British Medical Bulletin (1979) Vol. 35, No. 2, pp. 113-119

CONTROL OF EARLY DEVELOPMENT

E D Adamson & R L Gardner

FIG. 1 . Principal stages in early development of the mouse embryo Inner cell mass

CONTROL OF EARLY DEVELOPMENT E D ADAMSON PhD R L GARDNER MA PhD FRS Department of Zoology University of Oxford

Ectoplacental cone Extra-embryonic ectoderm

Importance of cell position in early differentiation Maternal genome Activation of the embryonic genome Cellular relations during cleavage a Determination of inner cell mass and trophectoderm cells b Control of trophoblast development c Commitment of primitive endoderm cells in the inner

Embryonic ectoderm

cell mass

Parietal endoderm

Molecular characterization of cell type Conclusion References

a: early embryo (at the 8-cell stage) progressing to compacted morula b: blastocyst [3\ days after coitus) still inside the zona pellucida c: zona-free implanting blastocyst 4J days after coitus d : post-implantation embryo, at the egg-cylinder stage, showing derivatives of the trophectoderm (solid black), the primitive endoderm (stippled), and the primitive ectoderm (hatched area)

It is really only within the last two decades that early mammalian embryos have become accessible to detailed experimental investigation. This period has witnessed both the formulation of media that will support fertilization and development in vitro up to the stage of implantation, and refinement of procedures for routine transplantation of such embryos into the genital tract of suitably prepared recipient females (Daniel, 1971, 1978). In consequence of these and other technical advances, studies on early mammalian development have progressed from a primarily descriptive phase to an analytical one. The aim of this paper is to summarize and integrate the principal findings that have emerged from investigations at the cellular and molecular level. The cellular studies have been addressed to three interrelated problems. The first has been to establish the normal fate of the various cell populations of the early embryo, and thereby to trace the developmental origins of the tissues of the later conceptus. The second has been to find out when these cell lineages can no longer be interconverted; or, in other words, when determination takes place. Finally, attempts have been made to elucidate the factors governing the commitment of cells to particular paths of subsequent differentiation. Molecular studies have been directed towards dissection of the role of the maternal versus the zygote genome in directing early development. More recently, they have also been used to search for gene products that might serve as more reliable markers for the various types of cells emerging during development than the morphological criteria that have had to be used hitherto.

development of the mouse embryo are illustrated in fig. 1. Cellular differentiation is first clearly discernible on the fourth day of development, with the formation of a blastocyst consisting of a monolayer of trophectoderm enclosing the blastocoele and inner cell mass (ICM). Within the next 24 hours both tissues undergo further regional differentiation, so that four cell populations are evident at the time of implantation. Implantation is followed by a period of rapid growth and complex tissue movements, which result in the ICM and part of the polar trophectoderm (extra-embryonic ectoderm, fig. Id) occupying most of the former blastocoelic cavity.

1

Work on a variety of non-mammalian species has led to the cytoplasm of the unfertilized egg being commonly regarded as a mosaic of determinative molecules. Generation of cellular diversity in early development is thus attributed to action of these determinants on nuclei parcelled into their respective territories during cleavage (Gurdon, 1974; Davidson, 1976). Clearly, if early mammalian development conformed to this pattern one would have to focus on events taking place in the egg before fertilization in order to understand how it was controlled. Early attempts to interpret differentiation of ICM versus trophectoderm in rodents on these lines centred on the demonstration of two cytoplasmic regions in eggs fixed for cytochemistry (reviewed by Dalcq, 1957). It was claimed that similar cytoplasmic differentiation was evident between quartets

Critical studies on cell fate and commitment depend on combining cells or tissues from embryos carrying prescribed genetic differences. Biochemical studies often need relatively large numbers of embryos. These requirements have led to adoption of the mouse for most of the work to be discussed. Indeed, apart from the rabbit, little has been done on embryos of other eutherian mammals. Some of the key steps in early 113

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Importance of Cell Position in Early Differentiation

