The human T transcription factor: A study of genetics and function

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The human T transcription factor:

a study of genetics and function

Charalambos Papapetrou

December 1998

Thesis submitted for the degree of Doctor of Philosophy

in the University of London

MRC Human Biochemical Genetics Unit

The Galton Laboratory

Department of Biology

University College London

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A b str a c t

The human T gene is a member of a family of transcription factors, the T-box genes, which play an important role during embryogenesis. T is expressed early in development in the primitive streak, axial mesoderm, notochord and tail bud and is

essential for normal mesoderm development and notochord differentiation. Mouse T

mutations are lethal and heterozygotes have features that resemble neural tube defects

and sacral agenesis in man.

This thesis describes the genetic analysis of the human T gene in healthy individuals and in neural tube defect and sacral agenesis patients. Eight new

polymorphisms were identified by a combination of SSCP and sequence analyses and

three of these involve an amino acid change in evolutionary conserved domains;

Glyl77Asp in the DNA binding domain and Gly356Ser and Asn369Ser in a

transcriptional activation domain. An association between an allele {TlVSy-l) and a particular haplotype (T363.G530.IVS7-2) and susceptibility to spina bifida was detected. Analysis of T in sacral agenesis patients identified a rare variant Ala338Val in the transcriptional activation domain, in a single patient and his mother. Overall it was

concluded that T may play a role in the aetiology of neural tube defects and sacral agenesis, but accounts for only a small proportion of the genetic component of

susceptibility to these disorders.

The proximal promoter of the human T gene was cloned, sequenced and compared with the mouse sequence in a search for conserved regulatory elements.

Several potential motifs were identified, the most significant of which was a 30 bp

region conserved not only between man and mouse, but also Xenopus.

In vitro DNA/protein binding studies were carried out to demonstrate that human T protein is able to bind to a DNA motif known to be the target for mouse T protein. In

addition, it was demonstrated that both human and mouse T bind their DNA target as a

dimer and could form a heterodimer. It was shown that the Asp 177 variant of the DNA

binding domain reduces the stability of T dimer formation. Mouse Tbx6 protein, another member of the T-box family, was shown to bind to the same DNA target, but it was

demonstrated that T and Tbx6 are not likely to bind this target as a heterodimer.

The human homologue of the TBX6 gene was cloned, sequenced and mapped to chromosome 16pl 1.2. Expression studies showed that TBX6 is expressed in the

notochord and tail bud in the early stages of development, but also in a wide range of

adult tissues. A novel member of the T-box gene family, designated T-like, was identified by sequence analysis of PGR products. Preliminary analysis suggests that

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Statement from Charalambos Papapetrou regarding contribution to work in each Results Chapter

C h a p te r 3 T he g e n o ty p e analysis o f the TIVS^ p o ly m o rp h ism in N T D fa m ilies and controls was divided 50:50 with M s Katie M orrison. All o f the data analysis was carried out by me.

T h e pape r M o rriso n et al. 1996 was largely w ritten by P ro fe sso r Y v o n n e H. E dw a rds, how ever, I con trib u ted sections in the M aterials and M e th o d s section and the section describing the association analysis.

I was entirely responsible for the finding o f p o ly m o rp h ism s in T e x o n s 2 and 3 and for the g en o ty p e analysis in N T D fam ilies and controls and for an a ly sin g the data. I was also responsible for the S S C P analysis o f T exon 1.

T h e g en o ty p in g o f the M T H F R therm olabile variant in the British N T D fam ilies and controls, the statistical analysis and the association studies with the T variants was also m y work.

T h e Papapetrou et al., 1996 p ap e r was w ritten by m e in the first instance and revised by P rofessor Y v o n n e H. Edw ards.

C h a p te r 4 F or 15 o f the 31 sacral agenesis cases I carried out all o f the g e n o ty p e analysis, this included S S C P and direct sequencing. For the re m a in in g 16, w hich w ere only sequenced, the w ork w as divided 50:50 with D r Felicity D ru m m o n d .

1 was entirely responsible for the search for p o ly m o rp h ism s in T e x o n s 4, 5, 6, 7, and 8 and the 5 ’U T R and also for the characterisation o f these p o ly m o rp h is m s by s eq u e n cin g and their analysis in controls. I also investigated the o c c u rre n c e o f the rare variant in exon 7 in N T D patients and controls by genotyping.

T he lam b d a clone co n tain in g the hu m an T p ro m o te r w as isolated and D N A pre pare d by Ms W e n d y Putt. M s Putt also carried out the S o u th e rn analysis and su b clo n ed the pro m o ter contain in g fragm ents. I was responsible for the seq u e n c in g o f the p ro m o te r and the s u b seq u e n t seque nce c o m p ariso n s and search for transcription factor binding sites.

C h a p te r 5 All o f the w ork d escribed in this C h ap ter w as carried out in person, i.e. preparation o f the in vitro synthesised protein, all F M S A s , cro sslin k in g e x p e rim e n ts etc.

T h e P apapetrou et al., 1997 p ap e r was written by m e with critical reading and revision by P rofessor Y v o n n e FI. E dw ards.

C h a p te r 6 I was responsible for the library screening to identify T B X 6 and fo r the isolation o f the clone and p reparation o f D N A . M s W e n d y Putt su b c lo n e d exon c o n ta in in g fragm ents and these w ere seq u e n ced as a jo in t effort. F IS H analysis was carried out by Dr M arg aret Fox using the D N A I had prepared. All o f the su b se q u e n t T BX6 analysis was carried out in person. All o f the R T - P C R e xpression studies w e re my ow n work.

I was responsible for the identification o f the T-like g ene and its su b seq u e n t characterisation by partial seque nce analysis and R T -P C R .

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Acknowledgements

M y first thanks go to Professor Yvonne Edwards, who supervised this

project and to whom I am m ost grateful for her invaluable help and endless

support, encouragem ent and enthusiasm throughout this work. Special thanks

also go to D r Jane Sowden for her support, useful discussions and help,

especially with the functional study. I would also like to thank everyone in the

laboratory for their support and help and for making a happy w orking

environm ent. I particularly thank W endy Putt for all her technical advice and

help with the cloning o f the hum an T prom oter and TBX6 gene and Katie M orrison for her help with the TIVSy polym orphism analysis. M y special thanks go to Dr Felicity Drum mond for her special friendship and support, as

well as technical advice, useful discussions and help with the sacral agenesis

work. Thanks also to James W ilson and Ira Islam for their valuable help. I

would also like to thank D r M argaret Fox for providing the FISH analysis. Dr

W illiam Reardon and Professor Robin W inter for providing the sacral agenesis

samples and our colleagues in Newcastle and The Netherlands for providing

the neural tube defects families. On a personal level, I would like to thank my

parents for all their love, encouragem ent and support throughout m y studies

and my girlfriend M arina for her love, support, patience and constructive

reading o f this manuscript.

