The role of bone morphogenetic protein-4 in the developing chick eye

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TH E ROLE OF BONE MORPHOGENETIC PROTEIN-4 IN

THE DEVELOPING CHICK EYE

LI M ING LEONG

THESIS SUBM ITTED TO TH E UNIVERSITY O F LONDON FOR TH E D EG REE OF Ph.D

Department of Biochemistry and M olecular Biology University College London

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A B S T R A C T

B o n e morphogenetic protein-4 (Bm p-4) is a member of the Transform ing

Growth Factor-6 (TGF-6) superfamily of secreted signalling molecules. In this w ork, I

have investigated its spatiotemporal distribution and possible function in the developing

chick eye. The chick homologue of B m p -4 is expressed throughout the developing

chicken optic vesicle between Ham burger and Hamilton stages 13 and 17. It then

show s a distinctive restriction to the dorsal quadrant o f the eye until stage 30, w hen

expression begins to fade, becom ing undetectable by stage 35. Transcripts o f the

homeobox-containing gene G H 6 show a similar dorsal restriction to B m p -4 , but

between stages 20-35. Overexpression of Bm p-4 in vivo results in no detectable change

in eye m orphology, but enlarges the expression dom ain o f GH6, indicating that the two

genes may form part of a gene cascade involved in patterning the eye. In vitro, BM P-4

appears to stimulate retinal cell proliferation. It has no effect on retinal cell

differentiation in vitro. In the chick, the patterning activities o f sonic hedgehog (shh)

are know n to involve activation of B m p expression in at least two instances; the

developm ent of the limb and midgut. I report here that shh is not expressed in the chick

eye at all during development, but is found in the ventral portion of the diencephalon

(forebrain).

The available data suggests a number o f roles for BMP-4 in chick eye

development. BM P-4 may specify dorso-ventral retinal polarity required fo r proper

retinal axon pathfinding, or establish a dorso-ventral gradient o f cell proliferation in the

developing optic cup. Alternatively, it may act as a hedgehog family member antagonist

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TABLE OF CONTENTS

TITLE PAGE

Page number

1

A B S T R A C T

C O N T E N T S

LIST OF ABBR EV IA TIO N S

LIST OF FIGURES

7

11

LIST OF TABLES

A C K N O W L ED G E M EN T S

15

16

CH APTER 1 - INTRO DUCTIO N AND AIM S 18

1.1 The Bone M orphogenetic Proteins 19

1.1.1 H ISTO RY AND RO LE IN BONE FO RM A TIO N 20

1.1.2 FA M ILIES W ITH IN T H E TO F^ SU PERFA M ILY

-A N OVERVIEW 21

1.1.2.1 The T G F-p subfamily 23

1.1.2.2 The A ctivin subfam ily 23

1.1.2.3The D VR group 23

1.1.2.4 O ther m ore divergent genes 26

1.1.3 STR U C TU R E OF TH E BM PS 27

1.1.4 BM PS IN B O N E FO R M A TIO N 31

1.1.5 BM PS AS SIGN A LLIN G M OLECULES IN D EV ELO PM EN T 34

1.1.5.1 BM P-1 34

1.1.5.2 B M P-2 36

1.1.5.3 BM P-3 38

1.1.5.4 B M P-4 38

1.1.5.5 BM P-5 43

1.1.5.6 BM P-6 43

1.1.5.7 BM P-7 44

1.1.5.8 BM P-8, 9 and 10 45

1.1.6 T H E BM P RECEPTO RS 45

1.1.7 D O W N STR EA M CO M PO N ENTS OF BM P SIGN A LLIN G 50

1.2 Eye D evelopm ent 50

1.2.1 EA RLY EV EN TS IN M ORPHOGENESIS O F TH E AVIAN

EYE 51

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1.2.2.1 M olecules that affect early phases of eve induction 1.2.2.2 M olecules that affect lens and retina induction 1.2.2.3 Patterning within the developing eve

1.2.2.3.1 The N aso-tem poral axis 1.2.2.3.2 The D orso-ventral axis 1.2.2.3.3 The Lam inar A xis 1.2.2.4 Cell death and patterning 1.2.3 CLUES FRO M D ROSO PHILA

1.2.3.1 hh and shh

1.2.3.2 dpp and B m p -21 B m p -4

1.2.3.3 H om eobox genes

57 62 64 65 70 74 77 77 79 81 82

1.3 A im s 85

C H A P T E R 2 - M A T E R IA L S A N D M E T H O D S

88

2.1 M aterials

2.1.1 EM BRY O S

2.1.2 B A C TER IA L STRAINS 2.1.3 PLA SM ID S

2.1.4 CELL CU LTU RE REA G EN TS 2.1.5 EN ZY M ES

2.1.6 RA DIOCHEM ICALS 2.1.7 SOLUTION S AND M EDIA

2.1.7.1 General M olecular Biologv 2.1.7.2 Plasm id Preparation

2.1.7.3 In-Situ Hybridisation

2.1.7.4 Bacterial Culture Media 2.1.7.5 Cell Culture

2.1.7.6 Im m unofluorescence assay 2.1.7.7 Solutions For Retroviral W ork 2.1.8 M ISC ELLA N EO U S

88

89 89 89 90 90 90 90 91 91 91 92 92

2 .2 M olecular Biology Techniques 92

2.2.1 RESTRICTIO N EN ZY M E DIGESTION 92

2.2.2 A G A R O SE GEL ELECTROPHORESIS 93

2.2.3 LARGE SCA LE PLAM ID PREPARATION 94

2.2.4 SM ALL SCA LE PLAM ID PREPARATION 96

2.2.5 TRA N SFO RM A TIO N OF E .C O LI W ITH PLASM ID D N A 97

2 .2.6 IN -S IT U HYBRIDISATION TO SECTIONS 98

2.2.6.1 Tissue and section preparation 98

2.2.6.2 Preparation of Slides and Sections 98

2.2.6.3 Probe Preparation 99

2.2.6.4 Pretreatm ent 100

2.2.6.5 H ybridisation 101

2.2.6.6 Post-hybridisation W ashes 102

2.2.6.7 D ipping, Exposure, Developm ent and Staining 102

2.2.7 IN -S IT U HYBRIDISATION TO W HOLE M OUNTS 103

2.2.7.1 Tissue preparation 103

2.2.7.2 Probe preparation 103

2.2.7.3 Pretreatm ent 104

2.2.1 A H ybridisation 104

2.2.7.5 W ashes and antibody reaction 105

2.2.7.6 C olour Reaction 105

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2.3 C e ll C u ltu r e T e c h n iq u e s 106

