Genetics of the early mouse embryo.

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GENETICS OF THE EARLY

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Annu. Rev. Genet. 1976.10:361-388. Downloaded from www.annualreviews.org by University of Nebraska - Lincoln on 07/18/13. For personal use only.

MOUSE EMBRYO Anne McLaren MRC Mammalian Development Unit, University College London, London, England

CONTENTS PHYSICAL BASIS OF GENE EXPRESSION . ... . ... .. ......... . .. . .. . .. ................ ... .... ... .. .. . . .. .. .

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DNA ............................................................................................................................ RNA ............................................................................................................................ Protein ..................... . ................................................................. ................... .............. SINGLE-GENE VARIATION .... ............ . .................... ......... ..... ................ ... ...... . . .. ....... ... Enzymes ...... ......... . .. ................... .. . . ........................ .. ... ........................ ................. ... . Antigens ........... .. . . ................ .. . ... ......................... ..... .... ..................... .... .......... ... .. . . ... Lethal Genes .............. ........... ..................................................................................... Yellow (A') ...... . .. .. ................ ... .. ......................... ..... ........................ . .. .

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T locus...............................................................•.•..•...............................................

tl'

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t"")

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to" .................................................................................................................. t' (also t'. "tl': and t'O) ....... . ..................... ..... . ............................................. .. t"' (also 16 other to alleles) .. .. .......... ........... ... ... ............. ........................ .......... t' (also t'. t"". t..... to")

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T.......................................................................................................................

........... ............ .......... .. ... ....................... ......... ... .... ..... .. . . ... ... ...... . .. . . .. .. ......... Conclusion .............................. ...................... . ... .. ............................................. Fused (Fu). Kinky (Fuki) .. ........... ........ .... ... ................. ...... ...... ........ . ..................... c locus ......................................... .. ...................... ..... ............................................. Short-ear lethals ........................... .. .. ..................... ............................................... ... Blind (Bid) .................................................. .......................................................... Velvet coat (Ve) .... .... ............................................................ .................................. Waved coat (We) .. ..................... ........... ...... .............. ... ........................................... Dickie's small eye (Dey) . .. .. . .. ..... .. . .. ... . . Hydrocephalus-1 (hy-1) ............................ ....... . ............ ... .... .. .. .......................... Oligosyndactylism (Os) ................... .................................. ... ........................... .. ... . . .. Tail-short (Ts) .......... .............................. ................... .................... .......... .. .. ........... Ovum mutant (am) ............................ .............................. ................................. ..... CHROMOSOME EFFECTS ...... .. .............. .. . . ............ .. ........... . . .. .. . .......... ... ... ....... ... ..... ..... Single Chromosomes ............... .... ... ..... .... .......... ................ .. ........... ......... ....... ... Chromosome Sets ..... ............................ .................... .............. .. ..... ............... .... ... CONCLUSIONS .......... .. ............. . . .......... .............. .. ............... ... . .. ..................... .... .. . .. .. . .... Other T alleles

tol (also to', tol'. to'l t07l, t>11)

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This article is concerned with genetic effects on the mouse embryo before organo genesis begins. It covers the first seven to eight days of life, the period during which the embryonic axis is established, up to and including the appearance of the primi tive streak. Implantation begins about four days post coitum, halfway through this period. As we shall see, a wide range of genetic defects lead to death during the first week of development; in humans, the equivalent stage of gestation is thought to be associated with very high embryonic mortality. The first part of the article considers briefly the biochemistry of early development insofar as it relates to gene expression; the second reviews gene effects, and the third covers chromosome effects resulting from abnormalities of both single chromosomes and chromosome sets. PHYSICAL BASIS OF GENE EXPRESSION To understand fully the genetics of the early embryo, we would need to know (a) when the embryonic genome is first transcribed, (b) when embryonic mRNA is first translated, (c) whether paternal genes in the embryonic genome are transcribed as early as maternal ones, (d) how long after fertilization maternal proteins, whether transmitted in the cytoplasm or synthesized from maternal mRNA, continue to be important in development. Some relevant information from studies on DNA, RNA, and protein synthesis is reviewed below. An illuminating discussion has recently been published by Epstein (50). DNA

The last DNA replication in male gametes takes place 3-5 weeks before fertilization (93, Ill). During the terminal stages of spermatogenesis, while the chromatin of the spermatids is condensing ( 1 1 2), the histones associated with the sperm DNA are replaced by protamines, rich in arginine (47.0%), histidine ( 1 2.2%), and cysteine (9.8%) (13). Unlike the basic chromosomal protein of human, rabbit, and guinea pig, that of mouse spermatozoa consists of two species. Autoradiographic studies after labeling with radioactive arginine (94) suggest that the protamine is removed during the course of sperm chromatin decondensation for pronucleus formation. This would allow the pattern of histone and nonhistone proteins to be reestablished under the control of the cytoplasm of the fertilized egg, and would be consistent with early participation of the paternal DNA in the developmental program of the embryo. The last replication of DNA in the egg nucleus takes place during fetal life, 1 3- 1 5 days post coitum (39). In the unfertilized egg, about one third of the total DNA is mitochondrial DNA (BOa). The inheritance and expression of mitochondrial genes are hard to investi gate (1 29a), as no mutations have yet been detected in the mammalian mitochon drial genome. However, analysis of the species-specific splitting patterns after restriction-endonuclease digestion suggests that all mitochondrial DNA in equine species hybrids is of maternal origin (80). The assay used would not detect a minor component ofless than 5%, so paternal mitochondria would not have been identified had they been present in their initial proportion (72 mitochondria per bull sper-


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matozoon, versus 106 in the egg). In any case, on the basis of inhibitor studies, Piko & Chase (1 30) have concluded that the mitochondrial genome does not play an important role in the development of the preimplantation embryo. The first DNA replication in the fertilized egg takes place 4-6 hr after insemina tion, and lasts about 4 hr ( 1 , 100). DNA synthesis during cleavage does not appear to be rigidly linked to cell division, as cell and nuclear division can be inhibited by cytochalasin-B while DNA synthesis and chromosome replication proceed normally (140). From the two-cell stage onwards, doubling time averages about 1 1 hr, and each DNA replication lasts about 6 hr (69, 10 1). Cleavage is thus very slow, in mice as in other mammals, compared with cleavage rates in lower vertebrates. A paternal effect has been reported (166) on the rate of cleavage in crosses between inbred strains of mice, from the two-cell stage onwards (see also the effects of the yellow gene, below). After implantation the rate of cell division increases sharply, more so in the ectoderm than in the primary endoderm (144). By 7.5 days post coitum (p.c.), doubling time in the ectoderm is down to 5-6 hr. Cell division ceases in the mural trophectoderm at implantation, and in the distal areas of the ectoplacental cone within the next few days, and is superseded by giant cell transformation. Endoredu plication (34, 60) of the entire genome (1 34) results in giant cells with chromosomes that are probably polytene (145), containing up to a thousand times the haploid DNA content (1 1). Unscheduled DNA synthesis after UV irradiation has been demonstrated in cleaving mouse embryos (128), so DNA repair mechanisms presumably operate. The high incidence of chromosome damage in preimplantation embryos cultured in low concentrations of 3H-thymidine (141) suggests, however, that the efficiency of DNA repair may be less than in adult cells. RNA

