01front.pdf - ResearchSpace - The University of Auckland

http://researchspace.auckland.ac.nz

ResearchSpace@Auckland

Copyright Statement

The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand).

This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use:

x Any use you make of these documents or images must be for research or private study purposes only, and you may not make them available to any other person.

x Authors control the copyright of their thesis. You will recognise the author's right to be identified as the author of this thesis, and due acknowledgement will be made to the author where appropriate.

x You will obtain the author's permission before publishing any material from their thesis.

To request permissions please use the Feedback form on our webpage.

http://researchspace.auckland.ac.nz/feedback

General copyright and disclaimer

In addition to the above conditions, authors give their consent for the digital copy of their work to be used subject to the conditions specified on the Library Thesis Consent Form and Deposit Licence.

Note : Masters Theses

The digital copy of a masters thesis is as submitted for examination and contains no corrections. The print copy, usually available in the University Library, may contain corrections made by hand, which have been

requested by the supervisor.

Dystrophin and the Dystrophin Associated Glycoprotein Gomplex

in the Zebrafish, Danio rerio

Stephen Peter Chambers

A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of

Philosophy in Biological Sciences, The University of Auckland, August 2004.

Abstract

Mutations affecting the dystrophin

gene

and the dystrophin

associated

glycoprotein

complex

(DGC)

genes

in

humans cause

chronic

muscle

wasting

diseases

collectively

termed muscular dystrophies

(MDs), often with

associated

cardiomyopathy. The

dystrophin gene

is

associated

with

the allelic,

X-linked

diseases Duchenne and Becker muscular dystrophy

(D/BMD), of

^which

DMD is fatal in early adulthood. The DGC is

associated

with the

autosomal recessive

limb

girdle muscular dystrophies

(LGMDs)

which result from mutations of the human cr-, F-, 6- and y- sarcoglycan

genes.

Dystrophin and the

DGC

genes encode a sarcolemmal complex

of

proteins that forms the structural linkage between the muscle cytoskeleton and extracellular matrix, which

is crucial for the

maintenance

of

muscle

integrity during normal activity. Dystrophin

and the DGC genes are evolutionarily conserved, underpinning the important role of this complex.

In terms of DMD modeling, the

mouse

has

served

as a suitable

vertebrate species

but

the pathophysiology

of the

disease

in the

mouse does

not entirely mimic

human

DMD. LGMD

modeling

is

served

by

a the

BIO

14.6 hamster

which is

a

traditional

model

of

cardiomyopathy, and mouse sarcoglycan gene

null shains.

The zebrafish

is

an established

model of

vertebrate development, and

is receiving

increasing attention

in

terms

of

human disease

modeling.

This thesis sought

to

examine the potential

of

the zebrafish as a further comparative model

for

^these MDs.

In

order

to

provide experimental support

to

realise

this

modeling potential, this thesis describes the

identification of

apparent orthologues

of

many

critical

members

of

the

DGC in

the zebrafish.

An

apparent zebrafish orthologue

of

the human dystrophin gene was

identified

that expresses a

400kDa

protein that is

localised

to

the

myofibre

membrane surface

of adult

zebrafish muscle.

Epitope mapping confirmed that zebrafish dyshophin retains all the

conserved functional domains reported

in the

human

protein. Dystrophin

expression

in the

zebrafish embryo was observed

at six

hours

post fertilisation (hp|,

although maternal transcript was present

in

the

zygote.

Dystrophin protein was localised at the myosepta

of

zebrafish embryos at 24 hours post fertilisation

(hp|.

Apparent zebrafish orthologues

of

human p-dystroglycan

and 9-, 6-,

^6-,

y-

and (-sarcoglycan were also

identified,

and

in

the case

of

B-dystroglycan ^and

p-,

and y-sarcoglycan were shown to

express proteins localised to the myofibre membrane surface

in

adult

zebrafish. Like

dystrophin,

zebrafish

B-dystroglycan

and

B-sarcoglycan genes express

proteins which localised to

the myosepta

of 24hpf

zebrafish embryos. Co-localisation

of

dystrophin

with

B-dystroglycan and F- actin confirmed that the zebrafish carries an intact DGC.

