Differentiation inhibiting activity (DIALIF ) and mouse development

DEVELOPMENTAL

BIOLOGY

151, 339-351

Differentiation

(19%)

Inhibiting Activity (DIA/LIF) and Mouse Development

AUSTIN G. SMITH,* JENNIFER *AFRC

Centre

for

Research, and tDe.partment

Genome

NICHOLS,*

University of Edinburgh, of Biochemistry, University

MORAG ROBERTSON,*

AND PETER D. RATHJEN?

King’s Buildings, West Mains Road, Edinburgh EH9 3JQ, United of Adelaide, Adelaide, South Australia 5001, Australia

Accepted

March

Kingdmn;

11, 1992

Analysis of the differentiation in culture of murine embryonic stem (ES) cells has resulted in the identification and characterization of the regulatory factor differentiation inhibiting activity (DIA). DIA specifically suppresses differentiation of the pluripotential ES cells without compromise of their developmental potential. DIA is identical to the pleiotropic cytokine leukaemia inhibitory factor (LIF) which has a broad range of biological activities in vitro and in viva It is produced in both diffusible and matrix-localised forms whose expression is differentially regulated. The compartmentalization of DIA/LIF and the modulation of its expression during stem cell differentiation and by other cytokines may be significant elements in the control of early embryo development. These features may also indicate general principles of the regulatory networks which govern stem cell renewal and differentiation in later development.0

1992 Academic

Press, Inc.

INTRODUCTION

During mammalian development the fate of a given cell is governed by a combination of ancestry and position. Ancestry, or lineage, defines the available options, whilst the immediate cellular environment specifies the particular pathway to be followed. Accumulating evidence indicates that the interactions between neighbouring cells which determine developmental decisions are mediated primarily via locally acting signalling polypeptides, or cytokines (Heath and Smith, 1989). The term cytokine is used in this review to encompass molecules originally identified and characterized as growth factors as well as factors which modulate other aspects of cellular phenotype including differentiation state (Nathan and Sporn, 1991). The identification of cytokines as instructive factors that control processes of cell diversification in vitro and in vivo marks a major advance in our understanding of vertebrate development. Perhaps the best example in mammalian development is provided by hematopoiesis where research over the past 20 years has succeeded in defining a number of potent cytokines which regulate the survival, proliferation, and differentiation of different lineages from a multipotential precursor (Clark and Kamen, 1987; Metcalf, 1989a). This achievement is due in large part to the availability of culture systems which have allowed the dissection of individual lineages and provided a range of in vitro assays for regulatory activities. The molecular analysis of determinative events in early embryogenesis imposes an even greater demand for appropriate in vitro models, due to the experimental inaccessibility of the postimplantation embryo. Cul-

tured murine embryonic stem (ES) cells provide such a system. ES cells are established cell lines derived from the pluripotential founder tissue of the mouse embryo, the inner cell mass/epiblast (Evans and Kaufman, 1981; Martin, 1981). They are nontransformed cells which retain the full potential of their tissue of origin. Thus on reintroduction to a host blastocyst ES cells participate fully in normal embryonic development, contributing differentiated progeny to tissues derived from all three foetal germ layers (Bradley et al, 1984) as well as to extraembryonic membranes (Beddington and Robertson, 1989). The ES cell progeny can also colonise the germ line and give rise to functional gametes which transmit the ES cell genotype. The genetic manipulation of ES cells can therefore be used as a route for transgenesis (Robertson, 1986) and in particular for introducing defined modifications into the genome via the isolation of homologous recombinants (Capecchi, 1989). From a developmental perspective, the complete integration of ES cells into host embryos implies that they are capable of responding to, and indeed of generating, the full repertoire of regulatory signals which direct murine embryogenesis. ES cells may therefore be exploited as an in vitro assay system for the identification and characterization of such regulators (Heath and Smith, 1988). CONTROL OF ES CELL DIFFERENTIATION DIFFERENTIATION INHIBITING ACTIVITY

BY (DIA)

ES cells were initially isolated and maintained by coculture on “feeder” layers of mitotically inactivated mouse embryo fibroblasts (Martin and Evans, 1975).

339

0012-1606192 $5.00 Copyright All rights

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

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DEVELOPMENTAL

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Originally the feeders were considered essential to maintain stem cell viability. Subsequently it was appreciated that addition of 2-mercaptoethanol to the cultures was sufficient to sustain cell survival in the presence of foetal calf serum (Oshima, 1978). Under these conditions ES cells underwent extensive differentiation when plated in the absence of feeders (Smith and Hooper, 1987). This implied that the primary role of the feeders in propagation of the stem cell phenotype was to supress differentiation. Medium preincubated with a feeder layer was found to reduce differentiation of pluripotential feeder-dependent embryonal carcinoma (EC) cells, the tumour-derived counterparts of ES cells, but this effect was marginal and did not allow serial cultivation of homogeneous stem cell populations (Smith and Hooper, 1983; Koopman and Cotton, 1984). However, the Buffalo rat liver (BRL) epithelial cell line, a rich source of cytokines, was shown to secrete a potent differentiation inhibitor (Smith and Hooper, 1987). The macromolecular activity, provisionally named differentiation inhibiting activity (DIA), suppressed the differentiation of EC and ES cells almost entirely and enabled their continuous propagation as monocultures. Significantly, the culture of ES cells in BRL cell-conditioned medium did not compromise their developmental potential: the cells retained the capacity for differentiation, both in vitro on withdrawal from DIA (Smith and Hooper, 1987) and in chimaeras where germ-line transmission was readily obtained (Hooper et al., 1987). DIA could therefore be employed to provide simplified and experimentally advantageous conditions for the propagation and manipulation of ES cells (Smith and Hooper, 1987; Smith, 1991). The complete reversibility of DIA action also indicated that it was not a selective agent but a normal regulatory signal which might play a significant role in development of the pluripotential ICM/ epiblast population in viva. Purification of DIA revealed that activity resided in a basic, relatively hydrophobic, single chain glycoprotein of M, 43,000 (Smith et ah, 1988) (Fig 1A). The protein was heavily glycosylated and digestion with N-glycanase disclosed a core polypeptide of M, 20,000. The purified factor was active at picomolar concentrations and ES cells were shown to express specific high affinity cell surface receptors (Smith et al., 1988; Williams et ak, 1988). Pure DIA was sufficient to maintain undifferentiated ES cell populations in continuous culture in mercaptoethanol-supplemented serum-containing medium (Fig 1B). Recombinant DIA was subsequently shown to fully sustain the pluripotential phenotype, including the capacity for germ-line transmission. It was also demonstrated that DIA could replace feeder layers in the isolation of new ES cell lines directly from cultured embryos (Nichols et ab, 1990; Pease et al., 1990). DIA thus

