herman lab university of oregon alex whitebirch how do neurons intrinsically regulate axon growth?

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