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?