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fulfills the essential function of a heterologous feeder layer in propagation of the stem cell phenotype. Indeed there have been several reports of germ-line transmission of targeted ES cells selected and maintained in the presence of DIA rather than on feeders (Thompson et al., 1989; Lufkin et ab, 1991). Nonetheless, the use of feeder cells may confer some advantage under suboptima1 culture conditions, for example by detoxifying media contaminants. It is also possible that uncharacterised serum factors are required in addition to DIA to maintain the stem cell phenotype. The ability to propagate undifferentiated ES cells for short periods in serum-free media supplemented with DIA alone suggests that other cytokines are not essential (A.G.S., unpublished), but optimization of conditions for serial cultivation in defined media is necessary to validate such a conclusion. The effect of DIA on ES cells appears to provide an example of deterministic control of stem cell behaviour. Thus at clonal density ES cells form colonies containing stem cells or wholly differentiated colonies depending entirely on the presence or absence respectively of DIA. However, in the presence of DIA there is always a low and somewhat variable level of differentiation. This background differentiation is not altered by increasing the concentration of DIA and is also observed in serumfree medium (A.G.S., unpublished). Such apparently random differentiation may be a culture artefact, or it could be an intrinsic property of stem cells. Since these two possibilities cannot be discriminated at present, it is equally valid to consider the action of DIA either as a direct inhibition of cellular differentiation or as a survival/proliferative effect specific for stem cells. DIA

IS A MULTIFUNCTIONAL LEUKAEMIA

CYTOKINE IDENTICAL INHIBITORY FACTOR

TO

The profound effect of DIA on self-renewal of early embryonic cells suggested that it might be active in other differentiating systems and prompted structural and functional comparison with known haematopoietic regulators. Studies in several laboratories revealed a relationship between DIA and a cloned murine cytokine, previously known as D-factor (Tomida et al, 1984) but now called leukaemia inhibitory factor (LIF) (Gearing et ab, 1987; Hilton et al., 1988), and its human homologue human interleukin for DA cells (HILDA) (Moreau et al., 1988). The biochemical properties of DIA and LIF were essentially indistinguishable and the availability of LIF cDNA clones led to the demonstration that purified recombinant LIF could effectively suppress ES cell differentiation (Smith et aZ., 1988; Williams et al, 1988). Subsequent transcriptional analysis (Rathjen et aZ., 1990b) and protein sequencing (A.G.S., unpublished) confirmed the identity of DIA with LIF. The murine, rat, and hu-

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REVIEWS

A -DIA Mr

+nlA

DIA

FIG. 1. Effect of purified BRL cell-derived differentiation inhibiting activity on murine ES cells. (A) Silver-stained ultrathin SDS-polyacrylamide gel of DIA purified from BRL cell supernatant by sequential hydrophobic interaction chromatography, reverse phase HPLC and cation exchange chromatography (Smith et al., 1988). (B) Phase contrast photomicrographs of (left) differentiated ES cells after 5 days of culture in nonsupplemented media, and (right) undifferentiated ES cells after 5 days of culture in media supplemented with purified BRL cell-derived DIA at 1 rig/ml. Bar = 10 pm.

man forms of DIA/LIF are highly conserved (Gough et al., 1988; Yamamori et al., 1989) and all are active on mouse ES cells. Interestingly, bacterially expressed recombinant protein is also functional (Gearing et ah, 1989) which indicates that the carbohydrate component is distinct is not required for in vitro activity. DIA/LIF from other characterised cytokines though it does show limited homology in amino acid sequence and gene structure to the tumour cell growth inhibitor oncostatin M (Rose and Bruce, 1991). The latter is also distantly related to interleukin-6 and granulocyte colony-stimulating factor (G-CSF) and it has been suggested that these four factors may constitute a structurally and functionally related family of cytokines (Rose and Bruce, 1991). The conjunction of myeloid and ES cell regulatory activities represented by DIA/LIF is a powerful demonstration of the range of action of individual cytokines. The involvement of common regulatory factors in difsystems is further underlined by ferent developmental the broad spectrum of biological activities now attributed to DIA/LIF (Table 1). It has effects on haematopoietic (Leary et al, 1990; Fletcher et al., 1990), osteogenic (Abe et al, 1986; Lorenzo et al, 1990), hepatic (Baumann and Wong, 1989), adipogenic (Mori et al., 1989), renal (Tomida et al., 1991; Bard and Ross, 1991), and neuronal (Yamamori et al, 1989; Murphy et ah, 1991) cells in vitro. Although the physiological significance of some of these assays is uncertain, it is apparent that

