Axon Targeting and Cell Fate in the Drosophila Eye Humera Ahmad Verni Logendran Herman Lab.
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Transcript of Axon Targeting and Cell Fate in the Drosophila Eye Humera Ahmad Verni Logendran Herman Lab.
Axon Targeting and Cell Fate in the Drosophila Eye
Humera AhmadVerni Logendran
Herman Lab
How do cells know their fate?
single-cell zygote
Cell division → Skin…
Neurons…
Muscle…Or blood cells…
The Drosophila eye contains different kinds of neurons
R1
R5
R4
R6
R3
R2
R7
© National Institute for Medical Research
R1-R8 express different rhodopsins and form synapsesin different brain layers
R1-6s express Rh1
Iris Salecker
R8s express Rh5/6
R7s express Rh3/4
R7s must receive several signals to meet their destination
8 8 52 8 523 4
8 523 4
1 68 52
3 4
1 67
precursors undergo
one moremitosis
larval development
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.
X
Homozygous mutant flies
Previous Method for ScreeningChemical mutagen
Dissect and examine R7 axon targeting and cell fate
Drawback of Screen I
Only identified two genes that controlled R7 development
What if genes important for R7 are also required for early development?
X
Mosaic mutants
Screen IIChemical mutagen
Dissect and examine homozygous mutant R7s
Heterozygous mutants
express FLP recombinaserandom
mutation
FRT recombination site
mitotic recombination
Creating Mosaic Animals
Homozygous mutant
Homozygous wild type
R7 Parents
Labeling mutant cells using MARCM
GFP transcription in homozygous mutant
cell
GFP not expressed
express FLP recombinase
mitotic recombination
Labeling mutant cells using MARCM
m/m cells express GFP Homozygous
mutant
Heterozygous
Gal80
Homozygous wild type
Gal80
Gal80
Gal80
Gal80
Gal80
* All cells express Gal 4-UAS/ GFP
Screen II has identified new genes important to cell fate and axon targeting
Wild Type Early StopLateral
Extension
1
23
45
67
Wild Type R1 R7 Transformation
Loss of
R7
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
FRT SiteDeletion
+
FRT Site Deletion
We attempted to recombine 55 deletions onto a FRT chromosome
Meiotic recombination
The closer the deletion is to the FRT, the less frequently recombination occurs
Recombination distance: 3
Recombination distance: 21
Recombination distance: 45
FRT2
FRT2
Del
TM6B (humoral)X
FRT2
Del
Meiotic recombination does not occur in males
TM3 (stubble)
TM6BX
FRT2 , Del
TM3
Genetic Scheme
Del
TM3
FRT2
TM3
TM3
+
Complementation Test: Determining whether the chromosome has the deletion
FRT2?
Del
FRT2?
TM6B
TM3
Del
TM3
TM6
FRT2?
TM3X
Del
TM6B
Wild-type
Hu
Sb
Sb, Hu
TM3
TM6B
TM3
Del
Dead
Hu
Sb, Hu
Sb
FRT2? Del
TM3X
FRT2? Del
Del
FRT2? Del
TM6B
Del
TM6B
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 ScreenDeletion #
Projected # of recombinants to be screened Actual # of recombinants scored
# of recombinants with deletion
# of recombinants with FRT
7563 7.47 17 2 2
7566 7.47 18 4 27564 7.47 22 8 17565 7.47 29 10 1
7569 7.7 16 0 07568 7.7 20 6 2 7567 7.7 25 9 17562 7.7 27 11 37570 7.7 28 9 47573 8.45 0 N/D N/D 7575 8.45 19 2 27576 8.45 19 0 07574 8.45 21 5 47577 8.45 21 4 0 7571 8.45 24 6 07572 8.45 30 15 37921 11.5 18 N/D N/D 7922 11.5 21 N/D N/D 7578 11.5 22 4 27583 14 28 10 77582 14 29 7 47580 14 30 4 37581 14 33 1 0 7584 14 33 5 37927 14.6 16 N/D N/D 7928 14.6 30 4 47587 14.6 33 3 17588 16.8 34 6 2 7745 19.5 19 N/D N/D 7929 19.5 25 N/D N/D7589 19.5 37 1 1 7591 19.5 38 0 17930 19.5 38 N/D N/D7924 20.6 17 N/D N/D
v
Results continued…Deletion #
Projected # of recombinants to be screened Actual # of recombinants scored
# of recombinants with deletion
# of recombinants with 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 89.8 22 6 27599 89.8 34 4 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?