The Primordial Germ Cells of Mammals: Some Current Perspectives

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

232, 194–207 (1997)

EX973508

REVIEW The Primordial Germ Cells of Mammals: Some Current Perspectives MIA BUEHR1 Centre for Genome Research, University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh EH9 3JQ, United Kingdom

1. INTRODUCTION

These are exciting days for those who study the biology of mammalian primordial germ cells (PGCs), the cells in the early embryo from which the definitive eggs and sperm arise. Answers to questions that have been discussed and debated for many years are beginning to emerge, and many old problems are now being solved by a combination of classical techniques and newer methods of transgenic manipulation and molecular analysis. Germ cells are the only cells that link the generations, and their unique capacity to transmit information from parent to offspring has interested biologists for many years. Not only are the PGCs of interest because of their role in heredity and reproduction, but their own developmental history is complex and intriguing in itself. In vertebrates (and many invertebrates) the primordial germ cells originate as a population of progenitor cells in very early development: indeed the germ line is often the first embryonic cell lineage to be established. After their origin, the primordial germ cells undertake a complex migration through the embryo and eventually become incorporated into the developing gonad. During this time they proliferate from a small number of cells to many thousands and are subject to many influences and stimuli which direct their determination, proliferation, survival, migration, guidance, and differentiation. The complex factors directing the ontogeny of germ cells are imperfectly understood, but in the past few years there have been significant advances in germ cell biology, some of which are summarized here. Because the complete history of mammalian germ cells is a long one, I have limited the scope of this review to studies of primordial germ cells from the time when they are first determined, through their migratory phase, to the time when they become incorporated 1

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2. ORIGIN AND SEGREGATION OF GERM CELLS

Markers of the germ line. The presumptive germ cells of some animals (e.g., Xenopus and Drosophila) contain a distinctive cytoplasmic inclusion, the germ plasm, which (in Drosophila at least) is the germ line determinant and which can serve as a convenient marker to trace the early ontogeny of germ cells. However, there appears to be no comparable germ plasm in mammalian PGCs. Electron-dense fibrillogranular material (‘‘nuage’’) has been described in the germ cells of some rodents [5] but its significance is not clear. Most postgastrulation mammalian PGCs (with the possible exception of those of rabbits [6] and some marsupials [7]) stain positively for the enzyme alkaline phosphatase (AP), and though not specific to the PGCs, the expression of AP has long been used to identify mammalian germ cells in histological sections and whole mounts [8–10]. There are several isozymes of AP (EC 3.1.3.1) [11, 12]: that expressed in the primordial germ cells is tissue nonspecific alkaline phosphatase (TNAP) [13]. Although AP is such a conspicuous marker of mammalian germ cells, its function in them is unknown, and the germ cells of mice homozygous

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in the developing gonad before they begin their differentiation as male or female gametes [in the mouse, this corresponds to development from about 7 days postcoitum (dpc) to 12.5 dpc]. In the great majority of cases research on mammalian germ cells describes their history in the mouse, and unless stated otherwise, the papers covered in this review use mouse germ cells as subjects. Such evidence as there is suggests that although there are some differences in details, the PGCs of most mammals are similar to those of the mouse [1]. Patterns of migration and proliferation similar to that in the mouse have been described in humans [2], rats [3], and other groups. In marsupials as well as eutherians germ cells have an extragonadal origin and reach the gonad by moving through the hindgut and up the dorsal mesentery [4].

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for a targeted disruption of the TNAP gene [12] are apparently normal. The precursors of the germ cells. Because no marker exists with which germ cells or their progenitors can be identified before gastrulation (TNAP is not a specific marker of PGC precursors [12]) there was for many years a lively controversy about the time that the definitive germ line was set aside from somatic lineages. It has long been established that the germ cells are derived from the epiblast of the pregastrulation embryo [1, 5, 14], but not until the work of Kirsty Lawson and her colleagues [15] have germ cell precursors been identified. When single cells of the pregastrulation epiblast were injected with a fluorescent dextran and the embryo was cultured for 40 h, the position of the descendants of the injected cell could be determined. In this way, a fate map of the 6 dpc (pregastrulation) and 6.5 dpc (early gastrulation) embryos could be constructed. By determining which cells contained both alkaline phosphatase and fluorescent dextran after the culture period, it was possible to identify the precursors of the primordial germ cells. Analysis of clones derived from single epiblast cells demonstrated that cells in the 6 and 6.5 dpc embryos which contribute to the germ line also give rise to substantial numbers of cells in the extraembryonic mesoderm: the germ line has therefore not segregated at these stages. Before gastrulation, at 6 dpc, the cells from which the PGCs will be derived appear to be evenly distributed in a belt encircling the part of the epiblast immediately adjacent to the extraembryonic region (the proximal epiblast) but at 6.5 dpc, PGC precursors are missing from the most anterior part of this belt. These putative precursor cells do not lie close together, but are scattered in the epiblast among cells that give rise only to such tissues as the extraembryonic mesoderm and the blood islands of the yolk sac. Although it has not been possible so far to track the precursors of the PGCs directly through gastrulation, it is believed that, together with the cells that surround them, they move through the primitive streak and into the extraembryonic region quite early in gastrulation when the primitive streak first appears [16]. Although Lawson’s work succeeded in identifying PGC precursors in the proximal epiblast, the question of whether cells from any part of the epiblast can contribute to the germ line remained unanswered. However Patrick Tam’s group [17] recently grafted regions of the epiblast from mice carrying a transgene onto unmarked epiblasts and cultured them for 48 h. By staining cultured embryos both for alkaline phosphatase and for b-galactosidase (the product of the transgene), they could determine whether the grafted cells contributed to the germ line. They found that cells