DIA/LIF has the potential to act on a variety of cellular targets of different lineages. This is further evidenced by its effects in viva. Adult mice with elevated serum levels of DIA/LIF have been generated both by the introduction of primitive haemopoietic cells genetically manipulated to overexpress DIA/LIF (Metcalf and Gearing, 1989) and by intraperitoneal injection of recombinant DIA/LIF protein (Metcalf et al, 1990). In the former study the animals developed a fatal wasting syndrome (cachexia) accompanied by altered calcium metabolism and bone formation. Some bone resorption was apparent, but there was an overall deposition of new bone, an increase in osteoblast numbers, and calcification of muscle tissue including the heart. By contrast, intraperitoneal administration of DIA/LIF resulted primarily in alterations to the haemopoietic system, in particular in enhanced thrombopoiesis characterised by elevated levels of platelets and megakaryocytes and increased numbers of spleen progenitors. There was no significant bone deposition in these mice. These varied pathophysiological effects emphasise that the normal expression and distribution of DIA/LIF are likely to be highly restricted. The complexity of the phenotypes, however, and their dependence on the route of delivery mean that the experiments do little to elucidate the physiological role(s) of DIA/LIF or to identify primary responsive cells. A unifying theme is not immediately apparent from the diverse biological effects of DIA/LIF. One possibil-

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TABLE 1 IS A PLEIOTROPIC REGULATOR OF DEVELOPMENTAL DECISIONS IN VITRO

Responsive population

Biological activity

Embryonic stem (ES) cells Renal cells/kidney rudiments Preadipocytes Da-la cell line Haematopoietic progenitors Primordial germ cells Sensoryneurones Ml cell line Sympathetic neurones Foetal calvaria Foetal long bones Hepatocytes

Inhibition of differentiation Inhibition of differentiation Inhibition of differentation Survival/proliferation Survival/proliferation Survival/proliferation Survival/proliferation Terminal differentiation Cholinergic differentiation Stimulation of bone resorption Inhibition of bone resorption Acute phase induction

ity is that it has been recruited for multiple unrelated functions. However, an argument can also be made in favour of a more defined role as a generalised stem cell factor (Smith and Rathjen, 1991b). Many of the actions of DIA/LIF appear to be mediated via stem cells or progenitor cells rather than direct effects on differentiated tissue. Thus the suppression of ES cell differentiation is a direct action on stem cells as are the survival/proliferative effects on primitive haematopoietic precursors (Leary et al., 1990) and primordial germ cells (De Felici and Dolci, 1991; Matsui et ab, 1991). In cultured kidney rudiments DIA/LIF specifically prevents nephrogenesis by reversible inhibition of the differentiation of mesenchymal progenitor cells (Bard and Ross, 1991). In viva DIA/LIF stimulates the production of ectopic bone, suggesting that it acts on osteogenic precursors (Metcalf and Gearing, 1989). These stem cells or their progeny may be responsible for the activation of osteoclasts detected in vitro, since the bone-resorbing osteoclasts do not express DIA/LIF receptors (Allan et al., 1990). Indeed the complex phenotype produced by ectopic expression of DIA/LIF in the whole animal is consistent with activity on a range of progenitor cell populations. It will therefore be of interest to investigate the involvement of DIA/LIF in other stem cell systems. DIA/LIF IS EXPRESSED AND IMMOBILISED

IN SOLUBLE FORMS

The demonstration that ES cell lines can be established de nova by direct culture of blastocysts in medium supplemented with DIA/LIF (Nichols et aZ.,1990; Pease et ah, 1990) confirms that this cytokine fulfills all the essential functions of fibroblast feeder cells for maintenance of the pluripotential state. However, the level of DIA/LIF in feeder cell supernatants is low and in some cases undetectable (Smith and Hooper, 1983; Rathjen et

Alternative Differentiation Lipoprotein HILDA

designation

inhibiting activity lipase inhibitor

Leukaemia inhibitory factor Cholinergic differentiation factor Hepatocyte simulatory

ah, 1990b). Secretion of soluble DIA/LIF

factor-III

is therefore unlikely to be the primary means by which feeders suppress ES cell differentiation. Closer examination revealed a requirement for direct co-culture and specifically for contact between the ES cells and the extracellular matrix (ECM) of the feeder layer. Differentiation was suppressed when ES cells were cultured on cell-free matrix preparations from feeder layers (Rathjen et al., 1990b). This is not a general ECM property, but is restricted to the matrix of particular cell types. The active component can be solubilized by extraction with acid or high concentrations of EDTA. Purification of this activity revealed a glycoprotein with similar biochemical properties to soluble DIA/LIF. This identity was confirmed by amino terminal sequence determination. Thus a major component of the mechanism by which feeders regulate ES cell differentiation is via incorporation of DIA/LIF into the ECM. The molecular origin of the differential distribution of DIA/LIF has been resolved by analysis of the transcripts expressed by embryo fibroblasts and other cell types (Rathjen et ab, 1990b). Two distinct transcripts were identified by ribonuclease mapping. The rapid amplification of cDNA ends (RACE) procedure (Frohman et al., 1988) was used to obtain cDNA clones corresponding to the 5’ regions of the two transcripts. The clones were identical throughout the second and third exons, but diverged upstream of the exon l/exon 2 boundary. One clone included and extended the previously described (Gearing et ah, 1988) 5’ sequence of the mRNA for soluble DIA/LIF, the “D” transcript. The other clone contained a distinctive 5’ untranslated region and a short stretch of alternative coding sequence. The effect of this change is to alter the amino terminal sequence of the primary translation product from MKVLAAGto MRCR-, whilst leaving the core hydrophobic secretory signal and the sequence for the mature