Extensive studies have been carried out on RNA metabolism in mouse gametes and early embryos. Some postmeiotic RNA synthesis has been demonstrated in both oogenesis (123, 163a) and spermatogenesis (89a, 1 12). No RNA synthesis has yet been detected in the zygote; newly synthesized RNA (ribosomal, 4S, 5S, and hete rogeneous) is first detected at the two- to four-cell stage (69). Studies using virus induced cell fusion suggest that nuclear RNA synthesis at the two- to four-cell stage is under the control of cytoplasmic factors (23). The rate oPH-uridine incorporation increases sharply between the morula and blastocyst stage: when allowance is made for changes with age in precursor uptake (50), little or no increase occurs in the rate of RNA synthesis per genome over the same period. In the rabbit, transfer RNA becomes extensively demethylated during the transition from morula to blastocyst ( 104). Experiments with inhibitors of RNA synthesis have yielded ambiguous results. Epstein (50) concludes that the results with actinomycin D, a-amanitin, and S-bromodeoxyuridine, as well as the increase in rate of RNA synthesis between the two- and four-cell stage, are indicative of, but fail to provide conclusive evidence for, participation of the embryonic genome in preimplantation development. There

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seems no doubt, however, that transcriptional activity occurs as early as the two-cell stage. RNA polymerase activity can be demonstrated throughout embryonic develop ment (1 14, 137), but neither polymerase I nor II shows any significant correlation with the amount or type of RNA synthesized at different preimplantation stages, suggesting that gene expression is not controlled by the availability of polymerases (1 38).


Protein

The total protein content of the mouse embryo decreases during cleavage, but some protein synthesis can be demonstrated from the unfertilized egg onwards. A striking increase in the rate of protein synthesis occurs at fertilization or parthenogenetic activation (97) From the eight-cell to the blastocyst stage, the rate of protein synthesis increases significantly for the embryo as a whole, though the rate of synthesis per cell remains more or less constant. Between the early and late blas tocyst stages, there is an increase in the rate of uptake of precursors, but none in the incorporation of precursors into protein (50). Several recent studies have compared qualitative patterns of newly synthesized proteins at different stages of mouse preimplantation development, using labeled proteins separated by polyacrylamide gel electrophoresis. A striking change of pattern occurs after fertilization (98, 157) or parthenogenetic activation (157); since the new pattern appears even in the presence of transcription inhibitors, it may result from the translation of a "masked" messenger RNA (98). The most extensive changes during cleavage occur between the two- and eight-cell stages (5 1 , 99, 157); some of the major protein bands are synthesized for less than 12 hr. Little informa tion is yet available on the identity of the various protein bands, though three of the major bands that appear identical in mouse, rat, and rabbit blastocysts have been tentatively identified as actin, tubulin, and myosin. A more sensitive system of two-dimensional electrophoresis has revealed differ ences in the pattern of protein synthesis between inner cell mass and trophectoderm, separated by microsurgery at the blastocyst stage ( 1 56). The activity of individual enzymes shows no consistent trend during early devel opment: some increase, some decrease, some do not change in activity (50). A recent histochemical study demonstrated that �5-3.B-hydroxysteroid dehydrogenase in the mouse, rat, hamster, and rabbit embryo appears first at the morula stage, and disappears again shortly after implantation (40). Whether for any enzyme the changes observed are associated with synthesis of new protein is not usually known, nor is it general1y known whether such synthesis involves transcription of the embryonic genome. The X-linked enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) is more informative. Eggs from normal mice with two X chromosomes have twice as much HGPRT activity as eggs from XO mice with only a single X chromosome, yet by the blastocyst stage HGPRT activity has increased to almost the same level, whatever the genotype of the mother (49). This result can be interpreted to indicate control by the embryonic genome from about the eight-cell stage onwards, though interpretation is complicated by a lack of information as to .


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when X-chromosome inactivation takes place. A similar study (95) on the X-linked enzyme phosphoglycerate kinase (PGK) in embryos of XX and XO mice suggests that embryonic enzyme is first produced between 84 hr and 6.5 days p.c., and that by this time dosage compensation is complete. The lactic dehydrogenase (LDH) locus is not activated until after implantation Unfertilized eggs contain a high level ofLDH-l (/3 subunits only) (7). This maternal enzyme is progressively degraded during the preimplantation period, and it is not until enzyme levels again increase, in the 6.5-day embryo or after trophoblast outgrowth in vitro, that the LDH-5 band (a subunits) appears. The relative propor tions of bands 5, 4, and 3 at 6--8 days of development is different in different regions of the embryo ( 1 1 3), in such a way as to suggest that /3 subunits of embryonic origin are synthesized earlier in the embryo proper than in extraembryonic or trophoblast tissue. The report that LDH-X, normally only found in the testis, can also be detected on the surface of blastocysts has yet to be confirmed (14). Biochemical differentiation must lead to developmental changes in the antigenic or surface properties of the embryo. A gonadotrophin-like molecule has been de tected on the surface of mouse morulae (167); a change in the surface glycoproteins is apparent at the blastocyst stage (13 1). Antiserum raised against blastocysts reacts with preimplantation embryos but not with unfertilized eggs or trophoblast out growths, and blocks development in vitro; antiplacenta antiserum, on the other hand, reacts with eggs, preimplantation embryos, and trophoblast outgrowths, but has no adverse effect on development (168). The occurrence during development of genetically characterized antigens is considered in the next section.


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SINGLE-GENE VARIATION Enzymes

Enzymes with known genetic variants that have been analyzed during early develop ment include isocitrate dehydrogenase (Id-l), supernatant malic enzyme (Mod-I), glucosephosphate isomerase (Gpi-l), and /3-glucuronidase (Gus). The first three involve electrophoretic variants; alleles at the Gus locus differ in levels of activity and heat denaturation kinetics. The Id-l (52, 1 70) and Mod-l (1 70) loci are apparently not activated till midterm (9.5 and 10.5 days p.c. respectively); maternal and paternal variants then appear simultaneously. Paternal gene expression for Gpi-l has been demonstrated in the late blastocyst (35) and perhaps as early as the eight-cell stage (27). /3-glucuronidase activity shows a small but significant increase between the two and eight-cell stage, and a lOO-fold increase between the eight-cell and blastocyst stage. A study (173) of the heat denaturation kinetics of hybrid embryos from C3H females that carry the heat-labile Gul' allele showed that both maternal and pater nal embryonic alleles were expressed during the second period; data on activity levels during early cleavage, obtained with a microfluorometric assay sensitive enough to measure the enzyme activity of single embryos, suggest that the paternal allele may already be functioning at the two- to four-cell stage.

