1. Understanding deafness in the Usher syndrome: A possible interaction between Grxcr1 and the zebrafish Usher complex Jennifer M. Panlilio Westerfield Lab

2. Roadmap I. Introduction to the Usher Syndrome and mechanosensation II. Grxcr1 as a possible protein partner of the Usher complex III. Results: Characterization of Grxcr1 depletion phenotype in zebrafish V. Future Directions

3. Overview of Usher Syndrome Most common form of hereditary deafblindness in humans Symptoms include issues with hearing, balance and vision Three clinical subtypes: Usher Type IUsher Type II Usher Type III Congenital hearing loss and severe deafness Severe balance defects Early onset retinitis pigmentosa Congenital hearing loss, less severe deafness Late onset of retinitis pigmentosa Progressive hearing loss Variable balance defects Late onset of retinitis pigmentosa

4. How Hearing Works: A look into the inner ear hair cells Sound Mechanoreceptor Stereocilia +Kinocilium Cell body Nerve fibers Actin Nicolson, Ann. Rev. Genet., 2005

5. GeneProtein USH 1BMyosin VIIa USH 1CHarmonin USH 1DCadherin 23 USH 1FProtocadherin 15 USH 1GSans USH 2AUsherin USH 2CVLGR1b USH 2DWhirlin USH 3Clarin-1 Usher protein complex: Pan and Zhang, Physiology,2011 Protein Myosin VIIa Harmonin Cadherin 23 Protocadherin 15 Sans Usherin VLGR1b Whirlin Clarin-1 Hars-1

6. Finding proteins interacting with the Usher protein complex: How are the Usher proteins transported? How is the complex is assembled? Pan and Zhang, Physiology,2011

7. Grxcr1 as a candidate Glutaredoxin cysteine-rich 1 in Inner ear of mouse in p5 Frolenkov et al. Nature Reviews, 2004 Odeh et al., Journal of Human Genetics,2010 Grxcr1: green Actin: red Grxcr1 is present in the stereocilia

8. Karolyi et al. Human Molecular Genetics, 2003 There are phenotypic similarities in the inner ear between Grxcr1 mutant and the Usher mutants Grxcr1 mouse mutantWild type mouse Usher mutant (Myo7a) mouse Grxcr1 as a candidate

9. Strategy Characterization of grxcr1 expression pattern Depletion of Grxcr1 to determine whether it is required in mechanoreceptor development Effects of depletion of Grxcr1 on the Usher protein complex

10. grxcr1 expression pattern Where is grxcr1 expressed? grxcr1 is expressed in the ears, eyes, brain and liver of 5 dpf larvae Brain Eyes Inner Ear Liver

11. Loss of Grxcr1 function Exon 1 2 3 4 2 3 4 Wt mRNA Pre-mRNA Splice blocker MO1-grxcr1 MO1-grxcr1 protein Wt protein Exon 1 2 3 4 MO1-grxcr1 mRNA

12. Loss of Grxcr1 function Splice blocker MO1-grxcr1

13. Exon 1 3 4 2 3 4 Wt mRNA 2 pre-RNA Exon 12 3 4MO2-grxcr1 mRNA Loss of Grxcr1 function Splice blocker MO2-grxcr1 MO2-grxcr1 protein Wt protein

14. Loss of Grxcr1 function Splice blocker MO2-grxcr1 Non injected MO-2 grxcr1

15. Defects in mechanoreceptor development following Grxcr1 depletion Wild type MO2-grxcr1 MO2-grxcr1 Actin Staining Anterior macula- sensory patch Hair cell supporting cell Nicolson, Ann. Rev. Genet., 2005

16. GeneProtein USH 1BMyosin VIIa USH 1CHarmonin USH 1DCadherin 23 USH 1FProtocadherin 15 USH 1GSans USH 2AUsherin USH 2CVLGR1b USH 2DWhirlin USH 3Clarin-1 Usher protein complex: Pan and Zhang, Physiology,2011 Protein Myosin VIIa Harmonin Cadherin 23 Protocadherin 15 Sans Usherin VLGR1b Whirlin Clarin-1 Hars-1

17. Depletion of Grxcr1 on the Usher complex in 5 dpf larvae Myo 7aa Myo7aa seems to be less concentrated apically in morphants Harmonin Harmonin seems to be depleted in morphants Wild type MO2- grxcr1 \

18. Conclusions grxcr1 is expressed in the eyes and inner ear like usher genes Grxcr1 may play a role in mechanoreceptor development Grxcr1 effects the localization of some Usher proteins

19. Future Directions How does Grxcr1 affect the Usher complex? -via study of changes of localization with other known Usher proteins Does Grxcr1 interact with the complex? -via Colocalization -via Coimmunoprecipitation

20. Acknowledgements Monte Westerfield Aurélie Clément Berde Blanco-Sanchez Judy Peirce Jeremy Wegner Sabrina Toro Jennifer Phillips Rachel Rae Drake Marcel Rockwell Peter O’Day Adam Unger NIHCD