CONTROL OF EARLY DEVELOPMENT E D Adamson & R L Gardner of blastomeres at the 8-cell stage, and between the ICM and trophectoderm of the blastocyst (Dalcq, 1957; Mulnard, 1965). If the relationship between cytoplasmic endowment and cell fate was indeed causal and irreversible as Dalcq and his colleagues suggested, commitment of cells should coincide with cytoplasmic segregation at the 8-cell stage. Kelly (1975, 1977) has demonstrated that this is not the case. Individual cells isolated from 8-cell mouse embryos can contribute to both ICM and trophectoderm derivatives when combined with groups of genetically dissimilar blastomeres. It is obviously possible to salvage the above type of instructive model of early development by postulating more subtle and complex arrangements of cytoplasmic determinants that could yield later segregation. However, results of several other types of experimental manipulation run counter to the hypothesis that egg organization per se is decisive in early differentiation of the mammalian embryo. These include investigations on the development of dissociated blastomeres (Tarkowski & Wroblewska, 1967; Sherman, 1975; Rossant, 1977) and on variable numbers of isolated blastomeres or intact embryos encouraged to aggregate in a variety of spatial configurations (Mintz, 1965; Hillman et al. 1972; Stern & Wilson, 1972). The principal findings may be summarized as follows:

2

Maternal Genome

Protein synthesis continues at a low level in the mouse zygote in the absence of measurable RNA synthesis (Epstein, 1975; Schultz & Tucker, 1977; Young et al. 1978). The conclusion that it depends on translation of mRNA made during oogenesis is reinforced by the fact that RNA polymerase (RNA nucleotidyltransferase) activity is not detectable until the 2-cell stage (Moore, 1975). Studies by Monk (1978) on the activity of the X-chromosome-coded enzyme hypoxanthine phosphoribosyltransferase (HPRT) in early embryos also indicate the persistence of stored maternal mRNA (or possibly a progressive activation of stored enzyme). Individual 8-cell mouse embryos were assayed for activity of both HPRT and the related autosomally coded enzyme adenine phosphoribosyltransferase (APRT). The ratio of HPRT:APRT activity in embryos from XO mothers was found to be half that of embryos from XX mothers, and was unimodal. In other words, regardless of whether they were XO, XX or XY in constitution (YO embryos appear to die earlier), embryos from XO mothers exhibited the same activity ratio. These findings, coupled with the observation of an over-all 20-fold increase in HPRT activity by the 8-cell stage, suggest that new enzyme molecules are being produced during early cleavage by translation of maternal mRNA. A method of measuring levels of mRNA utilizes the polyadenylate moiety found at the 3' end of most (but not all) raRNAs. Levey et al. (1978) estimated the amount of poly(A)containing RNA in 1-cell, 2-cell and blastocyst stage mouse embryos. The results suggested the presence of significant quantities of maternal mRNA during early development, but also that polyadenylation of RNA transcribed from the embryonic genome occurred as early as the 2-cell stage. Similar studies were undertaken in the rabbit by Schultz (1975) who identified poly(A)-containing sequences in the unfertilized egg, half of which were attached to ribosomes, and therefore presumed to be functioning. The unbound fraction fell to 30% by 10 hours of development. Inhibitors of RNA synthesis have been widely used by embryologists to examine the role of stored mRNA. Use of actinomycin D in such investigations has been questioned because of its lack of specificity. Low concentrations of aamanitin in vitro specifically inhibit RNA polymerase II, the polymerase responsible for mRNA synthesis, though it is not clear what concentration is needed for in-vivo specificity. Golbus et al. (1973) found that 60-70% of 1-cell mouse eggs were able to cleave in the presence of 10~ 3 M to 10~ 7 M aamanitin compared with untreated controls. In the rabbit, Manes (1973) found that a few cleavage divisions could occur in the presence of 10~*M a-amanitin, a dose which blocked [3H]uridine incorporation by 95%. Normally, at concentrations below lOOug/ml this agent does not affect the activities of RNA polymerases I and III, but Levey & Brinster (1978) found that prolonged incubation with the drug also impaired the synthesis and/or processing of rRNA. This probably explains the disruption of nucleolar morphology observed in 4-cell and morula stage mouse embryos incubated for 24 hours in low doses of a-amanitin (Golbus et al. 1973). Despite uncertainty regarding the specificity of action of a-amanitin, it is clear that at least one cleavage can take place in early rabbit and mouse embryos exposed to low doses of the drug. Under these conditions there is no effect on the amount of amino acid incorporated into protein until the experiments are conducted

