1. 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

2. 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

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

4. 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

5. 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

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

7. 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

8. 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

9. 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

10. 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

11. 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 

12. 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

13. 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

14. 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

15. 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

16. 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

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