herman lab university of oregon alex whitebirch how do neurons intrinsically regulate axon growth?
TRANSCRIPT
Herman LabUniversity of Oregon
Alex Whitebirch
How do neurons intrinsically regulate axon growth?
How is the the circuitry of the nervous system formed?
axon
dendrites
cell bodynucleus
synapse
Neuron with projecting axon
Neurons establish connections through the activity of the growth cone
Neuron with projecting axon
Neurons establish connections through the activity of the growth cone
Neuron with projecting axon
Neurons establish connections through the activity of the growth cone
Growth ConeLowery and Vactor. Nat Rev MCB 10 (2009)
Neuron with projecting axon
Neurons establish connections through the activity of the growth cone
Growth ConeLowery and Vactor. Nat Rev MCB 10 (2009)
Neuron with projecting axon
Neurons establish connections through the activity of the growth cone
How is the propulsive motion of growth cones regulated to allow new axons to establish a highly organized set of connections?
Growth cones are guided by external signals
Attractive signale.g. Netrins
Repulsive signal e.g. Ephrins
Cellular responses to axotomy demonstrate intrinsic regulation of axon growth
axon
synapse
Cell body
Liu K. et al. Annu. Rev. Neurosci. 34: 131-52 (2011)
Cellular responses to axotomy demonstrate intrinsic regulation of axon growth
axon
synapse
Cell body
Liu K. et al. Annu. Rev. Neurosci. 34: 131-52 (2011)
Retraction bulb
Growth cone
Some neurons
Other neurons
The Drosophila visual system provides a model of axon growth
R2
R1
R5
R4R3
R8R6
R7
Ttk69ProsperoPhalloidin
MARCM enables precise genetic manipulation of R7 cells
MARCM: mosaic analysis with a repressible cell marker
MARCM enables precise genetic manipulation of R7 cells
MARCM: mosaic analysis with a repressible cell marker
MARCM enables precise genetic manipulation of R7 cells
MARCM: mosaic analysis with a repressible cell marker
Tramtrack69 is necessary to restrict axon growth
Misexpression of Tramtrack69 is sufficient to terminate axon growth
WT
UAS
-ttk6
9
48 hr APF 48 hr APF24B10CD8-GFP
Misexpression of Tramtrack69 is sufficient to terminate axon growth
WT
UAS
-ttk6
9
48 hr APF 48 hr APF24B10CD8-GFP
What does Tramtrack69 do on a molecular level to restrict axon growth?
Tramtrack69 is a transcription factor
Transcription
Ttk69Ttk69 is a transcriptional repressor
RNA product No transcription
Tramtrack69 is a transcription factor
Ttk69Ttk69 is a transcriptional repressor
What genes is Ttk69 regulating in order to inhibit axon growth?
TranscriptionRNA product No transcription
TGF-β/Activin signaling pathway is necessary to inhibit axon growth
dSmad2ActivinBaboon
Ting C et al. Neuron 56 (2007)
Axon growth
Tramtrack69 acts upstream of Baboon to restrict axon growth
Nuc
lear
FLA
G-d
Smad
2 (fo
ld e
nric
hed)
2
1.5
1
WT
ttk69
n = 10
n = 10
dSmad2
Activin
Baboon
Axongrowth dSmad2
Activin
Baboon
Axon growthWild type ttk69
BaboonActivin
dSmad2
Antagonist
Axongrowth
Hypothesis: Tramtrack69 restricts axon growth by repressing an antagonist of Activin signaling
Wild type
BaboonActivin
dSmad2
Antagonist
Axongrowth
BaboonActivin
dSmad2
Antagonist
Axongrowth
Hypothesis: Tramtrack69 restricts axon growth by repressing an antagonist of Activin signaling
Wild type ttk69
BaboonActivin
dSmad2
Antagonist
Axongrowth
BaboonActivin
dSmad2
Antagonist
Axongrowth
BaboonActivin
dSmad2
Antagonist RNAi
Axongrowth
Hypothesis: Tramtrack69 restricts axon growth by repressing an antagonist of Activin signaling
Wild type ttk69 ttk69, Antagonist-RNAi
Double mutants showed no overgrowth phenotype
WT ttk69, Cripto-RNAittk69, Follistatin-RNAittk69
24B10SytGFP
Double mutants showed no overgrowth phenotype
WT ttk69, Cripto-RNAittk69, Follistatin-RNAittk69
24B10SytGFP
A perfect rescue?...
My “ttk69”
My “ttk69” chromosome lacked the ttk69 mutation
ttk69
My “ttk69,” Follistatin-RNAi
24B10SytGFP
Ttk69ProsperoPhalloidin
Ttk69
ttk69
Future Directions
Follistatin – GFP
Recombineering with BACs: bacterial artificial chromosomes
3. If there is a rescue….
1. Rebuilding ttk1e11 stock and repeating the experiment
2. Exploring the potential role of other activin antagonists in the tramtrack69 pathway
Acknowledgments
The Herman LabUniversity of OregonInstitute of Molecular Biology
Summer Program for Undergraduate Research
TorySashaEricJen JonSarahDaveErikaMike
Peter O’DayAdam Unger
NICHD R25 Summer Research Program
Acknowledgments
The Herman LabUniversity of OregonInstitute of Molecular Biology
Summer Program for Undergraduate Research
TorySashaEricJen JonSarahDaveErikaMike
Peter O’DayAdam Unger
Questions?
NICHD R25 Summer Research Program