i. Isolated blastomeres that continue to cleave typically form either miniature blastocysts or trophectoderma] vesicles. The former pattern of development tends to be exhibited by those that attain a higher cell number. Development of only ICM tissue has not been observed (Tarkowski & Wroblewska, 1967; Sherman, 1975). ii. Blastocysts formed by aggregated pairs of cleaving eggs exhibit a disproportionate increase in number of ICM cells (Buehr & McLaren, 1974) and absence of any consistent pattern of distribution of constituent cells between ICM and trophectoderm (Mintz, 1965; Garner & McLaren, 1974). iii. Multiple embryos arranged in spatial configurations that militate against extensive sorting out of cells before blastulation are nevertheless capable of forming integrated unitary blastocysts (Mintz, 1965). iv. Marked blastomeres or entire embryos placed in an outside location in artificial aggregates tend to contribute mainly to the trophectoderm and its derivatives, while those that are wholly enveloped contribute primarily to the ICM and its derivatives (Hillman et al. 1972). These results support the "inside-outside" hypothesis of Tarkowski & Wroblewska (1967), according to which commitment of cells to trophectodermal or to ICM differentiation depends on whether they occupy an external or internal position, respectively, in the early embryo. The major challenge presented by this hypothesis is elucidation of the nature of the positional cues and means by which they elicit stable differences between inside and outside cells. Rejection of a strict segregation model of early differentiation does not of course exclude the maternal genome from playing a crucial role. Thus, in principle, position could exercise control via posttranscriptional processes, by activation and/or inhibition, or by relocation of molecules synthesized before fertilization (see Johnson et al. 1977). This could lead in turn to cellular diversification, either directly or via selective transcription of the embryonic genome. Hence it is relevant to consider what is known at present about the involvement of maternal as against embryonic genomes in early development. 114

Br.Med.Bull. 1979

CONTROL OF EARLY DEVELOPMENT E D Adamson & R L Gardner TABLE I. Morphological and biochemical events from fertilization to implantation Time after coitus (days)

Biochemical characteristics and/of times of "nitlatkm

Stage

Morphological events

FertULzation

Unfenlhzed ovulated egg Zygote

Evidence for stored mRNA (see text) Low uptake of protein and RNA precursors* Pyruvate and lactate sole energy sources* High ATP levels and high ATP ADP ratio 4 High NAD* and NADH concentrations" High LDH and G6PD enzyme concentrations* K* and Ca 2 * necessary for mouse development, Ca** and Fe** for rabbit"

Membrane asymmetry*

2 call

Synthesis of high-molecular-weight RNA and tRNA* Many new proteins synthesized* Hamster embryo secretes tetrepeptide which inhibits ovulation* Phosphoenolpyruvate may be used as an energy source* Alkaline phosphatase appears in contacting membranes*

4 cell

Synthesis of rRNA In mouse* Synthesis of oocyte-type proteins declining* Cleavage still resistant to »-amanitln £ ' '

Mitochondria begin to transform to orthodox type' Reorganization of microvilli and cytoplasmic microtubules-7'1 Compaction ^Formation of tight junctions*

Vacuoles appear In cells 46 Formation of zonular Junctions'1 CAVHALKXI

8 cell

Rapidly Increasing rates of protein and RNA synthesis* Extensive membrane and transport changes'' Necessity for Ca24 during compaction" Highest turnover rates for ATP* Evidence for activity of embryonic genome (see text} Sensitivity to n-amanltliV Maximum expression of some embryonic antigens"-* Dramatic changes In specific activities of many enzyme^

