TISSUE & CELL, 1990 22 (2) 103-111 0 1990 Longman Group UK Ltd
Y. P. CRUZ”
MOSAICISM IN THE TROPHECTODERM Keywords:
Mosaicism,
MOUSE
cell allocation.
mouse embryo
ABSTRACT. The issue of mosaicism in the mouse trophectoderm is examined by reviewing two sets of evidence: one arguing for a mosaic, the other for a non-mosaic character. Evidence for mosaicism includes documented cellular contribution from the inner cell mass to the trophectoderm, and data that reveal the gradual pace of the allocation process that separates the inner cell mass and trophectoderm lineages. Evidence suggesting a non-mosaic character for the trophectoderm is based on the polarization process undergone by exterior cells in the eight-celled embryo, the heritability of the changes brought about by this process. and the formation of gap junctions between the resulting apolar, trophectoderm progenitor cells. Since inner-cell-mass cells are developmentally labile, spatially heterogeneous and translocate to the polar trophectoderm, it is concluded that the polar trophectoderm is a mosaic tissue.
Introduction
infection of cleavage blastomeres with a retrovirus (Soriano and Jaenisch, 1986). Integration of these molecular probes into the mouse genome occurs after the first round of mouse DNA replication, and the resulting transgenic animal is usually mosaic in both germ and somatic lines. The issue of mosaicism is an important one in the case of the mouse trophectoderm. This tissue results from the earliest differentiation event which, in the pre-implantation mouse embryo, partitions blastomeres between a strictly extraembryonic and a potentially embryonic fate. A mosaic origin for the trophectoderm would indicate that this differentiation event occurs gradually and is completed later than is now thought. There is some evidence suggesting a mosaic character for the trophectoderm; likewise, evidence to the contrary exists. I will examine these sets of evidence and argue that the trophectodertn is developmentally a mosaic tissue.
A mosaic tissue, organ or organism is composed of genetically dissimilar cells. Mosaicism can arise spontaneously; for instance, inactivation of one of the two X chromosomes during early development in female mammals renders every adult female a developmental mosaic with respect to the X chromosome. Mosaicism can also result from such experimental means as aggregating two or more cleavage-stage embryos (see, for instance, Garner and McLaren, 1974; McLaren, 1976; Rossant, 1976; Kelly et al., 1978; Kelly, 1979) or reconstituting embryos from microsurgically isolated component tissues (see, for instance, Gardner, 1968; Gardner et al., 1973; Gardner and Papaioannou, 1975; Rossant and Papaioannou, 1977; Papaioannou, 1982). In order to underscore their non-spontaneous origin, experimental mosaics are usually called chimeras (McLaren, 1976). Yet another type of mosaicism can be induced by the introduction of foreign genetic material into embryos, either by microinjection of DNA into one of the zygotic pronuclei (Wilkie et al., 1986) or by *Department,of 44074. Received
Biology,
Oberlin
College,
The Mouse Trophectoderm First, it is necessary to describe the mouse trophectoderm, its origin and its fate. The trophectoderm is a monolayer of flattened epithelioid cells that surrounds the inner cell
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mass and the blastocoele in the 32-64-celled (or 3-4-day-old) mouse embryo, or blastocyst. The region of the trophectoderm that faces the blastocoele, or embryonic cavity, is called the mural trophectoderm; that which overlies the inner cell mass, the polar trophectoderm. A series of gradual changes establishes the fate of the mural trophectoderm. Soon after cavitation of the embryo, mural trophectoderm cells are joined by tight junctional complexes (Enders and Schlafke, 1965; Calarco and Brown, 1969; Ducibella et al., 1975). These complexes allow the mural trophectoderm cells to accumulate fluid into the extracellular space that eventually becomes the blastocoele (Borland et al., 1977; Wiley, 1984). Subsequently, mural trophectoderm cells undergo numerous rounds of DNA replication (Zybina, 1970; Zybina and Grishenko, 1972; Barlow and Sherman, 1972) and only very rarely, mitosis (Zybina and Grishenko, 1970; Ilgren, 1980), resulting in uninucleate polyploid cells (Bower, 1987) with polytene chromosomes (Barlow and Sherman, 1974; Snow and Ansell, 1974; Zybina, 1977; Varmuza et al., 1988). The trophectoderm assumes a more friable appearance, and is later called the trophoblast (Snell and Stevens, 1966). Its enlarged, polyploid cells, the primary trophoblast giant cells, stimulate the uterine mucosa to form a decidual swelling during implantation (Gardner, 1972; Slack, 1983). The mural trophectoderm is clearly a differentiated tissue with an extraembryonic fate. The polar trophectoderm undergoes a similar differentiation sequence and acquires a similar fate. Like the mural trophectoderm, it facilitates implantation; additionally, it participates in the formation of the placenta. First, its cells proliferate, forming a rapidly enlarging tissue, called the extraembryonic ectoderm, which displaces the inner cell mass further into the blastocoele, and acts as a stem cell pool for all trophoblast types (Rossant and Lis, 1981). At its opposite end, the extraembryonic ectoderm forms a protrusion, called the ectoplacental cone, which later contributes to the fetal placenta (Gardner et al., 1973; Rossant and Papaioannou, 1977; Papaioannou, 1982; Rossant, 1986). The ectoplacental cone also produces secondary giant cells which are motile (Slack, 1983), phenotypically indistinguishable from
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primary giant cells (Varmuza et al. 1988) and serve to reinforce the trophoblast (Slack, 1983). The fate of the polar trophectoderm is clearly extra-embryonic. The remaining cells in the mouse blastocyst constitute the inner cell mass. Unlike the trophectoderm, the inner cell mass gives rise to both embryonic and extra-embryonic derivatives. The inner ceil mass is first observed as a clump of interior cells during the formation of the embryonic cavity, or blastocoele, in the 32-celled mouse embryo (Chisholm et al., 1985; Fleming and George, 1986). The number of cells in the inner cell mass increases gradually around the time of implantation, during which some of the cells on its blastocoelic surface differentiate into parietal, or primitive, endoderm cells (Snell and Stevens, 1966; Slack, 1983; Rossant, 1986). Embryonic reconstitution experiments indicate that parietal endoderm cells later contribute to the placenta and the yolk sac, but not to the actual embryo of later stages. The actual embryo (that is, the entire fetus), the amnion, allantois, and extraembryonic endoderm are all derived from the remainder of the inner cell mass (Gardner and Papaioannou, 1975; Gardner and Rossant, 1979; Gardner, 1982). Thus, the inner cell mass has both an embryonic and extraembryonic fate. The regional interface between the trophectodermal epithelium and the inner cell mass defines the polar trophectoderm. This confluence has been interpreted in two different ways. One of these interpretations proposes that the inner cell mass contributes cells to the polar trophectoderm, and ultimately, to the mural trophectoderm. In this view, therefore, the polar trophectoderm is a mosaic, composed of cells allocated to the trophectodermal lineage during blastocyst formation and of cells contributed subsequently by the inner cell mass. If so, the allocation of embryonic ceils between the very distinct trophectoderm and inner cell mass lineages continues past the onset of phenotypic divergence between them (Balakier and Pedersen, 1982; Cruz and Pedersen, 1985; Pedersenetal., 1986; Winkel and Pedersen, 1988). The alternative interpretation holds that, on the contrary, the trophectoderm and the inner cell mass lineages are segregated once embryonic cells enter the sixth cycle, that is, just after the fifth cleavage
