Identification of factors involved in the maintenance of embryonic stem cell self renewal and pluripotency

Intracellularly, the transcriptional regulatory circuit governed by OCT4, SOX2 and NANOG, have also been established as being crucial for maintaining ES cells in an undifferentiated stat

Chapter1

Introduction

1.1 Embryonic stem cells

1.1.1 Derivation and definition of embryonic stem cells

During the transition of an embryo from the morula to the blastocyst stage, a differentiation event partitions a developing embryo into its extraembryonic and embryonic components The outer layer of cells of the blastocyst is the extraembryonic components and forms an epithelium, the trophectoderm Descendents of the trophectoderm are restricted to the generation of the trophoblast components of the placenta The embryonic component, located on the interior is referred to as the inner cell mass (ICM) and this embryonic component comprises a stem cell population The ICM represents a unique transitory cellular structure that differentiates into epiblast (also known as embryonic ectoderm) derived cell types, namely the mesodermal, endodermal and ectodermal lineages in a regulated manner during the course of embryo development Alternatively, the ICM may differentiate into the primitive endoderm which gives rise to extraembryonic tissues instead of the pluripotent epiblast during this other differentiative event The three germ layers are the precursors to all tissues of the body Ectoderm develops into the epidermis, retina, brain and nervous system etc, while the mesoderm gives rise to the bones, muscles and most of the cardiac and circulatory system Endoderm gives rise to the respiratory organs and gastrointestinal tract Cells of the ICM also have the potential to develop into germ cells (Chambers and Smith, 2004)

Of great significance is the fact that explant cultures of ICM can generate pluripotent embryonic stem (ES) cell lines The derivation of pluripotent cell lines from blastocysts was first achieved in the murine system in 1981 by Martin Evans and Matthew Kaufman and independently by Gail R Martin (Evans and Kaufman, 1981; Martin, 1981) Subsequently, successful derivation and propagation of rodent, rabbit, primate and human ES cell lines have been reported (Fan and Collodi, 2006; Iannaccone

et al., 1994; Reubinoff et al., 2000; Thomson and Marshall, 1998; Vackova et al., 2007; Thomson et al., 1998)

While ES cells are generally considered an in vitro phenomenon and are not true

equivalents of the ICM, ES cells are at least ICM-like in terms of their ability to differentiate into cells of all three germ layers This pluripotent nature of mouse ES cells was first demonstrated by their ability to contribute to all tissues of adult mice, including the germ line following injection into host blastocysts Also, these cells can also form teratomas - benign tumours which consist of a mixture of differentiated cell types from the three germ layers following ectopic engraftment to immune-compromised mice

(Reubinoff et al., 2000) In the in vitro scenario, ES cells likewise display a remarkable

capacity to form a plethora of differentiated cell types of the three germ layers in culture

Another defining feature of ES cells is their acquired capacity to proliferate

indefinitely in vitro without undergoing senescence while retaining a normal karyotype

This characteristic, coupled with the potential to differentiate into more than 200 unique cell types render ES cells a very attractive cell source for potential use in regenerative

medicine The successful isolation and propagation of human ES cells in November 1998

by Thomson et al further brought this promise of ES cell based therapy one step closer to realization (Thomson et al., 1998)

1.1.2 Applications of ES cells

ES cells can contribute to the formation of chimeric organisms following engraftment into a host ICM, and continue to give rise to all three germ lineages This potential for germline transmission in chimeras, coupled with the amenability of mouse

ES cells to genetic manipulation enable the generation of knock-out mice As such, mouse ES cells have since found widespread applications in applied pharmacogenetics and basic functional genomics research (Pease and Williams, 1990; Tesar, 2005; Voss et al., 1997; Wolf et al., 1994)

As for the potential therapeutic application of ES cells, ES cell based therapies and transplantation have been the most frequently discussed This has yet to be realized

in clinic due to caveats such as less than optimal efficiency of in vitro differentiation, the

danger of tumorigenecity of transplanted tissues and graft versus host response triggered

in recipients upon recognition of non-autologous hES cells-derived cells as foreign Once these limitations can be overcome, it is likely that monocellular deficiency states such as Parkinson's disease and type I diabetes will be among the first examples of intractable diseases that can be rectified through ES cell based replacement therapies (Burns et al.,