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from the distal epiblast (which normally contribute to neurectoderm and surface ectoderm) could indeed give rise to germ cells when grafted to the proximal epiblast and that in the mouse at least, the ability to form germ cells is not restricted to specific epiblast cells. It is, however, not yet clear whether the germ cells are determined by their position in the embryo, through induction by other tissues, or by some other mechanism [16]. In the absence of any mutation which affects the germ cells at this stage, more experimental work is needed to define the influences operating on them at this critical stage in their development. When is the germ line determined, and how many founder cells are there? By finding that single cells of the 6- and 6.5-day epiblast can contribute to both somatic and germ lineages, Lawson’s work provides evidence that the germ line is not lineage restricted at this time. These results conflict with those of Soriano and Jaenisch [18] who conclude that the germ line is restricted very early and in a founding population of as few as 3 cells. Lawson, on the other hand, calculates that the founder population of the germ line cannot be much smaller than 45. Her results further suggest that lineage restriction takes place during the first 16 h of gastrulation, probably soon after the progenitor cells have left the epiblast, passed through the primitive streak, and moved to the extraembryonic region of the embryo at about 7.2 dpc. It is about this time that Ginsburg et al. can first detect alkaline phosphatase in putative early germ cells [10]. The alkaline phosphatase positive cells that Ginsburg and her colleagues describe in the extraembryonic region of the early 7 dpc embryo lie in a cluster posterior to the primitive streak, between the endoderm and the mesoderm of the ventral part of the amniotic fold. Apart from their positive reaction for alkaline phosphatase, they cannot be distinguished from other cells in the extraembryonic mesoderm. Because of the clustering and diffuse staining of these cells, accurate counts could not be made, but their number was estimated to be about 125, and about 8 of them contained a dense spot of alkaline phosphatase activity in the cytoplasm (possibly in the Golgi apparatus). In the 7.5 dpc embryo (Fig. 1a), the cluster of diffusely stained cells can be clearly seen above the newly formed amnion, at the base of the developing allantois posterior to the primitive streak, and there is little doubt that these cells, or at least some of them, are the primordial germ cells. The number of cells with dense cytoplasmic spots increases with developmental age, and the authors suggest that the establishment of a spot in a previously diffusely stained cell may reflect a maturation process. Lawson calculates that there are about 45 PGCs present in the embryo at the time the germ line is

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FIG. 1. Position of the PGCs in the embryo from 7.5 to 10.5 dpc. (a and b) At 7.5 and 8.5 dpc the PGCs (solid circles) lie at the base of the allantois. (c) At 9.5 dpc they are incorporated in the gut. (d) At 10.5 dpc they leave the gut, move up the dorsal mesentery, and enter the paired gonadal rudiments.

determined (about 7.2 days) while slightly later at 7.5 dpc expression of the transcription factor oct-4 (see section 5) is seen in 35 to 40 cells that appear to be PGCs [19]. However, Ginsburg et al. describe 125 cells in the 7.25 dpc embryo that express alkaline phosphatase and so may be PGCs. The discrepancy in numbers may be due to the difficulties of making accurate counts of PGCs at this stage or to differences in the mouse strains used. It is also conceivable that the progenitor population may undergo a rapid doubling in numbers after the determination of the germ line, but before the PGCs can be identified on the basis of alkaline phosphatase expression. Alternatively, not all the alkaline phosphatase positive cells that Ginsberg et al. describe may contribute to the germ cell population. Segregation of the germ cells in extraembryonic tissues. The temporary sequestration of the germ line in extraembryonic regions has been described in many

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vertebrates, but the reasons for it are not understood. The segregation of the germ line may be necessary to preserve its pluripotency at a time when many somatic cell lineages are being determined [15, 20, 21]. Monk et al. [22] suggest that the germ cells may be withdrawn from embryonic tissues so that they may escape the widespread tissue-specific methylation that occurs around the time of gastrulation (see section 7). Hematopoietic cells and PGCs: A common ontogeny? Several recent studies have drawn attention to the fact that there are significant similarities in the ontogeny of the germ line and the hematopoietic system. The work of Lawson and Hage [15] demonstrates that both PGCs and the cells of the yolk sac blood islands appear to originate from the same region of the pregastrulation epiblast, in the posterior part of the 6-day extraembryonic mesoderm. The presumptive blood island cells extend further distal along the egg cylinder than do the PGC progenitors, but the two areas overlap in the extreme posterior part of the epiblast, and in this region some (although not all) clones containing PGC progenitors also contribute cells to the blood islands. At later stages Medvinsky et al. [23] identify hematopoietic CFU-S (spleen colony-forming units) in the axial AGM (aorta–gonad–mesonephros region) of the 9–10.5 dpc embryo and point out that primordial germ cells are found in the same area. There are certainly parallels in the developmental histories of primordial germ cells and hematopoietic stem cells, and some pleiotropic mutations such as Dominant White Spotting (W), Steel (Sl), and Hertwig’s anemia affect both cell types. On the basis of studies in which PGC-containing regions of 7.5 and 8.5 dpc embryos were disaggregated and cultured, Rich [24] suggests that hematopoietic cells can be derived directly from PGCs in vitro, but the nature of the in vivo relationship between blood and germ cells has yet to be elucidated. 3. PROLIFERATION AND MIGRATION OF PRIMORDIAL GERM CELLS

After about 8 dpc the developmental history of the germ cells is well documented. In 1957, when the origin of the definitive germ cells was still a contentious subject, Mintz and Russell [9] published a study of the migrating germ cells in the mouse which, 40 years later, remains a standard reference in the field. Using alkaline phosphatase staining, they traced the movement of the PGCs in embryos from 8.5 to 13.5 dpc. At 8.5 dpc, most of the PGCs are in the stalk of the allantois and the yolk sac splanchnopleure at the posterior end of the primitive streak (Fig. 1b). In this position they lie at the margin of the open hind gut and become incorporated in the gut as it forms. By 9.5 dpc (Fig. 1c),

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TABLE 1 Mean Number of Primordial Germ Cells in Mouse Embryos of Different Ages According to Three Different Studies

Mintz and Russell [9] Tam and Snow [29] Buehr et al. [30]

Strain

821 dpc

921 dpc

1021 dpc

1121 dpc

1221 dpc

1321 dpc

C57 Bl/6 Q C3H/101

40 145 98

231 364 242

515 1012 858

2452 2999 —

4000 — —

— 25791 —

Note. The discrepancies in numbers are probably due mostly to strain differences and different counting methods.