MOSAICISM IN THE MOUSE TROPHECTODERM
which produces the 32-celled division, embryo, or nascent blastocyst. This interpretation concludes that the polar trophectoderm is unlikely to have a mosaic origin, since it is highly uncommon for an interior blastomere to gain access to the exterior environment, become polarized at this time, and so contribute to the polar trophectoderm (Johnson and Ziomek, 1983; Johnson, 1986). It is important to determine which of these interpretations is supported by available data in order to understand the basis for, and the sequence of events during, cell differentiation in the mouse blastocyst. Evidence for Mosaicism The polar trophectoderm would be a mosaic tissue if it were composed of cells originating from two or more lineages. If, in fact, the trophectoderm and the inner cell mass represent two lineages already established at the time they become phenotypically distinct in the 32-celled embryo, or nascent blastocyst, then there is evidence to support the notion that the polar trophectoderm is mosaic. Direct evidence of a cellular contribution from the inner cell mass comes from work with cell-labeling studies. Inner-cell-mass cells labelled in situ in mature blastocysts (average cell number 64) with microinjected markers were frequently detected (41%) after 24 hr in the polar trophectoderm (Winkel and Pedersen, 1988). The extent of this cellular contribution is considered large, approaching the rate at which inner cell mass cells and their descendants were detected in the parietal endoderm (45% of embryos, each with, on average, three labeled cells). In another study, using similar markers introduced mainly by endocytosis, a much smaller inner cell mass contribution to the polar trophectodetm was detected: only 10% of blastocysts incubated for up to 36 hr were positive for one or two, rarely three, lineage-crossed cells (from inner cell mass to polar trophectoderm, and vice versa). The embryos in this study were analyzed by serial sectioning, whole mounts and blastocyst disaggregation. The latter procedure was admittedly inefficient (Dyce et al., 1987), and along with the relatively small sample sizes, may have underestimated the actual extent of lineagecrossing. It is clear, however, that there is direct evidence, well into the sixth cell cycle,
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of inner cell mass cells contributing to the trophectoderm and, in fact, vice versa. Further evidence of an inner cell mass contribution to the polar trophectoderm is provided by chimera studies. A cellular contribution from the inner cell mass to the extraembryonic ectoderm, which is polartrophectoderm-derived, was detected in conceptuses developing from reconstituted blastocysts (Papaioannou, 1982). Additionally, a similar contribution, this time considered unusually large (4%), from the inner cell mass was detected in 13-E-day-old placentas arising from reconstituted embryos for which the inner-cell-mass donors may have been early cavitating blastocysts (Rossant and Croy, 1985). Undetected contamination of inner cell masses with trophectoderm cells could not be eliminated in this study, nor could incomplete enclosure of inner cell masses by the trophectodermal vesicles used during blastocyst reconstitution. These results, however, are compatible with an inner-cell-mass contribution to the trophectoderm well past the 32-celled stage. Indirect evidence also argues for an inner cell mass contribution to the polar trophectoderm. The number of cells in the inner cell mass has been found to decline relative to that in the polar trophectoderm, in spite of co-declining but comparable mitotic indices in these two tissues (Handyside, 1978; Copp, 1979; Handyside and Hunter, 1986). A higher rate of cell death in the inner cell mass relative to the polar trophectoderm has been proposed to explain these observations (Handyside and Hunter, 1986), as has cell recruitment from the inner cell mass to the trophectoderm (Cruz and Pedersen, 198.5). The latter conclusion is based on an analysis of the displacement rate of intracellularly marked cells from the polar to the mural trophectoderm in mature blastocysts incubated up to 48 hr. This conclusion is consistent with a model proposed subsequently for cell proliferation and fate in the inner cell mass (Winkel and Pedersen, 1988). Evidence for Non-Mosaicism The argument for a non-mosaic origin of the mouse trophectoderm is based mainly on the positional differences among the blastomeres of the mouse morula (Tarkowski and Wroblewska, 1967). Radioactively labeled,
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blastomeres occupying exterdisaggregated ior (‘outside’) or interior (‘inside’) locations maintain these positions ‘when allowed to reaggregate in vitro (Hillman et al., 1972). These positional restrictions apparently result from the persistence of the polarized phenotype acquired by blastomeres during the g-celled stage (Johnson and Ziomek, 1981; Johnson and Maro, 1986). Because of their position, exterior cells are expected to contribute more to the trophectoderm than to the inner cell mass; the interior cells, to the inner cell mass. The allocation of cells to an interior position appears to be the result of differentiative (Fleming 1987; Garbutt et al., 1987), or polarized (Balakier and Pedersen, 1982), divisions. Such cleavage divisions may be specifically attributed to the descendants of the early-dividing blastomeres in the 4-celled embryo (Garbutt et al., 1987). In the 816celled embryo, these divisions result in an outer polar cell derived from the apical region of the parent cell, and an inner apolar cell derived from the basolateral region (Johnson and Ziomek, 1981; Johnson 1986; Pickering et al., 1988). Polarization is largely stable (Johnson and Maro, 198.5; 1986) and depends, at least in part, upon cell contact (Ziomek and Johnson, 1980; Adler and Ziomek, 1986; Sobel and Goldstein, 1988). The manifestations of polarization include rearrangements of cell-surface (Handyside 1980; Lee 1987) and cytoskeletal (Johnson and Maro, 1984; Chisholm and Houliston, 1987) elements, and endocytotic (Reeve, 1981; Fleming and Pickering, 1985) and other membrane-bounded organelles (Maro et al., 1985). These changes facilitate intercellular flattening, a process mediated by a Cat+mediated adhesion system (Johnson, 1986). The active molecule in this system is uvomorulin (variously called E-cadherin, L-CAM, cell CAM 120/80, and gp 123) (Kemler et al., 1977; Hyafil et al., 1980, 1981; Damsky et al., 1983; Gallin et al., 1983; Shirayoshi et al., 1983; Peyrieras et al., 1983; Vestweber and Kemler, 1984; Yoshido-Noro et al., 1984; Johnson et al., 1986; Richa and Solter, 1986). Polarization is stabilized at the l&32-celled stages by the increased complexity of this and other factors that facilitate intercellular adhesion, such as surface glycosylation (Surani et al., 1981, 1983; Kimber and Surani, 1982; Bird and Kimber, 1984; Sato et al., 1984; Ras-