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F ig u res

Page

CHAPTER ONE

Introduction

Figure 1.1 Schematic representation of the T gene and its protein 19

Figure 1.2 The formation of the neural tube 38

Figure 1.3 Schematic representation of the neural tube closure 40

Figure 1.4 Intracellular pathways of the folic acid metabolism 48

CHAPTER THREE

Investigation of the human T gene as a candidate gene for susceptibility to

neural tube defects

Figure 3.1 Analysis of the human T intron 7 TIVSjC polymorphism 86

Figure 3.2 SSCP of the human T intron 7 TIVSjC polymorphism 95

Figure 3.3 SSCP analysis of the human T exon 1 coding sequence 97

Figure 3.4 Analysis of the human T exon 2 C363T polymorphism 99

Figure 3.5 Analysis of the human T exon 3 G530A (Glyl77Asp)

polymorphism 103

Figure 3.6 Diagnostic digest of mouse T exon 3 104

Figure 3.7 Cladogram illustrating possible ancestral Tchromosomes 113 Figure 3.8 Digestion analysis of the human MTHFR C677T

polymorphism 116

CHAPTER FOUR

Genetic analysis of the human T gene in sacral agenesis

Figure 4.1 SSCP analysis of T exon 6 129

Figure 4.2 SSCP analysis of T exon 4 (A) and exon 5 (B) 130

Figure 4.5 Analysis of the human T 5'UTR variant 135

Figure 4.6 Analysis of the human T exon8 G1176A variant 136

Figure 4.7 Analysis of the human T exon 8G1066A variant 138

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Figure 4.9 Analysis of the human T intron 7 AIVS7G variant 140 Figure 4.10 Analysis of the human T exon 7 C1013T (Ala338Val)

mutation 143

Figure 4.11 Restriction enzyme map of the 5' end of the human T gene 145 Figure 4.12 Comparison of the human and mouse 5'-flanking

sequences 146

Figure 4.13 Analysis of T exon 3 of a sacral agenesis patient 150

Figure 4.14 The human T gene and protein showing various

polymorphisms 151

CHAPTER FIVE

An investigation of the DNA binding properties of the human T protein

Figure 5.1 Summary of in vitro synthesis of truncated human T

protein 158

Figure 5.2 SDS-PAGE of in vitro synthesised T proteins 161

Figure 5.3 EMSAs using the Glyl77 and Asp 177 truncated human

T protein 163

Figure 5.4 Thermostability assay of truncated human T/BS.p

complexes 164

Figure 5.5 EMSAs using variable amounts of truncated human T

protein 166

Figure 5.6 Comparison of the DNA binding properties of Glyl77 and

Figure 5.7 Comparison of the DNA-binding properties of Glyl77 and

Figure 5.8 A. Formation of a heterodimeric T/BS.p complex between

full-length and truncated T proteins and B. thermostability

assay of homodimeric and heterodimeric T/BS.p complexes 170

Figure 5.9 A. EMSAs and B. SDS-PAGE of cross-linked truncated

human T protein 172

Figure 5.10 SDS-PAGE of in vitro synthesised truncated mouse Tbx6

protein (aa 1-288 and 46-288) 175

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Figure 5.13 The crystallographic structure of the T-domain with its

DNA target 181

Figure 5.14 Sequence of the N-terminal aa of Xenopus, mouse and

human T homologues and mouse Tbx6 185

CHAPTER SIX

Cloning of other T-box genes

Figure 6.1 Comparison of the deduced aa sequences of human and

mouse TBX6 proteins 188

Figure 6.2 The human TBX6 cDNA and 5'-flanking sequences 190

Figure 6.3 Sequence analysis of human TBX6 to demonstrate the

presence of two extra nucleotides compared to mouse Tbx6 193 Figure 6.4 Comparison of the deduced aa sequences of the human and

adjusted mouse TBX6 proteins 194

Figure 6.5 FISH on normal metaphase chromosomes 195

Figure 6.6 RT-PCR using RNA from human and mouse tissues and

human TBX6 specific primers and diagnostic digests 197

Figure 6.7 RT-PCR using human tissue RNA and human T specific

primers and diagnostic digests 199

Figure 6.8 RT-PCR using human tissue RNA and human T specific

primers within the T-box region and diagnostic digests 201

Figure 6.9 A. Comparison of the fetal testis RT-PCR product and human

T and B. comparison of the deduced aa sequences of the

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T a b le s

CHAPTER ONE

Introduction

Table 1.1 Main areas of expression of murine T-box genes

Page

35

CHAPTER TWO

Materials and methods

Table 2.1 Primers and annealing temperatures for the amplification of

the human T and MTHFR genes

Table 2.2 Primers and annealing temperatures for the amplification of

TandraXdcDNA

66

73

CHAPTER THREE

Investigation of the human Tgene as a candidate gene for

susceptibility to neural tube defects

Table 3.1 TDT using the TlVSy polymorphism 89

Table 3.2 Genotype and allele frequencies of the human TIVSj

polymorphism 91

Table 3.3 Contingency tables comparing TIVSj genotype distribution 93

Table 3.4 Conditions for SSCP analysis of the human T gene 96

Table 3.5 Genotype and allele frequencies of the human C363T

polymorphism 100

Table 3.6 TDT using the C363T (Serl21Ser) polymorphism 101

Table 3.7 Genotype and allele frequencies of the human Glyl77Asp

polymorphism 105

Table 3.8 TDT using the Glyl77Asp (G530A) polymorphism 107

Table 3.9 Contingency tables comparing Glyl77Asp genotype

distribution 108

Table 3.10 Haplotype frequencies 110

Table 3.11 Contingency tables comparing haplotype distribution 111

Table 3.12 Haplotype TDT 114

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Table 3.13 Genotype and allele frequencies of the human MTHFR C677T polymorphism

Table 3.14 TDT using the MTHFR C677T polymorphism

Table 3.15 Association analysis of the T TIVSyC and MTHFR C677T

alleles

117

119

120

CHAPTER FOUR

Genetic analysis of the human Tgene in sacral agenesis

Table 4.1 Conditions for SSCP analysis of the human T gene Table 4.2 Sequence variation identified in the human T gene

