Disruption of the talin gene arrests mouse development at the gastrulation stage.

Studies on cultured cells show that the cytoskeletal protein talin plays a key role in cell spreading and the assembly of cell-extracellular matrix junctions. To examine the role of talin in vivo, we have generated mice with a targeted disruption of the talin gene. Heterozygotes are normal, but no surviving homozygous mutant animals were obtained, proving that talin is required for embryogenesis. Mutant embryos develop normally to the blastocyst stage and implant, but there is a gross disorganization of the embryos at gastrulation (6.5-7.5 days post coitum), and they die around 8.5-9.5 days post coitum. The embryonic ectoderm is reduced in size, with fewer cells, and is incompletely organised compared with wild-type embryos. The mutant embryos show disorganised extraembryonic tissues, and the ectoplacental and excocoelomic cavities are not formed. This seems to be because embryonic mesoderm accumulates as a mass on the posterior side of the embryos and fails to migrate to extraembryonic regions, although mesodermal cells are evident in the embryo proper. Spreading of trophoblast cells derived from cultured mutant blastocysts on fibronectin and laminin is also considerably reduced. Therefore, the fundamental deficit in these embryos seems to be a failure of cell migration at gastrulation.     

Differential distribution of cubilin and megalin expression in the mouse embryo

Cubilin and megalin are cell surface proteins that work cooperatively in many absorptive epithelia to mediate endocytosis of lipoproteins, vitamin carriers, and other proteins. Here we have investigated the coordinate expression of these receptors during mouse development. Our findings indicate that while there are sites where the receptors are co-expressed, there are other tissues where expression is not overlapping. Apical cubilin expression is pronounced in the extraembryonic visceral endoderm (VE) of 6-9.5 days postcoitum (dpc) embryos. By contrast, little megalin expression is evident in the VE at 6 dpc. However, megalin expression in the VE increases as development progresses (7.5-9.5 dpc), although it is not as uniformly distributed as cubilin. Punctate expression of megalin is also apparent in the region of the ectoplacental cone associated with decidual cells, whereas cubilin expression is not seen in association with the ectoplacenta. Strong expression of megalin is observed in the neural ectoderm, neural plate and neural tube (6-8.5 dpc), but cubilin expression is not apparent in any of these tissues. At 8.5 dpc, megalin is expressed in the developing endothelial cells of blood islands, whereas cubilin is absent from these cells. Finally, cubilin, but not megalin, is expressed by a subpopulation of cells dispersed within the 7.5 dpc embryonic endoderm and having a migratory morphology. In summary, the co-expression of cubilin and megalin in the VE is consistent with the two proteins functioning jointly in this tissue. However, the differential distribution pattern indicates that the proteins also function independent of one another. Furthermore, the finding of megalin expression in blood island endothelial cells and cubilin expression in embryonic endoderm highlight potential new developmental roles for these proteins.     

Abnormal Development at Early Postimplantation Stage in Mouse Embryos After Preimplantation Genetic Diagnosis

Preimplantation genetic diagnosis (PGD) is an established procedure for the genetic analysis of embryos. To assess the effect of the procedure on early embryonic development, we generated a murine experimental system, including mice implanted with biopsied in vitro cultured embryos, control mice implanted with in vitro cultured embryos without biopsy, and mice with naturally conceived embryos. Embryos at the 7.5‐dpc stage were isolated from all three groups and the embryo implantation rate, the survival rate of implanted embryos, and the developmental stage of surviving embryos were carefully assessed and compared among all three groups. We found the implantation rate was similar between biopsied and control group embryos (67.92% vs. 66.67%). However, the survival rate of implanted embryos in the biopsied group (49.31%) was significantly lower than that of the control (60.91%) and normal groups (96.24%) at 7.5 dpc. In addition, the survival rate of control group embryos was significant lower than that of normal group embryos. Classification of the precise developmental stages of randomly selected live implanted embryos at 7.5 dpc revealed no differences among the three groups. Our results indicate that blastomere biopsy does not adversely affect embryo implantation. The PGD procedure, in particular blastomere biopsy, increases the rate of embryo death at 4.5–7.5 dpc, but does not affect the development of surviving 7.5 dpc embryos. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Experimental analysis of the transdifferentiation of visceral to parietal endoderm in the mouse.