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tan etal., 1985) and tight junctions (Ducibella etal., 1977; Magnuson etal., 1977). The latter result in a permeability seal that separates the interior from the exterior environments of the embryo (Wiley and Eglitis, 1981), and coincides with cytokinesis-independent Naf/ K+-ATPase expression (Watson and Kidder, 1988). The stability of these changes result in the persistence of the polarized phenotype. The relationship between cell position and fate suggests a predominantly epigenetic mechanism for the allocation of cells between the trophectoderm and the inner cell mass. This has been reinforced by studies involving size regulation in half- and quadruple-sized embryos (Rands, 1985, 1986), and is implicit in the perception of the trophectoderm as a non-mosaic tissue. This argument can be summarized as follows: The positions assumed by blastomeres in the eight-celled mouse embryo are stabilized by the acquisition of polar or non-polar phenotypes and by the formation of tight junctions. Polar cells and their similarly polar descendants become the trophectoderm; apolar cells and their descendants, the inner cell mass. Thus, polarization is considered to be the proximate differentiative event that separates the trophectoderm and the inner cell mass lineages (Fleming et al., 1984; Fleming and Pickering, 1985).
Mosaicism vs. Non-Mosaicism The question of mosaicism clearly revolves around the issue of cell allocation between the trophectoderm and the inner cell mass. The first visible sign of cell allocation is the acquisition of an interior position by a variable number of blastomeres in the highly asymmetric g-celled embryo Barlow et al., 1972; Handyside 1981; Johnson and Ziomek, 1981). The number of interior cells during the 16-celled stage is somewhat greater (Barlow et al., 1972; Copp, 1978; Handyside, 1978; Surani and Barton, 1984; Chisholm et al., 1985) but contributes only about 75% of the inner cells (Pedersen et al., 1986). A subsequent allocation event is thus predicted by this figure (Barlow et al 1972; Copp, 1978; Handyside, 1978, 1986; Surani and Barton, 1984, Chisholm et al., 1985), and has been shown to occur during the fifth cleavage, that is, division to the 32-celled stage (Pedersen
MOSAICISM
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al., 1986). Thus, the allocation of blastomeres between the trophectoderm and the inner cell mass is a two-step process: a qualitative separation into polar and apolar-cells during the 8 and 16-celled stages, and a quantitative regulation of lineage size during the l&32-celled stage (Fleming, 1987). The complexity of this allocation step is difficult to deduce from the structual simplicity of the blastocyst. The observed cellular contribution from the inner cell mass to the polar trophectoderm is unequivocal proof that the latter is a mosaic of cells. This contribution varies from large or substantial (Rossant an’d Croy, 1985; Winkel and Pedersen, 1988) to small or inconsequential (Papaioannou, 1982; Dyce et al., 1987). A recent attempt to characterize the extent of this contribution indicates that it may indeed by too small for detection (Y. Cruz et al., in preparation) by straightforward probabilistic means (Iannaccone etal., 1987). Nevertheless, the detection of an inner cell mass contribution to the polar trophectoderm using different approaches intracellular and cell-surface (chimeras, labeling) indicates that cells from the inner cell mass become an integral part of the polar trophectoderm, which thus must be mosaic by definition. This conclusion further implies that the trophectoderm and the inner cell mass remain developmentally labile until after the fifth cleavage division, that is, to the 32celled stage, and, perhaps, for some time beyond. It is important to note that the arguments reviewed above for non-mosaicism contain a proviso that could accommodate an occasional, albeit unlikely, contribution from the inner cell mass. Were an interior cell to be exposed to the appropriate environment, it could polarize and thus become part of the trophectoderm lineage (Johnson and Ziomek, 1983). The difficulty of determining the nature of such an appropriate environment is apparent from experiments involving extirpation of polar trophectoderm cells. The results of inflicting such injury, performed to simulate naturally occurring cell death, indicate that exposure to the external milieu along does not increase the likelihood that an interior cell will assume an exterior position (Dyce et al.. 1987). In spite of these results, however, these same experiments report a small inner cell mass contribution et
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(-1% of the cells in 10-20% of blastocysts examined) to the trophectoderm. The actual point of disagreement, then, appears to be the extent of this contribution and the persistence of developmental lability that such a contribution demonstrates. The resolution of the mosaicism dilemma can be facilitated by an understanding of the commitment events in the inner cell mass. Inner cell masses isolated from early (-32celled) blastocysts form blastocyst-like structures when cultured subsequently (Handyside, 1978; Hogan and Tilly, 1978; Spindle, 1978), and induce a normal decidual response when transferred to foster mothers (Rossant and Lis, 1979). The exterior cells of isolated inner cell masses can produce descendants with inner-cell-mass and trophectoderm characteristics, indicating that the cell population is heterogeneous, and that loss of developmental lability in this tissue is not coordinated (Nichols and Gardner, 1984). These combined results indicate that the inner cell mass is a heterogeneous tissue and remains pluripotent beyond the point at which it becomes morphologically recognizable in the blastocyst. The heterogeneity of inner cell mass cells probably has a complex basis. First, earlierdividing blastomeres are likely to contribute more to the inner cell mass than to the trophectoderm (Kelly et al., 1978; Graham and Deussen, 1978; Graham and Lehtonen, 1979: Spindle, 1982; Surani and Barton, 1984). Thus, the inner cell mass is composed of developmentally older cells with, probably, some variability in age among themselves. This would explain why the loss of developmental lability in this tissue is not coordinated (Nichols and Gardner, 1984). The increasing asynchrony of the pre-implantation cleavage divisions (Pendersen, 1986) can only increase the heterogeneity among the inner cells of a nascent blastocyst, particularly when five such divisions have elapsed. This suggests very strongly that differentiation in the inner cell mass, and therefore, cell allocation between it and the trophectoderm, consists of a series of spatially and temporally overlapping events. It is precisely this staggered differentiation process that could prolong the ability of the inner cell mass to contribute to the trophectoderm. How and when this contribution ceases in the intact embryo remains to be seen.