REVIEWS

FIG. 2. Modular organization of the murine DIA/LIF gene. Alternative first exons enable targetting of the same active protein to different extracellular locations. Arrows indicate putative transcription start sites. The filled bar in exon 2 represents the core hydrophobic secretory signal sequence and the asterisk marks the position of the amino terminus of the mature protein. The long 3’untranslated region of exon 3 is not shown. Relative locations of the D and M exons were determined from the genomic sequence (Stahl et al, 1990).

protein unchanged. Although the involvement of differential splicing cannot be excluded, the absence of sequence identity at the 5’ untranslated ends of the two transcripts suggests that the messages are transcribed from distinct promoters. The alternative “M” exon is not conserved between human and murine genomic sequences (Stahl et al., 1990) and a human DIA/LIF cDNA corresponding to the M transcript has not yet been defined, although a second mRNA species which diverges at the 5’ end of the coding sequence can be detected by RNase protection (AGS and PDR, unpublished). The presence of the M transcript in cultured cells correlates with production of matrix-associated DIA/LIF. Transfection of the two cDNAs into heterologous cells confirmed that the D transcript generates a soluble protein whilst the product of the M transcript is preferentially incorporated into the ECM. The modular organization of the murine DIA/LIF gene (Fig. 2) therefore constitutes a molecular strategy whereby the same regulatory factor can be targetted to alternative extracellular locations (Smith and Rathjen, 1991b). Consistent with a role in the generation of spatial diversity, the two DIA/LIF transcripts are differentially expressed in culture and during embryogenesis (see below). The potential significance of this is that there are important distinctions between the roles of soluble and immobilized factors (Rathjen et ab, 1990b; Jesse1 and Melton, 1992a). Indeed many cytokines are now known to be produced in both diffusable and cell- or matrix-associated forms (Massague, 1990; Smith and Rathjen, 1991). The principle feature of soluble cytokines is that they mediate short-range communication between cells which are not in direct contact. This is likely to be essential both for multilayered tissues and in situations where the signalling and responding populations are physically separated. Furthermore, the combinatorial capacity provided by the diffusion of cytokines emanating from distinct sources may make a significant contribution to the spatial organization of developmental processes. The recruitment of rare or dispersed target cells, for example during migratory or repair processes, can

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also be facilitated by the local activity of diffusible factors. However, the immobilization of cytokines, either on the cell membrane or in the extracellular matrix, can confer significant advantages in particular contexts. Important amongst these is the confinement of potent, often multifunctional, bioregulators to the vicinity of the relevant target cells, thereby restricting the potential for inappropriate responses. Localised interactions with matrix and cell surface components can also modulate biological activity, for example the binding of basic fibroblast growth factor (bFGF) to high affinity receptors is potentiated by heparin (Yayon et al., 1991), whilst the action of transforming growth factor-p (TGF-P) is inhibited by the proteoglycan decorin (Yamaguchi et al., 1990; Ruoslahti and Yamaguchi, 1991). Such interactions provide a further level of position-dependent specificity to cytokine function. Following the paradigm of the specification of cell fate in the acellular syncitial blastoderm of Drosophila (Anderson, 1989), concentration gradients of diffusible regulators or morphogens are considered powerful systems for the control of morphogenesis (Slack, 1991). However, gradients can also be formed by differential expression of localised signals. In fact a major attribute of immobilized factors during embryogenesis may be their capacity to convey positional information more precisely within the complex topology of the developing mammalian embryo. This is due not only to their fixed localization, which allows, for example, for a discontinuous distribution, but also to the fact that they permit a temporal discontinuity between production of a signal and its utilization. Thus a factor can be produced in a latent form and its activity revealed at an appropriate stage in later development following cell migration or by proteolytic release from the cell surface or matrix. Such mechanisms are also important in regenerative and repair processes in the adult, for example in the constitutive remodelling of mineralised bone (Canalis et ah, 1988) or in angiogenic responses to wounding or infection (Vlodavsky et al., 1987; Saksela and Rifkin, 1990). During development the flexibility and range of soluble signalling molecules are complemented by the precision and longevity of immobilized factors. Spatial and temporal control of cell fate can therefore by governed by the net input of cytokines from three sources: immobilized in the surrounding ECM, directly presented by adjacent cells, and diffusing from neighbouring tissues. Integration of these signals, which is also dependent upon the repertoire of receptors and signalling pathways expressed by the target cell, will determine the developmental outcome.

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FIG. 3. Stem cell renewal during ES cell differentiation. ES cells were cultured in the absence of DIA for 8 days. The arrow indicates a colony of ES cells growing on the background of differentiated cells.