2.3.1 EM B R Y O N IC DISSECTION 107

2.3.2 C U L T U R E OF RETINA L TISSUE 107

2.3.3 RADIO A CTIV E CELL PROLIFERATION A SSA Y 108

2.3.4 A N TIBO D Y DETECTION OF M ATURE NEURONS 108

2.4 R e t r o v i r a l W o r k 109

2.4.1 C U LTU R E OF AVIAN FIBROBLASTS 109

2.4.2 FIB R O B LA ST TRANSFECTION 111

2.4.3 CO N CENTRATION OF VIRAL SU PERN A TEN T 112

2.4.4 GAG STAINING TO ESTIM ATE V IRA L SPREA D 112

2.4.5 T IT E R IN G OF VIRUS 113

2.4.6 IN JE C TIO N OF VIRAL SUPERNATENT 113

2.4.7 G R A FTIN G OF TRANSFECTED FIBRO B LA STS 114

2.5 P h o to m ic r o s c o p y 115

C H A P T E R 3 - B M P -4 AND R E L A T E D S IG N A L L IN G

F A C T O R S IN T H E E Y E 117

3.1 I n tr o d u c t i o n 118

3.2 D is tr ib u tio n O f B m p -4 tr a n s c r ip ts in th e

d e v e lo p in g c h ic k eye 121

3.3 D is tr ib u tio n O f G h6 tr a n s c r ip ts I n th e

3.4 D is tr ib u tio n o f S h h tr a n s c r ip ts in th e

3 .5 D is c u s s io n 143

C H A P T E R 4 - E F F E C T O F B M P -4 O N C H IC K

R E T I N A L C E L L S IN C U L T U R E 157

4.1 I n tr o d u c t i o n 157

4.2 R e s u lts 160

4.2.1R E T IN A L CELL CULTURES 160

4.2.2 T H E EFFECTS OF BM P-4 AND BM P-2 ON C H IC K

R ETIN A L CELL CULTURES 161

4.2.2.1 Effects of BM P-4 on cell number 164

4.2.2.2 Cell proliferation experiments 170

4.2.2.3 Effects on retinal cell differentiation 186

4.2.3 CO M PA RISO N OF THE EFFECTS O F V ARIO U S G RO W TH FACTORS WITH THAT OF BM P-4 ON TH E

G R O W TH O F CH ICK RETINAL CELS IN CU LTU RE 197

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4.2.3.2 Cell proliferation experiments 220

4.2.3.3 Effects on retinal cell differentiation 220

4 .3 D is c u s s io n 237

4.3.1 G ROW TH OF CH ICK RETINA L CELLS IN CU LTU RE 237

4.3.2 RETINAL CELL REPONSE TO BM P-4 237

4.3.3 DOSE RESPONSE TO BM P-4 SEN SE V IRU S 241

4.3.4 A COM PARISON OF THE EFFECTS OF BM P-2

PROTEIN AND BM P-4 SENSE VIRUS ON R ETIN A L CELLS 243

4.3.5 M ITOGENIC EFFECTS OF BFGF. T G F -b l A N D EO F 244

4.3.6 IN SUM M ARY 248

C H A P T E R 5 - E F F E C T O F B M P -4 O N T H E C H I C K

E Y E I N - V I V O 250

5.1 I n tr o d u c t io n 251

5 .2 R e s u lts 255

5.2.1 USE OF RCASBPfAl VECTORS TO IN FEC T

C H ICK RETINA 255

5.2.1.1 Injections/Grafts into Embryos betw een stages 9-13 258

5.2.1.2 Iniection/Grafts into Embryos between stages 14-20 260

5.2.2 EFFECTS OF IM PLANTING BEADS C O N TA IN IN G

BM P-2 263

5.3 D is c u s s io n 274

5.3.1 RETROVIRAL DATA 274

5.3.2 EFFECTS OF BM P-2 PROTEIN 276

5.3.3 IN SUM M ARY 278

C H A P T E R 6 - D IS C U S S IO N 280

6.1 G e n e r a l D isc u ssio n 281

6.1.1 NON-POSITIO N AL M OLECULES EX PRESSED IN

GRADIENTS ACROSS THE RETINA 282

6.1.2 "SPERRY"-TYPE POSITIONAL M OLECU LES 284

6.1.2.1 “Sperry m olecules” and positional inform ation 284

6.1.2.2 Downstream candidates for BM P-4 positiional

specification 285

6.1.2.2.1The TOP antigens 286

6.1.2.2.2 Eph receptor tyrosine kinases 287

6.1.2.2.3 The 33kD protein 287

6.1.2.2.4 Cell adhesion molecules 288

6.1.3 M OLECULES TH A T INFLUENCE RETIN A L PO LA RITY 291

6.1.4 TH E IN TERACTION OF BMP-4 W ITH S H H A N D G H 6 294

6 .2 C o n c lu s io n s 296

6.3 F u tu r e W o r k 297

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7

LIST OF A BBR EV IA TIO N S

ADMP: AER: aFGF: AHD: ALK: ALSV: ALV: AMY: anti-BM P-4/RCAS: A/P: bFG F: BMP: BM P-4/RCAS: Brk: bp: C-terminal: CAM: CEF: CFU: CMS: CRABP: CRBP: DABCO: dhh: dig:

anti-dorsalising-m orphogenetic protein

apical ectodermal ridge

acidic FG F

aldehyde dehydrogenase

activin receptor-like kinase

avian leukosis and sarcoma virus

avian leukosis virus

avian m yeloblastosis virus

RCA SBP(A ) vector containing the m ouse Bm p-4 in the

antisense orientation

anterior-posterior

basic fibroblast growth factor

bone m orphogenetic protein

RCA SBP(A ) vector containing the m ouse Bm p-4 in the

sense orientation

BMP receptor kinase

base pair

carboxy terminal

cell adhesion molecule

chick em bryonic fibroblast

colony-form ing units

central nervous system

cellular retinoic acid binding protein

cellular retinol binding protein

diazobicyclooctane

desert hedgehog

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8

Dll: distaless

DMEM: Dulbecco's modified eagle medium

DPP: decapentaplegic

DSL: dorsalin

D/V: dorso-ventral

EGF: epidermal growth factor

Egr: early growth response

ems: empty spiracles

ELAV: embryonic lethal abnormal visual system

ELF: Eph ligand family

en: engrailed

ey\ eyeless

FG F: fibroblast growth factor

GDF: growth and differentiation factor

GDNF: glial-derived-neurotrophic factor

Gly: glycine

H BS: H EPES-buffered saline

H E S S : Hank's balanced saline solution

H H: hedgehog

H N F: hepatic nuclear factor

Hox: homeobox containing gene or protein

IG F: insulin-like growth factor

ihh: Indian hedgehog

IL: interleukin

kb: kilobase

LB: Luria-Bertani

Ld: Limb deformity

LTR: long terminal repeat

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MIS: M sx: MAV: N-terminal: NCAM: nrl:

N G F:

OP: otd: Pcix: PBS: PBST: PFA; prd: p.c.: PEG: POU: RA: RAGS: RAR: RCAS: RCASBP: RSV: RTK: RXR: scix: SCW:

Mullerian inhibiting substance

m uscle segm ent hom eobox

myeloblastosis-associated helper virus

amino-terminal

neural cell adhesion molecule

neural retina leucine-zipper

nerve growth factor

osteogenic protein

orthodenticle

paired-box containing

phosphate-buffered saline

phosphate-buffered saline with tween

paraformaldehyde paired box post-coitum polyethylene glycol Pit-Oct-Unc quail retina-specific retinoic acid

repulsive axon guidance signal

retinoic acid receptor

replication competent ALV LTR containing a splice acceptor

replication competent ALV LTR containing a splice acceptor and

p o l gene from Bryan high-titre strain

Rous Sarcom a virus

receptor tyrosine kinase

retinoid X receptor

saxophone

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10

se: short ear mutation

Sen serine

Sey: small eye

shn: schmirri

shs: short-sighted

Six: sine ocidis homeobox containing gene

SO Ho: sensory organ hom eobox

SHH: sonic hedgehog

TBE: Tris-Borate-EDTA

TESPA: 3-aminopropyl trietoxysilane

TFR: transferrin receptor

TGF: transforming growth factor

tkv: thick veins

TRAP: temporal retinal axon protein

twhh: tiggy-winkle hedgehog

UV: ultraviolet

VG: vegetal

Xbra: X enopus brachyiiry

Xbar: Xenopiis dualbar

X enopus forkhead

X h o x : X enopus hox

X vent: X enopus ventral

X m o x : X en opus m esoderm derived hox

WD: W D-40 domain of G-protein

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LIST OF FIG URES

11

Figure P age number

1.1 Structure o f TGF-P superfamily members

1.2 Type I and type II receptors for TGF-P superfamily members

1.3 Schem atic diagrams showing the development o f the chick eye

1.4 Schem atic diagram o f the chick eye at stage 35

1.5 The major axes o f the eye

29

47

52

58

59

3.1 Expression o f B m p-4 and GH6 in frontal head sections as detected by

Francis-West et a/(1994) and Stadler and Solursh (1994)

