A Stem-line Model for Cellular and Chromosomal Differentiation in Early Mouse Development

Differentiation

Differentiation (1981) 19: 71-76

0 Springer-Verlag 1981

Models and Hypotheses

A Stem-line Model for Cellular and Chromosomal Differentiation in Early Mouse Development MARILYN MONK

MRC Mammalian Development Unit, Wolfson House, 4 Stephenson Way, London NWl 2HE, England

Differentiation in mouse embryo development is represented formally by means of a stem-line model in which: 1. Individualchoices are structured so that only a fraction of the cells of a given population receive a signal to undergo a change of state that results in a departure from the stem line. 2. Development proceeds by a series of restrictions in potency of all the cells in the stem line. 3. The stem line harbours the germ cells. 4. The germ cells are returned to a state of totipotency by some event@) leading to meiosis.

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During development of the mouse embryo from fertilisation to gastrulation different tissues appear in ordered sequence in time and space: trophectoderm, inner cell mass (ICM), primary endoderm, and epiblast. Much information on these early cell lineage relationships has been provided by studies on cell fate and potency in mouse embryo chimaeras [l-31. The trophectoderm and primary endoderm give rise to extra-embryonic structures, the placenta, and various membranes. The ICM-epiblast (foetal) lineage harbours pluripotent cells capable of giving rise to the three definitive germ layers. This lineage undergoes several restrictions in potency prior to gastrulation, yet still contains precursors to the totipotent germ line [2-41. A stem cell mode of differentiation has been considered with respect to the derivation of these lineages [5] and to the pattern of X chromosome differentiation [ti] during development. This paper formally elaborates such a ‘stem-line’ model and describes its application to cell lineage relationships. Temporal and regional studies on X chromosome differentiation, [6-101 which are consistent with the two types of choice structure implicit in the model, are reviewed. An important feature in the model is the hypothesis that meiosis is a ‘dedifferentiation’event. That is, in addition to its accepted roles in genetic exchange and production of haploid gametes it is proposed that meiosis plays another crucial role in the restoration of totipotency to the germ line. The ‘stem-line’ model is presented as a ‘Catherine wheel’ to illustrate this idea. Some implications of the catherine wheel model are discussed.

to become B, while the rest remain as A, as distinct from the more common view of a bifurcation of population A into populations B and C. Figure 1 shows this distinction; in the stem-line model only a fraction of the cells receive a signal directing them to depart. Further subctivision of departed differentiated populations into different derivative cell types may depend on position effects. In order that development proceeds, it is an essential feature of the model that changes in state occur at various stages in the whole population of stem cells, e.g., A 4 in Fig. lb. Prior to such a ‘restriction in potency’, the ‘stem line’ continues to produce the same type of differentiated cell (indicated by dotted lines in Fig. lb). Restrictionsin potency of the stem cells may be simply a time-dependent process, external or intrinsic. In Fig. l b a restriction in potency is 0

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pluripotent s t e m line !

A. Formal Aspects of ‘Stem-line’ Model

In general terms the model proposes that development proceeds by a series of departures of different cell lineages from a population of pluripotent ‘stem-line’ cells. At each departure point the ‘stem’ population remains for some finite time in the same state, and thus is capable of further production of cells of the same differentiated type. In essence it is the decision of a proportion of the population of cells type A

-rc restriction in potency

1 okporture of dr fferentioted populotton

Fig. 1. a A bifurcation of population of cells A into populations B and C. These latter two cell populations bifurcate further as shown. b A stem-line model. Each choice is structured so that only part of population undergoes a change in state. Development proceeds by restrictions in potency involving all the cells in the stem line

0301-4681/81/0019/0071/$01.20

M. Monk: Differentiation in Early Mouse Development

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depicted as occurring at a time midway between two departures of differentiated cells; however it could occur at any time between them. (It cannot be coincidental with one or the other departure point or the bifurcation becomes A =t B and C). At the cellular level an actual departure of a differentiated cell may occur at the division of each stem cell. In essence, a stem-line model (Fig. lb) is distinguished from a conventional bifurcation (Fig. la) in that it assumes two types of choice structure, the departure (e.g., divergence of B) and the transition (e.g., A X ) . Studies on X chromosome differentiation support this assumption. Another feature of the model is that the stem line contains precursors to the totipotent germ line. Since the stem line has undergone several restrictions in potency, some event must occur to restore totipotency to the germ cells, and through this event the stem line completes a cycle which is envisaged as the catherine wheel model presented below (Fig. 4).

