Chimeras between parthenogenetic or androgenetic blastomeres and normal embryos: allocation to the inner cell mass and trophectoderm

A series of chimeras was generated by injecting single normal, parthenogenetic, or androgenetic blastomeres carrying transgenic markers under the zona pellucida of nontransgenic eight-cell embryos. These chimeras were cultured to the blastocyst stage and sectioned, and the transgenic component was detected by in situ hybridization. No statistically significant difference was found among the normal, parthenogenetic, and androgenetic chimeras in the number of chimeric blastocysts with a transgenic contribution to the inner cell mass (ICM), the trophectoderm, or both the ICM and trophectoderm. Since androgenetic and parthenogenetic cells were present in chimeras at a high frequency in both the ICM and trophectoderm at the blastocyst stage, but not in similar chimeras at late gastrulation, these cells must not respond normally to developmental signals subsequent to blastocyst formation and prior to late gastrulation.     

Gastrula organiser and embryonic patterning in the mouse

Embryonic patterning of the mouse during gastrulation and early organogenesis engenders the specification of anterior versus posterior structures and body laterality by the interaction of signalling and modulating activities. A group of cells in the mouse gastrula, characterised by the expression of a repertoire of "organiser" genes, acts as a source and the conduit for allocation of the axial mesoderm, floor plate and definitive endoderm. The organiser and its derivatives provide the antagonistic activity that modulates WNT and TGFbeta signalling. Recent findings show that the organiser activity is augmented by morphogenetic activity of the extraembryonic and embryonic endoderm, suggesting embryonic patterning is not solely the function of the organiser.     

Conditional activation of RhoA suppresses the epithelial to mesenchymal transition at the primitive streak during mouse gastrulation

Gastrulation is a pivotal event of mouse early embryogenesis. In telencephalin (TLCN)-Cre mice carrying the Cre recombinase gene inserted into the translational initiation site of the TLCN gene, Cre-mediated recombination took place at the postimplantation stage. To examine the role of RhoA signaling in early embryogenesis, we produced Rho36 mice carrying constitutively active RhoA(G14V) gene inducible by Cre recombinase and crossed with TLCN-Cre mice. In doubly transgenic embryos at the gastrulation stage, there appeared an abnormal bulge of cells protruded from the primitive streak region into the amniotic cavity. The bulged cell mass expressed the epiblast marker gene Oct3 and E-cadherin, but not the primitive streak marker gene T except for the basal portion. These results suggest that the conditional activation of RhoA signaling suppressed the epithelial to mesenchymal transition at the primitive streak during mouse gastrulation.     

Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation.

Mouse embryonic stem cells are pluripotent cells that are derived from the inner cell mass of blastocysts. When induced to synchronously enter a program of differentiation in vitro, they form embryoid bodies that contain cells of the mesodermal, hematopoietic, endothelial, muscle, and neuronal lineages. Here, we used a panel of marker genes with early expression within the germ layers (oct-3, Brachyury T, Fgf-5, nodal, and GATA-4) or a variety of lineages (flk-1, Nkx-2.5, EKLF, and Msx3) to determine how progressive differentiation of embryoid bodies in culture correlated with early postimplantation development of mouse embryos. Using RNA in situ hybridization, we found that the temporal and spatial relationships existing between these marker genes in vivo were maintained also in vitro. Studying the onset of marker gene expression allowed us also to determine the time course of differentiation during the formation of embryoid bodies. Thus, stages equivalent to embryogenesis between implantation and the beginning of gastrulation (4.5-6.5 d.p.c.) occur within the first two days of embryoid body differentiation. Between days 3 and 5, embryoid bodies contain cell lineages found in embryos during gastrulation at 6.5 to 7.0 d.p.c., and after day 6 in culture, embryoid bodies are equivalent to early organogenesis-stage embryos (7.5 d.p.c.). In addition, we demonstrate that the panel of developmental markers can be applied in a screen for stage- or lineage-specific genes. Reporter gene expression from entrapment vector insertions can be co-localized with expression of specific markers within the same cell during embryoid body formation as well as during embryogenesis. Our results thus demonstrate the power of embryoid body formation as an in vitro model system to study early lineage determination and organogenesis in mammals, and indicate that they will prove to be useful tools for identifying developmental genes whose expression is restricted to particular lineages.     

Is the anterior-posterior axis of the fetus specified before implantation in the mouse?