3.2 Diagram illustrating plane o f sectioning

3.3 B m p-4 expression in stage 13 eyes

3 .4 B m p -4 expression in a stage 15 eye

3.5 B m p -4 expression in a stage 17 eye

3.6 B m p-4 expression in a stage 19-20 eye

3.7 Diagrams illustrating B m p-4 expression in stage 20 and 24 eyes

3 .9 B m p-4 expression in a stage 24 eye (closeup)

2.1Q B m p -4 expression in a stage 2 8 eye

3.1 1 B m p -4 expression in a stage 3 0 eye

3.1 3 G H 6 expression in a stage 17 eye

3 .1 4 G H 6 expression in a stage 20 eye

3.15 G H 6 and B m p-4 expression in a stage 24 eye

3 .16 G H 6 expression in a stage 24 eye (closeup)

3 .17 G H 6 and Brnp-4 expression in a stage 28 eye

3 .18 G H 6 and Brnp-4 expression in a stage 30 eye

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3 .1 9 G H 6 and B m p-4 expression in a stage 35 eye 146

3 .2 0 shh expression in stage 24 forebrain 147

3.21 shh expression in stage 30 forebrain 148

3 .22 shh expression in stage 35 forebrain 149

3.2 3 shh expression in stage 17 forebrain 150

4.1 Neurofilam ent-labelled chick retinal cell culture 162

4.2 Graph sh ow ing changes in cell number over 8 days in culture 163

4.3 Graphs com paring cell number in stage 26 cultures treated with BM P-4/RC A S and controls 165

4.4 Bar charts sh ow ing changes in cell number in stage 26

BM P-4/RC AS treated cultures and controls 167

4.5 Graphs com paring cell number in stage 35 cultures treated with BM P-4/RC A S and controls 171

4.6 Bar charts show ing changes in cell number in stage 35

BM P-4/R C A S treated cultures and controls 173

4.7 Graphs com paring cell proliferation in stage 26 cultures treated with

BM P-2 protein, BM P-4/R C A S and controls 176

4.8 Bar charts show ing changes in cell proliferation in stage 26 cultures treated

with B M P -2 protein, BM P-4/RCA S and controls 178

4.9 Graphs com paring cell proliferation in stage 35 cultures treated with

BM P-2 protein, B M P-4/R C A S and controls 181

4.10 Bar charts show ing changes in cell proliferation in stage 35 cultures treated

with B M P -2 protein, BM P-4/RC A S and controls 183

4.11 Graphs comparing differentiation in stage 26 cultures treated with

BM P-2 protein, BM P-4/R C A S and controls 187

4.12 Bar chart and photographs showing differentiation in stage 26 cultures treated with

BM P-2 protein, BM P-4/R C A S and controls 1 day after treatment 189

4.13 Bar chart show ing differentiation in stage 26 cultures treated with

BM P-2 protein, B M P-4/RC A S and controls 2 days after treatment 192

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BM P-2 protein, BM P-4/RCA S and controls 3 days after treatment 193

4.16 Bar chart and photographs show ing differentiation in stage 26 cultures treated with

4.17 Graphs comparing differentiation in stage 35 cultures treated with

BM P-2 protein, BM P-4/RC AS and controls 201

BM P-2 protein, BM P-4/RC A S and controls immediately after treatment 203

BM P-2 protein, BM P-4/RC A S and controls 1 day after treatment 204

BM P-2 protein, BM P-4/RC A S and controls 2 days after treatment 207

BM P-2 protein, BM P-4/RC AS and controls 3 days after treatment 208

4.24 Graphs comparing changes in cell number in cultures treated

with various growth factors 216

4.25 Graphs comparing changes in cell proliferation in cultures treated

4.26 Graphs comparing changes in differentiation in cultures treated

4.27 Phase contrast and neurofilament-labelled photographs o f cultures

treated with various growth factors 1 day after treatment 229

4.28 Phase contrast and neurofilament-labelled photographs o f cultures

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5.1 Schem atic diagram o f RCASBP proviral genom e 253

5.2 Bar chart depicting percentage mortality o f embryos 3 days after injections/grafts 25 7

5.3 Stage 12 embryos microinjected with BM P-4/RCAS viral

supernatant and stained for virus-positive cells 259

5.4 Stage 24 embryo grafted with BM P-4/RC AS virus-producing

cells and probed for virus-positive cells 261

5.5 Stage 21 embryo grafted with control virus-producing cells and

probed for virus-positive cells 2 6 2

5.6 Stage 25 embryo grafted with BM P-4/RCAS virus-producing

cells and probed for virus-positive cells 2 6 4

5.7 Stage 28 embryo microinjected with BM P-4/RCAS viral

supernatant and stained for virus-positive cells 2 6 5

5.8 Stage 30 embryo microinjected with BM P-4/RCAS viral

5.9 Stage 35 embryos microinjected with BM P-4/RCAS viral

5.10 Stage 28 eye with BM P-2 soaked bead inserted and probed for G H 6 2 7 0

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LIST OF TABLES

Table Page number

1.1 Organisation o f the TGF-P superfamily 22

1.2 R ole o f BM Ps in vertebrate bone formation 33

1.3 Multiple roles o f BM Ps in patterning the developing embryo 35

1.4 Known positional m olecules in the developing retina 66

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A C K N O W L E D G E M E N T S

Firstly, m any thanks to my supervisor, Prof. Paul Brickell, for all his support,

guidance and advice during these three years.

I am grateful also to Prof. Cheryll Tickle of A natom y who not only allowed me

to w ork in her department but also provided wise counsel. Prof. David Latchmann

(M olecular Pathology) and Dr. Benny Chain (Imm unology) are also offered thanks for

kindly allow ing m e to work in their departments during the course of my PhD.

Thanks go out to all the researchers w ho have so kindly contributed reagents,

techniques and technical advice to this thesis. In particular, I would like to thank Dr.

Philippa Francis-W est for all her help with m any diverse aspects of my project and for

her vision and skill. I would also like to acknowledge Dr. Esther Bell, M iss Anne

Cizmeci (photographic techniques). Dr. Jim Cohen (immunocytochemistry), Dr. David

Darling, Dr. Peter M obbs (retinal cell culture). Dr. H erm ann Rohrer, Dr. Eduardo

Seleiro and Dr. Scott Stadler (GH6 DNA).

A s alw ays, thanks to all my friends in various departments of U C L for good

advice and camaraderie; Esther T., Amanda, Anne, Shanie and Nat plus others too

num erous to m ention.

M ost o f all, my deepest gratitude to friends not directly in the field w ho have

had to put up with me throughout these three years. My special thanks to my dearest

friend. M iss M argaret Marchetti, w hose loving support extended to listening to my

ranting diatribes on obscure embryological problem s in num erous London cafes. In a

sim ilar vein, I am grateful to Miss Sophia Prevezanou, M iss Neo Soek Y ing, Dr. H o

Kwok Ki and M iss Lim Meei Shan; thanks all for your patience and understanding - it

was m uch appreciated.

