Mentor: Jennifer Phillips, PhD University of Oregon- SPUR Program Investigating the role of gpr98 (ush2c) in zebrafish sensory cell development and function Kelsey Anbuhl Spring Hill College August 11, 2010 Westerfield Lab Mentor: Jennifer Phillips, PhD University of Oregon- SPUR Program

Introduction What is Usher Syndrome? Leading cause of deafblindness Example of Usher vision loss What is Usher Syndrome? Leading cause of deafblindness Varies in: Severity of congenital hearing loss Onset of vision loss Presence/ absence of balance defects http://www.retinaeyecenter.com/images/RPSample.jpg

Usher Syndrome: The Affected Cell Types Hair cell Photoreceptor cilia nucleus ribbon synapses 3

Molecular Basis of Usher Syndrome 12 known loci, 9 identified genes Gpr98 Transmembrane Proteins Cell Adhesion Proteins Scaffold Proteins Motor Protein after Reiners et al., 2006

Models of Human Usher Syndrome Mouse Model Stereocilia organization is perturbed in Usher mutant mice However, accessing the hair cells for analysis is difficult 5

Other Possible Models? Zebrafish! Accessible, homologous sensory organs Fast development 0 24 48 72 96 hpf Hair cell specification and maturation Auditory function Retinal cell specification and maturation Visual function 5dpf retina 5dpf ear 6

Research Project Gpr98 is a very large transmembrane protein Mutations in the GPR98 gene are responsible for usher syndrome type 2c (USH2C) Characterized by: moderate/severe congenital hearing loss onset of vision defects in young adulthood normal balance function Gpr98 PURPOSE: To develop a zebrafish model of USH2C to elucidate the role of Gpr98 in sensory cell development and function. 7

Localization of Gpr98 in hair and retinal cells Breed wild type zebrafish and obtain embryos Euthanize, fix, and section fish at early time points (2-6 days post fertilization) Hair cells: Gpr98 localizes at the base of the kinocilium, in the region of the stereocilia ankle links Gpr98 3d N = 5 Acetylated tubulin 8

At 6 dpf, Gpr98 is localized within the kinocilium Gpr98 Localization Hair cells: 6d Gpr98 Acetylated tubulin N = 5 Gpr98 only At 6 dpf, Gpr98 is localized within the kinocilium 9

Gpr98 Localization 4d 3d Retina: synapse synapse CC CC Gpr98 localizes in the region of the connecting cilium and the photoreceptor synapse 3d Gpr98 AT N = 5 synapse CC CC synapse 10

Morpholino Knockdown Technology Developing zebrafish model of USH2C Morpholino Knockdown Technology Ctl MO 2dpf 4dpf Morpholinos are molecules that modify gene expression Splice blocking morpholino interferes with pre-mRNA splicing Brief efficacy period exon intron Normally: With morpholino: Pre-mRNA mRNA 

Developing zebrafish model of USH2C Morpholino Knockdown Technology Ctl MO 2dpf 4dpf Brief efficacy period Morpholinos are molecules that modify gene expression Splice blocking morpholino interferes with pre-mRNA splicing Inject gpr98 morpholino into the embryo at the 1 cell stage

Comparing controls with gpr98 morpholino-injected larvae Hair cells 3d ctrl 3d MO 2d ctrl 2d MO 4d ctrl 4d MO At 2-4 dpf, zebrafish larvae treated with gpr98 morpholino (MO) have shortened kinocilia 13

5d ctrl 5d MO Comparing controls with gpr98 morpholino-injected larvae Hair cells 5d ctrl Phalloidin 5d MO Zebrafish larvae treated with gpr98 MO have bent stereocilia bundles at 5 dpf 14

Conclusions In zebrafish inner ear, we observed Gpr98 localization at the base of the hair cell kinocilium, in the region of the stereocilia ankle links. We also observed Gpr98 localization in the kinocilium at 6 dpf. In the zebrafish retina, we observed localization of Gpr98 in the region of the connecting cilium and the photoreceptor synapse from 4 to 6 dpf. Zebrafish larvae treated with a morpholino targeting gpr98 have shortened kinocilia at 2-4 dpf and bent stereocilia bundles at 5 dpf. The results indicate that Gpr98 may affect normal development of the kinocilia, which subsequently plays a role in orienting developing stereocilia. Zebrafish Gpr98 may also be required for the formation of the ankle link complex, as seen in mammalian studies. 15

Immediate: Long Term: Future Directions Co-label the hair cells with Phalloidin and Gpr98 Investigating the role of Gpr98 with cargo trafficking Interaction studies with other USH proteins Long Term: Creating an USH2C zebrafish mutant 16

Acknowledgments Westerfield Lab Monte Westerfield Jennifer Phillips Bernardo Blanco Judy Pierce Sabrina Toro Jeremy Wegner SPUR Program- Peter O’Day Blakely Strand 17