1. Herman Lab University of Oregon Alex Whitebirch How do neurons intrinsically regulate axon growth?

2. How is the the circuitry of the nervous system formed? axon dendrites cell body nucleus synapse

3. Neuron with projecting axon Neurons establish connections through the activity of the growth cone

4. Neuron with projecting axon Neurons establish connections through the activity of the growth cone

5. Neuron with projecting axon Neurons establish connections through the activity of the growth cone

6. Growth Cone Lowery and Vactor. Nat Rev MCB 10 (2009) Neuron with projecting axon Neurons establish connections through the activity of the growth cone

7. Growth Cone Lowery 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?

8. Growth cones are guided by external signals Attractive signal e.g. Netrins Repulsive signal e.g. Ephrins

9. 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)

10. 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

11. The Drosophila visual system provides a model of axon growth R2 R1 R5 R4 R3 R8 R6 R7 Ttk69 Prospero Phalloidin

12. MARCM enables precise genetic manipulation of R7 cells MARCM: mosaic analysis with a repressible cell marker

13. MARCM enables precise genetic manipulation of R7 cells MARCM: mosaic analysis with a repressible cell marker

14. MARCM enables precise genetic manipulation of R7 cells MARCM: mosaic analysis with a repressible cell marker

15. Tramtrack69 is necessary to restrict axon growth

16. Misexpression of Tramtrack69 is sufficient to terminate axon growth WT UAS-ttk69 48 hr APF 24B10 CD8-GFP

17. Misexpression of Tramtrack69 is sufficient to terminate axon growth WT UAS-ttk69 48 hr APF 24B10 CD8-GFP What does Tramtrack69 do on a molecular level to restrict axon growth?

18. Tramtrack69 is a transcription factor Transcription Ttk69 Ttk69 is a transcriptional repressor RNA product No transcription

19. Tramtrack69 is a transcription factor Ttk69 Ttk69 is a transcriptional repressor What genes is Ttk69 regulating in order to inhibit axon growth? Transcription RNA product No transcription

20. TGF-β/Activin signaling pathway is necessary to inhibit axon growth dSmad2 Activin Baboon Ting C et al. Neuron 56 (2007) Axon growth

21. Tramtrack69 acts upstream of Baboon to restrict axon growth Nuclear FLAG-dSmad2 (fold enriched) 2 1.5 1 WT ttk69 n = 10 dSmad2 Activin Baboon Axon growth dSmad2 Activin Baboon Axon growth Wild type ttk69

22. Baboon Activin dSmad2 Antagonist Axon growth Hypothesis: Tramtrack69 restricts axon growth by repressing an antagonist of Activin signaling Wild type

23. Baboon Activin dSmad2 Antagonist Axon growth Baboon Activin dSmad2 Antagonist Axon growth Hypothesis: Tramtrack69 restricts axon growth by repressing an antagonist of Activin signaling Wild type ttk69

24. Baboon Activin dSmad2 Antagonist Axon growth Baboon Activin dSmad2 Antagonist Axon growth Baboon Activin dSmad2 Antagonist RNAi Axon growth Hypothesis: Tramtrack69 restricts axon growth by repressing an antagonist of Activin signaling Wild type ttk69 ttk69, Antagonist-RNAi

25. Double mutants showed no overgrowth phenotype WT ttk69, Cripto-RNAi ttk69, Follistatin-RNAi ttk69 24B10 SytGFP

26. Double mutants showed no overgrowth phenotype WT ttk69, Cripto-RNAi ttk69, Follistatin-RNAi ttk69 24B10 SytGFP A perfect rescue?...

27. My “ttk69” My “ttk69” chromosome lacked the ttk69 mutation ttk69 My “ttk69,” Follistatin-RNAi 24B10 SytGFP Ttk69 Prospero Phalloidin Ttk69 ttk69

28. Future Directions Follistatin – GFP Recombineering with BACs: bacterial artificial chromosomes 3. If there is a rescue…. 1.Rebuilding ttk1e11 stock and repeating the experiment 1.Exploring the potential role of other activin antagonists in the tramtrack69 pathway

29. Acknowledgments The Herman Lab University of Oregon Institute of Molecular Biology Summer Program for Undergraduate Research Tory Sasha Eric Jen Jon Sarah Dave Erika Mike Peter O’Day Adam Unger NICHD R25 Summer Research Program

30. Acknowledgments The Herman Lab University of Oregon Institute of Molecular Biology Summer Program for Undergraduate Research Tory Sasha Eric Jen Jon Sarah Dave Erika Mike Peter O’Day Adam Unger Questions? NICHD R25 Summer Research Program