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Antigens

The general topic of antigen expression during early embryonic development in the mouse has recently been reviewed (24, 84). Some examples of the changes that occur in cell-surface antigens as development proceeds have been mentioned earlier. Here we consider only the time of expression of antigenic differences controlled by known genetic loci. For a recent discussion of the role in development of the H-2 locus and other loci affecting the cell surface, see Edidin (47). Histocompatibility antigens of the major H-2 locus can be recognized on 7.S-day but not 6.S-day p.c. embryos, within 2-3 days of transplantation to preimmunized hosts (126). Immunofluorescence techniques have demonstrated H-2 antigens on presumed inner cell mass derivatives in outgrowths from blastocysts in vitro, grown to a stage of development equivalent to about 7.5 days in vivo (75). Non-H-2 histocompatibility antigens, but not H-2 antigens, can be detected on much younger embryos, possibly as early as the two-cell, and certainly by the eight-cell, stage (76, 1 17, 124). The loci expressed early are thought to include H-3 and H-6 (1 25). Studies on the deleterious effects of specific antisera on the development of cultured embryos suggested that paternal as well as maternal histocompatibility antigens are present on the blastocyst (38); such antigens have been directly demonstrated on the surface of blastomeres from the six- to eight-cell stage onwards, using im munofluorescence techniques ( 1 1 7). By 7.5 days p.c., the embryonic sac is highly immunogenic, but the proliferating ectoplacental cone is not (1 33); this may indicate that histocompatibility antigens on trophoblast are masked in some way rather than absent, since the detection of antigens on trophoblast cells after attachment and outgrowth of the blastocyst in vitro seems to depend critically on the technique used (24, 32). Various immunological cross-reactions between early embryos and tumor cells have been reported (47). The most specific involves a surface antigen, the F9 antigen (4, 5), shared by preimplantation mouse embryos, primitive teratocarcinoma cells, ( 105), and spermatozoa of both mouse and humans (55). F9 antigen is thought to be produced by the normal allele of the t12 gene (4,5) (see below). If this identifica tion is correct, it should be absent from about 40 % of cleaving embryos from t121+ X t12 1+ intercrosses (the expected proportion of homozygotes is more than 25%, because of the segregation distortion associated with the t locus): such con firmation is eagerly awaited. It is also claimed that F9 antigen is structurally, and hence probably phylogeneti cally, related to H-2 histocompatibility antigen: the number of subunits appears to be the same, the molecular weights are similar, and both are associated with a J3-2-microglobulin-like moiety in the cell membrane (160). Lethal Genes YELLOW (A') The yellow allele (AY) at the agouti locus is the first recessive lethal (indeed, one of the first genes) to have been described in mice (81,90,91). It also appears, as we shall see, to be one of the first to be expressed in development. Early work claimed abnormalities at the morula and blastocyst stage (91); later studies



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suggested that the abnormalities begin at implantation, with all presumed homozy gous yellow embryos dead by the egg cylinder stage, and an abnormal decidual reaction in the uterus (1 32). Survival of the presumed homozygous embryos was not prolonged by removing the mother's ovaries 2.5 days p.c. in order to induce delay of implantation (G. R. Dunn, personal communication). When ovaries from AY/+ females were grafted to +1+ females, the homozygous agouti mothers showed a better developed decidual reaction; the embryonic abnormalities appeared at the same stage of development, but the embryos were larger and differentiation pro ceeded further, suggesting an effect of the uterine environment (1 32). A maternal effect in the same direction, allowing heterozygous yellow embryos to survive better in agouti than in yellow mothers, has also been reported ( 1 7 1 ). Further histological investigation was interpreted to indicate that the expression of the yellow gene was confined to the trophectoderm, affecting trophoblast giant cell transformation (46). It seems clear that expression of AY, at least in homozygous form, is influenced not only by the uterine environment, but also by the genetic background of the stock, and perhaps also by the cellular environment. In one recent study, using AG/Cam AY I a" mice (58), abnormalities were again never seen before implantation; microsur gical transfers of either normal inner cell masses to blastocysts from AYIa" X AYIa" matings, or (AYIa" X AYIa") inner cell masses (of which 25% should of course be homozygous yellow) to normal blastocysts, gave suggestive evidence that both inner cell mass and trophectoderm of homozygous yellow genotype were capable of normal development. In another stock, derived from Jackson Laboratory C57BLI 6J - AYla mice, 1 7-24% of embryos from (AYla X AYla) matings showed abnor malities after development to the morula stage either in vivo or in vitro, with one or more blastocysts apparently blocked at the eight-cell stage, and excluded from further development of the embryo (127). Preimplantation (AYla X AYla) embryos from a 6PB/RI - AYla stock repeatedly backcrossed to C57BL/6 show similar abnormalities in our laboratory (H. Paterson, personal communication). In ultra structure, the excluded blastomeres resembled those of normal 8-cell embryos, occa sionally 4-cell or 16-cell (3�}. If the zona pellucida was removed from the presumed AYlAY blastocysts, some outgrowth from the trophectoderm occurred in vitro but it was sparse and abnormal in morphology, and no further development of the inner cell mass was seen (129) (confirmed by H. Paterson, personal communication). Thus the earlier hypothesis (46), that embryonic death is solely a consequence of a failure in the timing of trophoblast giant cell transformation, was not validated. Time-lapse cinematography showed that the embryos with excluded blastomeres at the morula stage tended to be those that had entered the second cleavage division last (129). Thus AY in homozygous form, the paternal as well as the maternal allele, may begin to exert its effects as early as the two-cell stage. Another allele at the agouti locus, Ax, is also lethal at or soon after implantation when homozygous (G. R. Dunn, personal communication). T LOCUS The complex locus T presents so many intriguing genetic and develop mental features that it has been reviewed many times (16,17,19,42,43,67). It is situated on chromosome 1 7, near the complex H-2 histocompatibility locus, to