Morula

Active transport and fluid accumulation**•' Detectable levels of synthesized rRNA in rabbit* Inner cells have a higher labelling index with | ] H|thymldine m Synthesis of trophectoderm-specific proteins*-' Synthesis of ICM-specific proteins*-' Accumulation of type III collagen"

Blastocyst

Transient appearance of H-2 antigens on trophectodemy Flbronectin accumulation between ICM cells0 Stabilizing patterns of protein synthesis*

Late blastocyst

Primitive endoderm cells synthesize type IV collacjerr'

Outer surface non-adhesive**

3}

Overt differentiation of trophoblast andlCM Primitive endoderm differentiates

Hatching Retraction of microvilli'' Impbintstion Abbreviations G6PD glucose-6-phosphate dehydrogenase

ICM Inner cell mass

References a Reviewed by Van Blerfcom ft Manes, 1977 b Reviewed by Blggers & Stern, 1973; Wales, 1975 c. Reviewed by Epstein, 1975 rf'Kent, 1975 e Mulnard & Huygens, 1978

I g h ; * /

LDH lactate dehydrogenase

Reviewed by Pikd. 1975 Reviewed by Izquierdo, 1977 Reviewed by Ducibella, 1977 Reviewed by Johnson at el 1977, Johnson, 1979 Reviewed by Jacob. 1977 Reviewed by Borland, 1977

The mitochondrial endowment of the embryo appears to be maternal in origin (Hutchison et al. 1974), and these organelles have been considered a possible source of segregating controlling elements. Evidence for and against this view has been discussed by Manes (1975). Likewise, a developmental role has been postulated for viruses that are found in mouse embryos at various stages of development (reviewed by Jaenisch & Berns, 1977; Daniel & Chilton, 1978), including those induced to develop parthenogenetically (Biczysko et al. 1974). Finally, examination of metabolic pathways may reveal gradients as suggested by Baquer et al. (1975) in Hydra, and Landstrom & Levtrup (1975) in Xenopus. However, data currently available for mammals (see Table I and reviews by Biggers & Stern, 1973; Wales, 1975) cannot be formulated into a metabolic strategy of development at present.

after the 8-cell stage; this suggests that most early protein synthesis occurs on preformed maternal mRNA templates. In contrast, post-compaction cleavage stages are morphologically retarded in the presence of 11 ng/ml a-amanitin, and there is corresponding impairment of incorporation of uridine and methionine into acid-insoluble material (Johnson et al. 1977). Two-dimensional gel electrophoresis has revealed that many polypeptides synthesized by early rabbit embryos are probably identical to those made late in oocyte maturation. Only after the 8-cell stage does oocyte-type protein synthesis cease (Van Blerkom & McGaughey, 1978). In view of the insensitivity of protein synthesis to inhibition of transcription during early cleavage, these findings suggest that a considerable number of species of functional maternal mRNAs are conserved. Matings between DDK mice and those of other inbred strains have identified a maternal effect (ovum mutant) that leads to death of the majority of embryos at the blastocyst stage (Wakasugi et al. 1967; Wakasugi, 1973, 1974). Although the lethal effect is inherited as a single autosomal gene its pattern of expression is complex. Lethality is attributed to a defective interaction between the product of the maternal allele and the wild-type paternal genome.

3

Activation of the Embryonic Genome

Transcription of tRNA and heterogeneous RNA can be detected soon after fertilization in the rabbit (reviewed by Schultz & Tucker, 1977), but only at the 2-cell stage in the mouse; rRNA synthesis is evident 48-60 hours after fer115

Vol. 35 No. 2

m- Reviewed by Graham, 1971 n M Sherman end S Gay, personal communication p-Reviewed by Jenklnson & Brlllngton, 1977 v--Zetter& Martin. 1978 r Adamson & Ayers. 1979