Apart from cellular therapy, ES cell technology can also prove to be very useful

in many other aspects of medicine For example, the availability of ES cell lines, coupled with the recent development of various differentiative and purification regimes for the generation of a broad spectrum of lineages from ES cells, have opened up exciting

opportunities to model mammalian embryonic development in vitro The study of events

regulating the earliest stages of lineage induction and specification is tedious in mouse embryos and prohibited in the human embryo due to ethical concerns ES cell based models provide a convenient means around these limitations Understanding the events that occur at the first stages of development has potential clinical significance for preventing or treating birth defects, infertility and pregnancy loss A thorough knowledge

of normal development could ultimately allow the prevention or treatment of abnormal human development For instance, testing drugs on cultured human embryonic stem cells could help reduce the risk of drug-related birth defects (Seiler et al., 2006)

The understanding gained from the study of stem cell biology may also profoundly improve the treatment of cancer as mechanistic links between ES cell self-renewal and cancer stem cell proliferation might make it possible to improve the treatment of cancer by targeting inappropriately activated self-renewal pathways

Investigation of a number of human diseases is severely constrained by a lack of animal and cell culture models For instance, a number of pathogenic viruses including human immunodeficiency virus and hepatitis C virus grow only in human or chimpanzee

cells Primate and human ES cells might provide cell and tissue types that will greatly accelerate investigation into some of these viral diseases (Yamamoto et al., 2003)

In short, the prospect for medical application of ES cell technology is vast and promising One great impediment to the unleashing of the immense potential of ES cells

is however the current incomplete understanding of the molecular mechanism underlying self renewal and cell fate determination of ES cells

1.2 Molecular basis underlying ES cell self renewal

Despite the importance of stem cell self renewal, we are only beginning to understand how it is regulated ES cell self renewal is a complex process that involves both the proliferation and maintenance of pluripotency Self renewal is an intricate interplay instigated by instructive and permissive instructions provided by signaling molecules in the microenvironment and also by intracellular regulators such as transcriptional factors Multiple factors are required to act in concert to maintain the embryonic stem cell phenotype Some factors regulate only proliferation, while others regulate developmental potential and prevent differentiation Some determinants regulate proliferation and inhibit differentiation (Molofsky et al., 2004)

To date, several key signaling pathways, such as the LIF/gp130/STAT3, bone morphogenetic protein (BMP) and wingless-type MMTV integration site (WNT) have been established as important pathways maintaining ES cell self renewal and preventing

cell differentiation Intracellularly, the transcriptional regulatory circuit governed by OCT4, SOX2 and NANOG, have also been established as being crucial for maintaining

ES cells in an undifferentiated state

1.2.1 Extrinsic regulators and signaling pathways governing ES cell self renewal

1.2.1.1 LIF/gp130/STAT3 pathway

When mouse ES cells were first derived in the 1980s, they were propagated in culture with a layer of fibroblast in the presence of serum An indication that fibroblasts act by secreting a signal that inhibits ES cell differentiation was substantiated by the ability of Buffalo rat liver cell line conditioned medium to replace the fibroblast requirement (Smith and Hooper, 1987) Leukeamia inhibitory factor (LIF) was subsequently identified to be the active component of the conditioned medium through

co-fractionation (Smith et al., 1988) Fibroblasts carrying deletions in Lif were also found to

have reduced capacity to support ES cells This further supports the fact that LIF is a major determinant of the ability of feeders to support ES cell self renewal Today, most investigators culture mouse ES cells feeder free in the presence of serum with supplementation of LIF

LIF is a member of the IL6 family of cytokines that signals through the transmembrane receptor, gp130 LIF maintains ES cell self renewal through the activation of STAT3, a member of the signal transducer and activator of transcription

(Stat) family (Raz et al., 1999) Evidence strongly indicates that STAT3 is the key

transcription factor downstream of the LIF/gp130 pathway Forced expression of a

dominant negative Stat3 mutant caused mouse ES cell differentiation even in the

presence of LIF (Niwa et al., 1998) Also, point mutation of the tyrosine residue of gp130 responsible for STAT3 binding abrogated the ability of LIF to maintain self renewal It was also shown that mouse ES cells expressing a fusion molecule consisting of STAT3 and estrogen receptor could be maintained in the presence of estrogen derivative tamoxifen (Matsuda et al., 1999) which translocates the fusion LIF to the nucleus