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which it may be functionally related, in that both may be concerned with molecular recognition (3). The semidominant allele T (Brachyury) produces a short-tailed phenotype in the heterozygote ( TI+), and in the homozygote is lethal. Over 100 recessive alleles (or possibly chromosomal rearrangements) of independent origin have so far been identified, including many from wild populations, giving tail-less mice when combined with T ( TIF). Most are viable when homozygous (txIF) but at least 35 are lethals, ranging in time of death from before implantation up to birth. The recessive lethals fall into six "complementation groups," in the sense that alleles within a group all act at about the same stage of development, and heterozygotes between alleles of the same group die, while combinations of alleles from different groups are usually viable and have normal tails, but are male-sterile. The com plementation groups are considered serially, in order of time of death. t12 (also r32) Homozygous t121t12 embryos die before implantation, at about the 3D-cell stage (morula or early blastocyst). The nucleoli at this time appear abnormal, both by light (1 39) and electron microscopy (29). DNA, RNA, and protein synthesis is reduced, and RNA levels in the morula are low, both in vivo (1 39) and in vitro (108, 1 36). Preliminary DNAIRNA hybridization results (92) suggested that t12 might, involve deletion of a nucleolar organizer region. However, it seems that the nucleolar abnormalities are secondary effects of developmental arrest or degenera tion (29, 78), and since RNA synthesis and processing have been proved normal for 4S, 1 8S, and 30-32S RNA (53, 79), it is unlikely that nucleolar function is defective. Amino acid uptake and incorporation at the 8-16--cell stage also appear normal (53). The junctional complexes that characterize differentiation of the trophectoderm can be formed in t12It12 embryos (29), and development can proceed as far as the early blastocyst stage, but some presumed homozygotes arrest as early as the 8-1 2-cell stage, showing characteristic lipid droplets in the nuclei, as well as excessive amounts of cytoplasmic lipid (78). Aggregation chimeras, in which normal and homozygous t12 embryos were associated from the 8-cell stage, bear witness to the cellular autonomy of tl2lt12 lethality (107). Embryos homozygous for the r32 allele (the "w" in the superscript indicates that the allele was first identified in a wild population) die at the same stage as t121t12 embryos, and show similar abnormalities (1 39). t12 and r32 homozygotes also resemble one another in showing abnormally high levels of ATP metabolism during early cleavage (61), and synthesis of neutral lipid which is stored in abnormally large amounts as cytoplasmic lipid droplets (1 1 8) from the two-cell stage onwards. A recent study (77) confirmed that arrest of r321r32 embryos occurred at the eight-cell to morula stage, but on ultrastructural criteria concluded that the modal time of death was somewhat earlier than in t12It12 homozygotes (early rather than late morulae) and that the two types of mutant embryo could be distinguished by the nature of their nuclear inclusions, by the failure of the mitochondria in r321r32 embryos to undergo the normal four-cell structural transition to the adult form, and by the crystals often seen in r321r32 mitochondria. However, a final decision as to whether t12 and r32 are the same allele must await studies of both genes on the same genetic background.


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The original (I allele (distinct from that now termed tl, see below) was believed to be a preimplantation lethal (62), so probably also belonged in this complementa tion group. It is now extinct.


rw73 Homozygous rw73 embryos develop to the blastocyst stage and induce a decidual reaction in the uterus. The trophectoderm and ectoplacental cone fail to form the normal close association with the decidua, and development never pro gresses beyond the two-layered stage. The embryos are markedly retarded in growth, but may survive until 8-9 days p.c. (148). (6 (also, (0, "(I" and (30) (0/(0

embryos are classifiable soon after five days p.c., and are dead by seven days (63). The primary endoderm differentiates, but the egg cylinder does not lengthen normally and fails to segregate into embryonic and extraembryonic portions. The proamniotic cavity often fails to form, and no primi tive streak develops. In a study of partial complementation between recessive lethal (alleles (1 35), the time of death of to homozygotes was confirmed. "tJ" was found to be identical with to, and therefore almost certainly a different allele from the original tl (62). Heterozygotes between either to or " tJ" and tJ2 showed abnormalities from the primitive streak stage (7 days p.c.) onwards, with most mortality between 1 1 and 12 days p.c., but some embryos (12%) survived to birth. Defects of the brain and eye were particularly common; the underlying abnormality was thought to reside in the organization of the primitive streak, perhaps related to that seen in f"J8 (see below). t6, indistinguishable from to by complementation analysis, also causes death at the egg cylinder stage (1 19). Some mutant embryos can be identified as early as the late blastocyst stage by the presence of large lipid droplets in the cytoplasm. More dead cells than usual are seen, particularly in the polar trophoblast. Growth of the egg cylinder is associated with abnormalities in the arrangement of both the primary endoderm and ectoderm cells, and excessive cytoplasmic lipid and crystal-contain ing mitochondria. Similar features have been reported, though at an earlier stage of development, in tJ2 and f"32 homozygotes (77, 78). In contrast to the earlier description of to/tO embryos, some t6 homozygotes undergo segregation of the egg cylinder into embryonic and extraembryonic areas (1 1 9). Compared to control cultures, postimplantation cultures of embryos from inter crosses of t6 heterozygotes showed a 25% deficiency of outgrowths with develop ment of the inner cell mass (54). The authors suggest that some cell surface component is defective in the t6/t6 inner cell mass, leading to failure of cohesion and loss of the inner cell mass from the surface of the trophoblast in vitro. rw5 (also 16 other f" alleles) rw5, like other alleles in the same complementation group, interferes with the maintenance of the embryonic ectoderm (20). Separation of embryonic from extraembryonic ectoderm takes place, and the egg cylinder elongates normally, but after 6.5 days p.c. the embryonic region becomes increas ingly pyknotic, and finally disappears. The extraembryonic tissues survive 2-3 days,

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with yolk sac, amnion, chorion, and allantois essentially normal. Occasionally a small remnant of embryonic ectoderm persists, forming a small spherical "micro embryo" with neural tissue, notochord, and mesoderm. A similar picture is charac teristic of CBA-strain triploid embryos (1 72). Whether the primary defect is in the endoderm, leading to nutritional deficiency and hence degeneration of the ectoderm, or in the ectoderm itself, is not known. (9 (also (4, (wI8, (wJO, (52) (9 homozygotes can be distinguished histologically from their litter mates by 6 days p.c. (1 16). The egg cylinder is attached at the distal as well as the proximal end; the endoderm is abnormal, and shows no sharp distinction from the ectoderm. At 8 days, the ectodermal neural folds in the head are over developed, while the mesodermal trunk structures, including the somites, fail to differentiate. The allantois is also retarded. Death occurs between 8 and 9.5 days, accompanied by general retardation, and such features as overgrowth and abnormal differentiation of the head folds, dUplication of the neural tube and neural folds, and overgrowth and duplication of the allantois. 1"'18 homozygotes present a superficially similar appearance, with an apparently double neural tube (but no duplication of the allantois) (2 1). The abnormality of the neural tube is thought to be caused by a defect in the primitive streak: movement of cells through the primitive streak to form mesoderm is inhibited; hence cells accumulate and form overgrowth or bulging of the primitive streak, which may force the neural folds into a W-shaped structure, with duplicated neural folds. Ultrastructural studies on t9/t9 embryos ( 1 47) have shown that the presumptive mesoderm cells moving through the primitive streak do not establish normal cell contacts with one another, and are grossly deformed, with blunt processes instead of the normal stellate ones. The processes are deficient in microfilaments. On transplantation to the testis, r18/r18 embryos give rise to tumors composed almost entirely of ectodermal derivatives (2). Many of the tumors appear on histo logical examination to be malignant, resembling neuroepitheliomas. This suggests that the ectodermal precursors of mesoderm have undergone transformation, or perhaps that some interaction with mesoderm is required for the control of ectoder� mal growth and differentiation. T The semidominant T allele acts primarily on the components of the primitive streak and notochord (36, 74). Effects in the T/T homozygote are first seen at 8.5 days p.c. (four-somite stage), when large fluid-filled blebs appear under the dorsal skin. Abnormalities of the notochord, neural folds, and somites develop, leading to an absence or marked reduction of the posterior region of the body. Ultrastructural studies ( 147) show that cell contacts are abnormal, in that neuroepithelial cells form close associations, including specialized contact zones, with cells of unlike type (notochord and somites). Death occurs by 10.5 days p.c., as a result of gross abnormalities of the allantois that prevent establishment of the umbilical circulation (64). Heterozygous effects are not seen until about 11 days p.c.' On transplantation to the extraembryonic chick coelom, abnormalities could be detected in TIT embryos as early as the 6.5-day egg cylinder stage (64). In vitro,