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Acknowledgement This work was supported by NICHHD Grant Number 1 R1.5 HD24245-01. Part of this report was delivered at a symposium held
during the joint meeting of the American Society for Cell Biology and the American Society for Biochemistry and Molecular Biologyin January 1989.
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Manipulation of early mammalian development: what does it tell us about cell lincages’~, in Developmentu/ Biology: A Comprehensive .Synrh& (ed. R. B. L. Gwatkin). pp, 279-296. Plenum. New York and London. Johnson, M. H. and Mare. B. 1984. The distribution of cytoplasmic actin in mouvz X-ccl1 hlastomeres. J. Embrvol. Exp. Morphol.. 82,97-l 17. Johnson. M. H. and Mare. B. 19X5. A dissection of the mechamsms generating and stabilizing polarity m mouse X and Ih-cell blastomcres: the role of cytoskeletal elements. J. Embryo/. Exp. Morphol., 99,31l-334. Johnson. M. H. and Mare, B. 1986. Time and space in the mouse early embryo: a cell biological approach to cell diversification. in Experimentcri Appromhe.7 to Mammalian Development (eds. J. Rossant and R. A. Pedcraen), pp. 35-66. Cambridge University Press. New York and London. Johnson. M. H.. Mare. B. and Takeichi. M. 1986. 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Mare, B., Johnson, M. H., Pickering, S. J., and Louvard, D. 1985. Changes in the distribution of membranous organelles during mouse early development. J. Embryo/. Exp. MorphoL, 90,287-309. McLaren, A. 1976. Mammalian Chimaeras. University Press, Cambridge. Nichols, J. and Gardner, R. L. 1984. Heterogeneous differentiation of external cells in individual isolated early mouse inner cell masses in culture. J. Embryol. Exp. Morphol., 80,225-240. Papaioannou, V. E. 1982. Lineage analysis of inner cell masses and trophectoderm using microsurgically reconstituted mouse blastocysts. J. Embryol. Exp. Morphol., 68,9%109. Pedersen, R. A. 1986. Potency, lineage, and allocation in preimplantation mouse embryos, in ExperimenralApproaches 10 Mammalian Embryonic Development (eds. J. Rossant and R. A. Pedersen) pp. S-34, Cambridge University Press, New York and London. Pedersen, R. A., Wu, K. and Balakier, H. 1986. Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection. Dev. Biol.. 117,581-595. Peyrieras, N., Hyafil, F., Louvard. D., Ploegh, H. D.. and Jacob, F. 1983. Uvomorulin: a nonintegral membrane protein of early mouse embryos. Proc. Nat/. Acad. Sci. U.S.A., 80,6274-6277. Pickering, S. J., Mare, B., Johnson, M. H., and Skepper. J.. 1988. The influence of cell contact on the division of mouse f&cell blastomeres. Development, 103,353-363. Rands, G. F. 1985. Cell allocation in half- and quadruple-sized preimplantation mouse embryos. J. Exp. Zool., 236,67-70. Rands, G. F. 1986. Size regulation in the mouse embryo: I. The development of quadruple aggregates. J. Embryol. Exp. Morphol.. 94,13%148. Rastan, S., Thorpe, S. J., Scuddcr. P., Brown, S., Gooi. H.., and Feizi, T. 1985. Ccl1 interactions in preimplantation mouse embryos: evidence for involvement of saccharides of the poly-N-acetyllactosamine series. J. Embryol. Exp. Morphol., 87,115128. Reeve. W. J. D. 1981. Cytoplasmic polarity develops at compaction in rat and mouse embryos. J. Embryo/. Exp. Morphol., 62,351-367. Richa, J. and Salter. D. 1986. Role of cell surface molecules in early mammalian development. in Experimentu[ Approach to Mamma&m Embryonic Development (eds. J. Rossant and R. A. Pcdersen), pp. 29.3-320. Cambridge University Press, New York and London. Rossant. J. 1976. Investigation of inner cell mass determination by aggregation of isolated rat inner cell masses with mouse morulac. J. Embryol. Exp. Morphol.. 36,163-174. Rossant. J. 1986. Development of extraembryonic cell lineages in the mouse embryo, in Experimental Approaches to Mammalian Embryonic Development (eds. J. Rossant and R. A. Pedersen), pp. 97-120, Cambridge University Press, New York and London. Rossant, J. and Croy, B. A. 1985. Genetic identification of tissue of origin of cellular populations within the mouse placenta. J. EmbryoL Exp. Morphok, 86, 177-189. Rossant, J. and Lis, W. T. 1979. Potential of isolated mouse inner ccl] masses to form trophectoderm derivatives in viva. Dev. Biol., 70,249-254. Rossant. J. and Lis, W. T. 1981, Effect of culture conditions on diploid to giant cell transformation in postimplantation mouse trophoblast. J. Embryo/. Exp. Morphol.. 62,217-277. Rossant, .I. and Papaioannou, V. E. 1977. The biology of embryogenesis, in Concepts in Mammalian Embryogerwssis (ed. M. I. Sherman), pp. l-36. MIT Press, Cambridge, Mass. Sato, M., Muramatsu. T. and Berger, E. G. 1984. Immunological detection of cell surface galactosyltransfcrasc in preimplantation mouse embryos. Dev. Biol., 102,514-51X. Shirayoshi. Y., Okada. T. S. and Takeichi, M. 1983. The calcium-dependent cell-cell adhesion system regulates inner cell mass formation and cell surface polarisation in early mouse development. CeI, 35,631-638. Slack. M. J. W. 1983. From Eggto Embryo: Detwminarive Events in Early Developmenf. University Press, Cambridge. Snell, G. D. and Stevens, L. C. 1966. Early embryology. in Bio/ogy of the Laboratory Mouse (ed. E. L. Green). pp. 205-245. Dover Publications, New York. Snow. M. H. L. and Ansell, J. D. 1974. The chromosomes of giant trophoblast cells of the mouse. Proc. Royal Sot. London B, 187,93-98. Sobel, J. S. and