Investigation of the tissue specificity of the lethal yellow (Ay) gene in mouse embryos

The tissue specificity of the lethal yellow mutant was investigated by separation of blastocyst tissues. Embryos from experimental (Ay/ae X Ay/ae) and control (ae/ae X Ay/ae) crosses of the AG/CamPa inbred strain were recovered at 3.5 days post coitum, cultured for 24 hours, and then mechanically dissected into the component tissues of the blastocyst, the inner cell mass (ICM), and trophectoderm. These fragments were then cultured separately, with or without a feeder layer of inactivated fibroblasts, for an additional 3-5 days. Comparisons between experimental and control crosses indicated that the lethal Ay/Ay embryos were among the blastocysts successfully dissected but that both the ICM and trophectoderm from lethal embryos failed to develop further in vitro, either with or without feeders. With retrospective identification of the lethal embryos, it was found that at 4.5 days, after 1 day of culture, they had formed morphologically normal blastocysts but were frequently more fragile upon dissection and had smaller ICMs. Although none had hatched from the zona pellucida, some had ruptured it and were halfway out. With culture, lethal ICMs showed no development, and lethal trophectoderm usually attached but showed very limited outgrowth. Thus, no rescue of lethal tissue was shown with dissection and in vitro culture, and results are consistent with the gene affecting both tissues of the late blastocyst.     

Blastomeres of the mouse embryo lose totipotency after the fifth cleavage division: expression of Cdx2 and Oct4 and developmental potential of inner and outer blastomeres of 16- and 32-cell embryos

Sixteen inner or outer blastomeres from 16-cell embryos and 32 inner or outer blastomeres from 32-cell embryos (nascent blastocysts) were reaggregated and cultured in vitro. In 24 h old blastocysts developed from blastomeres derived from 16-cell embryos the expression of Cdx2 protein was upregulated in outer cells (new trophectoderm) of the inner cells-derived aggregates and downregulated in inner cells (new inner cell mass) of the external cells-derived aggregates. After transfer to pseudopregnant recipients blastocysts originating from both inner and outer blastomeres of 16-cell embryo developed into normal, fertile mice, but the implantation rate of embryos formed from inner cell aggregates was lower. The aggregates of external blastomeres derived from 32 cell embryo usually formed trophoblastic vesicles accompanied by vacuolated cells. In contrast, the aggregates of inner blastomeres quickly compacted but cavitation was delayed. Although in the latter embryos the Cdx2 protein appeared in the new trophectoderm within 24 h of in vitro culture, these embryos formed only very small outgrowths of Troma1-positive giant trophoblastic cells and none of these embryos was able to implant in recipient females. In separate experiment we have produced normal and fertile mice from 16- and 32-cell embryos that were first disaggregated, and then the sister outer and inner blastomeres were reaggregated at random. In blastocysts developed from aggregates, within 24 h of in vitro culture, the majority of inner and outer blastomeres located themselves in their original position (internally and externally), which implies that in these embryos development was regulated mainly by cell sorting.     

Functional analysis of trophoblast giant cells in parthenogenetic mouse embryos

Diploid mouse embryos containing only maternal DNA (parthenotes) fail, in part, because the inner cell mass does not induce the trophoblast to grow. In this study, we asked whether any of the defects in parthenotes may arise from alterations in trophoblast function. We examined the expression of genes important for normal trophoblast function and found several trophoblast genes that were expressed at normal levels in the primary trophoblast cells of parthenotes: E-cadherin, a cell adhesion molecule, was expressed normally in both the ICM and trophectoderm of parthenogenetic blastocysts and blastocyst outgrowths; the gene for Hxt, a basic helix-loop-helix factor that regulates trophoblast development, was expressed in both zygotic and parthenogenetic giant cells; placental lactogen-1, a hormone that is normally secreted by trophoblast giant cells, was expressed in most of both parthenogenetic and normal trophoblast cells; and the 92 kDa matrix metalloproteinase, gelatinase B, also known as MMP-9, was secreted at equivalent levels by both zygotic and parthenogenetic blastocyst outgrowths. However, once the outgrowths had developed, a subpopulation of trophoblast cells in parthenogenetic embryos had decreased DNA replication and significantly fewer nucleoli per nucleus than did zygotic embryos. Moreover, the parthenogenetic trophoblast cells growing out from blastocysts had a decreased viability in culture. These data suggest that, although parthenogenetic embryos are able to initiate primary trophoblast differentiation, the stability and continued differentiation of trophoblast giant cells may be abnormal. Our data support the hypothesis that the deficiency of secondary trophoblast giant cells may contribute to the decline of parthenogenetic embryos and suggest that the factors controlling this subset of trophoblast are distinct from those for primary trophoblast. Dev Genet 20:1–10, 1997. © 1997 Wiley-Liss, Inc.     