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TIT tissues grow well, with proliferation of fibroblasts, differentiation of cartilage, and heartbeat maintained for two months (48), so T appears not to be a cell lethal. In vitro recombination experiments suggested that the defect lay primarily in the

somites (IS). The correlation of abnormal notochord differentiation with abnormal neural tube development in TIT embryos suggested (62) the existence in mammals of an induc tive relationship between notochord and neural ectoderm, as in lower vertebrates.


Other T alleles Several other dominant T alleles have been described, producing homozygotes that are lethal at 7 days of gestation or soon after (1 7). In certain radiation-induced T-like mutations, the primary defect seems to involve a block to

cell division in the embryonic ectoderm at the egg-cylinder stage; embryonic en doderm is affected secondarily. The ectodermal abnormality can be recognized 6.5-7 days p.c; the embryo is dead by 8 days, but the ectoplacental cone and yolk sac continue to grow (22). THp (Hairpin) shows a peculiar kind of maternal effect, in that only those embryos receiving THp from the mother die in utero. THp 1+ embryos receiving the mutant allele from the father have short tails and normal viability, even when gestated in the uterus of a THp 1+ mother (83). ("'1 (also ("'3, ("'12, ("'21, ("'7J, ("'72) Alleles of this complementation group do not act before 9 days p.c., when homozygotes show abnormal cephalogenesis ( 18). The embryos may survive to birth. The time of gene action is thus later than that covered by this review. Conclusion It is tempting to seek a unifying explanation for the death of t recessive

lethal homozygotes at their various stages of embryogenesis. One such explanation, put forward by Bennett (16), argues that each lethal interferes with a successive stage of differentiation of the ectoderm: t12It12 involves a failure of differentiation of the peripheral cells of the morula into the trophectoderm; in f'1f' embryos, the egg cylinder ectoderm is unable to make the transition into embryonic and extra embryonic ectoderm; ("'SI("'s interferes with the subsequent growth and mainte nance of the embryonic ectoderm; ("'IBI("'IB affects the growth of the primitive streak, thus blocking the normal differentiation of mesoderm; and ("'11("'1 acts specifically on the nervous system. Several different t alleles (f', ("'1, ("'5, ("'32) have been shown by sperm cytotoxicity tests to be associated with specific cell-surface antigens expressed in spermatozoa ( 174). The surface antigen shared by early embryonic cells, primitive teratocar cinoma cells, and spermatozoa, and thought to represent the expression of the normal allele of tI2, has already been mentioned (see section on antigens). It is present from the two-cell to the blastocyst stage (that is, the stage at which the effects of homozygous t12 are seen). Many of the stage-specific developmental effects of particular t alleles are at least consistent with abnormalities of the cell surface, affecting cell recognition and differentiation. A strong case can therefore be made (16, 1 7, 1 9) for the T locus playing a major role in the organization of early development, by specifying cell-surface protein s that control a series of onto geneti-

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cally critical interactions between cells. Conclusive evidence for this view may emerge from immunological studies. An alternative explanation for the death of t12/t12, t"'35/t"'35, and to/to embryos, put forward by Hillman and her colleagues on the basis of direct biochemical and ultrastructural studies (6 1 , 77, 1 1 8, 1 19), invokes a defect in cellular energy metabo lism, associated with abnormal mitochondria, excessive levels of ATP synthesis, and excessive lipid accumulation. The metabolic imbalance could arise if carbohydrate energy sources were utilized more efficiently in mutant than in normal embryos (61). The Fused locus is situated near the T locus, on chromosome 17. Fu and FUki both cause tail defects in heterozygous condition. FUki homozygotes develop multiple embryonic axes at about 7 days p.c. (65). The neural ectoderm undergoes hyperplasia, bulging into the amniotic cavity and even through the membranes. Duplications of the neural tube, notochord, archenteron, somites, heart, and allantois are all sometimes seen, producing partial or even complete twinning. Death occurs at 8-10 days p.c. The primary effect of the gene is postulated to involve a disturbance of some organizer of axis development, analo gous to that found in Amphibia. Fu in homozygous condition is lethal only on certain backgrounds. From 9 days onwards, the nervous system shows a tendency to duplicate, and the notochord is sometimes branched ( 1 54).


FUSED (Fu), KINKY (Futi)

c LOCUS A series of overlapping deletions at the albino (c) locus has been induced by radiation. They are all lethal when homozygous, showing a range of biochemical, morphological, and ultrastructural defects. Two are expressed at the early egg cylinder stage, c25H and c6H ( 155). c25H, thought to be the largest deletion, has been detected cytologically (106), but its effect on development has not yet been described in detail. Homozygous c6H embryos could first be identified at 6.5 days p.c. (96). They were retarded in growth and development, and showed a characteristic spear-shaped extension of the distal endoderm into the maternal decidua. The ectoplacental cone was either very small or absent, with no mitoses to be seen; the embryonic ectoderm looked normal, though somewhat retarded in growth, but the extraembryonic ec toderm was sparse and disorganized, and the proximal endoderm was irregular and failed to show any differentiation into embryonic and extraembryonic regions. By 7.5 days, all germ layers were obviously abnormal. Formation of the primitive streak and differentiation of mesoderm never occurred, and by 8 days all the homozygous embryos were dead. As the authors point out, the developmental failure of both extraembryonic ectoderm and ectoplacental cone is consistent with the recent suggestion that both are derived from trophectoderm (59). Studies on isolated trophoblastic vesicles have shown that the presence of an inner cell mass is required for proliferation of the trophectoderm (59); perhaps in c6H/c6H embryos some aspects of the relation be tween inner cell mass and trophectoderm is disturbed. The unique extension of the distal endoderm, a derivative of the inner cell mass (59), is unexplained.