B. Cell Lineage Relationships The application of the model to cell differentiation in mouse embryo development is shown in Fig. 2 and its implications to current knowledge of cell lineages can now be discussed (see also [l-5, 11, 121). Firstly, the development of blastomeres isolated from cleavage stage embryos indicates that each blastomere is totipotent at least until the 8-cell stage [13]. The first overt sign of differentiation occurs when the ball of blastomeres of the morula separates into two distinct populations of cells - the outside sphere of trophectoderm cells and a small group of inner cells, the inner cell mass (ICM; see Fig. 3). The trophectoderm gives rise to extra-embryonic regions and the placenta, the embryo proper arising from among the cells of the ICM. The environmentaltrigger for this first ‘departure’ appears to be associated with inside or outside cell position in the morula [14, 151. However, as Rossant [2] has pointed out, we only require one signal, viz. ‘outside’, for the departure of trophectoderm; the inside cells, or stem-line cells, may be those protected from that signal. There is also a time component in the first differentiation event; if there are not enough cells to provide internal protection at the time it occurs, then all cells become trophectoderm, and the stem line (and embryo) is lost [14]. The departure of a trophectoderm

cell from the stem line may normally occur at cell division. Johnson et al. [16] have demonstrated intracellular polarization of several distinct properties at the 8tell stage, and these properties are distributed unequally to outside and inside daughter cells at division. If there is a tendency for a distinct fate for each daughter cell at division, it is subject to regulation, i.e., the system is labile. The model predicts a finite time in which we could observe the potential of the inside blastomeres to generate more trophectoderm cells if exposed to the outside stimulus. That isolated inner cells can indeed give rise to more trophectoderm has been well established [17-201. However, the first restriction in potency occurs prior to primary endoderm departure (see Figs. 2 and 3); inner cell masses (ICMs) isolated from older blastocysts are no longer able to regenerate trophectoderm either in vivo [21] or in vitro [22]. There is also evidence that outside cells are labile for some time. When aggregated with cleavage stage embryos outside cells isolated from late morulae can contribute to ICM derivatives [23]. The time of irreversible commitment of outside cells to trophectoderm may be very close to the time of restriction in potency of the internal blastomeres to ICM. There is no evidence that interaction with trophectoderm cells effects the subsequent restriction of the inside cells to ICM. Rather cell interaction occurs in the direction ICM to trophoblast [24] (see Fig. 2). The next departure is that of the primary endoderm from the ICM cell population (Figs. 2 and 3). As in the previous case the environmental trigger may be the outside position of the cells [25]. The primary endoderm gives rise to the visceral (proximal) and parietal (distal) endodermal layers of the yolk sac; it does not give rise to the definitive endoderm of the foetus [ll, 261. As in the case of trophectoderm departure, there is evidence that the inner cells, the ICM, retain their ability to produce more endoderm if the initial layer is stripped away [27]. Position effects influence further differentiation of the primary endoderm. Interaction between extra embryonic ectoderm and the proximal primary endoderm inhibits alpha-foetoprotein synthesis [a] (see Fig. 2), but again, there is no evidence for an effect of cell interaction on the restriction in potency of ICM to epiblast which may be a time dependent process as suggested above. Less is known about the time of origin of different populations of cells of the three definitive germ layers following implantation. The process of gastrulation in the mouse embryo is complex. It is in general agreed that mesodermal cells are the first to appear, invaginating through the primitive streak at 7 days’ gestation, and that definitive tmph.ctodum drrivd P gbntc.lls

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e.p.c. ext. emh I

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FQ. 2. A stem line model applied to cell lineage in early development. Abbreviations: troph. trophectoderm; giant = trophoblast giant cells; e.p.c. ectoplacental cone; ext.emb.ect. extraembryonic ectoderm; prim.endo. primary endoderm; prox.endo. proximal endoderm; dist.endo. distal endoderm. CelZ interactions. The continued presence of ICM is required for proliferation of overlying (polar) trophectoderm cells to form the extraembryonic ectoderm and ectoplacental cone [4, 241. AU cells of the primary endoderm are capable of synthesising

alphafoetoprotein (AFP);contact with extraembryonic ectoderm The inhibits AFP synthesis of overlying proximal endoderm [B]. mutual requirement of mesoderm and endoderm in ectopic transplants is described in [29]

Morula

Mastocyst

Late Blartocyrt

Postimplantation

E m b w (6d.)