Last and best o f all, I would like to thank my parents for their love, patience and

w isdom. Y ou w ere always with me in spirit if not physically and I couldn't have done

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17

For my parents in loving dedication

"All xue can do in life is try,

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CHAPTER 1 I N T R O D U C T I O N

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- ■ C ^ k ¥ m ^ \ ‘ W m O D Ü G TlO N AND AIMS . ' 19

Over the past few years, great strides have been made in identifying the many

signalling factors that regulate cell-cell interactions in embryogenesis. Among the many

large families o f grow th factor molecules that have been extensively researched is the

Transform ing G row th Factor-13 (TG F-6) superfamily which includes the Bone

M orphogenetic Proteins (BMPs).

These secreted factors have been implicated as regulators of m orphogenesis in

diverse em bryonic systems ranging from mouse skeletal element formation (Kingsley et

al, 1992; K ingsley, 1994a) to epiderm is formation in X enopus gastrula (W ilson and

H em m ati-Brivanlou, 1995) to chick limb patterning (Francis et al, 1994).

This thesis examines the role of one member of the BMP subgroup, B M P-4, in

the development o f the chick eye. I sought to do this by firstly investigating the

distribution of B m p-4 RNA in the developing eye and then looking at the effects of

BM P-4 on retinal cells in vitro and in vivo.

1.1 T he Bone M orphogenetic Proteins

The Bone M orphogenetic Proteins are a group o f at least ten signalling proteins

some o f w hich w ere originally identified by their ability to induce ectopic bone

form ation (Urist, 1965). Because of this functional approach to their identification, one

m em ber of this group is structurally unrelated to the others; BMP-1 has been identified

as Procollagen C-Proteinase (K essler et al, 1996; Reddi, 1996) whereas the other

BM Ps are members of the TG F-6 superfamily and share a closely-related carboxy-

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m k Y m K l -i n t r o d u c t i o n AND AIMS X 20

1.1.1 HISTORY AND ROLE IN BONE FORMATION

The majority of embryonic bone development takes place by the process of

endochondral bone formation which involves the laying dow n of a cartilage matrix

w hich then calcifies. This process can be mimicked by the implantation of

demineralised bovine bone extract in sites that would not normally give rise to bone,

such as rabbit intram uscular pouches (Urist, 1965).

Urist (1965) believed that a proteinaceous agent in the extract was responsible

for its bone-form ing activity and named this agent bone morphogenetic protein (B M P ).

At this time, it w as still unclear as to w hether BMP ability w as due to one or several

interacting com ponents of the extract (W ozney et al, 1988).

The process o f bone formation as mediated by BM Ps takes place as follows. By

day 3 after implantation of the extract, invasion by surrounding mesenchymal cells

takes place. The mesenchymal cells differentiate into chondroblasts and chondrocytes

producing a m ass o f cartilage (a condensation) w hich undergoes proliferation,

hypertrophy and finally calcification (Carrington and Reddi, 1991). At day 10, the

cartilage matrix begins to break down due to the activity of osteoblasts, derived from

mesenchymal cells, and osteoclasts, derived from bone m arrow haematopoietic stem

cells, w hich go on to form bone on the remnants of the cartilage matrix. At the same

time, the m arrow o f the developing bone becomes filled with blood vessels and

haematopoietic cells. The entire process o f ossicle formation is complete by day

21 (Zheng et al, 1994).

Largely because this bioassay of ossification remained the defining criteria for

BM P activity, separation of this activity from the rest of the extract remained

problem atic W ang and coworkers succeeded in extracting BM Ps in guanidine (W ang et

al, 1990). The initial cloning of the first three members of the BMP subgroup, BM Ps -

2, -3 and -4, (W ozney et al, 1988) led shortly to the cloning of another three B M Ps,

BM P-5, -6 and -7 (Celeste et a l , 1990). BM P-8 or Osteogenic Protein-2 w as later

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. r - C H A P T E R 1 - INTRODirCTlOMAm AIMS'': ; ; , / - 2 /

discovered, BM P-9 and BM P-10, has not yet been published, but patents have been

applied for in 1993 and 1994 respectively.

The BMP activities of BM P-1, -2 and -3 were demonstrated by expressing the

proteins in COS cells, showing that implanted cells resulted in cartilage formation

(Luyten et al, 1989). Similarly, Wang et at (1990) show ed that recombinant BM P-2

expressed in Chinese hamster ovary cells was capable of inducing cartilage and bone

form ation. Secreted BMP-4 has also been shown to have osteogenic activity

(H am m onds et al, 1991) as has BMP-5 in cell lines(Baylink et al, 1996).

Another BMP that has been shown to have bone-inducing potential is BM P-7

w hich has been shown to induce differentiation of chondroblasts and osteoblasts in

m urine clonal cell lines (Asahina et al, 1996). The other BM Ps have not been show n to

induce ectopic bone or cartilage, but several are thought to have various functions as

signalling molecules in morphogenesis (see later).

1.1.2 FAM ILIES W ITH IN THE TG F-p SUPERFAM ILY-AN OVERVIEW

All the BM Ps, except BM P-1, are members o f the Transform ing G row th

Factor-p superfamily. This is a large group of molecules (at least 36 members at tim e of

w riting), which are peptide growth factors capable o f regulating m orphogenesis at

m any stages o f embryonic development. Members o f the TG F-P superfamily have

been divided into distinct subgroups according to the sequence homology o f their

mature C-terminal domains (see Table 1.1). The major categories consist of : 1) The

TG F-p subfam ily 2) The Activin subfamily 3) The large dpp-Vg-related (DVR) group

(23)

Members of the TGF-jg superfamily

GROUP

FAMILY

SUBFAMILY

MEMBERS

TGF TGF 1.2,3,4 & 5

Activin Inhibin /3A, B

DVR

<

BMP/OP

<

dpp

60A

Radar

BMP-2, BMP-4 & dpp

BMP 5-8. 60A, UNIVIN. OP-3

Radar, GDF 5-7

V g l, dsl. BMP-9

BMP-3, GDF1, GDF3, nodal, screw, ADMP, BMP-10, GDF-10

Inhibin a, MIS, GDNF

T a b le 1 .1 The organisation of the TG F-6 superfamily into families, subfam ilies and

groups according to sequence identity. M ost o f the groupings are taken from K ingsley

(1994), while the organization o f the B M P /O P family is from Griffith (1996), Radar

(24)

1.1.2.1 The T G F-P subfamily

The first discovered m em ber o f this family, T G F -p i, w as isolated from human

platelets and placenta, and bovine kidney as a 25kD homodimer with a unique N-

terminal sequence. Other closely related isoforms have been found in vertebrates,

namely T G F -(32, p3, p4 and p 5 (reviewed in Sporn and Roberts, 1992; Mosesgf al,

1990). TG F-p4 is thought to be the chick homologue o f m am m alian T G F -pi (Burt and

Paton, 1992).

TG F-Ps have been show n to regulate multiple functions in animal cells,

including cell cycle progression, differentiation, adhesion, migration and extracellular

matrix production (reviewed in Sporn and Roberts, 1992). T G F -p i null mutant mice

suffer defects in yolk sac haematopoiesis and endothelial differentiation w ith those

surviving past birth dying a few weeks later from an inflammatory syndrome (Dickson

et al, 1995; Kulkarni et al, 1993).