128

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A b b r e v ia tio n s

aa amino acid

Ala alanine

AMPS am m onium persulphate

Arg arginine

Asn asparagine

Asp aspartic acid

bp base pair

BS3 B is(sulfosuccinim idyl)suberate

BSA bovine serum albumin

BS.p palindrom ic DNA target of T protein

C- carboxy

CBS cystathionine-p-synthase

cDNA com plem entary deoxyribonucleic acid

CEPH Centre d ’Etude de Polym orphism e Hum aine

C l confidence interval

Ci Curie

DNA deoxyribonucleic acid

cpm counts per minute

ddNTP dideoxynucleoside triphosphate

DAP difference in allele frequencies

DEPC diethylpyrocarbonate

dNTP deoxynucleoside triphosphate

dpc days post coitum

DTT dithiothreitol

ED TA ethylenediam inetetra-acetic acid

EM SA electrophoretic m obility shift assay

epd eukaryotic prom oter database

FETi Fisher’s exact test, one-sided

FISH fluorescent in situ hybridisation

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F R -a GCF Gin Gly GMS HAc HGM P HLA HNF-5 HPE IBD lie IVS kb L M APF2 M AZ M CK M HC M OPS m RNA MS M THFD

M TH FR

myc N-NTD P PAGE PBS PCR

folate receptor alpha

GC-binding factor

glutam ine

glycine

genome m ism atch scanning

acetic acid

hum an genom e mapping project

hum an leukocyte antigen

hepatocyte nuclear factor 5

holoprosencephaly

identical by descent

isoleucine

intervening sequence

kilo base

fast migrating, low band in EM SAs

m uscle actin prom oter factor 2

m yc-associated zinc finger protein

m otif from the m uscle creatine kinase prom oter

m ajor histocom patibility com plex

3-[A-m orpholino]propanesulfonic acid

messenger RNA

methionine synthase

methylene tetrahydrofolate dehydrogenase

methylene tetrahydrofolate reductase

m yelocytom atosis

amino

neural tube defect

probability

polyacrylam ide gel electrophoresis

phosphate-buffered saline

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pfu PGM PM SF PN K PPi psi PSM R RN A RN Ase rpm RT-PCR E SA SAP SB SEA SBO SDS sdw SE

SEdaf

Ser SHH SSC SSCP SSPE TA TB E TC A TD T

plaque form ing units

phosphoglucom utase

phenylm ethylsulphonylfluoride

polynucleotide kinase

saturated tetrasodium pyrophosphate

pounds per square inch

phage storage m edium

repression

ribonucleic acid

ribonuclease

revolutions per m inute

reverse transcriptase polym erase chain reaction

sum

sacral agenesis

shrimp alkaline phosphatase

spina bifida

spina bifida aperta

spina bifida occulta

sodium dodecyl sulphate

sterile distilled w ater

standard error

standard error of difference in allele frequencies

serine

sonic hedgehog

salt sodium citrate

single strand conform ation polym orphism

salt sodium phosphate EDTA

transcriptional activation

Tris-borate/ED TA

trichloroacetic acid

transm ission disequilibrium test

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TE Tris-ED TA

TEM ED N ,N ,N ',N '-tetram ethylethylenedi amine

TED transcription factors data base

Thr threonine

Tris Tris (hydroxym ethyl) am ino-m ethane

TRP-1 tyrosinase-related protein 1

UTR untranslated region

U slow migrating, upper band in EM SAs

uv ultraviolet

Val valine

VPA valproic acid

v/v volum e per volume

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CHAPTER ONE

I n tr o d u c tio n

T is a transcription factor that plays a vital role in norm al axial

developm ent and form ation and differentiation of posterior m esoderm .

The m ouse T gene was identified in 1927 as a loss o f function mutation,

Brachyury (D obrovolskaia-Zavadskaia and Kobozieff, 1927). Hom ozygous em bryos die at m id-gestation due to a defective allantois w ith severe posterior

abnorm alities and an undifferentiated notochord. H eterozygotes show

incom plete axial developm ent and have posterior abnorm alities and short tails.

The m ouse T gene is part of the r-complex, a region o f the proxim al half o f m ouse chrom osom e 17, which for m any years intrigued developm ental

biologists. A naturally occurring variant o f the r-complex, called the t-

haplotype, is defined by four non-overlapping inversions and characterised by

distortion of m ale transm ission ratio, em bryonic lethality and m ale sterility

(Silver, 1993). Originally, t-haplotypes w ere referred to as r-mutations or

t-alleles and w ere thought to be m utant t-alleles o f the T locus. H owever,

t-haplotypes are invisible them selves, because neither heterozygous +/t nor live- born hom ozygous t/t m ice show abnorm alities. Interestingly, T m utations expose the f-haplotype. M ice carrying a m utant T allele on one chrom osom e in com bination with a w ild type r-complex on the other chrom osom e have

short tails, whereas, animals carrying a m utant T allele in com bination with a r-haplotype are tail-less.

The nature o f the T-locus and the Brachyury allele rem ained obscure for over 60 years until in 1990, H errm ann et al. (1990) cloned the m ouse T

gene. The T gene was revealed to be a nuclear localised protein with a unique D N A binding dom ain, designated the T-box. The Brachyury allele (7^"“^^) was show n to be a 200 kb deletion encom passing the entire T gene. Since then, the hom ologues from various species as diverse| as sea urchin (Harada et a i , 1995) and m an (Edwards et al., 1996) have been cloned.

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W ork carried out in the last 5 years has shown that T is the founder m em ber o f a large fam ily o f T-box transcription factors. To date as m any as

13 m em bers have been cloned in m ouse and for most of these, orthologues

have been identified in man (see section 1.1.6). These genes all appear to be

critical to norm al developm ent. M utations in two o f the hum an T-box genes

(TBX3 and TBX5) have been associated with developm ental disorders. Thus far, no m utations of the Tgene have been identified in man and no clinical condition has been associated with abnorm al Texpression. However, the expression pattern o f T and the phenotype of Brachyury and other mouse m utants m ake the hum an T gene a good candidate for disorders that affect posterior axial developm ent. Examples of disorders w hich can be considered

in this category are neural tube defects, sacral agenesis and chordomas.

In subsequent sections of this chapter, the structure, expression,

regulation, function and role in em bryogenesis o f the Tgene will be described. Tw o disorders o f posterior axial developm ent, neural tube defects and sacral

agenesis, will be looked at in detail. Their aetiology, phenotype, genetics and

the possible role of T in their pathogenesis will be described.

1.1 TH E r G ENE

1.1.1 Structure of the T gene

The T gene has been cloned in m ouse (Herrm ann et al., 1990), Xenopus laevis {Xbra\ Smith et al., 1991), zebrafish Brachydanio rerio {Zf-T; Schulte- M erker et al., 1992), the ascidians H alocynthia roretzi (As-T; Yasuo and Satoh, 1994) and Ciona intestinalis {Ci-Bra', Corbo et al., 1997), sea urchin

H em icentrotus pulcherrim us {HpTa\ H arada et al., 1995), chicken (Ch-T;

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sequences vary in length, encoding proteins of 4 3 2 -4 3 6 amino acids (aa) with

the exception of amphioxus A m Bra (440 aa), ascidian A s-T (471 aa) and

zebrafish Zf-T (423 aa). The overall amino acid identity varies betw een

64% —91 % am ongst vertebrates and is lower betw een vertebrates and

invertebrates (40% -63% ). Across the T dom ain the identity is particularly

high even betw een species o f different subphyla of the chordates (for example,

78.5% identity betw een ascidian and mouse proteins) or species o f different

phyla (for exam ple, 70% identity betw een sea urchin and m ouse proteins).