The first event involved in the LIF signaling cascade involves binding of LIF to the LIF receptor (LIFR) that contains a long cytoplasmic tail with a homology to the gp130 The LIF/LIFR complex then recruits gp130 to form a trimeric complex (Zhang et al., 1997) Heterodimer formation of the LIF receptor and gp130 receptor then results in the activation of the tyrosine kinase JAK The activated JAK phosphorylates tyrosine residues of gp130 which then serves as a docking site for STAT3 STAT3 is then activated and translocated into the nucleus to elicit transcriptional responses that prevent differentiation (Niwa et al., 1998)

LIF and LIFR are expressed in blastocysts and expression of LIFR can be detected in the ICM However, mutant embryos deficient in LIF/gp130/STAT3 signaling

forms normal ICM Lif deficient mice exhibit normal development (Stewart et al., 1992), while Lifr deficient mice showed perinatal lethality (Li et al., 1995; Ware et al., 1995) Embryos deficient in gp130 die progressively between dpc 12.5 and term Stat3 deficient

embryos developed into egg cylinder stage until embryonic day 6.0 and rapidly degenerate between E6.5-7.5 Lack of phenotype pertaining to pluripotency in LIF/gp130/STAT3 suggests other mechanisms are normally responsible for maintenance

of pluripotency in vivo In fact, a role for LIF/gp130 becomes only apparent for the

prolonged survival of blastocysts during diapause Diapause is a physiological adaptation

to the presence of suckling litter that allows embryos to persist for several weeks without implantation Upon cessation of suckling, the embryos implant in the uterus and

development proceeds normally gp130 mutants however lose the epiblast component

after 6 days in delay and can no longer generate a foetus upon implantation (Nichols et al., 2001) This observation suggests LIF/gp130/STAT3 pathway is not fundamental for

pluripotency, but instead functions primarily to extend the period of pluripotency in vivo

The LIF/gp130/Stat3 pathway also appears to be dispensable for the maintenance

of pluripotency and self- renewal of ES cells (Daheron et al., 2004; Ying et al., 2003)

Even though Lifr -/- mouse ES cells are less pluripotent than wildtype, it was found that undifferentiated colony formation was not completely inhibited This indicates that there are LIFR-independent means by which fibroblast can support mouse ES cell self renewal Indeed, this may truly be the case as maintenance of several mouse ES cell lines does not require LIF Also, it has been reported that LIF is not necessary for human ES cell culture and that maintenance of pluripotency in human ES cells is STAT3 independent (Daheron

et al., 2004; Thomson et al., 1998) Furthermore, STAT3 is expressed in a wide range of cell types, and in some cases drives differentiation (Hirano et al., 2000) Therefore, other

core pathways and mechanisms that maintain pluripotency and ES cell self renewal may exist

1.2.1.2 Bone morphogenetic factor 4 and BMP signaling

In ES cell media without LIF, there is limited self renewal of mouse ES cells and the induction of neural differentiation It has recently been demonstrated by Ying et al that the requirement for serum can be replaced by Bone Morphogenetic Factors (BMP) 4 BMP4 treatment suppresses neural differentiation and in combination with LIF, is sufficient to sustain ES cell self renewal without feeder or serum factors (Ying et al., 2003) This concurs with the fact that BMP signaling inhibits premature neural differentiation in the mouse embryo (Di-Gregorio A et al, 2007) The mechanistic action

of BMP signaling has been attributed to the activation of inhibitor of differentiation (Id) genes by the downstream signal transducers, SMAD1/5/8 Forced Id1, Id2 and Id3

expression did not impair ES cell self-renewal nor block differentiation in the presence of

serum ES cells transfected with Id1, Id2 and Id3 can however self-renew in serum-free

culture containing LIF strongly, thereby suggesting that BMP/SMAD1/5/8 acts through Id1/2/3 proteins Id proteins exert a neuroectoderm lineage-specific block on ES cell differentiation by preventing precocious expression of proneural basic helix loop helix

transcription factors as the Mash genes It could also be that IDs exert their effect by

interaction with non bHLH proteins such as the PAXs

The role played by BMP signaling in embryonic stem cells is however disparate

in mouse and human (Varga and Wrana, 2005) BMP signaling appears to play a differentiation related role in the human context Firstly, analysis of a hES cell line cultured on Matrigel-coated plates in fibroblast-conditioned media showed that in the continuous presence of FGF signaling, BMP induces hES cells to differentiate into the trophoblast lineage (Xu et al., 2002) It has been found that blocking BMP activity in serum with the BMP antagonist Noggin does not maintain human ES cell self-renewal, but instead enhances neural differentiation by inhibiting non-neural differentiation (Pera

et al., 2004) Also, by culturing cells in the absence of conditioned media but in the presence of high levels of bFGF, and with noggin to block BMP signaling, hES cells can

be maintained in a pluripotent state (Xu et al., 2005) Thus, one common thread arising from analysis of mouse and human systems is that BMPs have maintained an evolutionarily conserved role to block neural differentiation in early embryo and ES cells