The mouse conceptus is generally held to be radially symmetrical about its embryonic-abembryonic axis from the blastocyst stage until the primitive streak appears at the beginning of gastrulation. However, this notion has been challenged by recent observations on conceptuses sectioned in utero which suggest that the blastocyst is already bilaterally symmetrical when it begins to implant. Accordingly, the blastocyst has been assigned an anterior-posterior axis which appears to persist through gastrulation and is claimed to coincide with the anterior-posterior axis of the future fetus in both orientation and polarity. In the present investigation the relationship between these two axes was examined in conceptuses dissected from the uterus early in gastrulation so that it could be determined more accurately than is possible in situ. The anterior-posterior axis of the conceptus and nascent fetus were found to be either parallel or antiparallel to each other, suggesting that while the orientation of the fetal axis may be specified at the blastocyst stage its polarity is not.     

cdx4/lacZ and cdx2/lacZ protein gradients formed by decay during gastrulation in the mouse

Expression of the mouse caudal genes cdx4 and cdx2 is examined by use of lacZ reporter constructs expressed in transgenic mouse embryos. During early gastrulation, up to at least 8.5 days of development, reporter mRNA distributions are apparently similar to those of endogenous cdx mRNAs. By 8.25 to 8.8 days, cdx/lacZ protein activities have become distributed as posterior-to-anterior gradients along the neural and mesoderm tissues. The gradients form by decay of activity as cells become distanced from the regressing tailbud. In situ hybridization studies indicate that the decay is primarily in cdx/lacZ protein activities rather than mRNAs. As gastrulation proceeds, the locations of the gradients regress progressively posteriorly along the growing axis. Our results indicate how cdx4 and cdx2 protein gradients might be generated by decay during normal development. The smoothness of the gradients that we detect shows that there cannot be extensive mixing of cells once they leave the tailbud to contribute to the growing axis. An enhancer element located in the first intron of the cdx4 gene is essential for correct transgene expression.     

Postimplantation development of mitomycin C-treated mouse blastocysts

Treatment of morula-stage mouse embryos with mitomycin C (0.004-0.5 microgram/ml) in vitro resulted in a decrease in the number of inner cell mass (ICM) cells at the blastocyst stage. The trophectoderm population was reduced only at the highest dosage (0.5 microgram/ml) tested. Postblastocyst development in vitro was retarded: Fewer embryos formed trophoblastic outgrowth, and the ICM was poorly developed. The embryo transfer experiments demonstrated that a reduction in ICM cell numbers diminished the potential of embryogenesis. The presence of a sufficient number of trophoblasts and ICM cells in the blastocyst is therefore a prerequisite for successful implantation and embryogenesis. The mitomycin-treated blastocysts with only 70% of normal ICM cells developed to egg cylinders that were about half normal size, but by days 12-14 the body size of the surviving embryo was similar to that of the control embryo. Morphogenesis was retarded during the early organogenesis stages, but only a slight delay was seen in the treated embryo on day 12. Such observation strongly suggests that a restorative phase of growth and morphogenesis has occurred during the immediate postimplantation period.     

MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.     

A novel retrovirally induced embryonic lethal mutation in the mouse: assessment of the developmental fate of embryonic stem cells homozygous for the 413.d proviral integration

A genetic screen of transgenic mouse strains, carrying multiple copies of an MPSV neo retroviral vector, has led to the identification of a recessive embryonic lethal mutation, termed 413.d. This mutation is associated with a single proviral insertion and when homozygous, results in the failure of the early postimplantation embryo at the gastrulation stage of development. Embryonic stem cell lines (ES cells) were derived from 413.d intercross embryos. Genotyping, with respect to the 413.d integration site, identified wild-type, heterozygous and homozygous ES cell lines. The differentiation abilities and developmental potential of the ES cell lines were assessed using a number of in vitro and in vivo assays. Results indicate that the ES cell lines, regardless of genotype, are pluripotent and can give rise to tissue and cell types derived from all three germ layers. Furthermore, analysis of midgestation conceptuses (10.5 p.c.) and adult chimeras generated by injecting mutant ES cells into host blastocysts, provides strong evidence that the mutant cells can contribute to all extraembryonic tissues and somatic tissues, as well as to functional germ cells. These results indicate that the homozygous mutant cells can be effectively 'rescued' by the presence of wild-type cells in a carrier embryo.     