EXPRESSION OF DIA/LIF DIFFERENTIATED

IN ES CELLS AND PROGENY

The differentiation of ES cells in culture suggests that the stem cells do not themselves produce DIA/LIF. However, a highly sensitive ribonuclease protection assay revealed that the DIA/LIF gene is expressed at low levels in undifferentiated ES cells (Rathjen et ah, 1990a). Analysis of four independent ES cell lines revealed a characteristic profile of DIA/LIF transcripts. The D transcript is barely detectable but a significant level of DIA/LIF-M message is present. This implies either that some post-transcriptional mechanism suppresses production of biologically active DIA/LIF in ES cells, or that ES cells, and by implication epiblast cells, may have a limited capacity for autocrine regulation. Evidence consistent with the latter is provided by the effect of cell density on ES cell differentiation. In the absence of DIA/LIF, differentiation of the entire population is only obtained if ES cells are plated as single cells at clonal density. If the ES cells are not thoroughly dissociated, however, or are allowed to form small colonies before withdrawal of DIA/LIF, the initial differentiation does not extend to all the cells and groups of stem cells persist surrounded by differentiated progeny. Under these conditions, the undifferentiated cells subsequently expand and repopulate the culture (Fig 3). They remain pluripotential, however, and dependent on DIA/LIF for continued propagation. Their expansion presumably occurs because differentiated ES cells pro-

duce DIA/LIF at relatively high levels (Rathjen et al., 1990a). Increased expression of both D and M transcripts is detectable at the onset of cellular differentiation, 24 hr after withdrawal from DIA/LIF. This production of DIA/LIF by newly differentiated cells provides a feedback mechanism for regulating the balance between differentiation and self-renewal in stem cell populations (Smith and Rathjen, 1991). Self-renewal will be promoted whenever stem cells and their immediate differentiated progeny remain in close proximity. The interactions observed in differentiating ES cell cultures may mirror aspects of the expansion and differentiation of the ICM/epiblast in the early embryo (Martin et al., 1977; Doetschman et ah, 1985; Smith and Rathjen, 1991). This raises the possibility that autocrine production of matrix-associated DIA/LIF might be a significant feature in the maintenance of pluripotency in the embryo whilst the ICM and epiblast remain multi-layered prior to and shortly after implantation. The subsequent transition of the proliferating epiblast to an epithelial monolayer may render the stem cells more dependent on, and responsive to, the production of DIA and other cytokines by differentiated neighbours. Moreover, if DIA/LIF is a generalised stem cell factor, then autocrine production by stem cells and/or expression by subsets of differentiated progeny may be an important homeostatic mechanism in continuously regenerating tissues such as bone marrow, gut, and skin where stem cells persist throughout adult life. EXPRESSION OF DIA/LIF DURING MURINE EMBRYOGENESIS

EARLY

The essential function of DIA/LIF in the isolation and propagation of ES cell lines combined with the pattern of DIA/LIF expression in ES cells and their differentiated progeny argues that this factor is likely to play a key role in the expansion of stem cells in the peri-implantation embryo. The demonstration that both D and M transcripts are present at the egg cylinder stage of development (Rathjen et al., 1990a) supports this conclusion. DIA/LIF mRNA is barely detectable in most embryonic and adult tissues, but relatively high levels of expression are detected by RNase protection in the 6.5 day conceptus. The transcripts are significantly downregulated by the onset of gastrulation at 7.5 days. Expression is predominantly localised to the extraembryonic portion of the egg cylinder which suggests that DIA/LIF could act in a paracrine manner to regulate self-renewal in the primitive ectoderm, but might also have some function in the expansion of the extraembryonic founder tissues. Although a broad range of both transformed and nontransformed cell types express appreciable levels of

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PPMG 1 2 3 DIA/LIF in vitro, the expression of DIA/LIF transn,ripts in vivo is highly restricted both during embryonic development and in the adult (unpublished data). However, low levels of transcription are sufficient to 0 generate significant biological activity (Rathjen et al., M 1990a,b). This presents a technical obstacle to the analysis of physiologically relevant expression and has particularly hindered efforts to resolve sites of mRNA synthesis at the cellular level by in situ hybridization. Detection of DIA/LIF transcripts in cultured blastocysts has been reported using PCR amplification of reverse transcribed RNA (Murray et aZ., 1990). The limitations of this approach require that the finding be substantiated, but it is consistent with the notion that DIA/LIF has a role in ICM development. The highest levels of DIA mRNA in the adult mouse are found in the female reproductive tract, particularly in the uterus. Similar amounts of both transcripts are detected by RNase protection. Expression is not depenGAPdh dent on the presence of embryos since comparable levels are present in uteri from nonoestrous virgin and pregnant females (Fig 4). However, transcript levels are lower (relative to the GAPdh loading control) during FIG. 4. Ribonuclease protection showing DIA/LIF transcripts in oestrous indicating that uterine expression may be re- mouse uterus. Ribonuclease protection assays were performed essentially as described (Rathjen et al, 1990a) on 10 pg total RNA prepared lated to hormonal status. Interestingly, uteri from pregnant animals in which implantation has been delayed no from freshly isolated uterine horns by the lithium chloride/urea antisense longer contain DIA/LIF transcripts (Bhatt et al., 1991). method (Auffray and Rougenon, 1980). A pP]UTP-labelled RNA probe was generated by in vitro transcription of linearised pDR2 Reversal of the delay is accompanied by appearance of (Rathjen et al, 1990b). This probe contains D exon-specific sequence in DIA/LIF mRNA. The latter observations indicate that addition to common sequence upstream of the unique .%a1 site and protects RNA fragments of 368 (D transcript) and 345 (M transcript) in certain circumstances uterine expression can be modnucleotides. The glyceraldehyde 3-phosphate dehydrogenase (GAPdh) ulated by the hormonal status of the mother. In situ loading control probe was produced by SP6 transcription of a 220bp hybridization studies on pregnant females have locaSfaNl-Hind111 fragment of coding sequence cloned in pGEM2. t, lised the source of DIA/LIF transcripts in the uterus tRNA; P, DIA/LIF probe; M, Ml3 sequencing ladder; G, GAPdh probe; prior to implantation to the endometrial glands (Bhatt lane 1, nonoestrous uterus; lane 2, oestrous uterus; lane 3, flushed et al., 1991). Transcripts are subsequently detected in uterus from pregnant female 3.5 days pc. the decidual swelling in the endometrial gland cells surrounding the newly implanted embryo (Fig 5). The function of these cells, which are derived from the bone The relative abundance of DIA/LIF transcripts in the marrow, is obscure. Their distribution in the nonpregreproductive tract and their localization to the endomenant uterus is uncertain, but they become identifiable as trial glands suggests that maternal DIA/LIF could be clusters of epithelial cells throughout the endometrium intimately involved in the maturation and implantation at 2.5 days of pregnancy. During implantation they be- of the early embryo. Alternatively these findings may come confined to the tissue adjacent to the conceptus reflect a potential role for DIA/LIF in controlling the such that by Day 6 they are completely absent from preparedness of uterine stem cells for implantation and interconceptual regions (Stewart and Peel, 1981). This is the ensuing decidual response. suggestive of some role in the uterine response to implantation. They are known to produce several other cyMODULATION OF DIA/LIF ACTIVITY AND EXPRESSION BY tokines including GM-CSF, M-CSF, and IL-6 (Pollard, OTHER REGULATORY FACTORS 1990; Robertson and Seamark, 1991). Subsequently the Individual cytokines can have profound effects on isogranulated endometrial cells aggregate in a structure known as the metrial “gland” which develops at the base lated cell types. However, the full potential of these molprocesses of each implantation site (Bulmer et cd, 1987). It is not ecules to orchestrate complex developmental which result in yet known whether DIA/LIF expression persists at arises from their mutual interactions synergistic and antagonistic modulations of biological these later stages.