Inform ation on the genomic structure of T is only available for the m ouse and hum an orthologues. The hum an T gene is 10 kb long and

com prises eight exons (Fig 1.1; Edw ards et a l , 1996). The overall structure appears to be sim ilar to that described for the m ouse T gene (Stott et al.,

1993). However, a direct com parison is not possible, since details on

exon/intron boundaries and sizes have only been reported for the hum an gene

and the m ouse genomic structure has only been referred to schem atically in

Stott et al. (1993) and H errm ann and Kispert (1994). Although genom ic clones o f other orthologues have been isolated (zebrafish, am phioxus, Ciona),

no inform ation on the genomic structure o f these genes has been published.

1.1.2 r expression

Expression o f the T gene is largely confined to the early stages of developm ent. M ost inform ation has been derived from the studies o f mouse,

Xenopus and zebrafish T genes with more lim ited inform ation from hum an, chicken and prim itive chordates.

In the mouse, T expression is first detected in the early prim itive streak at 6.5 dpc. T mRNA appears transiently in the nascent and migrating

m esoderm generated by the prim itive streak. Expression ceases as the cells

m ove aw ay from the streak to form paraxial or extraem bryonic tissues. The

allantois, w ith the exception of the very early basal com ponent, does not

express T. In the prim itive streak and its derivatives, the node, notochord and tail bud, expression continues at a high level until m orphogenesis is com plete

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1Kb

TBOX

229230

313 320

380

401 DNA binding domain

I_ _

Reguiatory domain

NLS

Figure 1.1

Schematic representation of the

T

gene and its protein, showing the positions of the DNA binding and

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(W ilkinson et al., 1990; Herrmann, 1991; Beddington et al., 1992; W ilson et al., 1993). A t later stages, T expression can be detected in the m ature notochord cells of the nucleus pulposus but not in adult tissues (intestine,

stomach, thymus, brain, testis, ovary, kidney, heart, adrenal; W ilkinson et a l,

1990).

Areas of T protein accum ulation overlap with those o f mRNA

expression but appear to be wider and not strictly correlated with the

com m itm ent of cells to mesoderm. In addition to the nascent and m igrating

m esoderm , T protein is also detected transiently in early endoderm and in

prospective neuroectoderm after 8.5 dpc, during the late stages o f the prim itive

streak. T protein is also present in m igrating extraem bryonic mesoderm,

possibly the precursor cells o f the allantois. W hile T protein accum ulates in

these non-m esoderm al sites, mRNA has not been detected. A nother difference

betw een T protein and mRNA distribution is that although T transcripts can be detected in the intervertébral notochord cells at 14.5 dpc, T protein disappears

in a rostrocaudal direction and is undetectable at this stage (K ispert and

Herrm ann, 1994).

The pattern of expression of the chicken T hom ologue (Ch-T) is very sim ilar to that in mouse. However, both C h-T transcripts and protein are detectable transiently in differentiated non-m esoderm al cells o f the

neuroectoderm and paraxial endoderm (Kispert et al., 1995b; Knezevic et al.,

1997).

Hum an em bryos are inaccessible during the very early stages of

developm ent, thus, although the expression pattern o f hum an T is likely to be sim ilar to that of the m ouse gene during gastrulation, data is not available. T

m RN A is readily detected in 13- and 14-w eek gestation hum an intervertébral

notochord, a stage of developm ent equivalent to 17.5 dpc in the mouse.

H um an T, like m ouse T, is confined to the notochord and was not detected in hum an adult gut, adult and fetal muscle, erythroid and intestinal cell lines and

14-w eek gestation spinal cord (Edwards et al., 1996).

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The patterns of expression o f the T hom ologues in Xenopus (Xbra) and zebrafish {Zf-T) are very sim ilar to that of the m ouse T. Xbra is expressed throughout the m arginal zone and in the notochord and posterior m esoderm .

The m arginal zone, the equivalent o f the prim itive streak, is m ade up of

undifferentiated cells that give rise to m esoderm and endoderm cells during

gastrulation (Smith et aL, 1991). Similarly, Z f-Tis expressed in the germ ring, w hich gives rise to m esoderm and endoderm , and the notochord and posterior

m esoderm . m RN A and protein accum ulate in the m arginal zone and germ

ring transiently during gastrulation, but in the notochord and posterior

m esoderm persist until later stages of developm ent (Schulte-M erker et a l ,

1992).

Therefore, it seems that in all vertebrates the expression of T

hom ologues is conserved and this extends to the cephalocordate am phioxus

w hose em bryos and larvae express two T hom ologues A m B ra-1 a.ndAmBra-2.

U sing a cRN A probe that does not distinguish betw een A m B ra-1 and Am Bra-2, expression was identified in presum ptive m esoderm in early stages of developm ent and in posterior m esoderm and the notochord in late gastrulation

and neurulation (Holland et al., 1995). However, in the protochordates

H alocynthia roretzi {As-T) and Ciona intestinalis {Ci-Bra) the expression pattern appears to be different. A s-T and Ci-Bra are expressed only in notochord cells and not m esenchym e cells, although notochord and

m esenchym e cells, as well as muscle and nerve tissue, are derived from the

same cell lineage (Yasuo and Satoh, 1994; Corbo et a l , 1997).

Little is know n about the expression of Tin echinoderm s. In the sea urchin, H pTa expression is detected transiently in the archenteron and then at a high level at the tip of the archenteron and secondary m esenchym e cells

m igrating from there (H arada et a l , 1995). However, it is not yet clear w hether the archenteron and secondary m esenchym e cells are prim itive

equivalents o f the prim itive streak and notochord.

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chordates. In Drosophila, for example, the Trelated gene Trg is expressed in the hindgut and anal pads (Kispert et a i , 1994).

The difference in T expression between the vertebrates and am phioxus on one hand, and the urochordates on the other, led to some interesting

speculations. It is possible that the prim itive streak and posterior m esoderm al

expression o f T in vertebrates and amphioxus, is related to evolutionary

progress and represents an acquired property associated w ith the possession of

a tail (H olland et a l, 1995). M oreover, it also seems possible that T

expressions in the prim itive streak and in the notochord w ere acquired

separately during evolution and m ay consequently be regulated by different

factors.