The development potential of parthenogenetically derived cells in chimeric mouse embryos: implications for action of imprinted genes

Parthenogenetic embryos of mice die shortly after implantation and characteristically contain poorly developed extraembryonic tissue. To investigate the basis of the abnormal development of parthenotes, we combined them with normal embryos to produce chimeras and examined the distribution of the parthenogenetically derived cells during preimplantation and early postimplantation development. The parthenogenetic embryos were derived from a transgenic mouse line bearing a large insert, which allowed these cells to be identified in histological sections using in situ hybridization. At the blastocyst stage, the parthenogenetic embryos contributed cells to the trophectoderm (TE) and inner cell mass (ICM) of chimeras. By 6.5 days, however, in almost every embryo, parthenogenetically derived cells were not detected in the extraembryonic trophoblast tissue descended from the TE. In contrast, parthenogenetically derived cells could contribute to all descendants of the ICM of 6.5-and 7.5-day chimeras, including the extraembryonic visceral and parietal endoderm. Quantitative analysis of the degree of chimerism in the embryonic ectoderm at 6.5-7.5 days indicated that parthenogenetically derived cells could contribute as extensively as normal cells. These results indicate that normal trophoblast development requires gene expression from the paternally inherited genome before 6.5 days of embryogenesis. Tissues of the ICM lineage, however, apparently can develop independently of the paternal genome at least to 7.5 days of embryogenesis. Comparison of these results with those of others suggests that the influence of imprinted genes is manifested at different times and in a variety of tissues during development.     

Developmental abnormalities of NT mouse embryos appear early after implantation

In mammals, cloning by nuclear transfer (NT) into an enucleated oocyte is a very inefficient process, even if it can generate healthy adults. We show that blastocysts derived from embryonic stem (ES) donor cells develop at a high rate, correctly express the pluripotential marker gene Oct4 in ICM cells and display normal growth in vitro. Moreover, the majority of them implant in the uterus of recipient females. We combine embryological studies, gene expression analysis during gastrulation and generation of chimaeric embryos to identify the developmental origin (stage and tissue affected) of NT embryo mortality. The majority died before mid-gestation from defects arising early, either at peri-implantation stages or during the gastrulation period. The first type of defect is a non-cell autonomous defect of the epiblast cells and is rescued by complementation of NT blastocysts with normal ES or ICM cells. The second type of defect affects growth regulation and the shape of the embryo but does not directly impair the initial establishment of the patterning of the embryo. Only chimaeras formed by the aggregation of NT and tetraploid embryos reveal no growth abnormalities at gastrulation. These studies indicate that the trophoblast cell lineage is the primary source of these defects. These embryological studies provide a solid basis for understanding reprogramming errors in NT embryos. In addition, they unveil new aspects of growth regulation while increasing our knowledge on the role of crosstalk between the extra-embryonic and the embryonic regions of the conceptus in the control of growth and morphogenesis.     

Co-expression of the HGF/SF and c-met genes during early mouse embryogenesis precedes reciprocal expression in adjacent tissues during organogenesis

Early experiments with cells in culture and recent targeting experiments have confirmed that the mesenchyme-derived growth factor hepatocyte growth factor/scatter factor (HGF/SF) is a paracrine agent that regulates the development of several epithelial and myogenic precursor cells during organogenesis. Here, we report the expression pattern of HGF/SF and its receptor, the product of the proto-oncogene c-met, during gastrulation and early organogenesis in mouse embryo. During gastrulation, the expression of HGF/SF and c-met overlaps. Initially the two genes are expressed in the endoderm and in the mesoderm along the rostro-intermediate part of the primitive streak and, later, in the node and in the notochord. Neither HGF/SF nor c-met is expressed in the ectodermal layer throughout gastrulation. During early organogenesis, overlapping expression of HGF/SF and c-met is found in heart, condensing somites and neural crest cells. However, a second and distinct pattern of expression, characterized by the presence of the ligand in mesenchymal tissues and the receptor in the surrounding ectoderm, is seen in the branchial arches and in the limb buds. At 13 days postcoitum (d.p.c.), only this second pattern of expression is observed in differentiated somites and several major organs (i.e., lungs, liver, and gut. The expression of the HGF/SF and c-met genes throughout embryogenesis suggests a shift from an autocrine to a paracrine signaling system. The shift takes place in early organogenesis and implies different roles of HGF/SF in development. During gastrulation, HGF/SF may affect the fate of migrating mesodermal cells and may play a role in axis determination, whereas during organogenesis, the expression patterns of HGF/SF and its receptor reflect the recently established roles in the growth of certain epithelia and the migration of specific myogenic precursor cells. © 1996 Wiley-Liss, Inc.     