Goldstein. E. G. 1988. Spcctrin synthesis in the prcimplantation mouse embryo. Dev. Biol.. 128, 284-289. Soriano, P. and Jaenisch, R. 1986. Retroviruses as probes for mammahan development: allocation of cells to the somatic and germ cell lineages. CeN, 46,1Y-29. Spindle. A. I. 1978. Trophoblast regeneration by inner cell masses isolated from cultured mouse embryos. J. Exp. Zool., 203,483-489. Spindle, A. 1982. Cell allocation in preimplantation mouse chimeras. J. Exp. Zool.. 219,361-367. Surani, M. A. H. and Barton. S. C. 1984. Spatial distribution of blastomcrcs is dependent on cell division order and interactions in mouse morulac. Dev. Biol., 102,335-343. Surani, M. A. H.. Kimher, S. J. and Handyside, A. 1981. Synthesis and role of cell surface glycoproteins in preimplantation mouse development. Exp. Cell Res.. 133,331-339.
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Surani, M. A. H., Kimber, tation mouse 205-223.
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S. J. and Osborne, by Compactin,
J. C. 1983. Mevalonate
an inhibitor
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arrest of preimplanExp. Morphol..
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Tarkowski, A. K. and Wroblewska, J. 1967. Development of blastomeres of mouse eggs isolated at the 4 and X-cell stage. J. Embryo!. Exp. Morphal.. l&155-180. Varmuza, S., Prideaux. V.. Kothary. R., and J. Rossant. 1988. Polytene chromosomes in mouse trophoblast cells. Development. 102,127-134. Vestwcbcr, D. and Kemler, R. 1984. Rabbit antiserum against puritied surface glycoprotein decompacts mouse prcimplantation embryos and reacts with specific adult tissue. Exp. CeN. Res., 152, 169-178. Watson, A. J. and Kidder. G. M. 1988. Immunofluorescence assessment of the timing of appearance and cellular distribution of Na/K-ATPase during mouse embryogenesis. Dev. BioL, 126,8@-90. Wiley, L. M. 1984. The cell surface of the mammalian embryo during early development, in Ultrastructure of Reproduction (eds. J Van Blerkom and P. M. Motta). pp. 99-204, Martinus Nijhoff. The Hague. Wiley, L. M. and Eglitis, M. A. 1984. Cell surface and cytoskcletal elements: Cavitation in the mouse preimplantation embryo. Dev. Biol., 86,49?-501. Wilkie, T. M., Brinster. R. L. and Palmiter, R. D. 1986. Gcrmline and somatic mosaicism in transgenic mice. Dev. Biol.. 118,9-18. Winkel. G. K. and Pedersen. R. A. 1988. Fate of the inner cell mass in mouse embryos as studied by microinjcctlon of lineage tracers. Dev. Biol., 127,14%156. Yoshido-Nero, C.. Suzuki, N. and Takeichi, M. 19X4. Molecular nature of the calcium-dependent cell-cell adhesion system in mouse teratocarcinoma and embryonic cells studied with a monoclonal antibody. Dev. Viol.. 101, l%27. Ziomek, C. A. and Johnson, M. H. 1980. Cell surface interactions induce polartzation of mouse &cell blastomcres at compaction. Cell, 21,935-942. Zybina. E. V. 1970. Anomalies of polyploidisation of the cells of the trophoblast. Dokl. Akad. Nauk. S.S.S.R.. 12, 1081-1093 Zybina. E. V. 1977. The structure of polytene chromosomes in the mammalian trophoblast cells. Tsirologivo 12, 5x5-595. Zybina. E. V. and Grischenko, T. 1970. Polyploid cells of the trophoblast in various sections of the white rat placenta., T~itologiya 12,585-595. Zybina. E. V. and Grischenko, T. 1972. Spcctrophotometric determination cells in the endometrium of the albino rat. Tsito@iya 14, 2824-290.
of the degree of polyploidy
of the decidual
Y. P. CRUZ”
MOSAICISM IN THE TROPHECTODERM Keywords:
Mosaicism,
MOUSE
cell allocation.
mouse embryo
ABSTRACT. The issue of mosaicism in the mouse trophectoderm is examined by reviewing two sets of evidence: one arguing for a mosaic, the other for a non-mosaic character. Evidence for mosaicism includes documented cellular contribution from the inner cell mass to the trophectoderm, and data that reveal the gradual pace of the allocation process that separates the inner cell mass and trophectoderm lineages. Evidence suggesting a non-mosaic character for the trophectoderm is based on the polarization process undergone by exterior cells in the eight-celled embryo, the heritability of the changes brought about by this process. and the formation of gap junctions between the resulting apolar, trophectoderm progenitor cells. Since inner-cell-mass cells are developmentally labile, spatially heterogeneous and translocate to the polar trophectoderm, it is concluded that the polar trophectoderm is a mosaic tissue.
Introduction
infection of cleavage blastomeres with a retrovirus (Soriano and Jaenisch, 1986). Integration of these molecular probes into the mouse genome occurs after the first round of mouse DNA replication, and the resulting transgenic animal is usually mosaic in both germ and somatic lines. The issue of mosaicism is an important one in the case of the mouse trophectoderm. This tissue results from the earliest differentiation event which, in the pre-implantation mouse embryo, partitions blastomeres between a strictly extraembryonic and a potentially embryonic fate. A mosaic origin for the trophectoderm would indicate that this differentiation event occurs gradually and is completed later than is now thought. There is some evidence suggesting a mosaic character for the trophectoderm; likewise, evidence to the contrary exists. I will examine these sets of evidence and argue that the trophectodertn is developmentally a mosaic tissue.