Disabled-2 is essential for endodermal cell positioning and structure formation during mouse embryogenesis

The signal transduction adapter protein Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The brain-specific Disabled-1 (Dab1) functions in positional organization of brain cells during development. Dab2 is widely distributed and is highly expressed in many epithelial cell types. The dab2 gene was interrupted by in-frame insertion of beta-galactosidase (LacZ) in embryonic stem cells and transgenic mice were produced. Dab2 expression was first observed in the primitive endoderm at E4.5, immediately following implantation. The homozygous Dab2-deficient mutant is embryonic lethal (earlier than E6.5) due to defective cell positioning and structure formation of the visceral endoderm. In E5.5 dab2 (-/-) conceptus, visceral endoderm-like cells are present in the deformed primitive egg cylinder; however, the visceral endoderm cells are not organized, the cells of the epiblast have not expanded, and the proamniotic cavity fails to form. Disorganization of the visceral endodermal layer is evident, as cells with positive visceral endoderm markers are scattered throughout the dab2 (-/-) conceptus. Only degenerated remains were observed at E6.5 for dab2 (-/-) embryos, and by E7.5, the defective embryos were completely reabsorbed. In blastocyst in vitro culture, initially cells with characteristics of endoderm, trophectoderm, and inner cell mass were observed in the outgrowth of the hatched dab2 (-/-) blastocysts. However, the dab2 (-/-) endodermal cells are much more dispersed and disorganized than those from wild-type blastocysts, the inner cell mass fails to expand, and the outgrowth degenerates by day 7. Thus, Dab2 is required for visceral endodermal cell organization during early mouse development. The absence of an organized visceral endoderm in Dab2-deficient conceptus leads to the growth failure of the inner cell mass. We suggest that Dab2 functions in a signal pathway to regulate endodermal cell organization using endocytosis of ligands from the blastocoel cavity as a positioning cue.     

Lethal nonagouti (ax): Description of a second embryonic lethal at the agouti locus

The time and mode of action of the homozygous a^x gene, lethal nonagouti, has been investigated on the inbred AX/Pa background. Heterozygotes were mated inter se to produce 25% homozygous embryos and heterozygotes were mated with homozygous, nonagouti mice to provide control litters. Comparisons of the frequency of mating success, the ratio of implantation sites to ovarian ovulation sites, and the average litter sizes between experimental and control matings all indicated that a^x/a^x embryos are not lost prior to implantation. Histological examination of pregnant uteri indicated that a^x/a^x embryos are first evident as abnormal blastocysts at 4.5 days post coitum (pc). These implant and develop to varying degrees, some differentiating trophoblast giant cells and a primitive endoderm layer. Growth is retarded and only small, disorganized clumps of tissue remain by 7.5 and 8.5 days pc. The time and mode of gene action of lethal nonagouti is thus different from its allele, lethal Yellow.     