Developmental characterization and chromosomal mapping of a LacZ transgene expressed in the mouse apical ectodermal ridge

The apical ectodermal ridge (AER) has an essential role in limb morphogenesis involving the specification of the proximal-distal axis of the limb. During the analysis of transgenic mice that harbor a LacZ transgene, we detected strong expression of beta-galactosidase within the AER of developing embryos. In this mouse line, called Z16, the bacterial LacZ gene is linked to a Herpes simplex virus immediate early promoter that is normally silent in mice. Embryos from other independent mouse lines harboring the same DNA construct exhibited no AER specific staining. Thus, it appears that the LacZ transgene in the Z16 line is expressed in the AER in response to regulatory influences from genomic DNA flanking the integration site. By fluorescent in situ hybridization, the transgene insertion site was mapped to chromosome 12. Hemizygous and homozygous transgenic mice appear normal and are fertile. AER specific beta-galactosidase staining was detected by 9.5 days post coitum in the forelimb and hindlimb bud. beta-galactosidase staining could be seen throughout the development of the limbs up to 14.5 days post coitum when expression was restricted to the distal-most regions of the digits of the hindlimbs. The loss of beta-galactosidase staining between digits correlated with the onset of programmed cell death, or apoptosis, in the digit interzones. LacZ expression in this transgenic line represents a useful marker for studying AER function in limb specification during mouse embryogenesis.     

Embryonic stem cells derived from morulae,inner cell mass,and blastocysts of mink: Comparisons of their pluripotencies

A characterization of cell lines that we derived from morulae (three lines), blastocysts (two lines), and the inner cell mass (ICM) is given. The karyotype of all the lines was normal; the genotype of four lines was XX, and four lines were genotypically XY. The pluripotencies and commitment status of the derived lines were estimated. First, there were not less than two-thirds of cells in the populations of the lines derived from morulae and the lCM with both Xs active; 70–100% of cells of the blastocyst-derived lines had one of the Xs in an inactive state. The activity of glucose-6-phosphate dehydrogenase (G6PD) in the lines (genotype XX) derived from morulae and ICM was found to be twofold higher than in lines with genotype XY, and G6PD activity was the same in the blastocyst-derived XX lines and XY lines. Second, when injected intraperitoneally into athymic mice, morulae- and ICM-derived cells gave rise to simple and complex embryoid bodies (EB) resembling to typical “cystic” mouse EBs. Third, when injected subcutaneously to athymic mice, the ICM- or morula-derived cells gave rise to typical teratomas containing derivatives of the three germ layers and components of organogenesis. Comparisons of cell lines of different derivations demonstrated that the pluripotencies of the ES cells derived from morulae or the ICM are higher than those of blastocyst derivation. © 1993 Wiley-Liss, Inc.     

Inhibition of trophoblast stem cell potential in chorionic ectoderm coincides with occlusion of the ectoplacental cavity in the mouse

At the blastocyst stage of pre-implantation mouse development, close contact of polar trophectoderm with the inner cell mass (ICM) promotes proliferation of undifferentiated diploid trophoblast. However, ICM/polar trophectoderm intimacy is not maintained during post-implantation development, raising the question of how growth of undifferentiated trophoblast is controlled during this time. The search for the cellular basis of trophoblast proliferation in post-implantation development was addressed with an in vitro spatial and temporal analysis of fibroblast growth factor 4-dependent trophoblast stem cell potential. Two post-implantation derivatives of the polar trophectoderm - early-streak extra-embryonic ectoderm and late-streak chorionic ectoderm - were microdissected into fractions along their proximodistal axis and thoroughly dissociated for trophoblast stem cell culture. Results indicated that cells with trophoblast stem cell potential were distributed throughout the extra-embryonic/chorionic ectoderm, an observation that is probably attributable to non-coherent growth patterns exhibited by single extra-embryonic ectoderm cells at the onset of gastrulation. Furthermore, the frequency of cells with trophoblast stem cell potential increased steadily in extra-embryonic/chorionic ectoderm until the first somite pairs formed, decreasing thereafter in a manner independent of proximity to the allantois. Coincident with occlusion of the ectoplacental cavity via union between chorionic ectoderm and the ectoplacental cone, a decline in the frequency of mitotic chorionic ectoderm cells in vivo, and of trophoblast stem cell potential in vitro, was observed. These findings suggest that the ectoplacental cavity may participate in maintaining proliferation throughout the developing chorionic ectoderm and, thus, in supporting its stem cell potential. Together with previous observations, we discuss the possibility that fluid-filled cavities may play a general role in the development of tissues that border them.