most are embedded in the wall of the hind gut (usually in the endoderm), although a few remain in the yolk sac and the base of the allantois. At 10.5 days (Fig. 1d) cells have begun to migrate out of the hindgut and into the dorsal mesentery which now suspends the developing gut from the dorsal body wall. The germ cells move up the mesentery and into the paired gonadal primordia which develop either side of the dorsal mesentery from cells of somatic origin [16]. A few germ cells have reached the gonads by 10.5 days and most by 11.5 days. By 12 to 13 days, the gonad appears to be fully colonized, and the PGCs soon begin the first steps of differentiation into the definitive male or female gametes: at 13.5 dpc germ cells in the female enter meiotic prophase, while testicular germ cells arrest in the G1 phase of the mitotic cycle at the same age [25]. Alkaline phosphatase activity in the PGCs begins to decline by 14 dpc in female embryos and by 15 dpc in males [14]. The early history of the germ cells appears to be identical in both sexes, and it is only after the cells colonize the gonadal primordium that sex-specific differences in their development appear. These differences are, however, determined by the sex of the somatic cells among which they develop and not by their own chromosomal constitution [26]. The primordial germ cells of birds are known to be carried to the gonads in the blood [27] and it has been suggested that mammalian germ cells might occasionally take the same route [14, 28]. However, there is as yet no confirmation that this can happen, and in all mammalian groups so far studied the evidence suggests that the germ cells make their way to the gonad by migrating through solid tissue. The development of PGCs in the mouse from 8 to 12 dpc is characterized not only by migration, but also by cell proliferation. Although information on the cell kinetics of mouse PGCs at early stages in vivo is limited, Lawson’s analysis of cell clones which include the PGCs suggests that after lineage restriction the doubling time of PGCs increases from less than 7 h to 16 h [15]. Several studies have been published in which the number of PGCs of early embryos is estimated [9, 29, 30] (summarized in Table 1), and Tam and Snow [29] calculate that their figures represent a fairly regu-

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lar cell doubling time of 16 h. At 13.5 dpc, the colonization of the gonad by germ cells is complete and at this time, at the end of about eight replicative cycles, proliferation gradually ceases as ovarian germ cells enter meiosis and those in the testis undergo mitotic arrest. Ectopic germ cells. Cells which resemble PGCs morphologically and which express alkaline phosphatase have for many years been described in locations distant from the gonads or the normal route of PGC migration. Occasional ectopic cells have been described in skin and mesenchyme of normal mouse embryos from 8 to 13 dpc [9]. Primordial germ cells can also be found in the adrenal gland of embryos and young mice of both sexes [31]. Recent evidence [32] indicates that the adrenals and the gonads arise from the same primordia, and at the time when the urogenital ridge is colonized by the PGCs, it has not yet separated from the mesonephros and the adrenal. As a result, a few germ cells may remain in the mesonephric region or adrenal rudiment [26]. They can remain here for as long as 3 weeks (until Postnatal Day 12) and can enter meiosis and differentiate as oocytes. Germ cells have also been described in the adrenal primordia of both human [33] and marsupial [4] embryos. PGCs that escape the normal migration route have been suggested as a possible cause of some human tumors [34], such as germinomas of the central nervous system [35]. In embryos homozygous for the mutations W and Sl up to 30% of migrating germ cells may be found in ectopic locations such as the allantois or the wall of the vitelline artery [9, 30, 36]. 4. MECHANISMS OF MIGRATION

For several days mouse germ cells move through the embryo, following a complex route through different tissues. The mechanisms governing PGC movement probably change during the migratory phase, and there seem to be at least two distinct phases. During the first (from about 7.5 to 9 dpc), the cells move from the posterior end of the primitive streak into the open hindgut and become incorporated in the gut wall. At this time the cells are probably caught up in the morpho-

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genetic movements of the developing gut, and from their original position between the endoderm and the extraembryonic mesoderm they appear to be swept passively along with the gut endoderm as it invaginates. At this time they show no morphological or ultrastructural features characteristic of actively migrating cells [37]. However, once in the hindgut endoderm (at about 9 dpc) the PGCs develop pseudopodia and other structures characteristic of cells capable of amoeboid movement [38, 39]. PGCs isolated at about this time appear motile in time-lapse photography [40] and are invasive in in vitro culture [41]. During this second phase of migration (9.5 to 12.5 dpc) when they move up the dorsal mesentery and into the gonad primordia, the germ cells are thought to move actively through the tissues in which they lie [5, 40]. However, though the PGCs at this stage appear to be capable of amoeboid movement, the factors controlling their movement are not well understood. There are several possible mechanisms which may guide the migrating germ cells, such as chemotaxis (attraction of cells by a substance produced in the developing gonad), contact guidance (cells moving along a preformed molecular pathway in the substratum), and differential adhesion (PGCs responding to cell–cell contacts to ‘‘maximize the strength of adhesion’’ [5]. All of these mechanisms have been demonstrated in other systems, but it is not yet clear what part they or other factors play in guiding the germ cells. Because the mammalian embryo is difficult to study at these postimplantation stages, most experiments on migratory germ cells have been done in vitro. The migratory phase of mouse germ cell development lasts for approximately 4 days (8.5 to 12.5 dpc), and when it is finished, virtually all of the germ cells lie in the gonadal rudiment. At this time the germ cells gradually lose their capacity to move in vitro, and their morphology no longer resembles that of migratory cells [40]. Their cell surface properties change markedly: adhesion to fibronectin appears to decrease [42, 43], and several cell surface antigens (including SSEA-1) are lost [40, 44]. Alkaline phosphatase activity also decreases. The first stage of germ cell development is now over: the cells are established in the gonads and, influenced by the sex of the gonad in which they develop, begin to undergo changes that will ultimately lead to their differentiation as functional gametes. Chemotaxis. Early studies [45] suggested that chemotropic substances released by the genital ridges, the target organs of migrating germ cells, could act as attractants of the PGCs, and more recently Godin et al. [46] examined the in vitro responses of migratory PGCs to chemotactic stimuli. They demonstrated that genital ridges from 10.5 dpc embryos produced diffusible factors that enhanced the proliferation of PGCs in