1.1.2.2 The Activin subfamilv

A ctivin protein is fromed either by disulphide-linked homodimers of Inhibin pA

or pB subunits or by a heterodim er composed of pA and p B subunit proteins (Mitrani

et al, 1990). The pA and pB subunits can also form dimers with a more distantly

related subunit called Inhibin a (Kingsley, 1994).

The activins are thought to regulate the formation of axial structures in the chick

(M itrani et al, 1990) and mesoderm induction and neurulation in X en opus (N ishim atsu

et al, 1992a) but not in mice (M atzuk et al, 1995).

1.1.2.3 The DVR group

The DVR group is the largest subdivision o f the TG F-P superfamily and

(25)

P-3, G D F-1, GDF-P-3, etc (see Table 1.1). Kingsley (1994) has pointed out that the

grouping is supported by percentage similarity scores but that the borders of the group

are som ew hat blurry.

The BMP family itself can be further broken dow n into three subfamilies, the

dpp fam ily, the 60A family and the newly discovered radar family. Each of these will

be discussed in turn. Members within each subfamily share 12-92% identity, while

m em bers of different subfamilies share only 40-60% identity (Storm et al, 1994).

The dpp subfamily is named after the Drosophila decapentaplegic (dpp ) gene

w hich w as originally identified as a series o f allelic mutations affecting imaginai disc

developm ent (Spencer et al, 1982). This gene regulates several aspects o f Drosophila

developm ent and is discussed in greater detail in section 1.2.3.2.

BM P-2 and BMP-4 are vertebrate homologues o f dpp and are 75% identical to

the D rosophila gene. The fly and vertebrate proteins are able to substitute for each other

on a functional level. A chimeric dpp-Bmp-4 transgene w as able to rescue dorso-ventral

patterning of null dpp mutants flies (Padgett et al, 1993), while dpp and the related

protein 60A (see later) can induce bone formation in rats (Sam path et al, 1993).

Like the dpp subfam ily, the 60A subfamily consists o f one Drosophila gene

and a num ber of vertebrate BM Ps. The original member, 60A, w as isolated as part o f a

search for TG F-p family m em bers in the fruit fly genome (D octor et al, 1992; W harton

et al, 1991). 60A protein is cleaved and then secreted as an N-terminal domain and C-

term inal homodimers and may play a role in mesoderm form ation and gut development

(Doctor et al, 1992). Bm p-5, -6, -7 and -8 are members o f this subfamily and show

approximately 75% homology between themselves and approximately 60% hom ology

to 60A. The recently discovered m olecules Univin (in sea urchins; Stenzel, 1994) and

OP-3 (Griffith et al, 1996) also belong to this subfamily.

It has been suggested that members of the 60A subfam ily may exert their effects

(26)

' ; ' :/;v. > .ICHAPTER 1 y im m m C T tO N AN D AIM S _ x'. ' " /: n

heterodimers with potent bone-inducing capacity (Aono et al, 1995) and B m p -2 and

B m p -7 h?Lve overlapping expression domains in vivo (Francis-W est et al, 1995; Lyons

et al, 1995). Furtherm ore, BM P-2, -4 and -7 appear to bind to common cell surface

receptors (see section 1.1.5).

The third family, radar, was named after the recently discovered zebrafish gene

product of the same name and contains Radar and G rowth and dijferenatiation factors

5-7 (G d f 5-7). Radar is most closely related to G df-6 and is expressed in the dorsal

region of the neural plate and retina. It is also found in neural crest and ventral posterior

m esenchym e (Rissi et al, 1995). Mutations in the G df-5 gene cause a condition called

brachypodism in mice and in hum ans, characterised mainly by shortening o f limb

skeletal elements due to faulty chondrogenesis (Storm et al, 1994; Thom as et al, 1996).

Apart from these three subfamilies, the BMP/OP family also contains the V g-1,

dorsalin (dsl) and Bmp-9 genes. The Vg-1 gene w as originally isolated due to its

asym m etric distribution in oocytes (W eeks and M elton, 1987) and is thought

to be involved in mesoderm and axis formation in the oocytes (Thom sen and M elton,

1993)D s l has been implicated in the regulation of cell differentiation in the neural tube

(Basler et al, 1993) while B m p-9 is thought to be involved in liver m orphogenesis

(Song et al, 1995).

A number of diverse molecules lie within the DVR grouping but outside the

BM P fam ily. This group is more heterogenous than the others discussed so far. B m p

-3, for exam ple, bears only 40-48% homology to the other BM Ps. Gdf-1 w as isolated

during a screen for novel (mammalian) members of the T G F-P subfam ily. The gene

has been show n to comprise two non-overlapping open reading frames encoding two

different proteins. One of these was detected exclusively in brain, spinal cord and

peripheral neurons in mouse embryos. The other is highly conserved between mice and

(27)

Another GDF, GDF-3, lacks a cysteine residue important for the process of

dimer formation in other members o f the T G F -p superfam ily. It is expressed in

developing skeletal tissue (Jones et al, 1992b) and in adult thymus, spleen, bone

m arrow and adipose tissue.

N od al has been localised to the node at the anterior portion o f the primitive

streak in mouse embryos and is thought to encode a signalling molecule essential for

m esoderm formation and organisation of axial structure in early mouse development

(Zhou et al, 1993).

Unlike the other genes in this group, screw (sew) is a D rosophila gene thought

to interact with dpp in specifying dorsal cell fates in the developing em bryo. Like sew ,

Anti-Dorsalising-M orphogenetic-Protein (ADMP) is thought to be involved in setting

up em bryonic polarity, but as its name implies, overexpression of admp m R N A in

X enopu s suppresses dorsal and anterior structures. Dorsalising factors like activin and

LiCl induce A d m p gene expression suggesting that the gene may act to restrict the

dorsalising activity of activin(M oos et al, 1995).

G df-10 is expressed in developing murine uterus, adipose tissue, brain, liver

and spleen, suggesting that it has multiple roles in em bryogenesis (Cunningham et al,

1995).

1.1.2.4 Other more divergent genes

The last group of molecules I will discuss are divergent molecules that do not

fall into any particular group within the TG F-p s u p e r f a m i l y . / « / i fa has already been

mentioned. Mullerian inhibiting substance (M IS) is a gonadal hormone and M IS

deficient male mice develop as pseudohermaphrodites w ith oviducts and uteri. Four

receptors for MIS have been cloned (He et al, 1993). A nother molecule, glial-derived-

neurotrophic factor (GDNF) was isolated due to its ability to promote the survival and

(28)

X^fAPTBR 1 > um O D Ü C T lO N AND AIMS f : : : :/ 27

1.1.3 STR U C T U R E OF THE BM PS

A s already mentioned, BM Ps are members of the TGF-B superfamily. With the

exception of GDF-3 which has six cysteine residues, the T G F-p C-terminal domain has

seven conserved cysteines. The members of this family are initially synthesised as

large precursor proteins containing an amino terminal signal sequence and a pro

dom ain. This pre-pro precursor is cleaved at a dibasic or RX X R site to release a mature

C -term inal peptide of 110-140 am ino acids (Fig. 1.1 A). Proteolytic cleavage of the pro

dom ain and dimer formation are required for activity (Dale et al, 1993; Thom sen and

M elton, 1994) and may be an important method o f regulation o f this class of

m olecules. The BM Ps are capable of both homodimer and heterodimer formation

(A ono et al, 1995). The hom odim eric form o f X enopus B M P-2 has been purified from

X e n o p u s em bryos and shown to induce alkaline phosphatase expression in osteoblast

cells (Shoda et al, 1994), while homodimers of X en o p u s BM P-4 and BM P-7, as well

as heterodim ers o f BM P-4/-7 have osteoinductive capabilities in rats, with the

heterodim er having the strongest activity (Aono et al, 1995). Aono et al (1995) also

suggested that native bone-inducing activity in bony matrix w as due to heterodimeric

B M P.