WNT signaling pathway to support the transient self renewal of both mouse and human

ES cells Activation of the WNT pathway by 6-bromoindirubin-3’-oxime (BIO), a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK3), maintains self renewal and markers of pluripotency in both mES and hES cells The actions of BIO are functionally reversible and BIO withdrawal leads to normal differentiation Future more,

the over-expression of Wnt1 or stabilized β-catenin or abrogation of the APC complex

leads to the inhibition of neural differentiation, mediated by the activation of downstream

target genes that includes Cyclins and c-Myc (Aubert et al., 2002)

However, an understanding of how exactly the various components of the WNT

signaling pathway affect the transcriptional activity of key pluripotency genes like Oct4 and Nanog, is still lacking It is also not yet known which of these WNT-regulated genes

are fundamentally important for sustaining ES cell self renewal

1.2.2 Intrinsic regulators and mechanism governing ES cell self renewal

1.2.2.1 Oct4

Oct4 (also known as Oct3 or Pou5f1) is a POU (Pit, Oct, Unc) family

transcriptional regulator of genes required in maintaining an undifferentiated pluripotent state Its expression is restricted to early embryos, primordial germ cells, undifferentiated

ES cells and embryonal carcinoma (EC) cells In vivo, expression of Oct4 occurs in the

unfertilized egg and the early embryo prior to segregation of the ICM from the

trophectoderm (Pesce and Scholer, 2001) Subsequent to the allocation of ICM cells,

Oct4 mRNAs and proteins are readily detected in the ICM, but downregulated in the trophectoderm Oct4 expression is maintained in epiblast of pre- and post-implantation

embryos before becoming restricted to the migratory primordial germ cells where it persists throughout the formation of genital ridges in both sexes (Nichols et al., 1998)

In vivo, Oct4 is necessary for pluripotency Zygotic expression of Oct4 is required for the development of the pluripotent ICM Oct4 deficient mouse embryos are

embryonic lethal Such mutant embryos can develop to a blastocyst-like stage, but lack genuine ICM The ICM is not pluripotent and is instead restricted to differentiation along

the trophoblast lineage Oct4 is also necessary for pluripotency in vitro ES cell lines cannot be generated from Oct4 -/- embryos (Nichols et al., 1998) Oct4 is highly expressed

in both human and mouse ES cells, and its expression is markedly reduced during

differentiation Niwa et al found that the precise level of Oct3/4 governs three distinct

fates of mouse ES cells A less than twofold increase in expression causes differentiation

into primitive endoderm and mesoderm In contrast, repression of Oct-3/4 mRNA level

by more that 50% induces a loss of pluripotency and differentiation to trophectoderm

(Niwa et al., 2000) OCT4 RNAi in human ES cells also recapitulates a similar

differentiation pattern of human ES cells to trophectoderm (Matin et al., 2004)

As a transcription factor, OCT4 can act to activate or repress gene expression in

ES cells Recent works have also confirmed that OCT4 is at the helm of the intricate

cascades of genetic events that orchestrates ES cell pluripotency For example, Utf-1 and

Nanog are pluripotency-related genes that have been shown to be regulated by OCT4,

through a direct cooperative interaction with another ES cell transcription factor, SOX2 (Rodda et al., 2005) An auto-regulatory circuit of the SOX2/OCT-3/4 complex also

contributes to maintaining robustly, the precise expression level of Oct3/4 itself in ES

cells (Chew et al., 2005)

The maintenance of Oct4 expression, although necessary, is however not sufficient in itself to sustain the pluripotent phenotype In mouse ES cells, Oct4 over-

expression is insufficient to block differentiation in absence of LIF OCT4 must act in combination with factors whose activity is influenced by the STAT3 pathway activated

by Leukemia Inhibitory Factor (LIF) to prevent ES cell differentiation

In summary, Oct4 is necessary for pluripotency both in vitro and in vivo The level of Oct4 controls the first cell fate decision (trophoblast vs epiblast lineage) undertaken by the mammalian embryo As for ES cells, the function of Oct3/4 is to

specify cell fate into pluripotent cells or primitive endoderm cells by blocking trophectodermal differentiation