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Radiation-induced mutations in the dilute short-ear re gion of linkage group II include a lethal mutant 5RD300 H (se'), thought to be a minute deficiency. Homozygous embryos die by 8 days p.c., and can first be detected 7 days 16 hr p.c., showing virtual absence of mesoderm, excess proliferation of extraembryonic ectoderm, and overgrowth of trophoblast giant cells (41).

SHORT-EAR LETHALS

Homozygous Bid embryos appear very retarded at 6.5 days p.c.; the egg cylinder is only one fifth of the normal size. The primitive streak fails to form, and the endoderm is very abnormal, with greatly enlarged cells in the proximal endoderm. By the end of the following day the embryos are dead (158). Dysoptic appears from breeding tests to be either identical with Blind, or a closely similar allele at the same locus (164). Growth and differentiation cease at 6-7 days, with no mesoderm formation. The embryos are small at this time, showing few if any mitotic figures, and are dead by about 9 days.


BLIND (Bid)

VELVET COAT (Ve)

The velvet coat mutation (Ve) appeared in strain C57BL/6J.

Homozygous Ve embryos can be identified at 5.5 days p.c., since both embryonic and extraembryonic ectoderm are either absent completely, or represented by one or more small vesicles of healthy looking cells. Proximal and distal endoderm appears normal, but Reichert's membrane is secreted as solid clumps rather than as sheets. One VelVe embryo developed to the equivalent of 8-9 days' gestation, with a neural plate too small to form a neural tube, and disorganized tissues, but most die before this stage (S. B. Diwan and L. C. Stevens, personal communication). WAVED COAT (Wc) The waved coat mutation appeared in strain C3H/J. Wei We embryos show the "triploid syndrome" (1 72) typical also of fWs homozygotes (see above). The embryonic ectoderm becomes pyknotic and degenerates, while extra embryonic ectoderm develops normally, to give rise to an intact yolk sac, chorion, and allantois (S. B. Diwan and L. C. Stevens, personal communication). DICKIE'S SMALL EYE (Dey) Dey is a semidominant mutation, found by Dickie in the C3H/HeJ strain, causing abnormal development of embryonic ectoderm. Homozygotes die early. At 6.5 days p.c., some presumed homozygotes are com posed only of a small knot of distal endoderm cells with Reichert's membrane, trophoblast giant cells, and a few ectoderm cells. Heterozygotes can sometimes be identified 7.5 days p.c. by retarded development, failure of the egg cylinder to elongate, and abnormal embryonic ectoderm. Some die between 7 and II days p.c., others die later in gestation, with abnormal brain and eye development, and some survive birth, with gross eye defects (D. S. Varnum and L. C. Stevens, personal communication). HYDROCEPHALUS-I (by-I) The original description of hy-l homozygotes claimed that the trophoblast was defective as early as 4--6 days p.c., so that maternal material was able to enter the yolk sac. Reichert's membrane developed abnormally, and ruptures were seen in both yolk sac and amnion. High fluid pressure led to hemorrhages from the capillaries at 9-1 1 days p.c., and to hydrocephalus later (26). This description has been questioned (73). The gene is now extinct.

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About 25% of late blastocysts (64-cell stage) from Os/+ X Os/+ matings showed some abnormal cells, with pale cytoplasm, pyknotic and fragmented chromatin, and no nuclear membrane or nucleoli. The abnormal cells often occurred in pairs, as though arrested immediately after cell division, and were found in trophectoderm, inner cell mass, and endoderm. By the following day the embryos were degenerating, and few embryonic cells remained (1 59). In our laboratory (H. Paterson, personal communication), presumed homozygous as em bryos appear retarded at 4.5 days p.c., and are reduced in size, but show a mitotic index about nine times that of controls. More than one third of the cells contain mitotic figures. Air-dried preparations show some normal metaphase plates with chromosomes regularly arranged, and some with groups of highly condensed scat tered chromosomes, as seen after prolonged Colcemid� treatment. In culture, Os/Os blastocysts show rapid degeneration of the inner cell mass, and a reduction in number of trophoblast giant cells. Both in vivo and in vitro, the giant cells that were present developed normally, suggesting that the gene exerts its primary action on the mitotic apparatus and does not affect endoreduplication. Since many of the structural proteins constituting the mitotic apparatus are synthesized at least one cell cycle in advance, Os probably acts no later than 3.5 days p.c.


OLIGOSYNDACTYLISM (Os)

TAIL-SHORT (Ts) The time of death of TslTs embryos has been established as late' morula/early blastocyst (H. Paterson, personal communication).

OVUM MUTANT (om) Females of the DDK inbred strain were fully fertile when mated to males of their own strain, but showed a much reduced litter size with males of strains KK, NC, and C57BL. The reciprocal crosses were fully fertile. The lowered fertility of outcrossed DDK females was associated with high embryonic loss 3-4 days p.c., just before or during implantation. Some embryos failed to form blastocysts; others formed small blastocysts that induced a decidual reaction in the uterus but failed to develop further. The dead embryos often· showed a defect in trophectoderm formation (163). When DDK ovaries were grafted to F\ females, eggs shed from the grafted ovaries and fertilized by non-DDK spermatozoa suffered just as much embryonic mortality (161). Thus the bar to normal development must arise from some incompatibility between DDK cytoplasm and spermatozoa of other strains, rather than from any interaction of F\ embryos with the DDK female reproductive tract. On the basis of breeding tests, Wakusagi (162) postulates that there exists a cytoplasmic factor in the egg and a factor in spermatozoa, both controlled by autosomal genes. The cytoplasmic factor of the egg interacts specifically with the gene of sperm origin to synthesize some substance necessary for the formation of trophectoderm. The symbol om (ovum mutant ) has been given to the DDK gene, and OM to the corresponding wild-type gene; sand S are the corresponding genes acting in spermatozoa. Homozygous om or OM females produce substances 0 and o respectively during oogenesis; these are stored in the egg cytoplasm. Heterozygous


375

(om/OM) females produce 0- and O-substance in equal amounts, and one or the other (at random) interacts irreversibly with the spermatozoa in any given fertilized egg. Of the four combinations 0 + s, 0 + S, 0 + s, and 0 + S, the last is almost always lethal to the embryo. The genetic data point to om and s being either identical or closely linked.