1.1.3 R egulation o f T expression

Tis required for normal m esoderm form ation and w hen over-expressed causes ectopic m esoderm formation. However, little is know n about how the

T gene is regulated and what factors determ ine prim itive streak and notochord expression. M ost work in this area has been carried out in X e n o p u s^ q c2l\xsq

the em bryos are easily manipulated and cultured.

In Xenopus, Xbra expression can be induced in anim al pole cells by vegetal pole cells, which are known to induce m esoderm form ation via grow th

factor-like signals. Xbra is activated by exogenous m esoderm inducing

proteins o f the fibroblast growth factor (FGF) and transform ing grow th factor-

13 (TGF-(3) families, bFG F and activin A respectively. Xbra expression is reduced w hen abnormal truncated versions of FGF and activin receptors are

over-expressed, presum ably because the active ligands are rem oved by the

truncated non-functional receptors (Smith et a l , 1991; Schulte-M erker and Smith, 1995). Xbra induction can occur in dispersed cells, is rapid and takes place even in the presence o f a protein synthesis inhibitor (cyclohexam ide).

This suggests that Xbra is an im m ediate early response gene of m esoderm induction (Smith et a l , 1991). Although FGF and activin can activate X bra

expression, continuous expression requires an intact FG F signalling pathw ay.

(25)

Expression o f Xbra declines when cells are dispersed and the intercellular link is diluted, but is m aintained when bFG F is added to the m edium o f the

dispersed cells (Isaacs et a l, 1994; Schulte-M erker and Smith, 1995). However, initial activation of Xbra by activin does not require a functional FG F signalling pathway (Schulte-M erker and Smith, 1995). Sim ilar pathw ays

are seen in other organisms. For example in chicken, C h-T is also induced by FG F and activin, but the induction is not an im m ediate early response

(Knezevic et a l , 1997). In zebrafish, Z f-T expression in anim al poles is induced by m arginal zone cells and exogenous activin A (Schulte-M erker et al., 1992). In Halocynthia, A s-T expression in the notochord can be induced by FG F (Nakatani et a l , 1996).

Em bryonic (e)FGF and Xbra can activate the expression o f each other

and eFG F maintains Xbra expression in dispersed animal pole cells (Isaacs et a l , 1994; Schulte-M erker and Smith, 1995). Xbra can activate its own expression but autoinduction requires protein synthesis and intercellular

signals (Rao, 1994; Tada et a l , 1997). This suggests that eF G F and Xbra are part of an autoregulatory loop in which X bra activates the expression o f

eFGF, which, in turn, maintains Xbra expression (Isaacs et a l , 1994; Schulte- M erker and Smith, 1995).

However, there is some evidence against the idea o f an autoregulatory

loop. Studies of chimaeric mice (see section 1.1.5) show ed

that T prom oter activity is m aintained in the prim itive streak and tail bud in

jBnuiiijBnuii carrying a T prom oter/lacZ construct up to the 14th day of gestation and thus, no T-dependent m echanism is required to m aintain

expression from this prom oter in T null regions. A ctivity from the T prom oterA acZ construct is apparent in null cells some distance (8 cell

diam eters) from neighbouring wild-type cells in the tail bud, and the level o f

expression o f the lacZ reporter gene is the same regardless o f the distance from T-expressing cells. This suggests that a T-dependant short-range

(26)

Furtherm ore, in embryos, which express a non-functional truncated

form o f the protein, Texpression ceases in early somite stage, indicating that up to this point w ild-type T is not required to m aintain its ow n expression

(Herrm ann, 1991; Kispert and Herrmann, 1994). The same is true for

zebrafish no tail mutants {ntl, functional T deficient), w hich continue to express m utant protein in the absence of functional protein (Schulte-M erker et a l , 1994). In Xenopus, expression o f a synthetic Xbra construct containing a repressor elem ent down-regulates Xbra expression in the notochord, but does not influence Texpression in ventral m esoderm (Conlon et a l , 1996). Thus, it seems that in the whole embryo the regulation of T is com plex and it is not clear w hether an autoregulatory loop involving T and FG F is in operation (Smith, 1997).

Recently, more evidence supporting the idea o f an autoregulatory loop

involving Xbra and eFG F in the notochord and dorsal m esoderm has emerged. It was shown that inhibition of Xbra function, by injection o f a synthetic Xbra

construct containing a repressor element, inhibits eF G F expression in the notochord and dorsal m esoderm of early embryos and FG F induced anim al

caps (Casey et a l , 1998). Thus, X bra function is required for m aintenance of eFG F expression. Furthermore, it was shown that induction of eFG F by X bra does not require protein synthesis or an intact FG F signalling pathw ay but is

cell autonom ous and occurs through binding of Xbra to eF G F 5'-flanking sequences (see section 1.1.4).

In Xenopus, 2.1 kb of Xbra 5'-flanking sequence and 50 bp of 5'- untranslated region is sufficient to direct expression in the m arginal zone but

not the notochord. Sequences within 381 bp from the transcription start site

are sufficient for induction of Xbra by activin and FGF. Response to activin is dose dependent and, at low levels activin induces Xbra expression, but at high levels suppresses Xbra. As yet, nothing is known about the factors w hich m ediate activin activation of T, however, suppression is m ediated by the hom eodom ain proteins goosecoid, M ix .l and Xotx2. These hom eodom ain proteins w ere shown to dow n regulate expression o f X bra by direct interaction

(27)

with B icoid and A ntennapedia binding sites in its 5'-flanking sequence

(Latinkic et a l, 1997). A second study also identified goosecoid as a repressor o f Xbra but associated with a different binding site in the 5'-untranslated region o f Xbra (A rtinger et a l , 1997).

In Ciona, Ci-Bra is only expressed in the notochord and transcriptional repression is essential for this specific expression pattern. A 434 bp m inim al

enhancer has been identified 5' of the putative TA TA elem ent which contains

three regulatory domains; a distal repressor elem ent that excludes expression

in m esenchym e and muscle, a central elem ent required for notochord

expression and proxim al elements which m ediate expression in ectopic

m esoderm al lineages. Activation of the central elem ent possibly involves a

regulatory elem ent related to the recognition sequence o f the Suppressor o f H airless [Su(H)] transcription activator. Su(H), is localised in the nucleus in response to activation of the Notch receptor (Corbo et a i , 1997). In addition, two binding sites for the Ciona snail gene (Ci-sna) were identified in the 434 bp enhancer. Ci-sna is expressed in the tail m uscles and it was suggested that it directly represses Ci-Bra expression in those m uscles (Fujiwara et a l ,

1998

^

In the mouse, 430 bp of proximal 5' flanking sequence is sufficient to

direct T expression in the prim itive streak. Two distinct regions have been identified; elem ents necessary for initiation o f expression in the first

gastrulating cells are found betw een nucleotides -4 3 0 and -2 8 0 and elem ents

required for expression in the later stages are found betw een nucleotides -2 8 0

and -1 9 0 . Thus far, sequences which confer expression in the head process

and notochord have not been identified in 8.3 kb o f 5' sequence and 5 kb o f 3'

sequence, suggesting that additional cw-acting sequences are necessary for T

expression in those regions. Thus, axial and non-axial expression o f T, and early and late prim itive streak expression, are regulated by different

(28)

1.1.4 T as a transcription factor

T protein is localised in the nucleus (Schulte-M erker et al., 1992; Cunliffe and Smith, 1994; Kispert and Herrmann, 1994; Schulte-M erker et al.,

1994). This localisation is mediated by nuclear localisation sequences w hich,

in the mouse, are betw een amino acids 137 and 320 (Fig 1.1; K ispert et al.,

1995a).