The visceral endoderm (VE) of isolated extraembryonic regions (ExEmbs) of 7 days postcoitum (dpc) prestreak mouse conceptuses have been shown to convert readily to parietal endoderm (PE). The present study addresses the following three unanswered questions. On what does conversion depend, how rapidly does it occur, and is it an enduring general property of a residual small population of relatively immature cells? In situ hybridization reveals that change in cell state occurs within 2 days of culture. Deprivation of the mesoderm also promotes it in later ExEmbs. Conversely, the conversion to PE in isolated 7 dpc ExEmbs is suppressed by grafting 8 dpc or 9 dpc mesoderm. Hence, the conversion provides an example of transdifferentiation that is promoted by the absence of extraembryonic mesoderm. The presence of mesoderm seems to be necessary to enable the VE to grow rather than convert to PE, as occurs if it retains contact with the extraembryonic ectoderm.     

Dynamic expression pattern of Hoxc8 during mouse early embryogenesis

The Hoxc8 expression pattern was examined in mouse embryos 7.5-12.5 days postcoitum (dpc) using whole-mount in situ hybridization and RT-PCR. The expression of Hoxc8 started between 7.5 and 8.5 dpc. A strong expression was detected in the ectoderm and mesoderm at 8.5 dpc. At 9.5 dpc, a distinct anterior boundary of Hoxc8 expression was established at the 10th and 16th somites in the neural tube and the paraxial mesoderm, respectively. This staggered expression pattern was maintained throughout the later stages. By 12.5 dpc, the forward progression of the Hoxc8 expression pattern was observed and the stain was weakened. In the ectoderm-derived neural tube, strong Hoxc8 expression was observed in the ventral horn and later in the ventral and mediolateral region of the mantle layer, indicating a possible association with the onset and progression of neural differentiation. In the case of the mesoderm-derivative cells, strong Hoxc8 expression was detected in the sclerotome on the way to the notochord and neural tube and mesonephros, suggesting a role of Hoxc8 in the formation of the vertebrae and ribs and the possible involvement in the differentiation into the kidney.     

Bmp4 is required for the generation of primordial germ cells in the mouse embryo

In many organisms the allocation of primordial germ cells (PGCs) is determined by the inheritance of maternal factors deposited in the egg. However, in mammals, inductive cell interactions are required around gastrulation to establish the germ line. Here, we show that Bmp4 homozygous null embryos contain no PGCs. They also lack an allantois, an extraembryonic mesodermal tissue derived, like the PGCs, from precursors in the proximal epiblast. Heterozygotes have fewer PGCs than normal, due to a reduction in the size of the founding population and not to an effect on its subsequent expansion. Analysis of beta-galactosidase activity in Bmp4(lacZneo) embryos reveals that prior to gastrulation, Bmp4 is expressed in the extraembryonic ectoderm. Later, Bmp4 is expressed in the extraembryonic mesoderm, but not in PGCs. Chimera analysis indicates that it is the Bmp4 expression in the extraembryonic ectoderm that regulates the formation of allantois and primordial germ cell precursors, and the size of the founding population of PGCs. The initiation of the germ line in the mouse therefore depends on a secreted signal from the previously segregated, extraembryonic, trophectoderm lineage.     

Fibroblast growth factor receptor 2 (Fgfr2) plays an important role in eyelid and skin formation and patterning.

Initiating as protruding ridges above and below the optic vesicle, the eyelids of mice grow across the eye and temporarily fuse in fetal life. Mutations of a number of genes disrupt this developmental process and result in a birth defect, "open-eyelids at birth." Here we show that a critical event for eyelid induction occurs at embryonic day 11.5 (E11.5) when the single cell-layered ectoderm in the presumptive eyelid territory increases proliferation and undergoes morphologic transition to form cube-shaped epithelial cells. Using embryos lacking the Fgfr2 Ig domain III (Fgfr2(DeltaIII/DeltaIII)) generated by tetraploid rescue and chimeric embryo formation approaches, we demonstrate that this event is controlled by Fgfr2 signals as the Fgfr2(DeltaIII/DeltaIII) mutation blocks these changes and results in embryos without eyelids. Fgfr2 and its ligands are differentially expressed in the ectoderm and underlying mesenchyme and function in a reciprocal interacting loop that specifies eyelid development. We also demonstrate that similar defects account for failure of skin formation at early stages. Interestingly, Fgfr2-independent skin formation occurs at E14.5 mutant embryos, resulting in much thinner, yet well-differentiated epidermis. Notably, mutant skin remains thin with decreased hair density after transplantation to wild-type recipients. These data demonstrate an essential role of Fgfr2 in eyelid and skin formation and patterning.     

Wtap is required for differentiation of endoderm and mesoderm in the mouse embryo.