Expression Pattern of E- and P-Cadherin in Mouse Embryos and Uteri during the Periimplantation Period

Periimplantation mouse embryos and uterine tissues were examined by means of immunohistochemistry for their expression of the Ca²⁺ dependent cell-cell adhesion molecules, E- and P-cadherin. E-cadherin was detected in all embryonic cells during periimplantation stages, and also detected in the uterine epithelium. When blastocysts attached to the uterine epithelium, E-cadherin was detected at implantation sites between the mural trophectoderm and the uterine epithelium on 5 day of pregnancy. P-cadherin was first detected in the mural trophectoderm on 4.5-day blastocysts, and then detected in the ectoplacental cone, giant cells and visceral endoderm from 5.5 day.
P-cadherin was also detected in the maternal uterine decidual cells from 5.5 day. After degeneration of uterine epithelial cells, giant cells make direct contact with uterine decidual cells, and P-cadherin was detected at contact sites between these cells.
Thus, the complicated process of implantation seems to be supported by temporal and spatial expression of the multiple classes of cadherins.     

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12-16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.     

Isolation and characterization of a bovine trophectoderm cell line derived from a parthenogenetic blastocyst

A bovine trophectoderm cell line was established from a parthenogenetic in vitro-produced blastocyst. To initiate the cell line, 8-day parthenogenetic blastocysts were attached to a feeder layer of STO fibroblasts and primary outgrowths occurred that consisted of trophectoderm, endoderm, and very occasionally epiblast tissue. Any endoderm and epiblast outgrowths were removed from the primary cultures within the first 10 days of culture by dissection. One of the primary trophectoderm cell cultures was chosen for further propagation and was passaged by physical dissociation and replating on STO feeder cells. The cell culture, designated BPT-1, was maintained in T25 flasks and passaged at a 1:3 split ratio for the first 15 passages approximately once every 2 weeks. Thereafter, the cell culture was passaged at 1:10-1:40 split ratios. Transmission electron microscopic examination showed the cells to be a polarized epithelium with apical microvilli, a thin basal lamina, and lateral junctions consisting of tight junctions and desmosomes. Lipid vacuoles and digestive vacuoles were also prominent features of the BPT-1 cells. Metaphase spread analysis at passage 59 indicated a near diploid cell population (2n = 60) with a mode and median of 60 and a mean of 64. BPT-1 cells secreted interferon-tau into the medium as measured by anti-viral assay and Western blot analysis. The cell line provides an in vitro model of parthenogenote trophectoderm whose biological characteristics can be compared to trophectoderm cell lines derived from bovine embryos produced by normal fertilization or nuclear transfer.     

Disruption of the cytokeratin filament network in the preimplantation mouse embryo

The distribution of the cytokeratin network in the intact preimplantation mouse embryo and the role of cytokeratin filaments in trophectoderm differentiation were investigated by means of whole-mount indirect immunofluorescence microscopy and microinjection of anti-cytokeratin antibody. Assembled cytokeratin filaments were detected in some blastomeres as early as the compacted 8-cell stage. The incidence and organization of cytokeratin filaments increased during the morula stage, although individual blastomeres varied in their content of assembled filaments. At the blastocyst stage, each trophectoderm cell contained an intricate network of cytokeratin filaments, and examination of sectioned blastocysts confirmed that extensive arrays of cytokeratin filaments were restricted to cells of the trophectoderm. Microinjection of anticytokeratin antibody into individual mural trophectoderm cells of expanded blastocysts resulted in a dramatic rearrangement of the cytokeratin network in these cells. Moreover, antibody injection into 2-cell embryos inhibited assembly of the cytokeratin network during the next two days of development. Despite this disruption of cytokeratin assembly, the injected embryos compacted and developed into blastocysts with normal morphology and nuclear numbers. These results suggest that formation of an elaborate cytokeratin network in preimplantation mouse embryos is unnecessary for the initial stages of trophectoderm differentiation resulting in blastocyst formation.     

