Download - Axon Targeting and Cell Fate in the Drosophila Eye Humera Ahmad Verni Logendran Herman Lab
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Axon Targeting and Cell Fate in the Drosophila Eye
Humera AhmadVerni Logendran
Herman Lab
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How do cells know their fate?
single-cell zygote
Cell division → Skin…
Neurons…
Muscle…Or blood cells…
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The Drosophila eye contains different kinds of neurons
R1
R5
R4
R6
R3
R2
R7
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© 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
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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
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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.
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X
Homozygous mutant flies
Previous Method for ScreeningChemical mutagen
Dissect and examine R7 axon targeting and cell fate
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Drawback of Screen I
Only identified two genes that controlled R7 development
What if genes important for R7 are also required for early development?
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X
Mosaic mutants
Screen IIChemical mutagen
Dissect and examine homozygous mutant R7s
Heterozygous mutants
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express FLP recombinaserandom
mutation
FRT recombination site
mitotic recombination
Creating Mosaic Animals
Homozygous mutant
Homozygous wild type
R7 Parents
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Labeling mutant cells using MARCM
GFP transcription in homozygous mutant
cell
GFP not expressed
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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
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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
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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
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Solution: Our Screen!• There is a library of molecularly defined deletions
– Know exactly which genes the deletion removes• Each deletion removes 10-50 genes
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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
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+
FRT Site Deletion
We attempted to recombine 55 deletions onto a FRT chromosome
Meiotic recombination
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The closer the deletion is to the FRT, the less frequently recombination occurs
Recombination distance: 3
Recombination distance: 21
Recombination distance: 45
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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
+
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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
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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.
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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
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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
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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
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Acknowledgements• Herman Lab
– Dr. Tory Herman, Jon Kniss, Jen Jeffress, Adam Miller, Eric Lyons, Scott Holbrook
• Peter O’Day• SPUR
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Questions?