The N-terminal region (leader and pro-domain) o f TGF-6 family members

exhibits a great am ount of variability (Kingsley, 1994) and can be disrupted in at least

one family m em ber, D SL, w ithout affecting biological activity (Basler et al, 1993).

Even w ithin the B M Ps, it is the C-terminal amino acid region that is most highly

conserved (Feng et al, 1994; Rosen and Thies, 1992). H ow ever, the pro-domain is

thought to be necessary for normal synthesis and secretion (Gray and M ason, 1990;

H am m onds et al, 1991; Thom sen and Melton, 1993). The im portance of the pro-region

is illustrated by the fact that creation o f a fusion protein containing the pro-region of

BM P-2 fused to the mature region of BMP-4 dramatically improved secretion of BMP-

(29)

ICHAFTERI '"'V'r2<5

The conserved cysteines in the mature protein play an important role in its

tertiary structure - the creation of the cystine or TGF-13 knot (Daopin et al, 1992;

Schlunegger and Grutter, 1992). This is an eight-member ring at the core of the

molecule formed by two disulphide bridges connecting four cysteine residues, w ith a

third disulphide bridge pointing directly through the ring (see Fig. I .I B ) . Finally, the

last cysteine residue in each m onom er subunit is necessary for the formation of

disulphide bonds between biologically active homodimers or heterodimers (M assague,

1990). Tw o TG F-6 family m em bers, GDF-3 and G D F-9 (Jones et al, 1992b;

M cPherron and Lee, 1993), lack this last cysteine but may be able to dimerise by virtue

of their hydrophobic contacts.

The three-dimensional structure of BM P-7 has been reported (Fig. I .I B ;

G riffith et al, 1996) and, like TG F-6, possesses the cystine motif and the beta-folds,

suggesting a common mechanism for receptor interaction.

It has been suggested that the protease activity of BMP-1 may have a role in the

activation o f the other BMPs, since its sequence is closely related to that of Drosophila

tolloid w hich has been implicated in the processing pathway of DPP, the Drosophila

ortholog o f BM P-2 and -4 (Childs and O 'C onnor, 1994; Finelli et al, 1994; Shimell et

al, 1991). This prospect seems less likely now that BM P-1 has been identified as

procollagen C-proteinase and is thought to play a more direct role in ossification

(K essler et al, 1996; Reddi, 1996). However, the BMP-1 protease may have m ore than

one function. Also, the Bm p-1 gene encodes two alternatively spliced transcripts, not

only for the BMP-1 pro-collagen protease, but also for mammalian Tolloid (which

encodes a putative astacin metalloendopeptidase domain). It has been suggested that

BM P-1 belongs to a separate class of structurally-related proteins containing CUB

(30)

: / -:v;=.<CHAPXER' 1 - m R O D U C T lO N A m AIMS = %.y:,.

Fig. 1.1 Structure o f TGF-B superfam ily members. (A) D iagram m atic view of TGF-B

related protein (from Kingsley, 1994). All members of the family are synthesised as

larger precursor molecules. The leader sequence targets the precursor to the secretory

pathway; while the variable pro-domain may assist in folding, synthesis or secretion.

The signalling molecule consists of homo- or hetero-dimers o f a mature w ell-conserved

C-terminal domain which is cleaved from the rest of the molecule at a RX XR site. The

mature fragment contains seven invariant cysteines. (B) Schematic draw ing o f the 3-D

structure of BM P-7 (from Griffith et al, 1996). Six cysteines form three disulphide

bonds characteristic of the cystine knot motif. Two at C ys-67-Cys-136 and C ys-71-

C ys-138, form a ring through which the third, Cys-38-Cys-104, passes. The four

strands of the antiparallel B-sheet, which emanate from the knot, form two finger-like

projections. The heel of the hand is formed by an a-h elix at the opposite end o f the

knot. The tube marked a l is the a helix while the solid thin lines indicate the

intrasubunit disulphide bonds. The N -term inus lacks the disulphide bond characteristic

(31)

CHAPTER \^lN T m D U C T J O N AND% ÎM S

m

Leader

(15-25 @a)

Pro-do main

(50-75 aa)

c c c c c c c

Mature domain

(110-140 aa)

H eel

Finger

(32)

1.1.4 BM PS IN BONE FORMATION

The initial discovery of the BM Ps w as due to their ability to induce the

form ation of bone when implanted in ectopic sites in the rat (see section 1.1.1) and their

effects on bone morphogenesis continue to be the subject of m uch research.

BMP-1 has recently been shown to be identical to Procollagen C-proteinase and

cleaves the C-propeptides of procollagen I, II and III to produce the collagen fibres of

the extracellular matrix (Kessler et al, 1996).

B m p-2 expression is localised to the precondensing mesenchyme o f the

developing mouse limb (Lyons et al, 1990) and both B m p -2 and -4 are found in the

developing cartilage elements of the chick limb (Francis et al, 1994), suggesting a role

for these BM Ps in embryonic skeletogenesis. B m p -2 and-4 have also been implicated

in orofacial primordia development (Bennett e ta l, 1995; Francis-W est et al, 1994).

B m p-3 is expressed in developing human perichondrium , periosteum and

osteoblasts and has been show n to have cartilage-inducing activity when expressed in

COS cells (Luyten et al, 1989; Vukicevic et al, 1994).

Mice carrying the short-ear {se) mutation have deletions in the B m p -5 gene

(Kingsley et al, 1992; Kingsley, 1994a). The m utant m ice suffer from a num ber o f soft

tissue alterations and skeletal defects. Kingsley (1994a) traced these defects to the early

stages of cartilage formation based on the alteration in the size and shape o f cartilage

condensations in the mutant mice. H ow ever, B m p -5 is also expressed later in

osteogenesis in the osteogenic stem cell layer surrounding the cartilage matrix

(K ingsley, 1994a) and Baylink et al (1996) have proposed that the defects in se mice

are due to impaired osteoblast differentiation.

The B m p -6 gene is expressed in hypertrophic cartilage in developing m ouse

em bryos(Lyons et al, 1989) and together with Bm p-1, 2 and 4 is expressed by cultures

o f foetal rat calvarial osteoblasts as they form mineralised bone nodules (H arris et al,

(33)

Q ^ \ Y I E K l B rm O D V C T IO N AN D A IM S

-B m p -7 m R N A is expressed at multiple sites in the m ouse embryo and is often

colocalised w ith Bm p-2, suggesting that the two BM Ps function co-operatively (Lyons

et al, 1995), perhaps in heterodimers. Mice deficient in B m p -7 have skeletal defects in

the skull, rib cage and hindlimbs (Luo et al, 1995; Dudley et al, 1996), show ing that

B m p -7 is required for proper skeletal developm ent. In addition, BM P-7 induces

chondroblastic and osteoblastic differentiation in murine clonal cell lines (Asahina et al,

1996).

BM P-8, 9 and 10 have not yet been show n to induce bone or cartilage

formation in vivo and their localisation in developing skeletal structures has not been

reported.

W hat is currently known about the osteoinductive properties of the BM Ps is

sum m arised in Table 1.1.