The consensus DNA binding site for mouse T protein was identified by

in vitro selection o f DNA binding sites from a pool o f random 26-m er oligonucleotides. This is a nearly palindrom ic consensus sequence o f 20 bp

with a core sequence A G GTG (TC/GA CACCTA G G T G T G A A A T T ). It was

shown that a half-site o f 12 bp (underlined) was not sufficient for binding in vitro and a m inim um o f six bases on one half-site in com bination w ith a com plete second half-site was required (Kispert and Herrm ann, 1993). C o

transfection experim ents showed that T can transactivate reporter genes

through binding to variously oriented and spaced half-sites. In general, T was

shown to transactivate better through binding to directly repeated dodecam ers

than inverted repeats, although, the highest activity was obtained through

binding to an inverted repeat of the dodecam er with a 24 bp spacer (K ispert et al., 1995a). A perfect palindrom e o f the half-site, flanked by Sm al half-sites (BS.p: 5 -G G G A A TTTCA CA CCTA G G T G T G A A A T T CCC-3 ) has been

w idely used in DN A /protein binding studies. Xenopus, zebrafish and

am phioxus T proteins also bind to the same BS.p DNA target in vitro (K ispert and Herrm ann, 1993; Herrm ann and Kispert, 1994; Holland et al., 1995).

T binds to its target DNA via the N-term inal 229 am ino acid residues,

the T dom ain or T-box (F ig l.l). Deletion of amino acids from the N- or C-

term inal o f this dom ain weakens or abolishes binding. In vitro D N A /protein binding studies have shown that T protein can bind to the BS.p palindrom e as

a m onom er, although, in this early work, the possibility that T can bind as a

dim er in vivo was not excluded (Kispert and Herrmann, 1993). D uring the course o f the project described here, the crystallographic structure o f the T

dom ain o f X bra in com plex with a 24 bp palindrom ic D N A target was

(29)

described and dem onstrated that T binds to DNA as a dim er (M üller and

Herrm ann, 1997). However, other studies have shown that X bra can regulate

transcription through binding to sites that com prise half of the BS.p

palindrom ic DNA target (Casey et al., 1998; Tada et al., 1998). These findings are discussed further in section 5.5.1.

Co-transfection experiments and the use o f heterologous systems

showed that T has a m odular structure. In addition to the N -term inal DNA

binding domain, the C-terminal half of the mouse protein contains two

transactivation (TA) domains, which act autonom ously and have an additive

effect; T A l, amino acids 2 3 0 -2 8 0 and TA2, amino acids 313-380. There are

also two repression (R) domains; R l, amino acids 2 8 1 -3 2 0 and R2, am ino

acids 4 0 1 -4 3 6 (Fig 1.1). The exact boundary betw een R l and TA 2 is not

know n (am ino acids 313-320; Kispert et al., 1995a).

Using a sim ilar approach it was shown that Xenopus Xbra and zebrafish Zf-T proteins also activate expression of reporter genes. However, a single

transactivation dom ain was identified in the carboxyl-term inal of these

proteins, m apping in X bra between amino acids 303 and 387, and in Zf-T

betw een am ino acids 312 and 407 (Conlon et al., 1996). Interestingly, the single transactivation domains of X bra and Zf-T map to the region of the

m ouse TA 2 domain, suggesting that this domain has been better conserved in

evolution than T A l.

The m odular structure o f T has been confirm ed using various m utant

forms o f the protein. For example, when proteins corresponding to the

zebrafish no tail m utations ntl^^^^ and ntf^^^ were used in cotransfection experim ents, no transactivation was observed (Conlon et al., 1996). ntl^^^° protein is 245 amino acids long and, although it retains its DNA binding

activity, it lacks a transactivation domain, ntl'’^^^ contains only 103 am ino

acids of the Zf-T protein, and thus lacks both a transactivation dom ain and an

(30)

the D rosophila engrailed protein is unable to activate transcription in transfection assays but competes with w ild-type X bra protein for D N A

binding (Conlon et a l , 1996).

This structure/function data for T protein bas been determ ined in vitro

and in cultured cell systems and therefore, should be treated with som e caution

since they m ight not necessarily mimic the situation in vivo. For exam ple, Rao (1994) showed in Xenopus animal cap assays that a truncated X bra protein o f 344 am ino acids, which lacks the C-terminal half o f the

transactivation dom ain, behaves like full-length X bra and induces m esoderm

form ation. Further truncation of X bra to 304 amino acids (B304) resulted in a

change from m esoderm to neural induction activity, w hich appears to

antagonise the m esoderm inducing activity o f full-length Xbra. Furtherm ore,

in the presence o f FG F or a low concentration of activin, the neuralising

activity o f B304 is increased.

The identification of target genes regulated by T has been relatively

slow. Very recently, using oocyte and cell culture transfection systems, X bra

has been show n to directly activate the expression o f eFG F (Casey et a l,

1998) and B ix l {Brachyury induced hom eobox gene 1; T ada et al., 1998). M is-expression o f B ix l can induce m esoderm or endoderm form ation in anim al pole tissue in a dose-dependent fashion and ventralisation o f dorsal

m esoderm in the m arginal zone.

1.1.5 The function of T and its role in embryogenesis

A m ajor source of inform ation about the in vivo function o f T protein comes from studies o f the phenotypes o f Tm utant and chim aeric mice.

The m ost com m only studied Tm utant is T Brachyury

w hich is due to a large deletion (1 8 0 -2 0 0 kb) encom passing the entire gene

and extensive flanking sequences. Hom ozygous m ice die at m id

gestation due to a defective allantois. The prim itive streak is thickened and

the node is not properly formed. As a result, somites posterior to the seventh

pair are absent or abnorm al and the notochord does not form. T acts in a dose

(31)

dependent fashion such that heterozygous m ice have short tails and the

notochord in the caudal region is branched or fused with the neural tube or gut

(Grüneberg, 1958). Thus, with the exception o f the allantois, the expression

pattern of T is consistent with the m utant phenotype (see section 1.1.2).