A mosaic tissue, organ or organism is composed of genetically dissimilar cells. Mosaicism can arise spontaneously; for instance, inactivation of one of the two X chromosomes during early development in female mammals renders every adult female a developmental mosaic with respect to the X chromosome. Mosaicism can also result from such experimental means as aggregating two or more cleavage-stage embryos (see, for instance, Garner and McLaren, 1974; McLaren, 1976; Rossant, 1976; Kelly et al., 1978; Kelly, 1979) or reconstituting embryos from microsurgically isolated component tissues (see, for instance, Gardner, 1968; Gardner et al., 1973; Gardner and Papaioannou, 1975; Rossant and Papaioannou, 1977; Papaioannou, 1982). In order to underscore their non-spontaneous origin, experimental mosaics are usually called chimeras (McLaren, 1976). Yet another type of mosaicism can be induced by the introduction of foreign genetic material into embryos, either by microinjection of DNA into one of the zygotic pronuclei (Wilkie et al., 1986) or by *Department,of 44074. Received
Biology,
Oberlin
College,
The Mouse Trophectoderm First, it is necessary to describe the mouse trophectoderm, its origin and its fate. The trophectoderm is a monolayer of flattened epithelioid cells that surrounds the inner cell
Oberlin OH
24 July 1989. 103
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mass and the blastocoele in the 32-64-celled (or 3-4-day-old) mouse embryo, or blastocyst. The region of the trophectoderm that faces the blastocoele, or embryonic cavity, is called the mural trophectoderm; that which overlies the inner cell mass, the polar trophectoderm. A series of gradual changes establishes the fate of the mural trophectoderm. Soon after cavitation of the embryo, mural trophectoderm cells are joined by tight junctional complexes (Enders and Schlafke, 1965; Calarco and Brown, 1969; Ducibella et al., 1975). These complexes allow the mural trophectoderm cells to accumulate fluid into the extracellular space that eventually becomes the blastocoele (Borland et al., 1977; Wiley, 1984). Subsequently, mural trophectoderm cells undergo numerous rounds of DNA replication (Zybina, 1970; Zybina and Grishenko, 1972; Barlow and Sherman, 1972) and only very rarely, mitosis (Zybina and Grishenko, 1970; Ilgren, 1980), resulting in uninucleate polyploid cells (Bower, 1987) with polytene chromosomes (Barlow and Sherman, 1974; Snow and Ansell, 1974; Zybina, 1977; Varmuza et al., 1988). The trophectoderm assumes a more friable appearance, and is later called the trophoblast (Snell and Stevens, 1966). Its enlarged, polyploid cells, the primary trophoblast giant cells, stimulate the uterine mucosa to form a decidual swelling during implantation (Gardner, 1972; Slack, 1983). The mural trophectoderm is clearly a differentiated tissue with an extraembryonic fate. The polar trophectoderm undergoes a similar differentiation sequence and acquires a similar fate. Like the mural trophectoderm, it facilitates implantation; additionally, it participates in the formation of the placenta. First, its cells proliferate, forming a rapidly enlarging tissue, called the extraembryonic ectoderm, which displaces the inner cell mass further into the blastocoele, and acts as a stem cell pool for all trophoblast types (Rossant and Lis, 1981). At its opposite end, the extraembryonic ectoderm forms a protrusion, called the ectoplacental cone, which later contributes to the fetal placenta (Gardner et al., 1973; Rossant and Papaioannou, 1977; Papaioannou, 1982; Rossant, 1986). The ectoplacental cone also produces secondary giant cells which are motile (Slack, 1983), phenotypically indistinguishable from
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primary giant cells (Varmuza et al. 1988) and serve to reinforce the trophoblast (Slack, 1983). The fate of the polar trophectoderm is clearly extra-embryonic. The remaining cells in the mouse blastocyst constitute the inner cell mass. Unlike the trophectoderm, the inner cell mass gives rise to both embryonic and extra-embryonic derivatives. The inner ceil mass is first observed as a clump of interior cells during the formation of the embryonic cavity, or blastocoele, in the 32-celled mouse embryo (Chisholm et al., 1985; Fleming and George, 1986). The number of cells in the inner cell mass increases gradually around the time of implantation, during which some of the cells on its blastocoelic surface differentiate into parietal, or primitive, endoderm cells (Snell and Stevens, 1966; Slack, 1983; Rossant, 1986). Embryonic reconstitution experiments indicate that parietal endoderm cells later contribute to the placenta and the yolk sac, but not to the actual embryo of later stages. The actual embryo (that is, the entire fetus), the amnion, allantois, and extraembryonic endoderm are all derived from the remainder of the inner cell mass (Gardner and Papaioannou, 1975; Gardner and Rossant, 1979; Gardner, 1982). Thus, the inner cell mass has both an embryonic and extraembryonic fate. The regional interface between the trophectodermal epithelium and the inner cell mass defines the polar trophectoderm. This confluence has been interpreted in two different ways. One of these interpretations proposes that the inner cell mass contributes cells to the polar trophectoderm, and ultimately, to the mural trophectoderm. In this view, therefore, the polar trophectoderm is a mosaic, composed of cells allocated to the trophectodermal lineage during blastocyst formation and of cells contributed subsequently by the inner cell mass. If so, the allocation of embryonic ceils between the very distinct trophectoderm and inner cell mass lineages continues past the onset of phenotypic divergence between them (Balakier and Pedersen, 1982; Cruz and Pedersen, 1985; Pedersenetal., 1986; Winkel and Pedersen, 1988). The alternative interpretation holds that, on the contrary, the trophectoderm and the inner cell mass lineages are segregated once embryonic cells enter the sixth cycle, that is, just after the fifth cleavage
MOSAICISM IN THE MOUSE TROPHECTODERM
which produces the 32-celled division, embryo, or nascent blastocyst. This interpretation concludes that the polar trophectoderm is unlikely to have a mosaic origin, since it is highly uncommon for an interior blastomere to gain access to the exterior environment, become polarized at this time, and so contribute to the polar trophectoderm (Johnson and Ziomek, 1983; Johnson, 1986). It is important to determine which of these interpretations is supported by available data in order to understand the basis for, and the sequence of events during, cell differentiation in the mouse blastocyst. Evidence for Mosaicism The polar trophectoderm would be a mosaic tissue if it were composed of cells originating from two or more lineages. If, in fact, the trophectoderm and the inner cell mass represent two lineages already established at the time they become phenotypically distinct in the 32-celled embryo, or nascent blastocyst, then there is evidence to support the notion that the polar trophectoderm is mosaic. Direct evidence of a cellular contribution from the inner cell mass comes from work with cell-labeling studies. Inner-cell-mass