Establishment of embryonic stem cell lines from preimplantation mouse embryos homozygous for lethal mutations in the t-complex

We have determined the frequency at which embryonic stem cell (ESC) lines can be established from inner cell masses (ICMs) isolated from blastocysts homozygous for lethal mutations in the mouse t-complex. Approximately one-third of the expected number, 3/29, of the ESC lines established from embryos obtained by inter-se mating of +/tw18 mice are homozygous for the tw18 haplotype. These tw18/tw18 ESC lines form a variety of cell types in vitro and in vivo, including mesodermal derivatives such as cartilage and muscle. On the basis of these and data from other studies, we suggest that the normal function of the gene represented by the tw18 lethal allele is required for multiplication/survival of mesodermal precursors in the embryo rather than the specification of the mesodermal lineage, and that the lethal effects of this mutation are expressed in only the highly structured environment of the early postimplantation embryo. In studies of the lethal tw5 haplotype, we found that 2/2 ESC lines obtained are mutant homozygotes. Analysis of these data, in conjunction with the results of our earlier study (Magnuson, T., Epstein, C. J., Silver, L. M., and Martin, G. R. (1982), Nature (London) 298, 750-753), suggests that homozygosity for the genes found in the tw5 haplotype does not reduce cell viability. By contrast, 0/16 ESC lines isolated from embryos obtained from matings of +/t0 mice are mutant homozygotes. Analysis of the genotypes of ICM-derived primary stem cell colonies suggests that t0 homozygous ICM cells are unable to undergo sufficient proliferation in vitro to give rise to ESC lines.     

Cytoplasmic projections of trophectoderm distinguish implanting from preimplanting and implantation-delayed mouse blastocytes

A scoring scheme was devised to characterize visually the morphological differentiation of whole-mount, unfixed mouse blastocysts. Embryos were recovered from groups of intact mice (implanting embryos) and mice ovariectomized on Day 3 of pregnancy (implantation-delayed embryos) every 3 h from 18:00 h on Day 4 until 12:00 h on Day 5. Blastocyst differentiation was assessed according to the presence of a zona pellucida, the appearance of the outer margin of trophectoderm cells, the visibility of the blastocoele and the relative size of the inner cell mass. The results obtained indicate that, during this period, implanting and implantation-delayed mouse blastocysts lose the zona as well as exhibit rounded trophectoderm cells, an enlarged inner cell mass and an increasing opacity of the blastocoele. In contrast, the trophectoderm cells of implanting blastocysts only exhibit extensive cytoplasmic projections, probably due to remodelling of the intracellular cytoskeleton. Growth of the inner cell mass appeared to precede the other morphological changes in the majority of blastocysts, and thus might be a prerequisite for further differentiation. The rate of blastocyst differentiation and the survival of embryos were adversely affected by the condition of delayed implantation, induced by ovariectomy. This study suggests that the appearance of cytoplasmic projections from trophectoderm cells is central to the control of blastocyst implantation.     

Trophectoderm cell subpopulations in the periimplantation mouse blastocyst

Trophectoderm of the preimplantation mouse blastocyst is composed of two cell subpopulations relative to their proximity to the inner cell mass. The polar trophectoderm overlying the inner cell mass proliferates to form the ectoplacental cone, and the mural trophectoderm endoreplicates and gives rise to giant cells. We examined specific differences in the two trophectoderm cell populations using a lectin (Dolichos biflorus) to detect cell surface characteristics and a simple sugar (D-Gal) to detect differences in incorporation. During the first day of delayed implantation, the mural trophectoderm presented twice as many lectin binding sites as did the polar trophectoderm. The mural trophectoderm of both nondelaying and delayed implantation blastocysts showed a greater rate of incorporation of the tritiated sugar by presenting more reduced silver grains in radioautograms. These results indicate that the mural trophectoderm and polar trophectoderm are two distinct cell types in the periimplantation blastocyst.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

Progressive expression of trophoblast-specific genes during formation of mouse trophoblast giant cells in vitro

