in the experimental
tissue. These cells appear to be located throughout the tissue, which is not expected (Figure 3A).
Pictures taken by the confocal microscope seem to confirm the presence of avidin throughout the
tissue (Figure 3B). The control strain 5 showed similar results of uniform coloration, and was
therefore not shown.
Figure 3. D. melanogaster strain 2387 under blue light. Figure 3A was taken under a compound
microscope and the smallest divisions on the scale are 0.1 mm. Figure 3B was taken with a
confocal microscope. The location of apoptosis should be seen, but the avidin-FITC dye appears
in many cells in both 3A and uniformly across the tissue in 3B.
The goat anti-mouse CY3 appears red under green light as seen in Figure 4A. It shows
the location of ELAV in the tissue. ELAV is usually expressed in the nuclei of the nerve cells in
the developing eye. It serves as a maker to show the location of the morphogenetic furrow and
the developed nerve cells. The location of the furrow can be seen by the blue arrow. The
confocal microscope also retrieved a picture of the goat anti-mouse CY3 dye (Figure 4B).
Figure 4. D. melanogaster strain 2387 under blue light. Figure 4A was taken under a compound
microscope and the scale’s smallest divisions are 0.1 mm. Figure 4B was taken with a confocal
microscope. The location of ELAV in the tissue can be seen. ELAV is typically found in
photoreceptor cells. The morphogenetic furrow can be seen by the blue arrow.
The goat anti-rabbit CY5 dye appeared under infrared light as seen in Figure 5. It shows
the location of phospho-histone H3 in the tissues, which is present in cells undergoing mitosis. It
also served as a marker for the morphogenetic furrow. The furrow highlighted by the blue
arrow.
Figure 5. D. melanogaster strain 2387 under
infrared light. It was taken with a confocal
microscope. The location of phospho-histone H3
can be seen, which serves as a marker for cells
undergoing mitosis. Phospho-histone H3 is used
by the cell to condense DNA into chromosomes.
The line of mitosis is prompted by the gene
lozenge and prompts the differentiation of
photoreceptor cells R1, R6, and R7. The location
of the morphogenetic furrow can be seen by the
blue arrow.
A combination of Figures 2, 3A, and 4A can be seen in Figure 6. The ELAV staining,
which shows the location of developing nerve cells, seems to correspond to the TUNEL staining
for apoptosis. This is indicated by the yellow arrow.
Figure 6. A composite of Figures 2, 3A, and
4A. The images were taken under a confocal
microscope, and the smallest divisions of the
scale are 0.1 mm. The location of ELAV
seems to correlate with the location of the
avidin-FITC dye. This is indicated by the
yellow arrow. This correlation is unusual,
because cells expressing ELAV are
developing photoreceptor cells, so they
would not be undergoing apoptosis. The
image was created using ArcSoft
PhotoStudio 5.5.
Figure 7 shows a combination of Figures 4B and 5. The location of the phosphor-histone H3
shown in blue correlates with the location of the differentiation of photoreceptor cells shown in
red. The morphogenetic furrow can be seen by the blue arrow.
Figure 7. A composite of Figures 4B
and 5. The location of the
morphogenetic furrow (blue arrow) and
the area without cell division before the
second wave of mitosis can be seen.
Discussion
Although the positive control confirmed that the TUNEL method of detecting apoptosis
was able to work, the method had less success when working with actual experimental tissue.
Both the experimental and control strains of Drosophila failed to show any distinct band of
apoptosis across the cell. A slight band was present in Figure 6, but this does not seem to
actually represent areas of apoptosis. First, the cells expressing ELAV directly correspond to the
cells that appear to express avidin-FITC. ELAV shows cells that are differentiated and actively
growing, so these cells would not be undergoing regulated cell death. Cells that would undergo
cell death would not have fates, and these cells clearly have fates. Second, avidin-FITC is
present in low levels in other areas of the tissue as well, especially in Figure 4, and it is highly
unlikely that all of these cells would be undergoing apoptosis. It is more likely that the TUNEL
staining failed to accurately depict the locations of apoptosis activity.
There are several possible reasons for the failure of the TUNEL staining. The tissue may
not have been properly permeablized by the Triton-X before the addition of the terminal
deoxynucleotidyl transferase cocktail. As a result, the biotinylated deoxyuridine triphosphate
would not have been able to reach the DNA in cells. The tissue may not have been washed
enough times to remove the TdT cocktail. The avidin-FITC may have been at a concentration
that was too high and showed overexpression in the tissue. Because neither the control nor
experimental tissue successfully showed the location of apoptosis, the hypothesis can be neither
confirmed nor denied based on the data obtained.
Although the TUNEL staining was not successful, other results could be obtained from
the data. As seen in Figure 7, development of photoreceptor cells occurs after the furrow passes.
Photoreceptor cells are present only above the furrow. Cells around the morphogenetic furrow
that are not yet differentiated are in cell cycle arrest. Some of these cells are needed by the
organism to form other photoreceptor cells or cone cells, but they are not to differentiate or
divide yet, so lozenge prevents the cells from dividing or dying. This can be clearly seen in
Figure 7 as well. These results correspond to previous research conducted on the development of
Drosophila eye discs (Escudero & Freeman, 2007; Wolff & Ready, 1991). Even though
apoptosis levels could not be determined based on the experiment, information concerning the
development of the Drosophila eye disc could still be obtained. Further testing must be done in
order to determine any differences in the occurance of apoptosis between the experimental strain
2387 and the wildtype strain 5. Future experiments include repeating this experiment such that
apoptosis is seen accurately and comparing the locations of cell birth and cell death between
control and experimental strains.
References
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splicing removes an Ets interaction domain. Development Genes and Evolution , 423–435.
Escudero, L. M., & Freeman, M. (2007). Mechanism of G1 arrest in the Drosophila eye imaginal
disc. BMC Developmental Biology .
Siddall, N. A., Behan, K. J., Crew, J. R., Cheung, T. L., Fair, J. A., Batterham, P., et al. (2003).
Mutations in lozenge and D-Pax2 invoke ectopic patterned cell death. Developmental Genes and
Evolution , 107-119.
Wolff, T., & Ready, D. F. (1991). The beginning of pattern formation in the Drosophila
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