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FIG. 5. Localization of DIA/LIF transcripts in endometrial glands by in situ hybridization. Bright field photomicrograph of decidual swelling at 5.5 days pc. Arrows indicate the endometrial glands. DIA/LIF transcripts were specifically detected by autoradiography following hybridization of tissue sections with a [gSS]-UTP-labelled antisense RNA probe essentially as described by Wilkinson and Green (1990). The probe was generated by in vitro transcription of linearized plasmid pDR1 (Rathjen et aL 1990b), which contains the conserved coding sequence of DIA/LIF-D and DIA/LIF-M, and therefore hybridizes to both transcripts. E, endometrium; M, mesometrium.

activity. The cytokine network thereby provides a powerful combinatorial communication system between different cell populations. The potential of DIA/LIF to synergize with other cytokines is indicated by its effects on cultured primordial germ cells (Matsui et aL, 1991) and haemopoietic progenitors (Leary et al, 1990). DIA/LIF enhances the survival/proliferative response of primordial germ cells to the Steel factor and of primitive blast cells to interleukin-3. Interestingly, interleukin-6 has a similar effect to DIA/LIF in the latter assay (Ikebuchi et ah, 1987) and in several other systems (see below). Indeed, the emerging picture in both haematopoietic and embryonic development is one in which the behaviour of a stem cell is not exclusively regulated by a unique factor, but rather is governed by the interplay of several cytokines, some of which are common to different systems. Other cytokines can also modulate the activity of DIA/LIF by regulation of its production. Phorbol esters enhance expression of DIA/LIF mRNA and protein by various tumour cell lines (Gascan et aL, 1990) whilst a variety of factors have been shown to induce expression of DIA/LIF transcripts and protein in established fibro-

blasts (Rathjen et aL, 1990a) and keratinocytes (A.G.S. and J. Pietenpol, unpublished) and in primary osteoblast cultures (Smith and Rathjen, 1991). Regulators shown to induce DIA/LIF transcripts include serum, epidermal growth factor, interleukin-la, interleukinl/3, tumour necrosis factor, bFGF, and members of the TGF-P family. Both D and M transcripts are inducible, though in general the D form is more responsive. Expression of the two transcripts can be differentially induced by different factors. The effects of different cytokines also vary according to the phenotype of the target cell. Thus TGF-P stimulates only a modest response in diploid MRC-5 human embryonic lung fibroblasts, but is a very potent inducer in C3H lOT1/2 established mouse embryo fibroblasts, whilst bFGF induces an intermediate response in both cell lines. Interestingly, the basal expression of DIA/LIF in ES cells is not affected by a variety of cytokines, including TGF-0 and bFGF (Rathjen et ak, 1990a and unpublished data). An important additional control element could be provided by specific repressors of DIA/LIF expression. It is significant therefore that the glucocorticoid dexamethasone inhibits the induction of DIA/LIF by bFGF

REVIEWS

in fibroblasts (Rathjen et al., 1990a) and by a variety of factors in osteoblasts (Smith and Rathjen, 1991). Such repression may be a means of preventing the inappropriate expansion of stem cells and promoting differentiation. Whether glucocorticoids are important repressors in viva remains to be determined, but clearly the potential exists for fine local modulation of the level of DIA/ LIF expression via a network of positive and negative regulators. The induction of DIA/LIF transcripts is rapid and occurs in the presence of protein synthesis inhibitors (Rathjen et ah, 1990a), indicating that the DIA/LIF gene is directly responsive to cytokine signalling. This suggests that production of DIA/LIF might be an important component of the pleiotropic response to molecules such as the TGF-fis. It is striking, for example, that specific cytokines which have been implicated in bone development activate expression of DIA/LIF in primary osteoblast cultures, whilst the cellular target of DIA/LIF itself appears to be the osteogenic precursors (Metcalf and Gearing, 1989; Rodan et ab, 1990). Such paracrine induction of DIA/LIF expression in differentiated cells could be a general mechanism for ensuring that stem cell populations do not become exhausted in situations which require their active recruitment, for instance during remodelling and repair processes. The observation that ES cells secrete a potent inducer of DIA/LIF expression (Rathjen et ab, 1990a) indicates that in some circumstances stem cells may even contribute directly to an inductive loop which guarantees self-renewal. The paracrine coupling of stem cells and differentiated cells may be a central mechanism for controlling the balance between self-renewal and differentiation in both the embryo and the adult (Smith and Rathjen, 1991). THE