cells labelled in situ in mature blastocysts (average cell number 64) with microinjected markers were frequently detected (41%) after 24 hr in the polar trophectoderm (Winkel and Pedersen, 1988). The extent of this cellular contribution is considered large, approaching the rate at which inner cell mass cells and their descendants were detected in the parietal endoderm (45% of embryos, each with, on average, three labeled cells). In another study, using similar markers introduced mainly by endocytosis, a much smaller inner cell mass contribution to the polar trophectodetm was detected: only 10% of blastocysts incubated for up to 36 hr were positive for one or two, rarely three, lineage-crossed cells (from inner cell mass to polar trophectoderm, and vice versa). The embryos in this study were analyzed by serial sectioning, whole mounts and blastocyst disaggregation. The latter procedure was admittedly inefficient (Dyce et al., 1987), and along with the relatively small sample sizes, may have underestimated the actual extent of lineagecrossing. It is clear, however, that there is direct evidence, well into the sixth cell cycle,
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of inner cell mass cells contributing to the trophectoderm and, in fact, vice versa. Further evidence of an inner cell mass contribution to the polar trophectoderm is provided by chimera studies. A cellular contribution from the inner cell mass to the extraembryonic ectoderm, which is polartrophectoderm-derived, was detected in conceptuses developing from reconstituted blastocysts (Papaioannou, 1982). Additionally, a similar contribution, this time considered unusually large (4%), from the inner cell mass was detected in 13-E-day-old placentas arising from reconstituted embryos for which the inner-cell-mass donors may have been early cavitating blastocysts (Rossant and Croy, 1985). Undetected contamination of inner cell masses with trophectoderm cells could not be eliminated in this study, nor could incomplete enclosure of inner cell masses by the trophectodermal vesicles used during blastocyst reconstitution. These results, however, are compatible with an inner-cell-mass contribution to the trophectoderm well past the 32-celled stage. Indirect evidence also argues for an inner cell mass contribution to the polar trophectoderm. The number of cells in the inner cell mass has been found to decline relative to that in the polar trophectoderm, in spite of co-declining but comparable mitotic indices in these two tissues (Handyside, 1978; Copp, 1979; Handyside and Hunter, 1986). A higher rate of cell death in the inner cell mass relative to the polar trophectoderm has been proposed to explain these observations (Handyside and Hunter, 1986), as has cell recruitment from the inner cell mass to the trophectoderm (Cruz and Pedersen, 198.5). The latter conclusion is based on an analysis of the displacement rate of intracellularly marked cells from the polar to the mural trophectoderm in mature blastocysts incubated up to 48 hr. This conclusion is consistent with a model proposed subsequently for cell proliferation and fate in the inner cell mass (Winkel and Pedersen, 1988). Evidence for Non-Mosaicism The argument for a non-mosaic origin of the mouse trophectoderm is based mainly on the positional differences among the blastomeres of the mouse morula (Tarkowski and Wroblewska, 1967). Radioactively labeled,
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blastomeres occupying exterdisaggregated ior (‘outside’) or interior (‘inside’) locations maintain these positions ‘when allowed to reaggregate in vitro (Hillman et al., 1972). These positional restrictions apparently result from the persistence of the polarized phenotype acquired by blastomeres during the g-celled stage (Johnson and Ziomek, 1981; Johnson and Maro, 1986). Because of their position, exterior cells are expected to contribute more to the trophectoderm than to the inner cell mass; the interior cells, to the inner cell mass. The allocation of cells to an interior position appears to be the result of differentiative (Fleming 1987; Garbutt et al., 1987), or polarized (Balakier and Pedersen, 1982), divisions. Such cleavage divisions may be specifically attributed to the descendants of the early-dividing blastomeres in the 4-celled embryo (Garbutt et al., 1987). In the 816celled embryo, these divisions result in an outer polar cell derived from the apical region of the parent cell, and an inner apolar cell derived from the basolateral region (Johnson and Ziomek, 1981; Johnson 1986; Pickering et al., 1988). Polarization is largely stable (Johnson and Maro, 198.5; 1986) and depends, at least in part, upon cell contact (Ziomek and Johnson, 1980; Adler and Ziomek, 1986; Sobel and Goldstein, 1988). The manifestations of polarization include rearrangements of cell-surface (Handyside 1980; Lee 1987) and cytoskeletal (Johnson and Maro, 1984; Chisholm and Houliston, 1987) elements, and endocytotic (Reeve, 1981; Fleming and Pickering, 1985) and other membrane-bounded organelles (Maro et al., 1985). These changes facilitate intercellular flattening, a process mediated by a Cat+mediated adhesion system (Johnson, 1986). The active molecule in this system is uvomorulin (variously called E-cadherin, L-CAM, cell CAM 120/80, and gp 123) (Kemler et al., 1977; Hyafil et al., 1980, 1981; Damsky et al., 1983; Gallin et al., 1983; Shirayoshi et al., 1983; Peyrieras et al., 1983; Vestweber and Kemler, 1984; Yoshido-Noro et al., 1984; Johnson et al., 1986; Richa and Solter, 1986). Polarization is stabilized at the l&32-celled stages by the increased complexity of this and other factors that facilitate intercellular adhesion, such as surface glycosylation (Surani et al., 1981, 1983; Kimber and Surani, 1982; Bird and Kimber, 1984; Sato et al., 1984; Ras-
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tan etal., 1985) and tight junctions (Ducibella etal., 1977; Magnuson etal., 1977). The latter result in a permeability seal that separates the interior from the exterior environments of the embryo (Wiley and Eglitis, 1981), and coincides with cytokinesis-independent Naf/ K+-ATPase expression (Watson and Kidder, 1988). The stability of these changes result in the persistence of the polarized phenotype. The relationship between cell position and fate suggests a predominantly epigenetic mechanism for the allocation of cells between the trophectoderm and the inner cell mass. This has been reinforced by studies involving size regulation in half- and quadruple-sized embryos (Rands, 1985, 1986), and is implicit in the perception of the trophectoderm as a non-mosaic tissue. This argument can be summarized as follows: The positions assumed by blastomeres in the eight-celled mouse embryo are stabilized by the acquisition of polar or non-polar phenotypes and by the formation of tight junctions. Polar cells and their similarly polar descendants become the trophectoderm; apolar cells and their descendants, the inner cell mass. Thus, polarization is considered to be the proximate differentiative event that separates the trophectoderm and the inner cell mass lineages (Fleming et al., 1984; Fleming and Pickering, 1985).