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culture and appeared to attract PGCs toward them. In 1991 the same group [47] investigated the effects of the cytokine TGFb1 on migratory PGCs in culture and found that it mimicked the chemotropic effect of genital ridges (although in contrast to medium conditioned with genital ridges, purified TGFb1 inhibited PGC proliferation). In addition, an antibody to TGFb1 blocked the chemotropic effect of whole genital ridges in culture. However, the role of TGFb1 in germ cell guidance in vivo is not yet clear. For instance, it is not known if the PGCs express a receptor for TGFb1 . Also, TGFb1 expression at this stage is not limited to the genital ridges, but extends over a large area of the dorsal body wall, and it seems unlikely that it alone can be responsible for the guidance of migrating cells to the gonad rudiments. Moreover, mice in which the TGFb1 gene has been disrupted [48] show many postnatal abnormalities, but no effect on gamete development is reported. A chemotactic stimulus may also be provided by the Steel factor (SLF). Keshet et al. [49] describe a gradient of SLF expression along the path of migrating PGCs in the 10.5 dpc embryo which is highest in the gonads and suggest that this may provide a mechanism for the control of ‘‘cell homing’’ through chemotaxis. However, soluble SLF appears to exert no chemotactic effect on germ cells in an in vitro assay [50] and most germ cells in embryos homozygous for Steel mutations (section 6) do migrate normally. Contact guidance and differential adhesion. As germ cells migrate, they contact several different cell types, and these cells or the matrix surrounding them may influence the movement of PGCs. There is evidence that in Xenopus embryos [51] extracellular matrix molecules such as laminin and fibronectin (both of which are present on the normal pathways of PGC migration in the mouse [52, 53]) guide the migrating germ cells. The mechanism by which substrate cells or molecules affect the migration of the PGCs over them is not clear. In Xenopus [51] the alignment of cells in the substratum may influence the direction of migration. Alternatively, differential adhesion or a change in the ability of the PGCs to adhere to substrate molecules might mediate the migratory ability of the cells [54]. The cell surface antigen SSEA-1 may be involved in such cell–cell interactions [55, 56]. Alternatively, integrin receptors expressed by PGCs at various developmental stages may mediate attachment to molecules of the extracellular matrix [39]. ffrench-Constant et al. [42] show that with increasing developmental age germ cells gradually lose the ability to adhere to fibronectincoated dishes, and they postulate that this changing pattern of adhesion could reflect the change from the passive transport of PGCs in the hindgut endoderm to the active phase of migration away from the endoderm.

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A partial loss of adhesivity to fibronectin during PGC migration (9.5–11.5 dpc) and a further reduction after gonad colonization (13.5 dpc onward) could favor PGC migration and later assist the cells to maintain their position in the gonadal rudiment. However, it has been reported [42, 57] that migration of PGCs in vitro is enhanced by exogenous fibronectin. The role of fibronectin in cell migration has yet to be fully elucidated, and a useful tool in this field will be the mice generated by George et al. [58] in which the fibronectin gene has been disrupted by gene targeting. Mice homozygous for this induced mutation begin to show abnormalities in many tissues (especially those of mesodermal origin) at 8 dpc, and defects accumulate as development proceeds. However, some embryos survive to 10.5 dpc or more, and studies of their PGCs will offer important clues on the role of fibronectin in germ cell migration in vivo. The Steel/c-kit signaling pathway (section 6) may also be involved in contact guidance of PGCs, although results obtained so far offer no clear picture of its role. In mice homozygous for some Sl or W mutations a significant proportion of germ cells are ectopically located [30, 36], which implies that the migratory ability of the cells is affected (though not abolished). SLF (the product of the Sl gene) may favor the adhesion of PGCs to the substrate in vivo, as soluble SLF seems able to do in vitro [59]. Some authors have suggested that membrane-bound SLF may mediate PGC adhesion to somatic cells and provide cues to guide their migration [55], and the interaction between the c-kit receptor and membrane-bound SLF appears to be responsible for the adhesion of spermatogonia to Sertoli cells [60]. Other mechanisms. Finally, cell contacts between the germ cells themselves may influence migration. In the past, most studies of germ cell migration assumed that the germ cells move independently of each other, but this is probably not the case. By using confocal microscopy of antibody-labeled germ cells in wholemount preparations, Gomperts et al. [56, 61] demonstrated the existence of extensive networks of interconnected germ cells during the migratory phase. These networks first appear in 9.5 dpc embryos, after the germ cells leave the gut endoderm, and by 10.5 dpc nearly all the migrating germ cells appear to be in contact with each other, linked by long, thin cytoplasmic processes. Gomperts and her colleagues also call attention to the fact that the first germ cells leave the gut at a time when the dorsal mesentery is not yet completely formed. These pioneers move almost directly from the gut into the dorsal midline and then to the developing gonadal primordia, suggesting that the interconnecting networks between germ cells could help to guide those leaving the gut at a later stage up

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the mesentery and into the gonads. De Felici et al. (personal communication) have also recently found that PGCs can adhere to each other in culture by cadherin-dependent mechanisms. The migratory and proliferative behavior of PGCs has been studied for over 20 years, and a variety of factors have been identified which can affect the behavior of PGCs in culture and which may also play important roles in vivo. However, a clear and unequivocal picture of the mechanisms controlling PGC behavior in the living embryo is still lacking, and it is not certain that all the factors identified as affecting PGC development in vitro will prove important in normal development. For instance, the cytokine TGFb1 affects PGC migration in vitro, but the germ cells of mice lacking a functional TGFb1 gene appear to migrate normally. Further, because conditions provided by a culture system are rarely if ever those encountered by cells in the embryo, the results of in vitro studies are often difficult to interpret. Perhaps the single unequivocal example of a system known to influence PGCs in vivo is the ligand–receptor complex of SLF and c-kit, whose effect is clearly demonstrated by the phenotype of homozygous W and Steel mice (section 7). The difficulty of interpreting in vitro observations however is emphasized by the conflicting results obtained from experiments examining the effect of SLF on cultured PGCs (section 9). In vitro studies have provided important insights into the biology of PGCs and will continue to supply information which can be obtained only by study of the isolated cell. However, other tools are now available with which to investigate the factors governing PGC development in the intact embryo. Among the most promising of these are mice carrying targeted mutations in which specific genes have been disabled or mutated. By removing or altering the function of a specific molecule and describing the influence of that mutation in vivo, a more direct examination of factors affecting PGC proliferation, migration, and differentiation will be possible. However, the possibility of alternative signaling pathways operating in the embryo may make analysis of these mutants difficult, and ultimately a combination of both in vivo and in vitro studies will probably be necessary before a clear picture of germ cell biology emerges. 5. GENES EXPRESSED BY PGCS