Wilms' tumor 1-associating protein (WTAP) was previously identified as a protein associated with Wilms' tumor-1 (WT-1) protein that is essential for the development of the genitourinary system. Although WTAP has been suggested to function in alternative splicing, stabilization of mRNA, and cell growth, its in vivo function is still unclear. We generated Wtap mutant mice using a novel gene-trap approach and showed that Wtap mutant embryos exhibited defective egg-cylinder formation at the gastrulation stage and died by embryonic day 10.5. Although they could form extraembryonic tissues and anterior visceral endoderm, Wtap mutant embryos and embryonic stem cells failed to differentiate into endoderm and mesoderm. The chimera analysis showed that Wtap in extraembryonic tissues was required for the formation of mesoderm and endoderm in embryonic tissues. Taken together, our findings indicate that Wtap is indispensable for differentiation of mesoderm and endoderm in the mouse embryo.     

Distribution of fibronectins and laminin in the early pig embryo

Fibronectins (FN) and laminin (LN) distributions were studied in the pig embryo by indirect immunofluorescence using antiporcine FN and antimurine LN antibodies. Extracellular FN are first detected in the early blastocyst before endodermal cell migration. They appear between the cells and on the blastocoelic face of the inner cell mass; thus, they are located at the interface of the trophectoderm and extraembryonic endoderm. Mesodermal cells migrate in a tridimensional network of fibrillar FN. These glycoproteins are also in the extraembryonic membranes (chorion and yolk sac wall) contiguous to the FN-rich basement membranes of embryonic ectoderm and endoderm. Extracellular LN appears in the blastocyst when the endoderm is already established as a continuous cellular monolayer, and is located between the trophectoderm and the extraembryonic endoderm, which produces it. Laminin also accumulates at the basal surface of the embryonic ectoderm at the onset of gastrulation. In the extraembryonic membranes, LN appears at the interface of the endoderm and mesoderm and at the interface of the trophectoderm and mesoderm. It is produced and secreted by extraembryonic mesodermal cells. Analysis of the distribution of these glycoproteins suggests that FN allow the migration of endodermal and mesodermal cells by providing them with a suitable substrate. When these cells become immobilized, they synthesize LN, probably to stabilize their interactions with the underlying extracellular material and epithelia.     

Protein kinase C mediated extraembryonic endoderm differentiation of human embryonic stem cells

Unlike mouse embryonic stem cells (ESCs), which are closely related to the inner cell mass, human ESCs appear to be more closely related to the later primitive ectoderm. For example, human ESCs and primitive ectoderm share a common epithelial morphology, growth factor requirements, and the potential to differentiate to all three embryonic germ layers. However, it has previously been shown that human ESCs can also differentiate to cells expressing markers of trophoblast, an extraembryonic lineage formed before the formation of primitive ectoderm. Here, we show that phorbol ester 12-O-tetradecanoylphorbol 13-acetate causes human ESCs to undergo an epithelial mesenchymal transition and to differentiate into cells expressing markers of parietal endoderm, another extraembryonic lineage. We further confirmed that this differentiation is through the activation of protein kinase C (PKC) pathway and demonstrated that a particular PKC subtype, PKC-δ, is most responsible for this transition.     

Development of mouse embryos in hanging drop culture

Mouse blastocysts were cultured in hanging drops for up to 6 days in order to study development under conditions that avoid the distortion of embryos typically seen when they are allowed to attach to a glass or plastic surface. The survival rate of embryos in hanging drops was equal to that of embryos attached to culture dishes and superior to that of embryos suspended in gyrating flasks. Development of the embryonic portion was similar to that in vivo and on culture dishes but slower than in vivo; the egg cylinder stage was reached after 8-10 equivalent gestation days (4 to 6 days in culture), while that stage is reached at 5.5 to 6 days in vivo. The trophectoderm, however, developed in a unique manner. The cells migrated away from the inner cell mass (ICM), similar to embryos on a culture dish, but without a surface on which to spread they clustered distal to the ICM. In vivo, trophectoderm remained covering the ICM. By 5 days in hanging drop culture the embryos had developed a segmented appearance with trophoblast giant cells at the abembryonic pole, extraembryonic cells not covered by vacuolated endoderm in the central region, and embryonic endoderm surrounding a developing proamniotic cavity in embryonic ectoderm at the embryonic pole. These observations suggest that the trophectoderm is able to follow a developmental program independent of that in the embryonic portion and that its behavior is dominated by the different adhesive properties of the trophoblastic and embryonic cells.     