Mosaicism vs. Non-Mosaicism The question of mosaicism clearly revolves around the issue of cell allocation between the trophectoderm and the inner cell mass. The first visible sign of cell allocation is the acquisition of an interior position by a variable number of blastomeres in the highly asymmetric g-celled embryo Barlow et al., 1972; Handyside 1981; Johnson and Ziomek, 1981). The number of interior cells during the 16-celled stage is somewhat greater (Barlow et al., 1972; Copp, 1978; Handyside, 1978; Surani and Barton, 1984; Chisholm et al., 1985) but contributes only about 75% of the inner cells (Pedersen et al., 1986). A subsequent allocation event is thus predicted by this figure (Barlow et al 1972; Copp, 1978; Handyside, 1978, 1986; Surani and Barton, 1984, Chisholm et al., 1985), and has been shown to occur during the fifth cleavage, that is, division to the 32-celled stage (Pedersen
MOSAICISM
IN THE MOUSE
TROPHECTODERM
al., 1986). Thus, the allocation of blastomeres between the trophectoderm and the inner cell mass is a two-step process: a qualitative separation into polar and apolar-cells during the 8 and 16-celled stages, and a quantitative regulation of lineage size during the l&32-celled stage (Fleming, 1987). The complexity of this allocation step is difficult to deduce from the structual simplicity of the blastocyst. The observed cellular contribution from the inner cell mass to the polar trophectoderm is unequivocal proof that the latter is a mosaic of cells. This contribution varies from large or substantial (Rossant an’d Croy, 1985; Winkel and Pedersen, 1988) to small or inconsequential (Papaioannou, 1982; Dyce et al., 1987). A recent attempt to characterize the extent of this contribution indicates that it may indeed by too small for detection (Y. Cruz et al., in preparation) by straightforward probabilistic means (Iannaccone etal., 1987). Nevertheless, the detection of an inner cell mass contribution to the polar trophectoderm using different approaches intracellular and cell-surface (chimeras, labeling) indicates that cells from the inner cell mass become an integral part of the polar trophectoderm, which thus must be mosaic by definition. This conclusion further implies that the trophectoderm and the inner cell mass remain developmentally labile until after the fifth cleavage division, that is, to the 32celled stage, and, perhaps, for some time beyond. It is important to note that the arguments reviewed above for non-mosaicism contain a proviso that could accommodate an occasional, albeit unlikely, contribution from the inner cell mass. Were an interior cell to be exposed to the appropriate environment, it could polarize and thus become part of the trophectoderm lineage (Johnson and Ziomek, 1983). The difficulty of determining the nature of such an appropriate environment is apparent from experiments involving extirpation of polar trophectoderm cells. The results of inflicting such injury, performed to simulate naturally occurring cell death, indicate that exposure to the external milieu along does not increase the likelihood that an interior cell will assume an exterior position (Dyce et al.. 1987). In spite of these results, however, these same experiments report a small inner cell mass contribution et
llJ7
(-1% of the cells in 10-20% of blastocysts examined) to the trophectoderm. The actual point of disagreement, then, appears to be the extent of this contribution and the persistence of developmental lability that such a contribution demonstrates. The resolution of the mosaicism dilemma can be facilitated by an understanding of the commitment events in the inner cell mass. Inner cell masses isolated from early (-32celled) blastocysts form blastocyst-like structures when cultured subsequently (Handyside, 1978; Hogan and Tilly, 1978; Spindle, 1978), and induce a normal decidual response when transferred to foster mothers (Rossant and Lis, 1979). The exterior cells of isolated inner cell masses can produce descendants with inner-cell-mass and trophectoderm characteristics, indicating that the cell population is heterogeneous, and that loss of developmental lability in this tissue is not coordinated (Nichols and Gardner, 1984). These combined results indicate that the inner cell mass is a heterogeneous tissue and remains pluripotent beyond the point at which it becomes morphologically recognizable in the blastocyst. The heterogeneity of inner cell mass cells probably has a complex basis. First, earlierdividing blastomeres are likely to contribute more to the inner cell mass than to the trophectoderm (Kelly et al., 1978; Graham and Deussen, 1978; Graham and Lehtonen, 1979: Spindle, 1982; Surani and Barton, 1984). Thus, the inner cell mass is composed of developmentally older cells with, probably, some variability in age among themselves. This would explain why the loss of developmental lability in this tissue is not coordinated (Nichols and Gardner, 1984). The increasing asynchrony of the pre-implantation cleavage divisions (Pendersen, 1986) can only increase the heterogeneity among the inner cells of a nascent blastocyst, particularly when five such divisions have elapsed. This suggests very strongly that differentiation in the inner cell mass, and therefore, cell allocation between it and the trophectoderm, consists of a series of spatially and temporally overlapping events. It is precisely this staggered differentiation process that could prolong the ability of the inner cell mass to contribute to the trophectoderm. How and when this contribution ceases in the intact embryo remains to be seen.
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Acknowledgement This work was supported by NICHHD Grant Number 1 R1.5 HD24245-01. Part of this report was delivered at a symposium held
during the joint meeting of the American Society for Cell Biology and the American Society for Biochemistry and Molecular Biologyin January 1989.
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