The primordial germ cells are in many ways highly specialized cells and express a specific repertoire of gene products including alkaline phosphatase, oct-4, DNA methyltransferase, stage-specific cell surface antigens, and others [62]. Some of these products (such as oct-4) may be concerned with the maintenance of

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totipotentiality. Some (e.g., cell surface molecules) may be necessary for normal migration and proliferation, while the function of others (for instance, alkaline phosphatase) is unknown. In the postgastrulation embryo, however, with the possible exception of oct-4 none of these gene products appears to be exclusive to the PGCs. Many cell surface antigens, such as SSEA-1, SSEA3, Forsmann’s antigen, and others, are expressed by PGCs at various stages of their development and have often been used to identify and manipulate germ cells [5, 39, 63–65]. These antigens are typically glycoconjugates of several different classes, and because their expression patterns change during the course of PGC development (and especially after the PGCs have reached the gonad) they are thought to play a role in specific recognition and adhesion interactions between the PGCs and their substrate [56, 66]. Perhaps the best studied of the cell surface antigens is SSEA-1 (stagespecific embryonic antigen 1), a carbohydrate antigenic determinant of a glycolipid expressed on a restricted number of embryonic and adult cell types [67], on lines of EC and ES cells, and on primordial germ cells from about 9 dpc through the migratory period until about 15.5 dpc, at which time the cells are in the gonad and have stopped proliferating [40]. Other proteins (such as the heat shock protein HSP 90 [68]) may also be expressed in PGCs in a stage-specific manner. The protein oct-4 (also termed oct-3 or oct 3/4), a member of the POU family of transcription factors, binds to the octamer motif in DNA [69]. It appears to be expressed only in totipotent cell lines (EC, ES, and EG cells), in mature oocytes, and in totipotent cells of the embryo such as the inner cell mass, the epiblast, and PGCs [70]. oct-4 expression is reported to be controlled by two different enhancers, one of which (the distal enhancer) directs expression in ES and EG cells, preimplantation blastomeres, and the totipotent cells of the germ line, while the other (the proximal enhancer) is active in the epiblast [19]. Although the pattern of oct-4 expression is not yet well characterized in animals other than mice, the limitation of its expression to totipotent cell types promises to make it a valuable marker of primordial germ cells in other species. 6. MUTATIONS AFFECTING PRIMORDIAL GERM CELLS

Although several mutations are known to affect germ cells in the embryo after 8 dpc, to date no mutation has been described which specifically blocks the initial establishment of the germ line. All mutations and chromosomal abnormalities so far described which allow development to postgastrulation stages appear to have no direct effect on the establishment of the germ cells:

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for instance, the number and distribution of germ cells from triploid and tetraploid mouse embryos of 9.5 and 11.5 dpc are normal [71, 72]. Mutations are known, however, which affect germ cells in embryos older than 8.5 dpc. These include Hertwig’s macrocytic anemia, teratoma, atrichosis, germ cell deficient, and most alleles of Dominant White Spotting and Steel. All of these affect the number of germ cells in the embryo and/or adult to a greater or lesser extent, all can cause sterility when homozygous, and all, with the possible exception of germ cell deficient, are pleiotropic. Hertwig’s macrocytic anemia (an: chromosome 4) [73, 74] causes both macrocytic anemia and a reduction in germ cell numbers in homozygotes, which (on some backgrounds) are viable but anemic and sterile. Although the number of germ cells is low, some proliferation does take place and a moderate number of cells enter the gonads, only to display degenerative changes by 12.5 dpc. Mice homozygous for the teratoma gene (ter: chromosome 18) on a 129/Sv or LTXBJ background have normal numbers of germ cells at 7.5 days, but after this time the cells do not proliferate normally. Germ cell migration, however, is normal, and on some backgrounds adult females may have a few litters [74, 75]. The atrichosis mutation (at: chromosome 10) reduces the number of germ cells in gonads of adult homozygotes and also the density of hair growth [74, 76]. Unlike the other mutations mentioned, germ cell deficient (gcd) is not a spontaneous mutation and does not appear to be pleiotropic. It arose as a result of the insertion of a transgene construct in chromosome 11, near the genes for the cytokines leukemia inhibitory factor (LIF) and oncostatin M (neither of which, however, appears to be affected by the insertion [77]). The mutation is recessive and causes a deficiency in the number of germ cells in homozygotes, which at 11.5 dpc number less than half those in wild-type embryos [78]. It is not clear, however, whether this deficiency is due to a reduced rate of proliferation or to abnormal migration. Dominant White Spotting (the W locus: chromosome 5) is one of the oldest mutations known in the mouse, and its effects on the embryo are well documented. The severity of the phenotype varies according to which of the many alleles is involved [79, 80], but most W homozygotes are sterile black-eyed white mice with a severe anemia that normally kills the neonate (although the anemia caused by some alleles is mild and homozygotes may be viable). The effects of the gene are obvious in three cell types. In homozygotes the development of the skin melanoblasts is affected (resulting in a white coat) but melanocytes of the pigmented layers of the retina and iris are normal. The number of hematopoietic stem

