1

An investigation of functional regions of the Zebrafish ortholog of human Usher

syndrome gene USH2A

Rachel Drake, University of MinnesotaJennifer Phillips, University of Oregon

Westerfield Lab

M. Pyron, 2003

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Overview

I. Introduction to Usher syndrome

II. Experimental Approach

III. Results

IV. Future Directions

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•Autosomal recessive syndrome

•Causes deafblindness

•Clinically variable presentation

Usher syndrome

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Usher type I Usher type II Usher type III

Visual Impairment

Onset in early childhood

Onset in late childhood

Variable

Hearing Impairment

Congenital Profound Congenital Moderate/ Severe

Progressive Variable

Balance Defect

Present Absent Variable

More Severe Less Severe

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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

Usher syndrome Type IIA (USH2A)

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What domains of the Usherin protein are essential for proper hearing and vision?

How does knockdown of ush2a affect Usherin localization in sensory cells?

Usherin is a large protein with multiple functional domains

Adato et. Al, 2005Human Molecular Genetics

Thrombospondin-type laminin G domain

Transmembrane domain

PDZ – domain binding motif

10 20 5030 40 60 70

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ush2a is expressed in zebrafish sensory cells

Eye: PhotoreceptorsEar: Hair cells

1. Nascone, 20072. J. Phillips

1

2

8dpf

5dpf

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Human Sensory Cell Anatomy

http://webvision.med.utah.eduhttp://what-when-how.com/acp-medicine/the-dizzy-patient-part-1

RetinaHair cells

9

10

I. Introduction to Usher syndrome

II. Experimental Approach

III. Results

IV. Future Directions

11

What domains of the Usherin protein are essential for proper hearing and vision?

How does knockdown of ush2a affect Usherin localization in sensory cells?

Thrombospondin-type laminin G domain

Transmembrane domain

PDZ – domain binding motif

Exon 4 Exon

69

introns introns

AAAA

AUnprocessed mRNA transcript:

Experimental Approach

C. Rouzer, 2010

GCCATAGCTAGCAATCGA

ATCG

Exon 69 Intron 69

Example Morpholino injection:

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2d W

T

6d

WT

3d

WT

6d

E69

3d

E69

2d

E69

Ladder

Results: E4 and E69 Morpholinos appear to specifically alter ush2a transcripts

Jen Phillips, Jeremy Wegner

E4 morphant fragmentsE69 Morphant fragments

8d W

T

8d E

41Kb L

adder

500 bp

500 bp

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Usherin is present in hair cells of uninjected fish through 5dpf

2dpf 4dpf

ac tubusherin

3dpf

5dpf

ac tubusherin

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Usherin is reduced in E69 Morphant Hair cells through 4 dpf

3dpf

4dpf

2dpf

Wild-Type E69 Morphantac tubusherin

ac tubusherin

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is present in retina of uninjected fish through 4dpf

Usherin

2 dpf 3 dpf4 dpf

ac tubusherin

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Disruption of splicing at exon 4 causes depletion of in photoreceptors at 5 dpf:Usherin

ac tubusherin

Wild-type E4 morphant

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is depleted in retina of E69 morphant through 4dpfUsherinE69 morphantWild-type

2 dpf

3 dpf

4 dpf

ac tubusherin

Morphant fish exhibit significant increase in cell death

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

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Caspase-3

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3dpf 4dpf 5dpf0

1

2

3

4

E69 morphants exhibit significant increase in cell death

Uninjected controlE69 Morphant

Ce

ll D

eath

(la

be

led

ph

oto

rece

pto

r m

ean

)

p<0.0001

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ConclusionsE4 and E69 morpholinos appear to specifically

alter ush2a transcript

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.

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I. Introduction to Usher syndrome

II. Experimental Approach

III. Results

IV. Future Directions

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Future Directions

Determine exactly how the transcript is altered.

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Ush2a Ush2c Ush2d

Ac tub

Ush2dbUsh2cUsh2daUsh2a

Determine interdependence of Usher type II proteins:

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Ush2d

Ush2c

Ush2a

?

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AcknowledgementsJennifer Phillips

Monte WesterfieldSabrina ToroJeremy WegnerJudy Peirce Aurélie ClémentBernardo Blanco

Peter O’Day Adam Unger

Jennifer PanlilioNicole VillegasDylan CalameMarcel Rockwell

NIH-1R25HD070817NICHD Summer Research Program at the University of Oregon

NICHD