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Axon Targeting and Cell Fate
in the Drosophila Eye
Humera Ahmad
Verni Logendran
Herman Lab
How do cells know their fate?
Neurons…
Cell division →
Skin…
single-cell zygote
Muscle…
Or blood cells…
The Drosophila eye contains different
kinds of neurons
R1-R8 express different rhodopsins and form synapses
in different brain layers
R1-6s express Rh1
R8s express Rh5/6
R7s express Rh3/4
Iris Salecker
© National Institute for Medical
Research
R7s must receive several signals to meet
their destination
larval development
8
2 8 5
1
6
8
2
5
3 4
2 8 5
3 4
precursors
undergo
one more
mitosis
1 7 6
2 8 5
3 4
Taking a genetic approach to identify
genes responsible for R7 development
• Two conventional screens using chemical
mutagens:
– Homozygous mutant flies
– Mosaic flies
• New approach: Systematic removal of small
regions of the Drosophila genome.
Previous Method for Screening
X
Homozygous
mutant flies
Dissect and examine
R7 axon targeting and cell fate
Chemical
mutagen
Drawback of Screen I
Only identified two genes that controlled R7
development
What if genes important for R7 are also required
for early development?
Screen II
X
Heterozygous mutants
Mosaic mutants
Dissect and examine
homozygous mutant R7s
Chemical
mutagen
Creating Mosaic Animals
FRT recombination site
random
mutation
express
FLP recombinase
Homozygous mutant
Homozygous wild type
mitotic recombination
R7 Parents
Labeling mutant cells using MARCM
GFP transcription in
homozygous mutant
cell
GFP not expressed
Labeling mutant cells using MARCM
m/m cells
express GFP
express
FLP recombinase
Homozygous
mutant
Gal80
Homozygous
wild type
Gal80
Gal80
Gal80
mitotic recombination
* All cells express Gal 4-UAS/ GFP
Gal80
Heterozygous
Gal80
Screen II has identified new genes
important to cell fate and axon targeting
3
4
2
1
7
5
6
Drawbacks of Screen II
• Laborious
– Mutation must be mapped to pinpoint gene
• Does not cover the whole genome
– Mutations are distributed randomly
• Not all results are significant
– Wild-type phenotype
Solution: Our Screen!
• There is a library of molecularly defined deletions
– Know exactly which genes the deletion removes
• Each deletion removes 10-50 genes
Solution: Our Screen!
• Creating stocks with specific deletions on the left
arm of chromosome III that will be screened with
MARCM
• Can systematically screen every gene in this
region of the genome
Deletion
FRT Site
We attempted to recombine 55
deletions onto a FRT chromosome
FRT Site
Deletion
+
Meiotic recombination
The closer the deletion is to the FRT, the
less frequently recombination occurs
Recombination
distance: 45
Recombination
distance: 21
Recombination
distance: 3
Genetic Scheme
FRT2
X
FRT2
Del
TM6B (humoral)
Meiotic recombination
does not occur in males
FRT2
X
TM3 (stubble)
Del
TM6B
FRT2
Del
FRT2 , Del
TM3
TM3
TM3
+
TM3
Complementation Test: Determining whether
the chromosome has the deletion
FRT2?
X
TM3
FRT2?
Del
FRT2?
Del
FRT2? Del
FRT2? Del
Wild-type
Hu
FRT2? Del
TM6B
TM3
TM3
Del
TM6
Sb, Hu
Dead
Hu
Sb, Hu
TM6B
TM3
TM3
TM6B
Del
TM6B
Sb
X
TM3
TM6B
Del
Del
Sb
FRT site verification
• Use PCR (polymerase chain reaction) to verify
the presence of FRT recombination site in
Drosophila’s DNA.
• If the FRT site is present, only then will a
region specific to this site be amplified.
Results: Our Screen
Deletion #
7563
Projected # of recombinants
to be screened
7.47
Actual # of recombinants scored
17
# of recombinants with
deletion
2
# of recombinants with
FRT
2
7566
7564
7565
7.47
7.47
7.47
18
22
29
4
8
10
2
1
1
7569
7568
7567
7562
7570
7573
7575
7576
7574
7577
7571
7572
7921
7922
7578
7583
7582
7580
7581
7584
7927
7928
7587
7588
7745
7929
7589
7591
7930
7924
7.7
7.7
7.7
7.7
7.7
8.45
8.45
8.45
8.45
8.45
8.45
8.45
11.5
11.5
11.5
14
14
14
14
14
14.6
14.6
14.6
16.8
19.5
19.5
19.5
19.5
19.5
20.6
16
20
25
27
28
0
19
19
21
21
24
30
18
21
22
28
29
30
33
33
16
30
33
34
19
25
37
38
38
17
0
6
9
11
9
N/D
2
0
5
4
6
15
N/D
N/D
4
10
7
4
1
5
N/D
4
3
6
N/D
N/D
1
0
N/D
N/D
0
2
1
3
4
N/D
2
0
4
0
0
3
N/D
N/D
2
7
4
3
0
3
N/D
4
1
2
N/D
N/D
1
1
N/D
N/D
v
Results continued…
Deletion #
Projected # of recombinants
to be screened
Actual # of recombinants scored
# of recombinants with # of recombinants with
deletion
FRT
7926
20.6
18
N/D
N/D
7586
20.6
24
6
2
7585
20.6
44
4
1
7593
23.2
49
3
1
7933
26.4
9
0
0
7594
28.3
49
12
2
7596
55.2
58
4
1
7595
55.2
69
11
1
7597
74.4
40
6
0
7934
7599
89.8
89.8
22
34
6
4
2
1
7601
89.8
44
6
2
7598
89.8
49
1
1
7600
89.8
51
6
0
7602
89.8
54
2
2
7607
151.2
52
1
0
7606
151.2
60
9
1
7608
458.2
51
5
0
7604
>1000
51
1
1
7605
>1000
51
2
0
7729
>1000
59
0
0
• 55 deletions used for the screen
• 34 chromosomes (thus far) containing FRT 2 site + deletion
Future Directions…
• Use verified chromosomes to create
homozygous mutant R7s
• Dissect retinas and brains
• Observe the effect in axon targeting and cell
fate.
• Determine which gene in the deletion is
required
Acknowledgements
• Herman Lab
– Dr. Tory Herman, Jon Kniss, Jen Jeffress,
Adam Miller, Eric Lyons, Scott Holbrook
• Peter O’Day
• SPUR
Questions?