The DGC in the

zebrafish

retina was also

studied

to provide a

basis

for

understanding the

diversity of DGC

expression and

function in

respect

of the pathology of the

human diseases

modelled. A DGC which

incorporates zebrafish utrophin

or

dystrophin

with

dystroglycan, was localised

to the glial cell layer (GCL)

and outer

plexiform layer (OPL) of the adult

zebrafish

retina. During

zebrafish retinal development, dystrophin and dystroglycan were

first

localised

in

the

GCL

at 48 hpf and the

OPL at72hpf.

ln

terms

of

targeting gene expression to achieve disease modeling outcomes,

two

strategies were used

targeting the

carboxyl-terminus

of

zebrafish dystrophin

transcript.

Peptide

nucleic

acid

(PNA)

masking

of

the splice donor site

of

zebrafish dystrophin exon

7l

resulted

in

mis-splicing

of

the transcript

which

conserved the reading frame

of

the

transcript.

Gene silencing

by

short interfering RNAs targeted against exon 53 and exon 68

of

zebrafish dystrophin caused temporary

loss of dystrophin transcript, and

delayed

dystrophin and

B-sarcoglycan

localisation at

the myosepta

of

affected embryos.

Together,

these

data

suggest

that dyshophin and the DGC in

zebrafish

may play a highly

conserved

functional role in

muscle architecture that,

when

disrupted,

could offer insight

into human neuromuscular disease processes.

Key words:

disease

modeling, dystroglycan, dystrophin,

dystrophin-associated glycoprotein complex, sarcoglycans, muscular dystrophy, peptide nucleic acids,

RNA

interference, zebrafish

ill

Acknowledgements

My first

and heart-felt thank

you

goes

to the

closest partners

in this

endeavour, professor Don Love, who managed to keep me going

till

the end, and my partner Lee who

lived

through

it with

me and is

still there.

No less so the many supporters who have helped

in

uncounted ways, but the closest collaboration _and support

in this work

came

from Andrew

Dodd,

I

thank

you too.

This

work would

have been impossible

without

the collaborations

of

Professor Glenn

Morris

and Dr.

Louise

Anderson

who both gifted

antibodies and

to whom I am

deeply

thankful. My

sincere thank

you

_also

to Dr. David

Bissett

for his gift of

utrophin antibodies and

for

discussion

of

his

pre-publication results. Thank you

also

to

Professor

Colin

Green,

Dr. Shiva Reddy

and Dr.

David

Crosssman who all helped this study

with

gifts of reagents and generous advice.

For your

friendly

advice and endless support;

Dr

Shane

Lavery,Dr Adrian

Turner,

Beryl

Davey,

Dr David

_{Saul and}

Dr Terry

Gruijters, thank

you all. For your

special support

with

confocal microscopy,

Hillary Holloway

and Jaqui Ross

of

the Biomedical lmaging Research

Unit,

thank you

too'

Giselle Maguire, Rupert Overall and Tamara Sirey were also part

of

this

work,

thanks

guys' Of

_{course there are}

all

_{the other people}

who

_{keep one's}

life

and sanity _together

by

their friendship, who are so many that

to list

them

all would

endanger the rainforests, so

my

apologies

if I

appear to have missed anyone. Special thanks to Steve F and

Murty;

'.Stevomix,, Corporation and saved

by

the

Lion;

also Bro Rich,

Michelle

and

Flynn;

Steve

p.

_and

family;

^paul _{and Megan,} Richie

M.,

Gus and all their familys; the Mahia,.boys',, _and all the rest

of

^you.

The sequence data used for gene predictions _{were produced} by the Danio rerio Sequencing Group at the Sanger Institute and can be obtained

from

ftp://ftp.sanger.ac.uldpub/zebrafish.

Grants

from

The

University of

Auckland _Research Committee, the

Maurice

and

phyllis

paykel Trust _{and the} Lottery Grants Board of

New

Zealandfinancially supported this work.

IV

Table of contents

1.1

Skeletal rurusele and museular

dystnophies...

.-.,.."..,.,.,,2

Sate.llitc cetls aud muso

A

clueter

sfmuscular

s.".,...,,..,....

1.2

DuchEnne

iirusculu

Clinioal

sxmptoms ^and pathophysio ogy of

DMD....

