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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 Varies in: • Severity of congenital hearing loss • Onset of vision loss • Presence/ absence of balance defects Example of Usher vision loss http://www.retinaeyecenter.com/images/RPSample.jpg Usher Syndrome: The Affected Cell Types cilia Photoreceptor nucleus Hair cell ribbon synapses 3 Molecular Basis of Usher Syndrome 12 known loci, 9 identified genes Transmembrane Proteins Gpr98 Scaffold Proteins after Reiners et al., 2006 Cell Adhesion Proteins Motor Protein 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 5 Other Possible Models? Zebrafish! Accessible, homologous sensory organs Fast development hpf 0 24 48 72 Hair cell specification and maturation Retinal cell specification and maturation 6 5dpf ear 96 Auditory function Visual function 5dpf retina 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 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 Acetylated tubulin Gpr98 localizes at the base of the 3d kinocilium, in the region of the stereocilia ankle links N=5 8 8 Gpr98 Localization Hair cells: Gpr98 Acetylated tubulin 6d Gpr98 only N=5 At 6 dpf, Gpr98 is localized within the kinocilium 9 9 Gpr98 Localization 4d Retina: Gpr98 localizes in the region of the connecting cilium and the photoreceptor synapse 3d AT Gpr98 N=5 synapse synapse CC CC 10 10 Developing zebrafish model of USH2C Morpholino Knockdown Technology 2dpf Morpholinos 4dpf are molecules that modify gene expression Ctl MO Ctl MO Splice blocking morpholino interferes with pre-mRNA splicing Brief efficacy period Normally: Pre-mRNA intron exon mRNA With morpholino: intron intron exon exon exon mRNA intron exon Developing zebrafish model of USH2C Morpholino Knockdown Technology 2dpf Morpholinos 4dpf are molecules that modify gene expression Ctl MO Ctl MO Splice blocking morpholino interferes with pre-mRNA splicing Brief efficacy period Inject gpr98 morpholino into the embryo at the 1 cell stage Comparing controls with gpr98 morpholino-injected larvae Hair cells 2d ctrl 3d ctrl 4d ctrl 2d MO 3d MO 4d MO At 2-4 dpf, zebrafish larvae treated with gpr98 morpholino (MO) have shortened kinocilia 13 13 Comparing controls with gpr98 morpholino-injected larvae Hair cells Phalloidin 5d ctrl 5d MO Zebrafish larvae treated with gpr98 MO have bent stereocilia bundles at 5 dpf 14 14 Conclusions 1. 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. 2. 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. 3. Zebrafish larvae treated with a morpholino targeting gpr98 have shortened kinocilia at 2-4 dpf and bent stereocilia bundles at 5 dpf. • 15 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 Future Directions Immediate: • • • 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: • 16 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 17
