1. Rachel Drake, University of Minnesota Jennifer Phillips, University of Oregon Westerfield Lab M. Pyron, 2003 1

2. Overview I.Introduction to Usher syndrome II. Experimental Approach III. Results IV. Future Directions 2

3. Autosomal recessive syndrome Causes deafblindness Clinically variable presentation Usher syndrome 3

4. Usher type IUsher type IIUsher type III Visual Impairment Onset in early childhood Onset in late childhood Variable Hearing Impairment Congenital ProfoundCongenital Moderate/ Severe Progressive Variable Balance Defect PresentAbsentVariable More Severe Less Severe 4

5.  Progressive photoreceptor degeneration  Congenital moderate hearing loss  Caused by mutation of the USH2A gene which encodes the protein Usherin  Most common subtype of Usher syndrome 5

6. 6

7. Adato et. Al, 2005 Human Molecular Genetics Thrombospondin-type laminin G domain Transmembrane domain PDZ – domain binding motif 10205030406070 7

8. Eye: Photoreceptors Ear: Hair cells 1. Nascone, 2007 2. J. Phillips 1 2 8dpf 5dpf 8

9. http://webvision.med.utah.edu http://what-when-how.com/acp-medicine/the-dizzy-patient-part-1 Retina Hair cells 9

10. I. Introduction to Usher syndrome II. Experimental Approach III. Results IV. Future Directions 10

11. 11 Thrombospondin-type laminin G domain Transmembrane domain PDZ – domain binding motif Exon 4 Exon 69 introns AAAAA Unprocessed mRNA transcript:

12. C. Rouzer, 2010 GCCATAGCTAGCAATCGA ATCG Exon 69Intron 69 Example Morpholino injection: 12

13. 2d WT 6d WT3d WT6d E693d E69 2d E69 Ladder Results: E4 and E69 Morpholinos appear to specifically alter ush2a transcripts Jen Phillips, Jeremy Wegner E4 morphant fragments E69 Morphant fragments 8d WT 8d E4 1Kb Ladder 500 bp 13

14. Usherin is present in hair cells of uninjected fish through 5dpf 14 2dpf4dpf ac tub usherin 3dpf 5dpf ac tub usherin

15. Usherin is reduced in E69 Morphant Hair cells through 4 dpf 3dpf 4dpf 2dpf Wild-TypeE69 Morphant 15 ac tub usherin ac tub usherin

16. is present in retina of uninjected fish through 4dpf 16 2 dpf 3 dpf 4 dpf ac tub usherin

17. Disruption of splicing at exon 4 causes depletion of in photoreceptors at 5 dpf: 17 ac tub usherin Wild-typeE4 morphant

18. is depleted in retina of E69 morphant through 4dpf 18 E69 morphantWild-type 2 dpf 3 dpf 4 dpf ac tub usherin

19. 8 dpf, E4 morphant Previously shown: Average Number of Labeled Photoreceptors in E4 morphant at 5dpf Uninjected Control.05±0.22 ush2aMO (E4)2.75±1.3 *** p<0.0001 Eberman et. al 2010 19 Caspase-3

20. 20 p<0.0001

21.  Conclusions  E4 and E69 morpholinos appear to specifically alter ush2a transcript 21  E4 and E69 morpholinos achieve a similar level of protein depletion in retina and hair cells.  Photoreceptor cell death in morphants appears to phenocopy the retinal degeneration of Usher syndrome.

22. I.Introduction to Usher syndrome II. Experimental Approach III. Results IV. Future Directions 22

23. Future Directions Determine exactly how the transcript is altered. 23

24. Ush2aUsh2cUsh2d Ac tub 24 Ush2db Ush2c Ush2da Ush2a Determine interdependence of Usher type II proteins:

25. 25 Ush2d Ush 2c Ush 2a ?

26. 26 Jennifer Phillips Monte Westerfield Sabrina Toro Jeremy Wegner Judy Peirce Aurélie Clément Bernardo Blanco Peter O’Day Adam Unger Jennifer Panlilio Nicole Villegas Dylan Calame Marcel Rockwell NIH-1R25HD070817 NICHD Summer Research Program at the University of Oregon NICHD