CHROMOSOME EFFECTS


Single Chromosomes

The lack of a single chromosome (monosomy) or its presence in excess of the normal diploid complement (trisomy) can have a profound effect upon development. In the case of X-chromosome inactivation the converse is also true; that is, the process of embryonic development has a profound effect upon a single chromosome. During the preimplantat'ion period, even large chromosome abnormalities and deficiencies do not prevent development to the blastocyst stage. When male rats were mated within a week of irradiation of the testis with 400-800 r, 60% of the blastocysts were aneuploid, sometimes lacking several paternal chromosomes (44). No correlation was found between chromosome number and cleavage rate. Many of the embryos died at or soon after implantation; those that survived to 12 days p.c. lacked gross chromosome aberrations; even in these, however, organogenesis was much impaired. A similar result was seen when dominant lethals were induced by treatment ofthe mother with pyrimethamine (45); a dose level that induced 70% aneuploidy still allowed the embryos to develop into morphologically normal blas tocysts. Monosomy and trisomy of genetic origin are also compatible with normal cleav age and blastocyst formation. The T6 chromosome translocation in mice leads to nondisjunction at meiosis and hence to the production of gametes with an unbal anced karyotype. Heterozygous males show abnormal spermatogenesis, and are sterile; but T6/+ females are fully fertile, and the cleavage rate proved to be normal, though some of the embryos had 41 and others 39 chromosomes (9). Most of the chromosomally abnormal embryos died at or soon after implantation, some at midgestation, with neural tube defects, and a few viable trisomic mice were born. An analogous but more detailed study has been carried out by Ford, Gropp, and their colleagues (56, 72). Male F 1 hybrids from the cross between Mus musculus and Mus poschiavinus have seven metacentric chromosomes that frequently give rise to nondisjunction at meiosis, producing a range of numbers of chromosome arms at second metaphase. On backcrossing to M. musculus, the distribution of numbers of chromosome arms in embryos 3.5 days p.c. proved not to differ significantly from that at second meiotic metaphase, showing that even gross genome unbalance (up to six chromosome arms lacking) interfered neither with fertilization nor with cleavage (56). Chromosome counts made at 10 days p.c. established that all the hypodiploid embryos had died by this stage of pregnancy; most of the hyperdiploid embryos were still surviving at 10 days, but none were born alive. Trisomy for


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particular chromosomes was shown to have specific morphological consequences in development (7 1, 72); for example "chefs hat" exencephaly and microphthalmia, without marked retardation, are characteristic of trisomy 1 2, while growth retarda tion with facial dysmorphy but no gross malformation is characteristic of trisomy 1 . Malformation is not in general thought to be the cause of death. Growth in vivo is affected, though trisomic cells grow normally in vitro; the placenta often shows hypoplasia and retardation, but failure of placental function is again not thought to be the cause of death of trisomic fetuses (37). Trisomy for chromosome 19, the smallest autosome in the mouse, is compatible with brief survival after birth (often with cleft palate), but many trisomy- 19 embryos die soon after implantation, and the remainder show reduced fetal and placental weights throughout gestation, together with degeneration of the ovaries in some individuals (1 65). Trisomy for chromosomes 8, 1 I , or 17 is lethal by 1 1 .5 days p.c. (9, 7 1 , 72), trisomy 1 by 1 4-15 days p.c., trisomy 12 by 1 5-16 days p.c. (72). Trisomic embryos from female mice heterozygous for the autosomal Robertsonian translocation TJ IEM die between 8 and 12 days p.c. (10). Among progeny of male mice heterozy gous for the T26 H reciprocal translocation, involving chromosomes 2 and 8, almost all the genetically unbalanced embryos (hyper- as well as hypodiploid) die within two days of implantation (25). Combined cytological and histological analysis sug gests that the larger the deficiency, the earlier the embryo dies. Although absence of one or more chromosomes seems to be compatible with cleavage and blastocyst formation, the same is not true of the absence of both members of a chromosome pair (nullosomy). At ovulation, 97% of oocytes from normal females have the normal haploid complement of 20 chromosomes; those from females heterozygous for the T6 chromosome translocation, on the other hand, showed 36.5% with either 19 or 2 1 chromosomes (88). The T 6 translocation affects chromosomes 14 and 1 5. When the oocytes were activated parthenogenetically (see next section) and eggs with a single pronucleus selected for study, the proportion of aneuploid chromosome counts was the same at first cleavage as at ovulation. The eggs were transferred to the reproduc tive tract of pseudopregnant females and recovered at the morula stage; all but one of the embryos with 1 9 chromosomes had disappeared, but the frequency of 2 1 chromosome counts was n o lower than before, and the mean cell number was similar to that of 20-chromosome embryos (88). Thus the addition of a chromosome to a haploid set does not hinder cleavage, but nullosomy, at least for chromosomes 14 and 15, appears to be incompatible with development beyond the two-cell stage. A similar conclusion has been reached for OY (i.e., X nullosomy) embryos. A study of preimplantation embryos from XO mothers found that just under 25% were arrested at about the two-cell stage ( l i S). These were interpreted as the OY products of fertilization, prevented from further development through lack of an X chromosome, but no chromosome preparations were made so the evidence remains circumstantial. In another study (P. Calarco and C. J. Epstein, personal communi cation), 17% of embryos from XO mothers lagged in development when compared



377

with litter mates, but did not cease development at any specific stage, and revealed no specific abnormalities on ultrastructural examination. There is also increased postimplantation mortality in litters of XO mothers, which may indicate a reduction in viability of XO embryos (1 1 5). However, results of in vitro culture suggest that all embryos from XO females, whatever their genotype, are inferior in their develop mental potential to embryos from their XX sisters (28). Cleavage and blastocyst formation were delayed; many embryos from XO mothers failed to form blastocysts and became grossly abnormal; a few (possibly OY?) died at the eight-c�ll stage in vitro. Lyon has comprehensively reviewed X-chromosome inactivation in mammals (102), and has put forward an ingenious and plausible hypothesis to explain its evolution (103). Chandra & Brown have proposed (33) that there exists an underly ing imprinting process, by which one of two genetically homologous chromosomes is predetermined to function differently from the other at some subsequent stage of development, and that this imprinting takes place in the mammalian egg at the time of fertilization, as it does in coccid insects. Recent information (Ia) on the number of X chromosomes inactivated, and their origin, in triploid embryos is at variance with the predictions of Chandra and Brown's model. In XX mouse embryos, neither X chromosome appears to have undergone inactivation by the late blastocyst stage (4.5 days p.c.), as judged by genetic function of chromosomes derived from them (57); the HGPRT evidence (see page 364), though suggestive of dosage compensa tion before implantation, is not entirely clear-cut. By 6.5 days p.c., activity measure ments on another X-linked enzyme, PGK, support the conclusion that dosage compensation is complete, in that the levels of activity are no higher among embryos from XX than from XO mothers (see section on protein, above). From the blastocyst stage (40-50 cells) onwards, one X chromosome is reported to stain differently from the other, with quinacrine mustard or even Giemsa (1 50); labeling experiments with tritiated thymidine suggest that the heterochromatic X is replicating in the middle part of the S-period by 6.5 days p.c., and at the end of the period, as in the adult, by 7.5-8.5 days p.c. In the embryo, maternal and paternal X chromosomes are thought to be inactivated at random, but in the extraembryonic membranes it is apparently always the paternal X chromosomes that are inactivated ( 1 5 1). Chromosome Sets