The exact mechanism of endochondral ossification induced by the B M Ps is yet

unknow n, but a number of clues to the pathway exist (for review, see R osen and

Thies, 1992).

The BM Ps are secreted signalling molecules and are thought to act in an

autocrine or paracrine manner to regulate skeletal development in the embryo (Chen et

al, 1995). Recombinant human BM P-2 has been demonstrated to enhance cell

proliferation (Yamaguchi et al, 1991) and stimulate gene expression of interleukin-6

(IL-6) and TG F-B l in a human osteoblast-like cell line (Zheng et al, 19 9 4 ). TG F-61

production by monocytes in response to recombinant human BM P-4 is thought to be

instrumental in the differentiation of mesenchymal cells into chondrocytes

(Cunningham et al, 1992); while IL-6 stimulates osteoclast maturation (K urihara, 1990)

and bone resorption (Lowick et al, 1989).

Recently, osteoblast treatment with BM P-2 has been shown to shift TGF-13

action away from DNA synthesis and towards stimulation o f collagen synthesis by

altering TGF-B binding to its receptors (Centrella et al, 19 9 5 ). TG F-Bl and BM P-2

have also been shown to stimulate chondrogenesis in a chondroblast cell line by altering

(34)

I ' ' / / - --y ; i Q n h Y ï m X - m R O D Ü C T l O H Â m AIM S

BM P g » n a I n d u e * * b o n e / c a r ti la g a '’ E x p r e s s io n in d e v e lo p in g s k e le ta l s tr u c t u r e s E ffects of k n o c k o u t o n b o n e fo rm a tio n BM P-1 C a r t ila g e

B M P -2 B o th

B M P-3

B M P - 4

B M P-5

BMP-6

B o th B o th

B o th

N at rep o rted

BMP-8 Not re p o rte d BMP-9 Not rep o rted BMP-10 Not rep o rte d

C o n d e n s in g m e s e n c h y m e C o n d e n s in g p r e c a r til a g in o u s m e s e n c h y m e , lim b a p ic a l e c to d e r m a l rid g e . In te rd ig its, o r o f a c ia l p r im o r d ia a n d to o t h b u d s . L a ter in p e r ic h o n d r iu m a n d p e r i o s t e u m

P e r ic h o n d r iu m , p e r i o s t e u m , o s t e o b l a s t s A p ica l e c to d e r m a l rid g e a n d

m e s e n c h y m e of early lim b b u d s . A lso In o ro fa c ia l p r im o r d ia

C o e x te n s iv e w ith m a n y r e g i o n s of c o n d e n s in g m e s e n c h y m e . L a ter fo u n d in p e r ic h o n d r iu m a n d p e r io s te u m o f d iffe re n tia tin g sk e le ta l e le m e n ts

H y p e r t r o p h i c c a r t i l a g e a n d

o s t a o b l a s t s

C o n d e n s i n g p r e c a r t i l a g i n o u s

m e s e n c h y m e , i n t e r d i g i t s . L a t e r

in p e r i c h o n d r i u m .

Not rep o rted Not rep o rted Not rep o rted

N ot re p o rte d N ot re p o rte d

N ot r e p o r te d No sk ele tal- sp e c ific d e f e c ts

Sm all ex tern al ea r. s c o llo p in g of th y ro id c a rtila g e , th in n in g of tr a c h e a rings. S h o rt w id e sk u ll, v e r te b r a l p r o c e s s e s r e d u c e d o r a b s e n t, lo n g b o n e s h a v e a lte r e d c u r v e s but s m a lle r w id th s

Not reported

Hind limb polydactyly. failure of seventh pair of ribs to fuse to sternum , cranial m e m b ra n o u s b o n e s not fully developed

Not reported Not reported

Not reported

T a b le 1 .2 Table summarising the role o f the Bone Morphogenetic Proteins in

vertebrate bone formation. Their ability to induce ectopic formation of bone and/or

cartilage, the pattern of expression of their transcripts in developing skeletal tissue and

(35)

I--- ' 1 - INTRODVCTION A m ÂÎMS

bone formation BM P-2 has also been show n to stimulate the in vitro migration of

osteoblasts and osteosarcoma cells and might therefore play a role in chemotactic

recruitm ent of osteoblasts during bone formation (Lind et al, 1996).

In addition, the Hox, P ax and Gli genes have been implicated in skeletal

developm ent and BM Ps have been show n to induce sequential H ox and M sx gene

expression in the process of ectopic bone formation (limura et al, 1994). If a parallel

may be drawn to related members of the TG F-6 family, TG F-6 itself has been show n

to induce the expression of a novel zinc-finger protein which is restricted to osteoblasts

and skeletal muscle cells (Subramaniam et al, 1995) and may mediate downstream

signalling in bone formation.

The BM Ps are of great interest because of their potential clinical applications in

stimulating bone healing (Rosen and Thies, 1992), how ever, much remains to be

discovered about the complex molecular cascades that accompanies endochondral

ossification.

1.1.5 BM PS AS SIG N A LLIN G M OLECULES IN DEVELOPMENT

A part from their function in the formation o f the skeleton, the BM Ps have been

im plicated in numerous other developmental processes.

Table 1.3 summarises the known m R N A expression patterns, effects o f m ouse

knockout/dom inant negative experim ents, naturally-occurring mutants and postulated

roles o f B M Ps in em bryogenesis outside of endochondral ossification.

1.1.5.1 BMP-1

BM P-1, the only non-TG F-6 related member of the family, may play a role in

axis formation in the em bryo. Transcripts o f the murine gene are distributed at low

(36)

BMP g e n * E x p re s s io n p a tte rn in so ft ti s s u e Effects of m o u s e k n o ck o u ts P r o p o s e d r o le s B M P. I

B M P-2

B M P .3

8 M P -4

B M P-5

B M P-6

B M P-7

B M P-8

B M P-9

B M P-10

M e s o d e r m , d e v e lo p in g s p in a l c o r d a n d m id /tiin d b ra in

M y o g en ic la y er of h e a rt c u s h io n s , to o th b u d s , hair & w h is k e r follicles, s e n s o ry p la c o d e s , d e v e lo p in g ovary a n d te stis .

In lu n g ep ith eliu m , kidney tu b u le s a n d in testin a l m u c o s a

M e s o d e rm , e p id e rm a l e c to d e rm , d o r s a l n e u ra l tu b e , lateral s o m ite s . L a ter in h e a rt, w h isk er follicles, to o th b u d s , b ra n c h ia l a rc h e s , d ie n c e p h a lo n , h in d b ra in , ey e.

D ev e lo p in g lu n g m e se n c h y m e , u re te r, b la d d e r , in testin e, a n d te le n c e p h a lo n . A dult lu n g e n d liver.

D e v e lo p in g o o c y te s , m e n in g e s , h e a rt, p la c e n ta , n eu ral tu b e a n d sk in & g u t ep ith elia. A dult a n d fetal b rain .

D e v e lo p in g n o to c h o rd , s u r f a c e e c to d e r m , h e a rt, g u t, kidney, e y e a n d b ra n c h ia l a rc h e s.

P a tte r n n o t re p o rte d

D e v e lo p in g h e p a to c y te s

N o t r e p o r te d

N ot re p o rte d

A b n o rm al m e s o d e r m a n d yolk s a c b lo o d v e s s e l form ation. D is o rg a n isa tio n of s tr u c tu r e s p o s te rio r to th e heart.