Sim ilarly, zebrafish hom ozygous no tail mutants (functional T null alleles) are lethal and lack a tail, a differentiated notochord and more than one third o f the

m ost posterior somites (H alpem et al., 1993; Schulte-M erker et al., 1994). These observations suggest that T plays a role in norm al posterior m esoderm

form ation and notochord differentiation.

The short-tail phenotype of the heterozygous m ice can be

rescued by the addition of a single copy o f a T transgene, verifying the dose

dependent action of T. Furtherm ore, transgenic studies suggest that a dosage

o f T higher than normal can increase embryonic lethality (Stott et al., 1993). It was proposed that over-expression of T could lead to aberrant cell type specification in the mesoderm, eventually leading to death. Interestingly, the

prom oter used to drive the expression of the transgene in these experim ents

has subsequently been shown to drive expression only in the prim itive streak

(Clem ents et al., 1996).

Studies o f other T mutants, which are not due to large deletions, also highlight the im portance of T dosage and led to hypotheses that T interacts with other proteins. is a m utant due to an insertion o f a retroviral-like

elem ent in the seventh exon and 7^ involves a 19 bp deletion in exon 8

(review ed in Herrm ann and Kispert, 1994). Both these m utations alter the

open reading fram e resulting in proteins that retain their D N A binding

property but are truncated at the carboxy terminus of the regulatory domain.

T^*® is lacking the TA 2 and R2 domains and T^ is lacking the R2 dom ain (see

previous section). Both and T m utant m ice exhibit a m ore severe phenotype than mice; homozygotes have no somites, the rostral

boundary o f em bryonic defects extends into the cervical region and

(32)

(M acM urray and Shin, 1988; Herrmann, 1991; K ispert and Herrm ann, 1994).

In addition, the T / + phenotype, but not the 7^/7^ phenotype, is dependent on the num ber of wild type T copies and two are required to rescue the tailless phenotype (M acM urray and Shin, 1988; Stott et a l , 1993). It is possible that

and proteins antagonise the wild type protein, effectively reducing the

am ount o f functional protein. There is an alternative explanation for the m ore

severe phenotype o f and ' f . If T interacts with other proteins to form a transcriptional complex, then a truncated T protein, which nevertheless binds

DNA, could interfere with the assembly or function o f this transcriptional

com plex. This interference in not a feature of mutants w hose T protein is

com pletely absent.

Further clues about the way that T protein exerts its influence on

m esoderm cell differentiation and m igration come from studies o f chim aeric

m ice generated by injecting embryonic stem (ES) cells into

blastocysts. chimaeras with high levels (>70% ) o f null alleles

are alm ost indistinguishable from intact hom ozygous mutants, w hereas low

level chim aeras show a range of defects that can vary from a severe

phenotype, sim ilar to that of mice, to localised defects in the tail

(truncation, branching) or allantois. In all chimaeras there is a rostrocaudal

increase in T null cell contribution and the level of chim aerism in the

neuroectoderm is higher than that in paraxial mesoderm. W here heterozygous

jSnuii/_^ cells w ere used to generate chimaeras, 7^”“^V+x+/+ mice are norm al with m ild tail defects (kinking, abnormal tail tip) and cells can colonise

rostral regions efficiently. The phenotypes of these chim aeric m ice support

the idea that T acts in a dose dependent, cell autonom ous w ay (Rashbass et al.,

1991; Beddington et a l , 1992; W ilson et al., 1993; W ilson et al., 1995). Careful analysis o f chimaeras and com parison with wild type and

jBnuiiijBnuii j^ytants showed that the cells fail to m igrate aw ay from the prim itive streak and subsequently accum ulate in the node, prim itive

streak and later in the tail bud, leading to abnorm alities in the notochord and

(33)

posterior m esoderm (Rashbass et a l , 1991; Beddington et al., 1992; W ilson et a l , 1993; W ilson et a l , 1995). It was suggested that T affects cell m igration by regulating genes involved in cell adhesion, an idea supported by other

observations. Brachyury m ouse mutants have a thickened prim itive streak and a reduced m esoderm /ectoderm ratio, which appears to be due to abnorm al cell

m ovem ent during gastrulation (Y anagisawa et a l , 1981). In a separate series o f cell culture experim ents (reviewed in W ilson et a l , 1993), it was shown that w hen cells were com pared with + /+ cells, they show reduced

m otility on extracellular m atrix and form smaller aggregates. However,

jSnuiiijBnuii tiave the same rate o f DNA synthesis and m itotic index as wild type cells, and in culture, they have the same differentiative capacity and

their life-span extends beyond the tim e of death in the embryo.

Thus, it seems unlikely that the abnormal behaviour o f cells in vivo can be explained by differential cell growth, aberrant cell differentiation, or low

survival time.

Interestingly, a 5 integrin deficient mice resem ble hom ozygous

Brachyury m ice (Yang et a l , 1993). a5(3l integrin is a cell surface receptor that binds fibronectin and is thought to be involved in m any cellular processes

including cell migration. In hom ozygous a 5 integrin null m ice the anterior

part o f the embryo from somites 1 -1 0 develops normally, but the posterior

regions are highly defective. Posterior somites are not form ed, paraxial

m esoderm is reduced and the neural tube is kinked. The notochord is formed,

but it is not know n if it is functional.

Analysis of chimaeras of wild-type cells and cells carrying a

w ild type T transgene, showed that the expression o f T in cells restores their normal behaviour in the prim itive streak and tail bud.

Furtherm ore, these experiments suggested that the level o f T expression determ ines the speed with which cells move away from the prim itive streak.

(34)

streak, w hereas cells with low levels of T tend to stay in the prim itive streak

for a longer time and populate the tail bud (W ilson and Beddington, 1997).

M uch o f w hat has been proposed regarding T and m esoderm

differentiation and migration refers to the situation in the prim itive streak and

node. However, it is not clear how this applies to the notochord or tail bud.

A lthough, the supply of cells to the axial mesoderm, and thus the notochord, is

directly dependent on the prim itive streak and node, it is likely that T protein

m ay have a distinct function in the differentiation and survival of notochordal

cells. D isruption o f these functions would lead to the notochord abnorm alities

seen in zebrafish and mouse T mutants and chim aeric m ice (Beddington et al.,

1992; W ilson et al., 1995). Similarly, although the prim itive streak is directly related to the tail bud, it has been proposed that T has a distinct role in tail bud function and affects tail differentiation and elongation. It follows from this

proposal that the abnormal tail m esoderm form ation seen in m ice is

a prim ary outcom e o f T deficiency and not a secondary response to a

degenerate notochord (W ilson et al., 1993; W ilson et al., 1995).