The spontaneous occurrence of haploid and polyploid embryos has been reported (12); in particular, "silver" strain mice were found to have a high incidence of triploid embryos, which developed up to the blastocyst stage. It is not known whether spontaneous haploids arise by parthenogenesis, gynogenesis, or androgene sis. Early studies on experimentally induced haploidy and polyploidy have been reviewed by Beatty (12) and Astaurov (6). Renewed interest in parthenogenetic development (70, 85, 1 52) followed the successful experimental activation of mouse eggs by electrical stimulation in vivo (1 53), or hyaluronidase treatment and culture in vitro (68). Parthenogenones show


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an increased mortality at all stages of development (1 69), but may survive to the early somite stage either in the uterus (1 53, 1 69), or in an ectopic site (68). Eggs of the LT strain often undergo spontaneous activation; these parthenogenetic em bryos also die soon after implantation (149). Variations in the technique and timing of experimental activation determine whether the resulting embryos are haploid, diploid, or haplodiploid mosaics. Eggs activated in vitro in medium of normal osmolarity give rise mainly to haploid embryos, either by extrusion of the second polar body if the eggs are recovered 19-20 hr after human chorionic gonadotrophin (ReG) injection (uniform haploids), or by immediate cleavage if recovery is delayed 5-6 hr (mosaic haploids, with female pronucleus and second polar body giving rise to two genetically distinct haploid cell lines). If medium of low osmolarity is used, extrusion of the second polar body may be suppressed, giving rise to a potentially heterozygous diploid embryo. Diploidiza tion of a uniform haploid during cleavage can result in a totally homozygous diploid embryo. Both haploid and diploid parthenogenones proved capable of inducing a decidual cell reaction in the uterus, though the proportion that did so was higher for diploids (86). When activation was induced by an alternative technique, involv ing heat shock and cytochalasin B, over 90% of the eggs showed suppression of the second polar body and developed as diploids (8). Notwithstanding their diploid condition, survival was not observed beyond the early egg cylinder stage, either in inbred or in randomly bred strains. Little is known of the cause of death of parthenogenones, whether haploid or diploid (1 52). Electrical activation apparently fails to release all the cortical gran ules, with the result that the zona reaction may be abnormal (109). Ultrastructural abnormalities during cleavage have been reported, following in vitro activation (146), but apparently normal development may continue for some days after im plantation. When transferred to ectopic sites (82), both haploid and diploid par thenogenones gave rise to growths containing a wide range of cell types, so the failure of development in the uterus cannot be due to an inability to undergo cytodifferentiation. Lethal genetic factors have been proposed as an explanation for developmental failure of haploids, on the grounds that haploid embryos from an inbred hamster strain developed better than those from a randomly bred strain, which might be expected to carry more deleterious genes (87). More than 90% of the haploid eggs in randomly bred hamsters failed to develop beyond the two-cell stage. It seems unlikely, however, that lethal genes can provide a full explanation for the death of haploid embryos, since even in highly inbred mouse strains no haploids survive beyond the egg cylinder stage. The possible importance of genetic heterozygosity has also been raised by the observation that heterozygous diploid and even mosaic haploid parthenogenones cleave better than uniform haploids derived from FI hybrid eggs (89). Again, however, heterozygous diploid parthenogenones do not survive beyond the egg cylinder stage. Possibly some component of sper matozoa other than chromosomes is needed for normal embryonic development; possibly the disturbance in nucleocytoplasmic ratio in haploids interferes with gene expression. Microsurgical removal of one pronucleus from the fertilized egg could allow the



379

consequences of haploidy to be assessed, independently of the effects of parthenogen etic activation ( 1 10). Only gynogenetic embryos were recovered, perhaps because more damage is inflicted on the egg by removal of the female than the male pronu cleus. Cleavage was slow, and although cell numbers of up to about 80 were eventually reached, blastocyst formation was rarely seen. The author suggests that haploid cells may possess little or no ability to differentiate into trophectoderm and to secrete blastocoel fluid; the successful development of apparently haploid blas tocysts after parthenogenetic activation ( 169) might be due to some degree of haplo-diploid mosaicism. It seems unlikely, however, that diploid cells would have been entirely missed in the parthenogenetic blastocysts, had they been present in sufficient numbers to support blastocyst formation. Triploid embryos are common in CBA-strain mice ( 1 72); the embryonic ectoderm fails to develop, giving an "extraembryonic" embryo, with normal amnion, chorion, and allantois, but little or nothing remaining of the embryonic part of the egg cylinder. This so-called "triploid syndrome," similar to that shown by tw5 homozy gotes (see above, section on T locus), turns out to depend on the genetic background of the strain used, rather than on triploidy as such. When triploidy was induced in A-strain mice by treatment with cytochalasin B, development was retarded, but normal egg cylinders were formed ( 1 22). The sex chromosome constitution was determined in seven triploids: four were XXV and three xxx. Neither the sponta neous nor the induced triploids survived beyond midterm. In humans, triploid embryos are occasionally carried to term (12 1 ), but most undergo spontaneous abortion in the first trimester (3 1 , 1 20). The genetic background seems equally influential in determining how tetraploid embryos develop. Tetraploidy in embryos of certain FI strain combinations, whether induced by treatment with cytochalasin B or by virus-assisted cell fusion, gave rise in (CBA/H X C57BL) F I X (CBA/H X C57BL) F 1

Steroidogenic capabilities of the early mouse embryo.

Tissue morphodynamics shaping the early mouse embryo.

The Dynamics of Morphogenesis in the Early Mouse Embryo.

Action of lead on early divisions of the mouse embryo.

Role of the extracellular matrix protein thrombospondin in the early development of the mouse embryo.

Recognition of the CDEI motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo.

Understanding the molecular circuitry of cell lineage specification in the early mouse embryo.

The functional importance of the oviduct in neonatally estrogenized mouse females for early embryo survival.

The effect of various assisted hatching techniques on the mouse early embryo development.

Insertional mutation of a gene involved in growth regulation of the early mouse embryo.

The Dynamin 2 inhibitor Dynasore affects the actin filament distribution during mouse early embryo development.

Mapping global changes in nuclear cytosine base modifications in the early mouse embryo.

Changes in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo.

Isolation of Mouse Embryo Fibroblasts.

Heritable L1 retrotransposition in the mouse primordial germline and early embryo.

Correction to: 'Developmental plasticity, cell fate specification and morphogenesis in the early mouse embryo'.

Cephalic neurulation and optic vesicle formation in the early mouse embryo.

Melatonin-related genes expressed in the mouse uterus during early gestation promote embryo implantation.

Detecting cardiac contractile activity in the early mouse embryo using multiple modalities.

Thymus colonization in the developing mouse embryo.

SHP-1 arrests mouse early embryo development through downregulation of Nanog by dephosphorylation of STAT3.

YODA signalling in the early Arabidopsis embryo.

Physiological factors and the early embryo.

Intercellular communication in the early human embryo.