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cells is drastically reduced (causing anemia) and as there are few or no germ cells in the gonad, the animals are sterile (although one allele has been described in which homozygotes are fertile [79]). Heterozygotes commonly have a forehead blaze, a white belly, and white feet, and the coat may be mixed with white. It is not yet clear whether the germ cells or hematopoietic stem cells of heterozygotes differ from those of wildtype animals: heterozygotes are healthy and fertile, but there is some evidence that in heterozygotes for Extreme Dominant Spotting (We), the number of germ cells may be lower than normal [30]. The deficit in primordial germ cells of W/W embryos has been well described [9, 30]. At about 8 dpc, homozygous mutants as well as heterozygotes and wild-type embryos have approximately 100 germ cells. However, at all later stages when the number of germ cells in the other two genotypes increases rapidly, germ cells in homozygous mutants show no sign of proliferation. Germ cells in We/We embryos also tend to adhere together in clumps rather than becoming distributed widely in the tissues in which they lie. As many as 30% of We/We germ cells are found in ectopic sites, but nonetheless some find their way into the gonad. The distinctive phenotype of W/W mice is mimicked by animals homozygous for mutations at the Sl locus (chromosome 10), but a considerable body of experimental evidence [79] shows clearly that mice homozygous for W mutations have defective melanoblasts, germ cells, and hematopoietic stem cells, while in Sl/ Sl animals the defect does not lie in the stem cells but rather in the microenvironment in which the stem cells are located. Although it has been clear for some time that the products of the W and Steel genes must affect different parts of the same developmental mechanism, only recently has the molecular basis of this mechanism been elucidated [81]. The product of the W gene is the transmembrane tyrosine kinase receptor c-kit [82], which is expressed at high levels in melanocytes, hematopoietic stem cells, and primordial germ cells, as well as in other tissues. In situ hybridization has shown that the c-kit receptor is expressed in primordial germ cells from the time they can first be distinguished in 7.5day embryos until the time that gamete differentiation begins in the gonad at about 13.5 dpc [83]. c-kit also appears to be expressed in some of the tissues through which the germ cells pass, such as the mesenchyme surrounding the dorsal aorta. After the c-kit receptor had been identified as the product of the W gene, it came as no surprise when the ligand for c-kit was shown to be the product of the Steel gene [84–86]. The Steel factor (SLF) is a transmembrane growth factor with cytoplasmic, transmembrane, and extracellular domains. As expected, SLF is ex-

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pressed in stromal cells of the skin, bone marrow, and gonad, with the highest level of expression in the gonads, the destination of the migrating germ cells [49]. Alternative splicing generates two different forms of SLF, one with a proteolytic cleavage site in the extracellular domain. If this is cleaved, the extracellular domain is rapidly released as a soluble growth factor. The second form has no cleavage site and is either cleaved very slowly or remains membrane-bound [87]. c-kit and SLF appear to be components of a signaling pathway required for normal development of melanoblasts, hematopoietic stem cells, and germ cells. There is much discussion as to whether this pathway is involved in cell proliferation, migration, or viability [81]. The activation of tyrosine kinase receptors similar to c-kit is often associated with a mitogenic signal [88], and the most conspicuous difference between W/W, Sl/ Sld, and wild-type embryos is the very low number of germ cells in homozygous mutants [9, 30, 36], which probably reflects a proliferation defect (although reduced germ cell viability cannot be ruled out). Although mutant germ cells may be somewhat retarded in migration and many are ectopic, the fact that some W/W and Sl/Sld cells survive for several days, follow the appropriate migratory route, and find the gonads indicates that their ability to survive and migrate is not totally abolished. However, the clumping of germ cells characteristic of We/We animals suggests that their ability to adhere normally to the substrate may be impaired, and this defect may affect migratory ability. 7. X INACTIVATION AND IMPRINTING

In somatic cells of female mammals, a single X chromosome (either paternal or maternal) is inactivated [89]. In the female germ line, X inactivation can be demonstrated in postmigratory germ cells [90], but the inactivation is reversed in the oocyte by 13 dpc as the cell enters the first meiotic prophase [14, 90]. Both X chromosomes are then active throughout oogenesis and at early developmental stages of the female embryo, but one becomes inactivated at some time after implantation. Several studies have attempted to specify the time in development at which X inactivation takes place in the germ lineage. After studying the expression patterns of the X-linked enzyme phosphoglycerate kinase, Marilyn Monk and her colleagues [91, 92] concluded that X inactivation takes place before the germ line and the germ layers are determined and is probably complete in all cells of the embryo at gastrulation (about 6.5 dpc). However, recently Patrick Tam’s group [93] was able to visualize X inactivation more directly by examining female embryos heterozygous for an Xlinked transgene expressing lacZ. If all cells expressed b-galactosidase (the transgene product), both X chro-

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mosomes were assumed to be active, while staining of 50% of cells indicated that only one was active. The results indicated that although X inactivation had taken place in a few 7.5 dpc germ cells, 85% of the germ cells at this time appeared to contain two active X chromosomes. X inactivation did not occur simultaneously in all PGCs and did not appear to be complete until relatively late, at about 9.5 dpc. However, interpretation of these results is complicated by the fact that there is a time lag between the inactivation of a chromosome and the disappearance of the protein product of a gene on that chromosome. The disappearance of b-galactosidase in these embryos would therefore be expected to take place at some time after functional X inactivation has occurred. The methylation of cytosine nucleotides in CpG sequences, especially in the promoter region of genes, has long been associated with the inactivation of transcription, whereas a low degree of methylation of genes has often been assumed to indicate genetic activity. Immediately after fertilization, most genes of the mouse embryo undergo active demethylation [94] and by the blastocyst stage most CpG sites in the embryo (apart from those affected by imprinting) are unmethylated. Sometime before gastrulation, there is a global de novo methylation throughout the embryo which is followed at various times by gene-specific demethylation, usually at a time when active transcription begins [94, 95]. In a study of the methylation status of germ cells, Monk et al. [22] report that at 12.5 dpc germ cells from both male and female embryos are highly undermethylated compared to other somatic tissues and embryonic stages examined. Two days later the female PGCs remain highly undermethylated, while the methylation of male cells has increased somewhat. More recently, Kafri et al. [94] find that the primordial germ cells appear to escape the global methylation that occurs around the time of gastrulation and remain undermethylated until they begin to differentiate as gametes, when they adopt a methylation pattern specific to eggs or sperm. The methylation of genes plays a part in the establishment and maintenance of imprinting [96, 97], although changes in chromatin configuration may also be important. The erasure of the old imprint and establishment of the new presumably take place in the germline, probably during gametogenesis [98], but neither the cause nor the mechanism of initiation of imprinting is known. 8. IN VITRO CULTURE OF PGCS