CONTROL OF EARLY DEVELOPMENT tilization in both species (reviewed by Epstein, 1975). One- and two-dimensional electrophoreses have demonstrated that the greatest changes in protein synthetic patterns occur between the 1-cell and 2-cell stages (reviewed by Van Blerkom & Manes, 1977). One concludes, therefore, that to make new proteins, new mRNA templates are being utilized. However, convincing evidence that these newly functioning mRNA templates are transcribed from the embryonic genome is first obtained at the 6-8-cell stage. Paternal variants for the enzymes glucosephosphate isomerase (GPI) and {3-glucuronidase were detected in 8-cell and 6-8-cell mouse embryos, respectively, by Brinster (1973) and by Wudl & Chapman (1976). Also at the 8-cell stage, cell-surface antigens determined by the paternal genome appeared (Muggleton-Harris & Johnson, 1976). Embryonic gene products are shown to be essential to development in the numerous examples of early lethal mutants (reviewed by Chapman et al. 1977). None of the above data demonstrates a role for a known embryo-coded polypeptide in an essential step in development or differentiation. Rigorous evidence of this sort will be hard to come by, given the difficulties of obtaining sufficient material. However, an extremely sophisticated approach to solving such a problem has been rewarding. Johnson and his colleagues combined two-dimensional electrophoresis of newly synthesized proteins from embryos incubated in the presence or absence of a-amanitin to show that a few (but only a few) of the proteins that appear for the first time during blastulation are susceptible to transcriptional block and, since blastulation is also blocked, these proteins were thought to be important to that developmental process. Moreover, having shown that some of these proteins were unique to the trophectoderm or to the ICM it was concluded that they may also underlie the process of differentiation (reviewed by Johnson et al. 1977). In fact trophectoderm- and ICM-specific proteins are detected as early as the 8cell to morula stage in the mouse (Van Blerkom et al. 1976; Handyside & Johnson, 1978). In summary, although maternal mRNA continues to be expressed during very early development, and transcription begins as early as the 1—2-cell stage, it is not clear which activity is more crucial either to development or to the production of new cell types. It is tempting to assign the inherited factors to the role of providing "household" molecules needed for growth, such as has been documented in sea urchins and amphibia, at least for histones and microtubular protein (reviewed by Davidson, 1976), since none of the factors so far identified in the mammalian egg is an obvious regulatory molecule. The role of producing new cell types may then be accorded to transcriptional activity, but whatever controls this must act through positional signals.

E D Adamson & R L Gardner

significance in establishing disparate microenvironments between inside and outside cells. It is clear from the above observations that cell surfaces are altered radically during cleavage. Recently, much attention has been directed towards elucidating the nature of these changes. Particular interest has centred on the complex T locus which is located close to the H-2 region on chromosome 17 in the mouse. Many mutants of this locus are lethal or semi-lethal from the morula stage onwards (for recent reviews see Bennett, 1975; Hillman, 1975; Sherman & Wudl, 1977). One hypothesis is that the T region specifies cell-surface components that mediate critical cell interactions at various steps in differentiation (Gluecksohn-Waelsch & Erickson, 1970; Bennett et al. 1972). Bennett and Jacob and their colleagues postulate that the Tregion genes have functions similar to those of the histocompatibility genes, and that their products are embryonic equivalents of H-2 antigens. They have obtained evidence that a gene product controlled by or closely linked to the T/t complex is similar to an antigen identified on nullipotent F9 teratocarcinoma cells. Consistent with a role for the F9 antigen in cell-surface interactions is the finding that compaction is inhibited if cleaving embryos are cultured in the presence of antiF9 Fab fragments (Kemler et al. 1977). The effect is specific, because cleavage continues and can lead to formation of normal blastocysts if exposure to the antibody fragments is limited to 48 hours. However, several of the t mutants are morphologically abnormal at a stage much earlier than that of developmental arrest. Abnormal accumulation of nuclear and cytoplasmic lipid and of nuclear fibrillo-granular bodies occurs as early as the 2-cell stage in tn/tn homozygotes, which die at the late morula stage. Some of these defects can be explained by the excessive rates of ATP metabolism found in mutant embryos (Hillman, 1975). Hence, it has yet to be established whether the primary lesion in T mutants resides in intermediary metabolism or in altered cell-surface components. Finally, changes in the lipid composition of the cell membrane are also evident during cleavage (reviewed by Pratt, 1978). a Determination of Inner Cell Mass and Trophectoderm Cells Obviously, the fact that single blastomeres of 8-cell embryos can produce both ICM and trophectoderm cells (Kelly, 1975, 1977) places commitment at a later stage. The earliest at which it could take place on the inside-outside hypothesis would be when inside cells are first seen, namely between the 8- and 16cell stages (Barlow et al. 1972). Unitary blastocysts are no longer formed by aggregation if one or both members of a pair of embryos are within approximately 8 hours of blastocyst formation (Burgoyne & Ducibella, 1977). Blastulation is first evident in the light microscope in embryos consisting of between 28 and 33 cells (Smith & McLaren, 1977). However, loss of ability to aggregate may be due to development of zonular junctions between outside cells (Ducibella & Anderson, 1975; Ducibella et al. 1975) rather than to cell determination, since late morulae and pairs of early blastocysts can form morphologically integrated embryos after partial dissociation (Stern & Wilson, 1972). The finding mentioned earlier, that postponement of compaction by culturing embryos for 2 days in anti-F9 Fab fragments is also compatible with formation of normal blastocysts, likewise argues for late commitment. More rigorous investigation of the timing of commitment depends on the isolation of pure populations of inside and outside cells. Microsurgical techniques developed for this