Early development of the brain and spinal cord in dysraphic mice

Summary The development and closure of the neural folds was studied in C57BL/6J and loop-tail (Lp) mutant mice by means of scanning electron microscopy on a series of embryos ranging in age from 7.5 to 9.0 days of gestation. The normal embryos (C57BL/6J; +/+; Lp/+) showed a transitional zone of flattened cells lying between the surface ectoderm and neuroepithelial cells at the apices of the neural folds in the presumptive hindbrain and spinal cord, and ruffles occurred at the boundary between the flattened cells and surface ectoderm in regions of the folds which were about to fuse. In the abnormal loop-tail homozygotes (Lp/Lp) which exhibit dysraphism, the ruffles were arranged erratically along the zone of flattened cells. Moreover, at the stage when the folds became apposed and fused in the normal embryos, the abnormals showed ruffles extending the entire length of the unfused folds, thereby distinguishing the abnormals from retarded n normal embryos. Within the neural groove of the hindbrain region, the lateral neuroepithelial cells of the abnormal dysraphic embryos exhibited more flattened surfaces and fewer villous projections than in the normal embryos. The abnormal embryos also lagged behind their normal littermates in converting the body axis from the initial V-shape to the C-shaped configuration.This research was supported by NIH grant no. HD09562 from the National Institute of Child Health and Human Development, USPHS     

The developmental fate of androgenetic, parthenogenetic, and gynogenetic cells in chimeric gastrulating mouse embryos

Both a maternal and a paternal genomic contribution are necessary for completion of embryonic development in the mouse. Parthenogenetic embryos, with only a maternally inherited genome, and androgenetic embryos, with only a paternally inherited genome, fail to develop to term, and these two types of isoparental embryos fail in development in characteristic ways. In this paper we describe the construction of chimeras between single androgenetic, parthenogenetic, and gynogenetic blastomeres and normal eight-cell embryos. We allow the development of the chimeras to reach the late-gastrulating-stage embryo and then analyze the tissue distributions of the isoparental component. The isoparental embryos are derived from a transgenic mouse line carrying plasmid and mouse beta-globin sequences. The isoparental cells are detected in histological sections of chimeras by DNA-DNA in situ hybridization to the transgene, using a biotinylated DNA probe with an enzymatic detection system. We found strong tissue preferences for the androgenetic, parthenogenetic, and gynogenetic cells in chimeras. Androgenetic cells contributed strongly to all trophectoderm-derived tissue, with only a rare contribution to any tissues of the embryo proper, extraembryonic mesoderm, or extraembryonic endoderm. Parthenogenetic cells shared a developmental fate similar to gynogenetic cells, contributing to all tissues of the embryo proper and to the extraembryonic mesoderm, but only rarely to the extraembryonic endoderm or to any trophectoderm-derived tissues.     

Ultrastructure of mouse egg-cylinder

Summary The mouse egg-cylinder prior to and after mesoderm formation was studied by means of electron microscopy. The ultrastructural appearance of the proximal entoderm of both embryonic and extraembryonic segments suggests an intensive absorptive and nutritional activity. Numerous pinocytotic vacuoles, microvilli, primary and secondary lysosomes and fair amounts of rough endoplasmic reticulum and free ribosomes were the most important characteristics of these cells. After mesoderm formation, the extraembryonic entoderm showed the aforementioned characteristics even more prominently, while the cells of embryonic entoderm became flattened and depleted of microvilli and of almost all organelles. The cells of the extraembryonic and embryonic ectoderm prior to and after mesoderm formation had the same ultrastructural appearance as mesodermal cells. The cytoplasm of these cells was replete with free ribosomes, but other organelles such as mitochondria and rough endoplasmic reticulum were few in number. The architecture of all cells of the egg-cylinder except those of the extraembryonic entoderm suggested a very low level of differentiation. The criteria and possibilities for the determination of the degree of differentiation on the ultrastructural level and possible differences in protein synthesis in extraembryonic entoderm as compared with other parts of the embryo are considered.     

Rb is critical in a mammalian tissue stem cell population

The inactivation of the retinoblastoma (Rb) tumor suppressor gene in mice results in ectopic proliferation, apoptosis, and impaired differentiation in extraembryonic, neural, and erythroid lineages, culminating in fetal death by embryonic day 15.5 (E15.5). Here we show that the specific loss of Rb in trophoblast stem (TS) cells, but not in trophoblast derivatives, leads to an overexpansion of trophoblasts, a disruption of placental architecture, and fetal death by E15.5. Despite profound placental abnormalities, fetal tissues appeared remarkably normal, suggesting that the full manifestation of fetal phenotypes requires the loss of Rb in both extraembryonic and fetal tissues. Loss of Rb resulted in an increase of E2f3 expression, and the combined ablation of Rb and E2f3 significantly suppressed Rb mutant phenotypes. This rescue appears to be cell autonomous since the inactivation of Rb and E2f3 in TS cells restored placental development and extended the life of embryos to E17.5. Taken together, these results demonstrate that loss of Rb in TS cells is the defining event causing lethality of Rb(-/-) embryos and reveal the convergence of extraembryonic and fetal functions of Rb in neural and erythroid development. We conclude that the Rb pathway plays a critical role in the maintenance of a mammalian stem cell population.