Because they are relatively few in number, change their position in the embryo quickly, and are always to be found mingled with somatic cells, mammalian germ

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cells are difficult to collect in large numbers or as pure samples for culture or analysis. However, several protocols have been developed for the collection, purification, and culture of mouse PGCs [63, 99–102] and germ cells isolated by these methods have been successfully used in many studies of germ cell biology. Early studies on germ cell culture [54, 103] established that primordial germ cells isolated during or shortly after the migratory phase and cultured in the absence of somatic cells survived only for a few days. The addition of substrates such as fibronectin, laminin, and type IV collagen improved cell viability to some extent but survival was still poor [42, 43, 57]. The use of feeder layers such as STO cells [40] enabled cultures to be maintained for 7 days or so, and further work has shown that with suitable feeder cells and the addition of appropriate cytokines to the medium, primordial germ cells can be maintained for a considerable time and under some conditions can give rise to cell lines (EG cells) similar to embryonic stem cells [59, 104] (section 10). Germ cells in migratory and early gonadal phases die rapidly when cultured without feeder layers and certain growth factors, and apoptosis has now been identified as the mechanism of death [120–122]. The death of ectopic germ cells in the intact embryo also appears to be apoptotic [120]. However, apoptosis in cultured germ cells can be blocked for a few hours by the addition of soluble SLF and LIF [120], so the ability of SLF and LIF to maintain the survival of cultured germ cells may be due to their ability to suppress apoptosis. Although cultured PGCs have yielded valuable information about factors controlling many aspects of their biology, there must be significant differences between germ cells cultured in vitro and those in the intact embryo. Because germ cells cannot successfully be cultured in the absence of feeder layers, direct effects of experimental treatments on germ cells are often impossible to distinguish from indirect effects on feeder cells, and undefined factors produced by the feeders can make the interpretation of results difficult. 9. PGCS AND CYTOKINES IN VITRO

The germ cells divide constantly as they migrate, but proliferation stops as the cells enter meiosis or mitotic arrest in the gonad, and many in vitro studies have attempted to define the role of a variety of factors on the initiation, maintenance, and control of germ cell proliferation. It can however be difficult to distinguish between cell proliferation and cell survival, especially when cells are grown on feeder layers. Although incorporation of BrdU or tritiated thymidine may be used to estimate proliferation rates, the addition of a survival

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factor that is not itself a mitogen may permit cells to respond to another mitogenic factor in the system [105]. Because germ cells respond to many cytokines and other factors that often act in synergy, it can be difficult to dissect out the specific function of a single compound, and many of the factors discussed in the following paragraphs may only exert their effects in concert with other molecules. The response of PGCs to some factors may prove to be an artifact of the culture system, and not all may have a role to play in normal germ cell development. Steel factor. Analysis of the phenotype of Steel and W embryos suggests that the signaling pathway controlled by the c-kit receptor and its ligand SLF in vivo principally affects the proliferation of germ cells during the migratory and early gonadal phases (although it has not been possible to rule out the possibility that germ cells in mutant embryos can proliferate but do not survive). However, studies of in vitro effects of SLF do not always confirm this. Some groups [50, 106, 107] report that germ cells survive and proliferate only when cultured on feeder layers that produce the membrane-bound form of SLF, but on the basis of BrdU incorporation studies conclude that under these conditions SLF acts principally as a promoter of survival and not as a mitogen. On the other hand, in the presence of LIF, SLF can act as a mitogen [59, 104]. There may therefore be a factor missing from culture systems that in the intact embryo acts in conjunction with the c-kit/ SLF system not only to maintain the germ cells but also to promote proliferation. Alternatively, the activation of the c-kit receptor under simple culture conditions might be insufficient to stimulate proliferation: although the activation of tyrosine kinase receptors often provides a mitogenic signal [88], low-level stimulation may promote survival [108]. Whatever its role in vivo, it is clear that SLF is necessary for the survival of cultured germ cells, and the absolute requirement of germ cells for feeder layers may reflect their need for a source of membrane-bound SLF (although feeder layers can produce other factors as well). Membranebound SLF is far more efficient at supporting PGC survival than is the soluble form, an observation which may explain the sterility of mice homozygous for the Steel–Dickie (Sld) allele which express only soluble SLF [50, 106]. If SLF acts as a survival factor, the restriction of its expression to cells along the normal route of germ cell migration might provide a way to eliminate potentially tumorigenic ectopic germ cells. The fact that adrenal germ cells, although few in number, can persist through embryonic development and into postnatal life [31] indicates that survival in some ectopic sites is possible: it is, however, not known whether or not SLF is expressed in the adrenal at these stages.