I n c r e a s e d I n c id e n c e of lu n g c y s ts , liver g ra n u lo m a s , h y d ro tic k id n e y s & u reter, d is p la c e d o v a r ie s & ir re g u la r in te s tin a l folding

N ot re p o rte d

F ailu re of n e p h r o g e n e s is in th e d e v e lo p in g k id n e y , m icro p h th a lm ia or a n o lp h th a lm ia

N ot re p o rte d

N ot r e p o rte d

E m b ry o n ic ax is form ation

M e s o d e rm in d u c tio n , n e u ro n a l sp e c ific a tio n , e p ith e lia l- m e s e n c h y m a l in te ra c tio n s , cell fate d e te rm in a tio n

S ig n allin g m o le c u le s in lu n g , k id n e y a n d in testin a l m o r p h o g e n e s is

Call fate sp e c ific a tio n , e p ith e lia l- m e s e n c h y m a l in te ra c tio n s , a p o p to s is , s ig n a llin g m o le c u le s in axis fo rm a tio n , n e u ro tro p h ic factor, m e s o d e r m a n d a n d e p id e r m a l in d u c e r

M e d ia te s

e p ith e lia l- m e s e n c h y m a l in te ra c tio n s

N e rv o u s ti s s u e m o r p h o g e n e s is

S ig n a llin g m o le c u le in k id n e y a n d e y e d e v e lo p m e n t

U nknow n

R e g u la to ry ro le in h e p a tic g ro w th a n d fu n ctio n

U nknow n

Table 1.3 Table summarising the multiple roles of Bone M orphogenetic Proteins in

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I

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6

%

4

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vUMS;

_____

2

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in the floor plate o f the spinal cord and mid/hindbrain from about 9.5 days p.c. The

latter pattern o f expression is regulated by the transcription factor H N F-33 (Sasaki and

Hogan, 1994). H ow ever, the recent discovery o f th e identity o f B M P -1 as Procollagen

C-proteinase (K essler et al, 1996) has raised the possibility that BMP-1 has no role in

patterning at all.

1.1.5.2 B M P-2

B M P-2 expression has been detected in a myriad o f non-skeletal tissues

including the limb, heart, to o th buds, hair and w hisker follicles (Lyons et al, 1990),

sensory placodes (A nthony Graham, unpublished com munication) and ovary and testis

(Nishimatsu É?/a/, 1992).

In the developing limb-bud, there are three m ajor sources o f signalling

molecules responsible for dictating outgrow th and axis specification. The apical

ectoderm al ridge (A ER) is thought to sustain outgrow th o f the limb by maintaining a

zone o f undifferentiated, proliferating mesenchymal cells at the tip o f the bud; the

progress zone (reviewed in Tickle, 1995). FGF-4 has been implicated as a ridge signal

candidate m olecule that can maintain outgrow th following removal o f the A E R (C ohn

et al, 1995; N isw ander and M artin, 1993; V ogel and Tickle, 1993).However, the

expression of F g f-4 is detected only after considerable outgrow th has occured and

another FGF, F gf-8, has been show n to be expressed prior to limb outgrow th in the

m ouse and chick and can also substitute for the A ER in stimulating limb outgrow th

(M ahm ood et al, 1995). Interestingly, B M P-2 counteracts the effect o f FG Fs in limb

buds devoid o f ectoderm (N isw ander and M artin, 1992).

A group o f cells in the posterior mesenchyme, the polarising region, are

thought to pattern the anterior-posterior axis. Bm p-2 transcripts are found in posterior

limb mesenchyme and can be activated in anterior mesenchyme by the application o f

retinoic acid o r polarising region grafts which also induce mirror-image duplication

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X -ÎN rR O D U C n O N AND AIMS:::

---recom binant human BM P-2 using a retroviral vector is able to induce ectopic F G F -4 in

the anterior ridge, followed by ectopic expression o f the H ox-D genes in the anterior

mesenchym e (Duprez et al, 1996). Partial digit duplications were also observed. This

may imply that BM P-2 has a role in maintenance o f the ridge, and possibly in

patterning the anterior-posterior axis.

B M P-2 transcripts in the heart are localised to the myocardium at the time of

formation o f the endocardial cushions (Lyons et al, 1990). The formation o f the

cushions is thought to be initiated by a signal from the myocardium (Potts and R unyon,

1989) and Lyons et al (1990) have postulated that this signal is BM P-2. Interestingly

enough, the closely related gene dpp has been show n to determine the com petency of

mesoderm al cells to develop into cardiac tissue (Frasch, 1995) implying that its

vertebrate counterparts may well be important morphogens in heart development.

BM P-2 is detected fairly late in tooth development and together w ith B M P-4, its

expression pattern is suggestive of a role in epithelial-mesenchymal interactions (Vainio

et al, 1993). It has been shown to induce odontoblast differentiation w hen applied

together w ith dentin and postulated to induce ameloblast differentiation as well (Vainio

et al, 1993).

In the process of hair follicle formation, BM P-2 is first detected in the epiderm al

placode and becom es restricted to non-dividing cells around the base of the hair shaft in

the mature follicle (Lyons et al, 1990). W hen the closely-related BM P-4 protein is

ectopically expressed in the outer root sheath, animals display progressive balding after

the first grow th cycle suggesting that the role of BM P-2 in normal follicle developm ent

is to term inate cell proliferation and turn on trichocytic markers (Blessing et al, 1993).

B M P-2 is able to induce the expression o f the neural cell adhesion molecules

N CA M and L I in neuroblastoma cells (Perides, 1994) and can induce a distinct set o f

neuropeptide m arkers in cultured sympathetic neurons (Fann and Patterson, 1994). The

neuropeptide genes induced change with membrane depolarisation possibly allowing

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I : c h X p x e r i m rR O D U cnoH a n d a i m s

-BM P-2 has also been put forward as a mesoderm -inducing factor synthesised

by the extraembryonic endoderm (Rogers et al, 1992). F9 cells resemble cells o f the

blastocyst inner cell mass when undifferentiated and extraembryonic endoderm w hen

differentiated. When differentiation is induced by retinoic acid (RA), B m p-2 m RN A

levels increase and BMP-2 treatment o f such cells can alter growth and m orphology.

BM P-2 also represses its own expression in a negative feedback loop (Rogers et al,

1992). The action of RA on BM P may be mediated by the RAR-alpha and the RAR-

gam m a receptors (Rogers, 1996).

X enopus Bm p-2 transcripts are detected in the oocyte and blastula, but not after

gastrulation (Nishimatsu et al, 1992) and the gene, like BM P-4, may play a role in

m esoderm form ation in the X enopus embryo.

1.1.5.3 BMP-3

BM P-3 is expressed in a hum an lung carcinoma cell line (Wozney et al, 1988),

and has been detected in human foetal lung tissue . It has also been found in developing

human intestinal mucosa and kidney mesonephric tubules (Vukicevic et al, 1994). This

suggests that it may have a regulatory role in the m orphogenesis of hum an lung, kidney

and intestine.

1.1.5.4 BM P-4

B m p-4 transcripts are found in many of the same organs that contain B m p -2

transcripts, but usually the spatiotemporal localisation within the organ is ju st slightly

different. Outside o f the developing skeleton, B m p -4 has been found in the heart, the

w hisker follicles (Lyons et al, 1991), the teeth (Vainio et al, 1993), the branchial arches

(Francis-W est et al, 1994; Wall and Hogan, 1995), the diencephalon and hindbrain

(Lyons et al, 1991; Graham et al, 1994) and the eye (this thesis; also Francis-W est et