The im portance o f T in m esoderm form ation is also highlighted by

expression studies in Xenopus. O ver-expression of Xbra in animal caps cause the ectopic form ation of posterior mesoderm. M esoderm al markers that are

indirectly activated by X bra are Xsna, a zinc-finger transcription factor,

m uscle-specific actin and Xhox3, a hom eobox transcription factor expressed in posterior m esoderm (Cunliffe and Smith, 1992). The type o f m esoderm

form ed depends on the dose o f Xbra RNA, with low am ounts inducing m ésothélial smooth m uscle and m esenchym e and high am ounts inducing

somitic m uscle (O'Reilly et a l , 1995).

Interestingly, the outcom e of Xbra over-expression is influenced by co expression with a variety o f other transcription factors and inducers, im plying

that in vivo several genes co-operate with Xbra to specify dorsal m esoderm . These collaborating factors probably include Pintallavis, a m em ber o f the forkhead/H N F3p transcription factor fam ily (O'Reilly et al., 1995), noggin, a bone m orphogenetic protein (BMP) antagonist and Xwnt-8, a m em ber o f the

(35)

w nt fam ily o f secreted glycoproteins (Cunliffe and Smith, 1994). Changing

the level of co-expression and the order o f expression of these factors

influences whether muscle, notochord and/or neural tissue are form ed and

w hether dorsalisation of m esoderm occurs.

Recently, expression studies in the ascidian Halocynthia roretzi have shown that the role o f T in notochord form ation is probably conserved in all

chordates (Yasuo and Satoh, 1998). O ver-expression of A s -T is sufficient for notochord differentiation of notochord precursors in a cell-autonom ous way.

However, in a sim ilar way to Xbra, the response to over-expression in non notochord lineages depends on the presence of other factors.

1.1.6 The T-box transcription factors

r is a m em ber of a growing fam ily of transcription factors, the T-box

genes. Thus far, 13 T-box genes have been identified in m am m als; T, Tbxl-8, TbxlO, T bxlS, T brl and Eomes (Herrm ann et al., 1990; Bollag et al., 1994; Bulfone et a l , 1995; Agulnik et a l , 1996; A gulnik et al., 1998; Law et al.,

1998; W attler et al., 1998). The hom ologues of 8 of these genes have been cloned in hum ans; T, TBXl-3, TBX5, TBXIO, TBX15 and T B R l (Bulfone et a l ,

1995; Cam pbell et a l , 1995; Edwards et a l , 1996; Bam shad et a l , 1997; Basson et a l , 1997; Chieffo et a l , 1997; Li et a l , 1997; A gulnik et a l , 1998; Law et a l , 1998). The identification of T-box genes in other species as

divergent as C.elegans, Drosophila, Xenopus, chicken etc. suggests that the T- box fam ily of transcription factors is evolutionarily conserved. Phylogenetic

analysis and com parison o f the genomic structure o f the T dom ain have shown

that the T-box genes can be divided into subfamilies. It has been proposed

that m ost anim al species are likely to have at least five T-box genes and these

will be related to the T, T b xl, Tbx2, Tbx6 and T brl of the m ouse (Papaioannou and Silver, 1998).

The characteristic feature of T-box proteins is the DNA binding

domain, a conserved region o f 180-200 amino acid residues, w hich although

(36)

relative to the 5' end. Both DNA-binding and gene activation properties have

been directly dem onstrated in T hom ologues from mouse, Xenopus and

zebrafish (Kispert and Herrmann, 1993; Herrm ann and Kispert, 1994; K ispert

et a l , 1995a; Conlon et a l , 1996), in mouse Tbx2 (Carreira et a l , 1998) and in Xenopus V egT (Zhang and King, 1996; Tada et a l , 1998). DNA binding has been dem onstrated for Drosophila omb (Pflugfelder et a l , 1992) and am phioxus A m Bra (Holland et a l , 1995) and transcriptional regulation has been shown for the zebrafish spadetail, spt (Griffin et a l , 1998). A lthough there is no other direct evidence for the transcriptional properties o f other T-

box proteins, it is likely that they will all have DNA -binding and

transactivation properties.

M ost inform ation on the expression patterns o f different T-box genes

comes from mouse and chicken, but it is reasonable to assum e that these

patterns would be sim ilar in other species, taking into account differences in

m orphology and tim e of development. Indeed, w here orthologues in different

species have been identified, the expression patterns are similar.

The T-box genes have distinctive but overlapping patterns of

expression. They are generally most abundant during em bryonic

developm ent, although some persist in adult tissues (for exam ple Tbx2 and

Tbx3, Table 1.1). A t the time of murine organogenesis there are at least two T-box genes expressed in all the m ajor organs, but not necessarily in the same

cell type (Bollag et a l , 1994; Bulfone et a l , 1995; A gulnik et a l , 1996; Chapm an et a l , 1996b). For example, Tbx2-5 are expressed in lung

m esenchym e, whereas, T bxl is expressed in lung epithelium . An overlap in the expression pattern of different genes is evident in m em bers o f the same

subfam ily; for exam ple Tbx2 and Tbx3 are coexpressed in kidney and

peripheral nervous system (PNS), whereas, Tbx4 and Tbx5 are coexpressed in extraem bryonic m esoderm and heart atrium (Table 1.1).

The im portance of the T-box genes in hum an developm ent has been

highlighted by the identification o f disease-associated m utations in two genes.

M utations in TBX3 cause Ulnar-M am m ary syndrome, an autosom al dom inant

(37)

Stage & Tissue T Tbxl Tbx2 Tbx3 Tbx4 Tbx5 Tbx6 T brl

Blastocyst

Inner cell mass - - - + - -

-Gastrulation

Extraembryonic mesoderm - - - + + +

-Embryonic mesoderm + + - - - - +

Notochord + - - - - -

-Organogenesis

CNS - - + + - - - 4

-PNS - - + + - - -

-Body W all, thorax - - - - - + -

-Cartilage/skeleton - + + + - - -

-Eye - - + + - + -

-Genital papilla - - + + + + -

-Genital ridge - - + + - - -

-Heart

Atrium - - - - + + -

-Ventricle - - - - - + -

-Kidney - - + + - - -

-Limbs

Forelimb - - + + + + -

-Hindlimb - - + + + - -

-Lungs

M esenchym e - - + + + + -

-Epithelium - + - - - - -

-Mammary gland

M esenchym e - - + - - - -

-Epithelial buds - - - + - - -

-Mandible m esenchym e - - + + + + -

-M axilla m esenchym e - - + + - - -

-M uscle - - + + - - -

-Optic vesicle - - + + - + -

-Otic vesicle - + + + - - -

-Tail + - - - + - +

-Adult

Brain - - - + +

Heart - - + +

Kidney - - + +

Liver - +

Lungs - - +

Ovaries - - + +

Testes - + -

The human T transcription factor: A study of genetics and function