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LIF. The cytokine LIF (also known as DIA or differentiation inhibiting activity) is a pleiotropic cytokine which is an inhibitor of differentiation in embryonic stem cells [109] and which can enhance the survival [110] and, in combination with SLF, the proliferation of cultured germ cells [59, 104, 107]. However, Cheng et al. [111] report that even in the absence of SLF exogenous LIF can act directly on PGCs to promote their proliferation in culture. LIF normally acts through a heterodimer of the low-affinity LIF receptor and the gp130 signal transducer, and cultured PGCs die in the presence of antibodies to either [111, 112]. The effects of LIF upon PGCs are not understood: it might act primarily as a mitogen, a survival factor, or an inhibitor of differentiation. Mice lacking a functional LIF gene are fertile and produce viable embryos (although their uteri cannot support implantation) [113] and mice homozygous for a targeted disruption of the LIF receptor [114] have normal PGCs. Alternative ligands exist, however (such as interleukin 6 [115]), which do not require the LIF receptor subunit and which may be capable of activating the gp130 signaling pathway in these animals. Other factors. The cytokine bFGF (FGF-2), when added to cultures of germ cells in the presence of membrane-bound SLF and LIF, is a potent mitogen [104] and extends the period of germ cell proliferation long beyond that of PGCs in the embryo. For this reason, it is questioned whether this factor is directly involved in the normal development of germ cells in vivo [105]. Kawase et al. [116] present evidence that tumor necrosis factor alpha (TNFa) stimulates the incorporation of BrdU in early (7.5 to 8.5 dpc) cultured germ cells, but not in 10- to 12.5-day cells. However, TNFa was most effective in the presence of SLF and LIF. Koshimizu et al. [117] report that retinoic acid promotes the survival and proliferation of 8.5, 11.5, and 13.5 dpc germ cells in culture. Finally, there is good evidence that cyclic AMP can provide a mitogenic stimulus to cultured germ cells. De Felici and his colleagues added forskolin to germ cell cultures [107, 118] which raised levels of intracellular cAMP and caused a significant increase in BrdU incorporation by germ cells at migratory stages. More recently, this group [119] reports that pituitary adenylate cyclase activating peptides (PACAP) are able to stimulate PGC proliferation by activating adenylate cyclase. As in many studies of factors influencing cultured germ cells, however, there appears to be a complex relationship operating between SLF, LIF, and other cytokines and biologically active peptides. The results of these in vitro studies suggest that the initiation and maintenance of germ cell proliferation are dependent on a combination of cytokines such as SLF and LIF acting in synergy with other factors, some

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of which probably have yet to be defined. Whether or not all of these factors will prove to be of importance in regulating PGC proliferation in the intact embryo is, however, not clear. There is also little information about the mechanism that stops mitosis once the cells are in the gonad. TGFb1 , produced by the gonads and implicated as a chemotropic factor involved in cell migration, is an effective inhibitor of proliferation [47] and is a candidate for the factor that ends the initial phase of germ cell proliferation. 10. CELL LINES DERIVED FROM PGCS

Embryonal carcinoma cells. In the 1960s it was found that genital ridges from 11.5–12.5 dpc male mouse embryos, when transplanted to ectopic sites in an adult host, could develop into teratocarcinomas [123] and that the EC cells isolated from these tumors had apparently originated from the germ cells. Later it was found that teratocarcinomas could also be derived from the epiblast of embryos aged 6.5 to 8.5 dpc [124]. However, by transferring fragments of epiblast to ectopic sites, Beddington [125] showed that anterior parts of the 7.5 dpc epiblast (which do not normally contain PGC precursors) were also capable of generating EC cells. The ability to give rise to EC cells is therefore not limited to PGCs or their immediate precursors in the epiblast. Embryonal germ cells. The ability of SLF (especially the membrane-bound form) and LIF to support the survival and to some extent the proliferation of cultured germ cells has made it possible to study many aspects of germ cell biology in vitro. However, so far no true PGC cell lines have been produced. The proliferative capacity of migratory germ cells in culture is limited (resembling the proliferative capacity of germ cells in vivo) and in cultures older than 7 days PGC proliferation slows and the number of germ cells decreases [104]. However, the addition of bFGF to cultures prolongs indefinitely the capacity of germ cells to divide and makes it possible to obtain cell lines from migratory (8.5–9.5 dpc) and early gonadal (12.5 dpc) germ cells [59, 104, 126] taken directly from the embryo without being derived from teratocarcinomas (as have EC cells). These cell lines have been termed embryonic germ cells (EG cells) and are in many ways similar to EC cells and embryonic stem cells (ES cells). They can be derived from 8.5 dpc germ cells of both sexes, but from 12.5 dpc male cells only [126]. Germ cells in newly established cultures seem to require the presence of LIF, bFGF, and both soluble and membrane-bound SLF, but after a short time they lose their requirement for bFGF and SLF. Like ES and EC cells, EG cells express alkaline phosphatase and oct-4 and can be ag-

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gregated in vitro to form embryoid bodies. They can give rise to teratocarcinomas when transferred to appropriate ectopic sites and can also contribute to chimeras and populate the germ line [126, 127]. Recently [117] it has been reported that EG cells can also be derived with a combination of LIF and either forskolin or retinoic acid. In this system, LIF can be replaced by oncostatin M or interleukin 6, cytokines which like LIF act through the receptor molecule gp130. The activation of the gp130 signaling pathway therefore appears to be as important for EG cells as it is for ES cells. Although EG cells are derived directly from germ cells, they appear to be a different cell type. Cultured PGCs are generally found singly or in small groups, much as they are in the intact embryo. EG cells, on the other hand, form tight colonies and have apparently lost the ability to migrate [128]. Germ cells require the presence of bFGF and SLF in the medium: established lines of EG cells do not. EG cells proliferate indefinitely in culture, while PGCs do not. In contrast to EG cells, migratory germ cells taken directly from the embryo and injected into a blastocyst have so far proven incapable of contributing to a chimera [34, 105] (though one group reports low levels of chimerism from rabbit blastocysts injected with gonadal germ cells [129]), suggesting that the immediate developmental potential of PGCs may be less than that of inner cell mass cells or EG cells. This may however be simply for technical reasons, such as suboptimal conditions of PGC collection and transfer. 11. CONCLUSIONS

In this review it has been possible merely to touch on some of the exciting studies which in the past few years have extended our knowledge of the biology of the primordial germ cells. Through a combination of classical methods and newer molecular approaches we now understand much about the origin of the mouse PGCs and the factors influencing their development. The precursors of the PGCs have been mapped, and experimental evidence now exists showing that the germ line in the mouse is determined at a relatively late stage. The complex factors influencing the proliferation, migration, and survival of the PGCs are being unraveled. Furthermore, primordial germ cells have given rise to a new cell type, the EG cell, that like the ES cell retains totipotency through many passages in vitro. At the time of writing, true ES cells have only been derived from a few strains of mice, and as a result the powerful technique of gene targeting is confined to the mouse. Should it prove possible to derive EG cells from nonmurine species, gene targeting would be possible in many other groups, dramatically expanding our

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understanding of genetics and development and having important commercial implications for the future. I am grateful to Anne McLaren, Massimo De Felici, and Austin Smith for their stimulating and constructive comments on the manuscript.

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Received October 24, 1996 Revised version received January 24, 1997

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