,!....:.i...i...t.,.r..i...,,10' Skeletal rnuscle danrage

DMD

also affeots the heart, brain and netina

Beckermuscular

dysfiophy.

.'...!q...,...L3

Fenale

Loss of,dystroph,in protoin is assooiated srith DN{D pathology....,....rq...,.r..r,..i..,i....:.,...,....,.,.15 The

stucture-firnction

relationship

in dystrophin

...15 The

dysfrophinprotein

exists as a variety of isoforms...rlr!t!...!....r..'...,i.1.!,r,..d...,..-..,17

l.

The dy.strophin-associated glycoprotein

complex.

...,...,22

fte

sarooglycan-sarOospan oomplex.r....r.,.,r...,...,..,ti.r!.!ir,..r...,r.,,...t.r...!i...ir..r...!.,,...,"....".-.23

1.5

Dyshobrevin and the synhophins

...,.

^...24

Dystrophin related

proteins

...25

Disease Models of

MD...

...26

Animal

models

of dyshophinopathy....

...26

The mdx

mouse...

...,...26

Other

MD

model

species...

^...27

TheZebrafish as a Potential Model of

DMD

...29

Is Another Model

of DMD Required?

...29

Zebrafish Development and

Genetics.

...29

Comparative Muscle Physiology in

Zebrafish...

...30

Are the technology requirements for disease modeling available in the zebrafish?...30

Summary and aims of this

thesis...

...32

The zebrafish as a platform

for

disease

modeling..

...32

2

^{Materials and}

Methods...

...34

2.1 Materials..

...35

2.1.1

Materials and

Suppliers..

...35

Bacterial

sfains

and

p1asmids...

...35

Enzymes and molecular biological

reagents

...35

Antibodies, histochemistry reagents and

stains...

^...35

Zebrafish..

...36

List of suppliers...

...36

2.1.2

Solutions and

Media...

...39

General

solutions...

^...39

Zebrafish solutions, media and

supplements...

...39

Bacterial media, antibiotics and other

additives...

...39 1.6

r.7

VI

Phylogenetic tree

building....

^...48

2.2.3

Histological

Methods.

...49

Cryopreservation and sectioning of

tissue

^...49

Preparation

of

zebrafish embryos and adult eyes for

cryosectioning...

^...49

lmmunohistochemistry of cryosectioned

tissue

...50

Wholemount

immunohistochemistrv.,...

...50

Pre-adsorption

of

secondary

antibodies

...51

Direct labelling

ofprimary antibody...

...51

Scanning confocal

microscopy

^...51

Electron

Microscopy...

...51

2.2.4

Zebrafish

Methods...

...52

Zebrafish

husbandry

...52

Zebrafish

breeding...

...-...53

Construction

of

embryo injection

trays...

...53

Microinjection

needles

...

...54

Preparation of

PNA

for

microinjection

...54

Microinjection

of zebrafish

embryos

...54

3

Dystrophin in

Zebrafish

...55

3.1 Identification

and sequence analysis

of

zebrafish

dystrophin

...56

3.2

Gene tree analysis of zebrafish

dystrophin

...,..61

3.3

Detection of dystrophin protein in

zebrafish

...61

Immunolocalisation of zebrafish dystrophin

protein

^...62

Epitope mapping the zebrafish dystrophin

protein

...64

3.4 Discussion...

...66

vill

4

^{The DGC}

in 2ebrafish...

...68

4.I

Identification and sequence of zebrafish DGC genes

...

...70

Zebrafish EST sequence

ana1ysis...

...70

Prediction

of

sarcoglycan sequences from the zebrafish

genome

^...74

4.2

Gene tree analysis of the zebrafish DGC

...

...77

4.3

Detection of DGC proteins

in

embryonic and adult zebrafish

...

...79

4.4

Dystrophin colocalises with DGC proteins in

zebrafish

...81

4.5 Discussion

...82

Non-muscle dystrophin and DGC in the

2ebrafish...

...84

Molecular diversity of the

DGC...,

...84

Dystrophin and the DGC in the zebrafish

eye...

...87

Zebrafish

utrophin

^...90

The DGC

in

the adult zebrafish

retina

...92

The DGC

in

the developing zebrafish neural

tube...