DIA/LIF

RECEPTOR

Cellular responses to DIA/LIF are initiated by binding to specific cell surface receptors which have been detected on a variety of responsive cell types (Smith et al, 1988; Williams et aZ., 1988; Hilton et ab, 1988; Allan et ab, 1990; Hilton et ak, 1991). A DIA/LIF receptor cDNA has recently been isolated by expression cloning in Cos cells (Gearing et al, 1991). The encoded protein is a member of the haematopoietin receptor superfamily (Cosman et ah, 1991). The latter is a group of structurally related receptors for functionally diverse ligands, including prolactin, growth hormone, and ciliary neurotrophic factor in addition to several haematopoietic cytokines. Members of the family are glycoproteins with a single transmembrane domain. They are characterised by the presence in the extracellular ligand-binding domain of a homologous stretch of around 210 amino

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acids which contains four highly conserved membranedistal cysteine residues and a membrane-proximal TrpSer-X-Trp-Ser motif. They also possess one or more fibronectin type III modules, domains implicated in molecular adhesion. These shared properties are likely to reflect general features in ligand binding and/or association with cell surface components. Expression of the cloned DIA/LIF receptor generates predominantly low affinity binding sites whereas responsive cells express high affinity receptors (Gearing et ab, 1991). This could indicate the existence of a separate high affinity receptor. It is more probable though that an additional component is involved which converts the low affinity receptor to a high affinity form. Subunits of this nature have been described for several other cytokine receptors (reviewed by Nicola and Metcalf, 1991). In some cases these subunits appear to be shared between receptors that bind distinct ligands and this may explain how different cytokines can induce equivalent biological responses in some systems. Competition between ligand-binding receptors for affinityconverting and signal-transducing subunits could be an important mechanism for integrating cytokine input at the cell surface prior to activation of signalling pathways. An unexpected property of many of the haematopoietin receptors is that they can be generated as secreted extracellular proteins via alternative splicing (Mosley et al., 1989; Goodwin et aZ., 1990). The DIA/LIF receptor falls into this class, as murine cDNAs have been identified which do not include transmembrane and cytosolic domains. These cDNA clones encode an extracellular binding protein (Gearing et aZ., 1991). Secreted receptors can act as natural antagonists of cytokine action by directly competing for ligand with transmembrane receptors and/or by promoting the systemic clearance of secreted cytokines (Goodwin et al., 1990). If the extracellular DIA/LIF receptor functions in this manner to reduce DIA/LIF activity it could be an important determinant of early embryonic differentiation. However, there is also evidence that extracellular receptors may facilitate the appropriate presentation of a ligand to a signal-transducing complex (Taga et aZ., 1991; Gearing and Cosman, 1991). In this context it may be significant that the secreted DIA/LIF receptor retains two fibronectin III domains and there is a suggestion that it may be predominantly produced in surface-associated rather than soluble form (Gearing et aZ., 1991). This could have the effect of potentiating localised activity of DIA/LIF. Clearly the extracellular receptor is likely to be an important parameter in DIA/LIF action and further investigation is required to clarify its role. In general there is little sequence similarity between the cytoplasmic domains of the haematopoietin recep-

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tors. This implies that their mechanisms of signal transduction may be unrelated which would be consistent with the diverse functions of their ligands. However, comparison of the deduced protein sequence of the DIA/LIF receptor with other members of the family revealed significant homology throughout the transmembrane and cytoplasmic domains to gp130, the putative signal transduction subunit of the IL-6 receptor (Taga et al, 1991; Hibi et al., 1990). This is particularly striking in view of the overlapping biological activities of DIA/LIF and IL-6. The two factors exhibit only IO12% homology at the protein level and the respective actions of DIA/LIF on ES cells and IL-6 in immune regulation are quite distinct, but they exhibit similar activities in several other systems. Thus both cytokines induce differentiation of Ml cells and are active in acute phase reactions, bone remodelling, haematopoietic stem cell proliferation, and thrombopoiesis (for review of IL6 see Hirano et ah, 1990). These shared activities are suggestive of some convergence of signalling mechanisms. The nature of the DIA/LIF primary signal transduction mechanism remains obscure, however. In common with other members of the haematopoietin receptor superfamily, neither the DIA/LIF receptor nor gp130 exhibit identifiable kinase or other catalytic domains. Postreceptor events initiated by DIA/LIF have been investigated in Ml cells. Rapid phosphorylation of specific proteins has been reported; a 27-kDa presumptive heat shock protein becomes labelled on serine and threonine (Michishita et al, 1991), whilst tyrosine phosphorylation of a 160-kDa protein is detected within 5 min (Lord et ah, 1991). The latter also occurs in response to IL-6 and both cytokines induce a group of immediate early genes which includes members of thejun family of transcription factors (Lord et al., 1991). This shared response, involving both transcriptional and post-transcriptional gene activation, is specific to DIA/LIF and IL-6 and indicates that the two factors utilise equivalent signalling mechanisms in Ml cells. However, Ml cells do not appear to reflect any normal biological response to either DIA/LIF or IL-6 (Metcalf et ab, 1988; Metcalf, 1989b), so it is probable that signalling pathways in these cells are disrupted at one or more levels. It is therefore necessary to evaluate in other systems the role of components implicated in Ml cell differentiation. The induction of terminal differentiation of Ml cells appears to be the antithesis of inhibition of ES cell differentiation. The activity of DIA/LIF as a determinant of both processes implies either that distinct signalling pathways are activated in different cell types, or that a common set of primary responses may be interpreted in a fundamentally different manner according to nuclear phenotype. It will be intriguing to compare the mecha-

nism of DIA/LIF action in ES cells with that in differentiated somatic cells such as Ml cells and hepatocytes. FUTURE PROSPECTS