1.2.2.2 Sox2

Sox2 is a member of the HMG-domain DNA binding protein family It has been demonstrated that Sox2 has a role to play in maintaining epiblast pluripotency, as gene targeting used to inactivate Sox2 in the mouse causes defective primitive ectoderm

(Avilion et al., 2003) Recent findings reported by Kuroda et al and Rodda et al also shed light on the fact that Sox2 is critical for the ES cell state SOX2 is known to play key

roles in transcription of several OCT4 targets such as Fgf4 (Ambrosetti et al., 1997) Also, Nanog is regulated by OCT4, through a direct cooperative interaction with SOX2

Recently, it has been found that unlike OCT4, SOX2 appears to be localized in both the nucleiand cytoplasm in pre-implantation embryos and ES cells This subcellular localization pattern suggests that SOX2 shuttles between these two subcellular compartments and that its nuclearlocalization regulates its transcription activity SOX2 contains two nuclear localization signals(NLS) Li et al found that ablation of these two NLS results in a dominant-negativeform of SOX2 that loses its ability to cooperate with OCT4 When stably expressed, this dominant-negative form of mouse SOX2 induces progressive polyploidy and trophectodermal differentiation in ES cells Knockdown of

Sox2 by small interfering RNA (siRNA) similarly induces trophectoderm differentiation

and polyploid formation in mouse ES cells The notion that SOX2 operates primarily in

pluripotent cells as a co-factor of OCT4 is reinforced by the fact that the mutant Sox2 phenotype mirrors that of Oct4 deficient mutants In essence, SOX2 maintains ES cell

self renewal byshuttling between the nucleus and cytoplasm in cooperation withOCT4 to prevent trophectoderm differentiation and polyploidformation in ES cells (Li et al., 2007).

1.2.2.3 Nanog

Nanog is a homeodomain transcription factor that has been shown to confer upon

mouse ES cells, the ability to self renew without STAT3 activation (Chambers et al.,

2003; Mitsui et al., 2003) Identification of Nanog was first reported by both Mitsui et al

and Chamber et al independently in 2003 Mitsui et al employed digital differential display to compare expressed sequence tag libraries from mouse ES cells and those from

various somatic tissues Nanog was among the handful of genes that were found to be highly enriched in ES cells Interestingly, Mitsui et al found Nanog to specifically confer

upon mouse ES cells, the ability to self renew in the absence of LIF for a short period of

time when overexpressed As for Chamber et al, they first isolated Nanog through expression cloning They similarly found that Nanog can bypass completely the requirement for STAT3 activation and that maintained expression of Oct4 is integral to

its ability to sustain ES cell self-renewal Chambers et al further showed that

overexpression of Nanog can even cause mouse ES cells to be more resistant to induction

of differentiation with compounds such as 3-methoxybenzamide or all trans-retinoic acid

Since then, human NANOG has also been characterized and implicated to play a role in human ES cells Overexpression of NANOG in human ES cells has been shown to enable

feeder-free growth while inducing primitive ectoderm features (primitive ectoderm is the pluripotent population in the embryo derived from the ICM) (Darr et al., 2006)

In vivo, Nanog expression is detected in pluripotent embryonic tissues but not in adult tissues During mouse embryonic development, Nanog is first detected in the

compacted morula It then becomes localized to the ICM of the blastocyst, and is

subsequently restricted to the epiblast Just prior to implantation, Nanog is

down-regulated Chambers et al also reported that epiblast expression of Nanog persists in implantation delayed blastocysts, a favoured source for ES cell derivation Later during

embryo development, Nanog mRNA is present in primordial germ cells in E11.5 gonadal

ridges, the very cells from which pluripotent EG stem cells can be generated The

knockout phenotype of Nanog -/- embryo is a lack of epiblast and primitive ectoderm

formations Nanog deficient embryos only produce parietal endoderm like cells instead This is indicative of the absolute requirement for Nanog in directing both ICM and primitive ectoderm development Also, in contrast to Oct3/4 null ICM, no trophoblast differentiation was observed This data indicates that Nanog is essential for maintenance

of pluripotency at a stage after the first differentiative event of the ICM which requires