...96

Discussion

...97 5.1

5.2 5.3

6 6.1

6.2 6.3

IX

7

The zebrafish as a model for r,nuscular dyshophy

Identifieation of a zebrafish dysnopbin

or&ologue ..,.r...!.,...qri ,*.-..I15

The zebrafish

carrissDGC

genes which _{express a} fimctional cornplox that is localised

to

Targsted gene knockdoum of the zebrafish

DGe ..,..,,.

..-...120

List of figures and tables

Figure

1.1

Skeletal muscle is the principar tissue affected by

DMD

...3

Figure

1.2

The myogenic

program

_...6

Table

1.1

Proteins implicated

in

different forms of muscular

dystrophy..

...9

Figure 1.3

Effects of muscular

dystrophy

...1I Figure

1.4 Multiple

transcripts are derived from _the dystrophin

gene...

...1g Figure

1.5

The dystrophin protein (Dp)

variants...

_...19

Figure

1.6

Molecular model of the dystrophin associated glycoprotein complex (DGC)

in humans.

..,,...'...,,...,22

Table

1.2

Vertebrate models of muscular

dystrophy...

...2g Figure

1.7

Myogenesis and muscle anatomy in

zebrafish

_...31

Figure

1.8 Flow

diagram of the components of this

thesis

_...33

Table

2.1

Bacterial strains and

p1asmids...,.

...35

Table

2.2 Erzpes

and molecular biological

reagents

....,...35

Table

2.3

Antibodies and histochemical reagents used in this

thesis...

...36

Table

2.4

General

solutions

...39

Table

2.5

Zebrafrsh solutions, media, and supplements

...

...39

Table

2.6

Bactenal _media, antibiotics _and

additives...

...39

Table

2.7

Histochemistry

solutions.

...39

Table

2.8

Summary

of

EST

clones...

...40

Table

2.9

Oligonucleotides _and their uses

...

...41

Table

2.10

PNAs, and their sequences and molar extinction co-efficients..,... ...42

Figure

3.1

Nucleotide and deduced amino acid sequence

of EST fc33bl2

...,..57 Figure

3'2

Annotated alignment of zebrafish and human dystrophin C-terminal peptide

sequences.

...5g

Figure

3.3

The map location of the zebrafish dystrophin locus on linkage group I ...,...60

Figure

3.4

Radial gene tree showing the dystrophin, utrophin and DRP2 proteins

in

vertebrates and

Drosophila

melanogaster.

...

...61

Figure

3.5

Western blot

of

dystrophin in zebrafish

muscle

...62

Figure

3.6

Immunolocalisation

of

dystrophin

in

adult zebrafish and human muscle...63

Figure

3.7

Immunolocalisation

of

dystrophin

in

the24 hpf to 48 hpf zebrafish embryo ...64

Figure

3.8

Epitope mapping of zebrafish

dystrophin

..,...,...65

Figure

4.1

Nucleotide and deduced amino acid sequence of EST

fb83d01

...71

Figure

4.2

Nucleotide and deduced amino acid sequence of EST

fc49ell

...72

Figure

4.3

Nucleotide and deduced amino acid sequence of EST

fi50e09...

...73

Figure

4.4

Annotated amino acid sequence alignment

of

zebrafish and human dystroglycan ...74

Table

4.1

Summary of dystroglycan and sarcoglycan sequences used

in

gene tree construction 75 Figure

4.5

Annotated amino acid sequence alignment of zebrafish and human sarcoglycans...76

Figure

4.6

Radial gene tree analysis dystroglycan proteins in vertebrates, Drosophila melanogaster and Caenorhabditis

elegans

...77

Figure

4.7

Radial gene tree analysis of sarcoglycan proteins

in

vertebrates, Drosophila melanogaster and Caenorhabditis

elegan^r...

^...78

Figure

4.8

Immunolocalisation of components of the DGC in adult zebrafish and hrunan muscle. ...80

Figure

4.9

Wholemount immunolocalisation of components of the DGC in 24 hpf zebrafish

embryos.

...81

Figure

4.10 Colocalisation

of dystrophin and the DGC

in

zebrafish

embryos

...82

Figure

5.1 Diversity

of the DGC in muscle and non-muscle tissues

...

...86

Figure

5.2

Architecture of the adult zebrafish

retina

...88

Figure

5.3

Development of the zebrafish

retina...