Characterization of the expression and function of DIA/LIF in cultured ES cells has implicated this factor as a regulator of pluripotential stem cell differentiation in the early mouse embryo. The observation that DIA/ LIF supports the de nova derivation of ES cell lines and the demonstration that DIA/LIF transcripts are present in the blastocyst and egg cylinder provide good evidence for a role in early development. It is noteworthy that relatively high levels of DIA/LIF-D mRNA are expressed in the egg cylinder, suggesting that the primitive ectoderm may effectively be bathed in a solution of DIA/LIF. Transcripts are still present at 7.5 days post conceptus, although in reduced amounts. These observations suggest that the onset of mesodermal differentiation is unlikely to be triggered solely by withdrawal of DIA/LIF. Rather it may be initiated by the localised production in or around the primitive streak of either an antagonist of DIA/LIF, such as the extracellular receptor, or a specific differentiation inducing factor which is effective in the presence of DIA/LIF. Precise information on the cellular distribution of transcripts and protein in the pre- and early postimplantation embryo is now required to understand the details of DIA/LIF activity during this period. The production of DIA/LIF in a matrix-associated form is an important mechanism for achieving specificity of action in viva. The nature of the interaction between DIA/LIF and the ECM has yet to be determined, however. Nor is it clear how differential localization of the DIA/LIF-D and DIA/LIF-M products is realised. The mature protein sequence is apparently identical in both cases, and the overall degree of glycosylation is similar. The alternative first exons therefore presumably act as targetting motifs which may direct appropriate subcellular compartmentalization of the mRNAs or proteins and/or induce specific post-translational modifications that promote matrix association. Short peptide sequences have been described which determine localization of proteins to the lumen (Munro and Pelham, 1987) or membrane (Nilsson et ah, 1989) of the endoplasmic reticulum, to the nucleus (Kalderon et aZ., 1984), to the nucleolus (Siomi et ah, 1988), or to the cell surface (LaRochelle et al, 1991). Such targetting domains constitute part of the mature protein, however. In contrast, the alternative amino termini of DIA/LIF-M and DIA/LIF-D are apparently removed along with the rest of the leader sequence. It will be of interest to establish whether the M exon of DIA/LIF can direct incorporation of heterologous proteins into the extracellular matrix.

REVIEWS

Data on the in situ localization of DIA/LIF transcripts and protein are essential for determining its potential functions throughout development and in particular for analysing whether DIA/LIF has a general role as a stem cell renewal factor. Several lines of evidence suggest that expression will generally be at a low level and highly localised, which may render detection problematic. A fruitful strategy may be to integrate a reporter gene into each of the alternative first exons by homologous recombination. This should enable resolution at the cellular level of sites of DIA/LIF transcription. Gene targeting will also allow a functional definition of the requirements for DIA/LIF in the early embryo via inactivation of the different forms. A complementary approach, which may prove more informative about actions of DIA/LIF in later development, is the ectopic expression of DIA/LIF in transgenic mice produced either by pronuclear microinjection or via ES cells. If DIA/LIF is a general stem cell factor, it will be important to examine the consequences of tissue-specific deregulation of DIA/LIF expression for haemopoietic and epithelial stem cells in neonatal and adult mice. Analysis of the central role played by DIA/LIF in regulation of the stem cell phenotype of ES cells has yielded significant insights into modes of stem cell regulation. These mechanisms are potentially operative not only in the early embryo, but also on lineage-specific stem cells in later development and in stem cell homeostasis in the adult. At a molecular level, the identification and characterization of DIA/LIF and its receptor afford the prospect of defining the mechanisms whereby the phenotype of a pluripotential stem cell is maintained. Ultimately this should contribute to a molecular explanation of cellular diversification in mammals. We acknowledge the encouragement and assistance of colleagues in Oxford, Edinburgh, and Adelaide. Duncan Davidson is thanked for invaluable help with the in situ hybridizations, Dean Evans and Jennifer Pietenpol for unpublished data, and Frank Johnston and Graeme Brown for photography. We are grateful to Bill Skarnes for thoughtful comments on the manuscript. Much of the work cited was carried out in the laboratory of John Heath, Department of Biochemistry, University of Oxford and was supported by the Cancer Research Campaign. Note added in proox Since this article was accepted for publication, it has been reported that DIA/LIF and oncostatin M bind to a eommon high-affinity receptor comprising the DIA/LIF transmembrane receptor and gp130 (Gearing et al, Science 255,1434-1437). The shared involvement of gp130 in both DIA/LIF and IL-6 high-affinity receptors is therefore likely to underlie the overlapping biological activities of these cytokines. REFERENCES Abe, E.,Tanaka, H., Ishimi, Y., Myaura, C., Hayashi, T., Nagasawa, H., Tomida, M., Yamaguchi, Y., Hozumi, M., and Suda, T. (1986). Differ-

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