4 Cellular Relations during Cleavage Blastomeres are roughly spherical, and mutual contact is therefore minimal up to the 8-cell stage. Thereafter, the relationship between them changes dramatically, contact becoming so intimate and extensive that cell boundaries can no longer be readily resolved. This phenomenon of compaction, which persists throughout the remainder of cleavage, is accompanied by formation first of focal and then of zonular tight junctions between outside cells (Ducibella & Anderson, 1975; Ducibella et al. 1975). Numerous other changes are initiated at this stage, including the organization of the cortical cytoskeleton, and the distribution of mitochondria and microvilli (Table I) (see also Ducibella, 1977). Interest in compaction lies in its possible 16

Br.Med.Bull. 1979

CONTROL OF EARLY DEVELOPMENT

blastocoelic surface of the ICM by 4j days after coitus seems to originate from this tissue rather than by ingrowth of trophectoderm (Rossant, 1975b, 1977). Its cells are distinguishable from those of the underlying primitive ectoderm both morphologically (Enders et al. 1978) and by their pattern of colonization of host blastocysts (Gardner & Papaioannou, 1975; Gardner & Rossant, 1979). Differentiation of primitive endoderm versus ectoderm appears from blastocyst injection experiments to mark the divergence of two stable cell lineages, with the endoderm destined to form only extra-embryonic endoderm. and the ectoderm the entire fetal soma and germ line, as well as the extraembryonic mesoderm of the allantois and visceral yolk sac (Gardner & Papaioannou, 1975; Gardner & Rossant. 1976. 1979). Similar conclusions have been reached about the fate of primitive endoderm and ectoderm from studies on their development as ectopic grafts (Diwan & Stevens. 1976; Skreb et al. 1976). Various observations have been interpreted to suggest that cell position may play a similar role in initial differentiation within the ICM—as has been proposed for trophectoderm compared with ICM (Gardner & Johnson, 1975; Gardner & Papaioannou, 1975; Rossant, 1975b, 1977). It is of interest in this context that the ICM also undergoes compaction before differentiation into outer endoderm and inner ectoderm (Enders et al. 1978). However, it has yet to be demonstrated that early ICM cells are bipotential, and, if so, that an external location precipitates endodermal differentiation, and an internal one ectodermal differentiation.