...89

Figure

5.4 Amino

acid sequence alignment of zebrafish and human

utrophin.

...91

Figure

5.5

Immunolocalisation of the DGC in the adurt zebrafish

eye...

...93 Figure

5'6

Time course of dystrophin and DGC localisation in the eye of zebrafish embryos....95 Figure

5.7

Dystrophin and the DGC

in

96

hour

and,7 day zebrafish

embryos

...96 Figure

6.1

Conceptual scheme of

pNA

induced mis_splicing

...,..

...101 Figure

6.2

Predicted zebrafish dystrophin intron:exon boundaries and

pNA

_binding sites ...102 Figure

6'3

Analysis of dystrophin intron:exon boundary sequences in the zebrafish genome...l03 Figure

6.4

RT-PCR assay to analyse splicing

events...

_...104

Figure

6.5

RT-PCR amplification of dystrophin exons

70-72in

pooled embryos by

RT-pCR.105

Figure

6'6 PNA

induced mis-splicing

of

exon 71 of the zebrafish dyshophin gene

transcript..l06

Figure

6.7

Confinnation of reaction conditions _for RT-PCR amplification of exon

s

51-54of the

zebrafish dystrophin gene transcript

...

...107 Figure

6.8

Analysis of

PNA

2410 injections targeting exon 53 of the zebrafish dystrophin gene

transcript..

...10g

Figure

6.9

Analysis of

PNA 24ll

injections targeting exon 53 of the zebrafish dystrophin gene ...108 Figure 6.

l0

Histological _analysis of dystrophin gene targeting

in

the

zebrafish

... I I ¹

xill

List of abbreviations

The most frequently used abbreviations in this

work

include:

2MeAO

2'-O-Methyl-antisense oligonucleotides

AAV

adeno associated virus

AmP ampicillin

AP

anterior_posterior (axis)

.A.TP

adenosine triphosphate

bP

base pairs

BMD

Becker muscular dystrophy

BSA

bovine _serum albumin

Ca*

calcium

ion

CK

creatine kinase

CMD

congenital muscular dystrophy

DGC

dyshophin associated glycoprotein complex

DMD

Duchenne muscular dystrophy

DMSO

dimethyl _sulphoxide

DNA

deoxyribose nucleic acid

dNTP

deoxynucleoside triphosphates

dPf

days post

fertilisation

DRPI

dyshophin-related protein

I

^(utrophin)

DRP2

dystrophin-related protein 2

DTT dithiothreitol

E.

coli

Escherichia

coli

EST

expressed sequence tag

EF

embryonic _fissure

g

gram

GCL glial

cell layer

GZ

germinal _zone

hpf

hours post fertili^sation

IgG

immunoglobulin _{G (also G1,}

G1,

etc)

ILM

inner

limiting

membrane

INL

inner nuclear layer

IPL innerplexifonn

layer

kb

L LGMD M MD

mg

ml rnM NEWI

ng

nl OFL oligo ONL

OPL PAGE PE PBS PCR PEG

pH PNA

Q-PCR RNA.

rpm RT-PCR

siRNA

SDS SGCA SGCB SGCD SGCE SGCG SGCZ

kilobases

lihes

limb girdle _muscular dystrophy

molar

muscular dystrophy

milligram

millilitre millimolar

North East Wales Institute nanografir

nanolitre

optic fibre layer oligonucleotide outer nuclear layer outer

plexiform

layer

ployacrylamide gel electrophoresis pigmented epithelium

phosphate buffered saline polymerase chain reaction polyethylene

glycol

potantial _hydrogen peptide nucleic acid quantitative PCR ribose nucleic _acid revolutions _per minute reverse hanscription

pCR

short

interferingRNA

sodium dodecyl sulphate a-sarcoglycan

p-sarcoglycan 6-sarcoglyoan e-sarcoglycan y-sarcoglycan (-sarcoglycan

TAE

Tris-acetate-EDTA

TBE

Tris-borate-EDTA

Tween

20

polyoxyethylene sorbitan monolaureate

Tet

tetracvcline

tlg

^microgram

PL microlitre

Pm

micrometre (micron)

PM

^micromolar

vlv

volume _by volume

wlv

weight by volume

XVi