Oct3/4

In vitro, Nanog expression is likewise confined to pluripotent cell lines such as ES

cells, EG cells and EC cells It is now known that Nanog is regulated by OCT4, through a

direct cooperative interaction with SOX2 in ES cells Nanog null ES cells lose pluripotency and differentiate into the extraembryonic endoderm lineage A proposed

mechanism is that Nanog prevents differentiation of ES cells into primitive endoderm through the transcriptional repression of differentiation promoting genes such as Gata6 Gata6 overexpression was found to be sufficient in inducing extraembryonic endoderm

differentiation NANOG also maintain ES cell self renewal by regulating ES cell specific

genes For example, the Nanog consensus sequence was found in the enhancer region of

Rex-1, an ES cell related gene The transcriptional regulation of Rex-1 by NANOG was

also subsequently demonstrated (Shi et al., 2006)

1.2.3 Current dogma of ES cell self renewal

Figure 1 summarises the current paradigm used to explain how NANOG, OCT3/4, SOX2, and STAT3 cooperate to maintain pluripotency Preimplantation embryos and ES cells require both OCT3/4 and NANOG to prevent differentiation into trophectoderm and primitive endoderm, respectively However, these two transcription factors of normal expression level are not sufficient for ES cell self-renewal In the mouse system at least, additional factor(s), such as STAT3 activated by LIF, are required to support prolonged maintenance of pluripotency

The current dogma is that transcription factors, NANOG, OCT4 and SOX2 are key regulators of ES cell fate These factors are at the top of the hierarchy of the ES cell regulatory network and they keep ES cells undifferentiated by either the activation of target genes that encodes pluripotency and self renewal mechanism, or the repression of target genes that promote differentiation The regulatory regions of hundreds of genes that are expressed or repressed in the undifferentiated ES cell state are found to be co-occupied by OCT4, NANOG and SOX2 (Boyer et al., 2005; Loh et al., 2006) Maintenance of the ES cell state is hence dependent on the precise level and

transcriptional control of Oct4, Sox2 and Nanog, as perturbations will kick off drastic

changes in the downstream cascade, leading to differentiation

Two landmark papers published recently have elucidated the binding landscape of OCT4 and NANOG in mouse ES cells and OCT4, NANOG and SOX2 in human ES cells (Boyer et al., 2005; Loh et al., 2006) These location maps are useful guides that can aid

in the identification of additional components in the ES cell self renewal regulatory network Also, if and what roles most putative OCT4, SOX2 and NANOG targets play in

ES cells are still very much a mystery at present In this thesis, the functional dissection

of the role(s) of target genes such as Lefty1 and Lefty2 in ES cells is described

Figure 1 Model proposed by Smith et al (2003) to describe the current dogma governing

ES cell self renewal Nanog and Oct4 are both essential to sustain ES cell identity, whereas Stat3 has an accessory function Oct4 serves to block differentiation into

trophoblast but tends to promote differentiation into primitive endoderm and germ layers

Nanog (and activated Stat3) may block this differentiation effect of Oct4

Stat3

LIF Nanog

Oct4

1.3 Lefty - a Nodal antagonist

1.3.1 Roles of Lefty in embryo development

Lefty proteins are members of the TGF-ß superfamily The TGF-ß family has

implicated in regulating cell growth, differentiation, apoptosis and cellular patterning amongst many other functions in embryonic development The Lefty proteins are atypical TGF-ßs Typically, TGF-ß family members act as dimers and bring together two transmembrane serine/threonine kinases, the type I and IIreceptors The assembly and oligomerization of these receptorslead to phosphorylation of receptor-regulated SMADs (R-SMADs)and release them from their docking site on the receptor andallow them to heterodimerize with a common SMAD, SMAD4 Thesecomplexes accumulate in the nucleus, where they interact withother transcription factors, bind to DNA, and activate transcriptionof TGF-ß-responsive genes Also, typical TGF-ß family members have a characteristic signature motif that is comprised of a series of seven cysteine residues at their C-termini All of these residues are used for the formation of intrapeptide bonds with the exception of the third cysteine residue from the C terminus of mature proteins Active dimerized TGF-ß ligands are linked by the formation of disulfide bonds between

the fourth cysteine residues at the C terminus of the proteins LEFTY and its Xenopus

homolog, Antivin however lack the cysteine residue necessary for the formation of intermolecular disulfide bond Therefore, LEFTY appears to belong to a subgroup of the TGF-ß superfamily with an unpaired cysteine residue that does not exist as a dimer For