purpose could be applied only to expanded blastocysts containing approximately 60 or more cells 3$ days after coitus (Gardner, 1971; Gardner & Johnson, 1972). Trophectoderm and ICM tissue isolated at this stage exhibited different properties (Gardner, 1975) and did not appear to contain any persisting labile cells in a variety of in-vivo and in-vitro assays designed to reveal them (Gardner & Johnson, 1972, 1973, 1975; Rossant, 1975a,b, 1976). An elegantly simple procedure developed recently by Solter & Knowles (1975) has enabled isolation of inside cells from earlier pre-implantation embryos. The intact embryos are treated with rabbit anti-mouse serum and, after being rinsed to remove unbound antibody, exposed to complement. Provided that the zonular junctions between outside cells are complete, the reagents cannot gain access to inside cells, and only the outer ones are lysed. Such immunosurgically isolated early ICMs have been found to form blastocyst-like structures capable of producing trophoblastic giant cells (Handyside, 1978; Hogan & Tilly, 1978; Spindle, 1978) and, more compellingly, to yield morphologically normal postimplantation embryos in utero (J Rossant, personal communication). Results on ICMs isolated immunosurgically from expanded blastocysts are in accord with the earlier microsurgical data in two studies (Handyside, 1978; Spindle, 1978) but not in a third (Hogan & Tilly, 1978). However, it remains to be established unequivocally that all trophectoderm cells are eliminated by immunosurgery. Thus it appears that inside cells are committed after blastulation, perhaps close to the 60-cell stage. The stage at which outside cells cease to repond to inside conditions by forming ICM cells has yet to be established.

5

b Control of Trophoblast Development Mitotic activity ceases before implantation in the mural trophectoderm (Copp, 1978). Mural cells nonetheless continue to replicate their DNA and thereby gradually transform into mononuclear giant cells (Barlow & Sherman, 1972; Sherman et al. 1972). Hence the majority of trophectoderm cells are already post-mitotic by the late blastocyst stage. Thereafter, additional mural cells are recruited from the polar trophectoderm (Copp, 1978, 1979), which persists as a proliferative centre and appears to generate all further trophoblastic cells of the conceptus (Gardner et al. 1973: Gardner & Papaioannou, 1975; Rossant, 1977). Prevention of formation or deletion of the ICM results in an exclusively mural pattern of giant transformation by trophectoderm cells (Gardner & Johnson, 1972; Ansell & Snow, 1975; Sherman, 1975). Normal proliferation can be sustained by recombining trophectoderm with ICM tissue before the onset of giant-cell formation (Gardner et al. 1973). Diploid trophoblastic tissue of the post-implantation conceptus also responds to isolation by ceasing cell division and initiating DNA endoreduplication (Rossant & Ofer, 1977). Thus, the presence of the ICM or its derivatives appears to be required for maintenance of trophoblastic cell proliferation until day 9, and possibly throughout the remaining 11 or 12 days of gestation. Nothing is known about how the ICM exercises control, though it evidently does so by means of a localized interaction with overlying trophectoderm cells at the blastocyst stage. This interrelationship is of particular interest as a means by which development and growth of the placenta may be coordinated with that of the embryo.

Molecular Characterization of Cell Type

Criteria such as size, DNA content and prominence of endoplasmic reticulum have been used to identify some of the types of cells emerging during differentiation. However, classification of others is based principally on their location within the embryo. This clearly poses problems of interpretation in studies on cell commitment in culture or ectopic grafts in which organization of the embryo is invariably disturbed. It is for this reason that strenuous efforts have been made in recent years to identify molecules exhibiting tissue specificity in distribution. An obvious difficulty has been the small amount of material

TABLE I I . Biochemical markers* of post-implantation embryonic tissues9 Tissue Trophedoderm Parietal endoderm

Visceral endoderm of egg cylinder

Marker ( \ ( s I

Pfasminogen activator0 Steroid hormones' Collagen type IV d and fibronectin* accumulate in Reichert's membrane Plasminogen activator 6 n-Fetoproteir/ Transferring Acid phosphaiass* Plasminogen activator detected in 8-day embryos, later than the above tissues'

I

• Markers refer to identified mecromotecular products which may be used to characterize tissues at particular stages of development References' a. Reviewed by Graham. 1977 b Strickland et al 1976 c: Chew & Sherman. 1975 d Adamson & Ayers 1979 e.Wartlovaara el al 1978

c Commitment of Primitive Endoderm Cells in the Inner Cell Mass The monolayer of primitive endoderm discernible on the 117