Flg. 3. Diagrammatic representation of mouse-embryo development from cleavage to day7 of pregnancy

M. Monk: Differentiation in Early Mouse Development

endoderm appears a day later at the headfold stage. Consequently for the purposes of completing the illustration I have arranged the departures in the order of mesoderm, endoderm, and ectoderm (Figs. 2 and 4). It should, however, be pointed out that there is no good evidence either for this order or that the departures arise sequentially rather than at the same time. More information on these questions will be gained from examinations of the range of cells derived from ectopic transplants [29], and from in vitro culture of different regions of the embryo isolated at different stages of development. The application of the stem-line model to early mouse development (Fig. 2) presents us with an obvious and significant problem concerning the departure or origin of the germ line (see also [4, 91). In the model the stem line has undergone a number of restrictions in potency. Either the germ line must depart much earlier, at the blastomere stage, before any restrictions in potency have occurred, or there must be some mechanism of restoring totipotency to these cells. Papaiannou et al. [5] considered this problem and suggested that fertilisation (or parthenogenetic activation) might cause expression of totipotency. For reasons presented below it is suggested here that some event associated with meiosis causes a derestriction in potency in the germ line. Through meiosisthe stem line is rejuvenated and the germ cells are once again totipotent. Consequently from generation to generation the process is cyclic; it is depicted as the catherine wheel model in Fig. 4. In the linear representation of the stem line, departures of differentiated cells are projected forward with respect to the time axis. The dynamics of a catherine wheel require that differentiated cells are thrown off the wheel which, as it turns, leaves these populations behind. Figure 4 shows the catherine wheel set in motion by fertilisation. To discussour results on X chromosome differentiation I have drawn the cycle for XX female cells; however, the model is equally applicable to X Y Cells.

C. X Chromosome Inactivation and Reactivation It has been known for some time that only one X chromosome is active in somatic cells of adult female mammals [6,30-321. The other X chromosome is genetically inactive, heterochro-

MEIOSIS

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x; %-

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primordial gtrm celb

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x; x; Fig. 4. Circularisationof the stem line model into a catherine wheel as shown for XX female embryos. In this form of the model, totipotency is restored to the germ line via meiosis

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matic, and late replicating in the Sphase of cell division. However, two X chromosomes are active in XX germ cells during oogenesis in mice; this has been established by comparing XX and XO eggs for gene dosage of several X-linked enzymes [33-35). Similarly, Gartler et al. [36] have demonstrated the hybrid G6PD (glucose-6-1phosphate dehydrogenase, E.C. 1.1.1.49) AB band in o w e s from human females heterozygousfor the A and B electrophoretic variants of this X-linked enzyme. On the other hand it appears that the X and Y chromosomes are inactive in the later stages of spermatogenesis. They become condensed [37], late replicating [38], and genetically inactive [39]. More recently X chromosome differentiation has been studied in early development. Both X chromosomes are active in the mid-cleavage stages of female embryonic development, at least with respect to two enzymes studied [6-8,40-42] XX embryos have double the activity of these enzymes compared with XY embryos. However, by the blastocyst stage one X chromosomehas become inactive in all or most of the cells [6], though there is both genetic [43] and biochemical evidence [7] to suggest that two X chromosomes remain active in ICM cells of female embryos. It was suggested [6] that X chromosome differentiation was coupled to cellular differentiationoccurring sequentially in the departing differentiated populations in the manner of the stem-line model discussed here. Further evidence for this idea was obtained by Monk and Harper [9]. They dissected 6-day-old embryos (Fig. 3) into extra embryonic ectoderm, endoderm, and epiblast regions and showed that whereas levels of HPRT activity in the former two regions were equivalent in male and female embryos, there remained evidence for two active X chromosomes in female epiblast. In so far as X chromosome differentiation is a measure of cell differentiation,these observationslend support to the stem-linemodel since the departure events that produce trophectoderm and primary endoderm are accompanied by X chromosome inactivation and are threrefore different from those that restrict the potential of blastomeres to ICM,and ICM to epiblast, where the two X chromosomes remain active. It is noteworthy that X chromosome differentiation also accompanies differentiation of teratocarcinoma stem cells [44, 451. The paternally derived X is preferentially inactivated in cell populations differentiating early (see Fig. 4) and in the tissues derived from them [46-491. There appears to be some memory mechanism recalling the origins of the two X chromosomes, perhaps related to the fact that the paternal X chromosome was previously inactive during spermatogenesis and sequestered away from ‘meiosis’ in the sex vesicle. Whatever the memory mechanism, it is assumed to be lost by the time the three definitive germ layers are formed when X chromosome inactivation is thought to become random. In Fig. 4 the germ line undergoes restrictions in potency while in the stem line and, in terms of X chromosome differentiation, the germ line also departs as a differentiated population. It is now known that X chromosome inactivation occurs in female primordial germ cells of the mouse some time before day 12 of gestation. Monk and McLaren [lo] found that XX,XO, and X Y germ cells at 11.5 days were equivalent with respect to X-linked HPRT activity. Evidence was presented that the inactive X chromosome was reactivated by day 13 of gestation. Peter Johnston (personal communication) has also obtained good evidence for Xchromosome inactivation in premeiotic female germ cells in female embryos heterozygous for the A and B electrophoretic variants of X-linked PGK

