Transcript Herman Lab - SPUR - University of Oregon

Using Drosophila to investigate
axon targeting
Lori Evans
Herman Lab
Institute of Molecular Biology
University of Oregon
SPUR Program
Brain wiring is complicated
a human brain
ZOOM IN on:
randomly labeled neurons
Brain wiring is organized into
layers
a human brain
Brain wiring is organized into layers
a human brain
How do neurons grow and connect during
development?
Each neuron extends its axon to
seek its target
cell body
axon
growth cone
Each neuron extends its axon to
seek its target
cell body
axon
growth cone
How can we study axon target
selection?
Drosophila photoreceptor axons go
to different brain layers
Fly eye
~ 800 ommatidia (facets)
Ommatidium
8 neurons
7
8
Targets in the
fly brain
Drosophila photoreceptor axons go
to different brain layers
Fly eye
~ 800 ommatidia (facets)
Ommatidium
8 neurons
7
8
Targets in the
fly brain
What does the fly visual system
really look like?
Drosophila photoreceptor axons go
to different brain layers
7
8
lamina
R8
medulla
R8
R7
R7
Drosophila photoreceptor axons go
to different brain layers
7
8
R8
lamina
R7
medulla
R8
R7
How can we identify genes required for R7 axons to choose
the R7 target layer?
We look for mutations that disrupt
R7 axons
Wild type R7s
We use mosaic
animals
R7s are
m/m
m/+
R8
R7
5-2 mutant R7s send axons to the
wrong layer
5-2 mutant R7s
wild type R7s
MAb24B10
GFP
R8
R7
Adult Phenotype
5-2 mutant R7s send axons to
wrong layer
wild type R7s
5-2 mutant R7s
MAb24B10
GFP
R8
R7
My main question of interest: What gene
does the 5-2 mutation disrupt?
5-2 mutation is on 3R chromosome
XX in ♀
XY in ♂
or
1
2
2L
2R
3
4
3L
3R
5-2 mutation is on 3R chromosome
XX in ♀
XY in ♂
or
1
2
2L
2R
3
3L
3R
4
How can we determine where the 5-2 mutation is on the
3R chromosome?
We used two methods to map the
5-2 mutation
• Deficiency Mapping
• Recombination Mapping
Deficiency kit with deletions of 3R
chromosome used to map 5-2
5-2 mutant
?
Deficiency Kit for 3R Chromosome
Deficiency kit with deletions of 3R
chromosome used to map 5-2
5-2 mutant
?
Deficiency Kit for 3R Chromosome
What is complementation testing?
5-2 failed to complement Df #5414
5-2 mutant
5-2 mutant
5-2 mutant
Df
5-2 mutant
Df
5-2 mutant
Df
5-2 mutant
Df #5414
dead
alive
alive
alive
dead
Gaps exist along the 3R chromosome
5-2 mutant
5-2 mutant
dead
5-2 mutant
Df
?
alive
5-2 mutant
Df
?
alive
5-2 mutant
Df
?
alive
5-2 mutant
Df #5414
dead
Gaps exist along the 3R chromosome
5-2 mutant
5-2 mutant
dead
5-2 mutant
Df
alive
5-2 mutant
Df
alive
5-2 mutant
Df
alive
5-2 mutant
Df #5414
dead
We can use recombination to test
whether or not this lethal mutation
causes the R7 phenotype
cu sr
sr
e
ca
e
ca
e
ca
ca
sr
e
We can use recombination to test
whether or not this lethal mutation
causes the R7 phenotype
cu sr
sr
e
ca
e
ca
e
ca
ca
sr
e
We can use recombination to test
whether or not this lethal mutation
causes the R7 phenotype
cu sr
sr
e
ca
e
ca
e
ca
ca
sr
e
How did I create recombinant chromosomes?
♀
5-2
ru cu ca
TM 3
ru cu ca
♀
♂
5-2
ru cu ca
ru cu ca
ru Pr ca
recomb (5-2)
♂
♂
D ; TM 3
ru Pr ca
D ; TM 6B
D ; recomb (5-2)
♀
I set up
several
generations of
crosses
♂
Y ; TM 6B
♂
D ; recomb (5-2)
♂
Y ; TM 6B
D ; TM 3
D ; recomb (5-2)
hist ♀
Y ; TM 6B
♀
D ; TM 6B
D ; recomb (5-2)
D ; recomb (5-2)
Y ; TM 6B
♂
Y ; TM 6B
hist ♂
These are the numbers of each type of
recombinant chromosome I found
cu sr
sr
e
e
ca
ca
ca
32
63
e
50
ca
sr
e
10
Preliminary results have been analyzed
sr
R8
R7
e
ca
Future Work
Hypothesis Correct
• Identify gene
– Use smaller deficiencies
Hypothesis Incorrect
• Look at other
recombinants
• Find 5-2 mutation
causing phenotype
• Identify gene
Conclusion
The 5-2 gene identified to be involved in axon
target selection in Drosophila may also play a
role in axon target selection in humans.
Acknowledgments
Herman Lab
Tory Herman
Adam Miller
Eric Lyons
Jennifer Jeffress
Heather Seymour
Jon Kniss
Scott Holbrook
Stefan Buetow
*Lola, Bert, Bode, Jobie*
SPUR
Peter O’ Day
Beth Roy
SPUR Participants
SPUR Funders