Vol. 35 No. 2

E D Adamson & R L Gardner

* Dziadek & Adamson. 1978 g. E D Adamson. unpublished observations h. Solter et al 1973 / V C Bode and M A Dziadek, tn preparation

CONTROL OF EARLY DEVELOPMENT E D Adamson & R L Gardner available for biochemical analysis or immunization. One solution has been to use mouse-embryo-derived teratocarcinoma cells (see Sherman & Solter, 1975) because they can be grown on a large scale in culture (Jacob, 1978). Another has been to use sensitive two-dimensional dectrophoresis to resolve polypeptides synthesized by isolated tissues of the early embryo. In this way, Van Blerkom et al. (1976) have identified several ICM- and trophectoderm-specific polypeptides. Other studies have been concerned with the synthesis and/or localization of known molecules (see Table II for a summary). One particularly interesting finding relates to the production of a-fetoprotein by visceral endoderm. When isolated, the whole tissue seems to engage in synthesis of this molecule, while in the intact embryo the part embracing the extra-embryonic ectoderm (see fig. Id) does not Dziadek (1978) has obtained evidence that this is owing to an inhibitory effect of the latter tissue.

6

Conclusion

We have discussed some of the recent work that seems particularly relevant to the problem of control of cellular differentiation in early mammalian development Two points clearly emerge from this brief survey. First, major gaps in knowledge remain at both the cellular and molecular levels of analysis. Second, the knowledge that we do possess is based on detailed studies in only two species. However, as pointed out by Van Blerkom & Manes (1977), the little that has been gleaned from other mammals is in general accord with what has been learnt in the mouse and rabbit ACKNOWLEDGEMENTS

We wish to thank Dr C F Graham and Mrs J I Brown for help in preparing the manuscript, and the Medical Research Council and The Royal Society for support

REFERENCES

Gardner R L & Johnson M H (1972)/. Embryol. Exp. Morphol. 28,279-312 Gardner R L & Johnson M H (1973) Nat. New Btol. 246,86-89 Gardner R L & Johnson M H (1975) In: Cell patterning, pp. 183-196 (Ciba Foundation symposium n j . no. 29). Elsevier; Excerpta Medica; NorthHolland, Amsterdam Gardner R L & Papaioannou V E (1975) pp. 103-132* Gardner R L & Rossant J (1976) In: Embryogenesls in mammals, pp. 5—25 (Ciba Foundation symposium n-s. no. 40). Elsevier; Excerpta Medica; North-HoUand, Amsterdam Gardner R L & Rossant J (1979) / . Embryol. Exp. Morphol. 52 (In press) Gardner R L, Papaioannou V E & Barton S C (1973) / . Embryol Exp. Morphol 30,561-572 Garner W & McLaren A (1974)/. Embryol. Exp. Morphol. 32,495-503 Glueckiohn-Waelsch S & Erickson R P (1970) Curr. Top. Dev. Btol. 3 , 2 8 1 316 Golbus M S, Calarco P G & Epstein C J (1973)/. Exp. Zool. 186,207-216 Graham C F (1971) Symp. Soc. Exp. Btol 23,371-378 Oraham C F (1977) pp. 315—394t Gurdon J B (1974) The control of gene expression In animal development. Oxford University Press, London Handyside A H (1978)/. Embryol Exp. Morphol. 45,37-53 Handyiide A H & Johnson M H (1978) / . Embryol Exp. Morphol 44, 191199 H01manN(1975)pp. 189-206* Hillman N, Sherman M I & Graham C (1972) / . Embryol. Exp. Morphol. 28, 263-278 Hogan B & Tflly R (1978)/. Embryol. Exp. Morphol. 43,93-105 Hutchison C A i n , Newbold J E, Potter S S & EdgeU M H (1974) Nature (London) 231,536-538 Izquierdc L (1977) In: Johnson M H, ed. Development in mammals, vol. 2, pp. 99-118. North-HoUand, Amsterdam Jacob F (1977) Immunol. Rev. 33,3-32 Jacob F (1978) Proc. R. Soc. London, B, 201,249-270 Jaenisch R & Berns A (1977) pp. 267-314J Jenkinson E J