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(phosphoglycerate kinase, E.C. 2. 7. 2. 3; [50]). Only the B form (carried by the preferentially active Searle’s X translocation [51]) was present on day 13 of gestation. However, on day 14, when most of the cells had entered meiosis, the A form appeared. Evidence for a cycle of X chromosome inactivation and reactivation (at the time of meiosis) had been obtained earlier in human female foetuses [52] although there was a contradictory report [53]. Given that we have established that inactivation of an X chromosome is associated with differentiating populations departing from the stem line, I would like to suggest that reactivation of the silent X coincident with meiosis might accompany cellular dedifferentiation, or in other words, restoration of totipotency of the germ line. Reactivation of the X chromosome in the germ line is one situation where the inactive state of the X chromosome in XX cells is known to be reversed. Another example of reactivation is that of the inactive, paternally derived X following fertilisation. As mentioned above, the X and Y chromosomes of the male are sequestered away from meiosis in the sex vesicle, and reactivation of paternally derived sex chromosomes must occur in the egg cytoplasm at fertilisation. The two postulated ‘sites’ of reactivation of the maternal X and paternal X, meiosis and egg cytoplasm respectively, could underly the non-random inactivation of the paternal X in tissues differentiating early in development. Other experimental attempts to reactivate the X chromosome [54, 551 have been unsuccessful despite mutagenesis and strong selective procedures (reviewed in [6]).

D. Teratoeardwma Cells in the pluripotent stem line, the ‘inner circle’ in Fig. 4, are potentially immortal and capable of producing tumours or teratocarcinomas, when injected into syngeneic hosts (reviewed in [56-581). Several observations are pertinent here. The first is that transplantable teratocarcinomascan be derived from embryo transplants with increasing efficiency up to about the time of gastrulation (7days’ gestation) when there is a marked decline [59]. In the model the number of stem cells capable of tumour formation is increasing until gastrulation and thereafter a decrease in number occurs when only the germ cells retain tumourogenk potential. The second observation is that there are a number of types of teratocarcinoma differing in the range of derivative differentiated tissues produced perhaps according to the progress in potency restriction in the stem line; some may not produce trophoblast for example, or trophoblast and yolk sac endoderm [56]. It is interesting that testicular teratocarcinomas in male mice of strain 129 do not arise by development through the blastocyst stage, yet resemble egg cylinder-like structures when first seen [60]. There is evidence that they arise from X Y primordial germ cells in 129 male embryos at about the time female XX primordial germ cells would enter meiosis. The significance of this observation is unknawn. E. Implications of the Catherine Wheel Model

The catherine wheel model is only of some value if it presents a useful framework for future experiments. Properties perhaps common to the ‘inner circle’ include morphological and histochemical characteristics, cell surface antigens, and resistance or sensitivity to a range of viruses. Inner cell mass cells, epiblast, primordial germ cells, and teratocarcinoma stem cells

M. Monk: Differentiation in Early Mouse Development

show morphological similarities [61-631. Histochemically these cell populations are all strongly alkaline phosphatase positive [64-661.They may also share a common metabolism [67]. Cell surface studies however have produced a complicated story (reviewed in [a]). Are there surface antigens common to the inner circle? Despite a wealth of antisera and monoclonal antibodies prepared against preimplantation embryos, germ cells and teratocarcinoma cells, no consistent picture emerges which might confirm this idea. Most intriguing is the suggestion here that reactivation of the silent X in the germ line occurs at meiosis. Is meiosis a ‘dedifferentiation’ event, a ‘derestriction in potency’? The inactive state of heterochromatin may be a function of DNA superstructure, supercoiling or packing, controlled by the quantity and types of non-histone and histone proteins [69,70]. If this were so, the unravelling mechanisms required in preparation for synapsis and genetic exchange specific to the meiotic process might remove superstructural constraints on genetic function. Acknowledgemen& I should like to thank A. McLaren, M. Harper, and A. McMahon for their enthusiasm and interest in the ideas presented in this paper and A. McLaren, J. Rossant, and M. Snow for helpful comments during the preparation of the manuscript.

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Received November 1980/Accepted January 1981