The expression of a battery of trophoblast-specific mRNAs was studied during trophectoderm development in vivo and in vitro to assess the use of these mRNAs as markers of trophoblast differentiation and to examine lineage relationships between various trophectoderm derivatives. In situ hybridization of sectioned day 6.5–18.5 mouse embryos localized mRNAs for mouse placental lactogens I and II and mouse proliferin (PLF) to trophoblast giant cells and proliferin-related protein mRNA to the spongiotrophoblast and giant cell layers. A fifth marker, cDNA 4311, was found only in spongiotrophoblast. Day 3.5 blastocyst outgrowths and day 7.5 diploid extraembryonic ectoderm (EX) and ectoplacental cone (EPC) were then cultured to produce polyploid giant cells in vitro. Cultures were processed for in situ hybridization after 2, 4, or 6 days. EX and EPC both formed secondary giant cells, which expressed all markers in the same sequence as was observed in vivo, and primary giant cells in blastocyst outgrowths expressed the early giant cell markers PLF and PL-I on days 4 and 6 of culture. EPC progressed through the sequence 2 days ahead of EX, indicating commitment of EPC to giant cell formation. These results suggest that EX, EPC, and primary and secondary giant cells all share in a common pathway of differentiation and that the highly ordered sequence of gene expression characteristic of this pathway occurs similarly in vivo and in vitro. © 1993 Wiley-Liss, Inc.     

Establishment of a bovine blastocyst-derived cell line collection for the comparative analysis of embryos created in vivo and by in vitro fertilization,somatic cell nuclear transfer,or parthenogenetic activation

Tools and methods for analyzing differences in embryos resulting from somatic cell nuclear transfer (NT) in comparison to those derived from normal fertilization are needed to define better the nature of the nuclear reprogramming that occurs after NT. To this end, a collection of bovine blastocyst-derived cell lines was created. In vitro expanded or hatched blastocysts, used as primary culture tissue, were from NT; in vitro maturation, fertilization, and culture (IVF); or parthenogenetic (P) activation. Also, five in vivo-fertilized and developed blastocysts were collected by uterine flushing on the eighth d postfertilization. Whole blastocysts were physically attached to STO feeder layers to initiate all of the cell lines generated. The majority of the cell lines in the collection are trophectoderm, 38 NT-derived, 6 in vivo-derived, 20 IVF-derived, and 13 P-derived. Trophectoderm identity was ascertained by morphology and, in many cases, interferon-tau production. Several visceral endoderm cell lines and putative parietal endoderm cell lines were also established. At approximately 5% efficiency, epiblast masses from NT and IVF blastocysts survived and were isolated in culture. Two epiblast masses were also isolated from P blastocysts. Spontaneous differentiation from the epiblast outgrowths resulted in the establishment of fibroblast cell lines. The use of the trophectoderm cell lines as a comparative in vitro model of bovine trophectoderm and placental function is discussed in relation to NT reprogramming.     

Fate of tetraploid cells in 4n<-->2n chimeric mouse blastocysts

Previous studies have shown that tetraploid (4n) cells rarely contribute to the derivatives of the epiblast lineage of mid-gestation 4n<-->2n mouse chimeras. The aim of the present study was to determine when and how 4n cells were excluded from the epiblast lineage of such chimeras. The contributions of GFP-positive cells to different tissues of 4n<-->2n chimeric blastocysts labelled with tauGFP were analysed at E3.5 and E4.5 using confocal microscopy. More advanced E5.5 and E7.5 chimeric blastocysts were analysed after a period of diapause to allow further growth without implantation. Tetraploid cells were not initially excluded from the epiblast in 4n<-->2n chimeric blastocysts and they contributed to all four blastocyst tissues at all of the blastocyst stages examined. Four steps affected the allocation and fate of 4n cells in chimeras, resulting in their exclusion from the epiblast lineage by mid-gestation. (1) Fewer 4n cells were allocated to the inner cell mass than trophectoderm. (2) The blastocyst cavity tended to form among the 4n cells, causing more 4n cells to be allocated to the hypoblast and mural trophectoderm than the epiblast and polar trophectoderm, respectively. (3) 4n cells were depleted from the hypoblast and mural trophectoderm, where initially they were relatively enriched. (4) After implantation 4n cells must be lost preferentially from the epiblast lineage. Relevance of these results to the aetiology of human confined placental mosaicism and possible implications for the interpretation of mouse tetraploid complementation studies of the site of gene action are discussed.     

Maintenance of the inner cell mass in human blastocysts from fragmented embryos

The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5-10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.