Search

We previously reported the requirement of Polycomb Repressive Complex 2 (PRC2) for spermatogenesis through transcriptional repression of somatic genes and meiosis-specific genes. To characterize how PRC2's two methyltransferase subunits, EZH1 and EZH2, regulate histone H3 lysine 27 (H3K27) methylation during germ cell development, we generated mouse models with a germline ablation of EZH1 and/or EHZ2. Only the combined loss of EZH1 and EZH2 caused a depletion of global H3K27me3 marks and meiotic arrest in spermatocytes. Genome-wide analysis of H3K27me3 in spermatogenic cells revealed that a noncanonical EZH1-PRC2 could establish and maintain this histone mark on somatic genes and certain meiotic genes. Consistent with it having active enhancers in testis, Ezh1 was not only abundant in highly differentiated spermatocytes but also in actively proliferating progenitor and stem germ cells. Taken together, our findings suggest that the expression level of Ezh1 determines the restoration of H3K27 methylation in the absence of the canonical EZH2-PRC2.

The prevalent model for the generation of axial polarity in mouse embryos proposes that a radial to a linear transition in the expression of primitive streak markers precedes the formation of the primitive streak on one side of the epiblast. This model contrasts with the models of mesoderm formation in other vertebrates as it suggests that the primitive streak is initially established in a radial pattern rather than a localized region of the epiblast. Here, we examine the proposed correlation between the expression of Brachyury and Wnt3, two genes reported as expressed radially in the proximal epiblast, with the movements of proximal anterior epiblast cells at stages leading to the formation of the primitive streak. Our results reveal that neither Brachyury nor Wnt3 forms a ring of expression in the proximal epiblast as previously thought. In embryos dissected between 5.5 and 6.5 dpc, Brachyury is first expressed in the distal extra-embryonic ectoderm and subsequently on one side of the epiblast. Wnt3 expression is evident first in the posterior visceral endoderm of 5.5 dpc embryos and later in the posterior epiblast. Lineage analysis shows that the movements of the proximal epiblast do not restrict Brachyury expression to the posterior epiblast. Our data suggest a model whereby the localized expression of these genes in the posterior epiblast, and hence the formation of the primitive streak, is the result of local cell–cell interactions in the future posterior portion of the egg cylinder rather than regionalization of a radial pattern of expression in proximal epiblast cells.

Several lines of evidence suggest that the extraembryonic endoderm of vertebrate embryos plays an important role in the development of rostral neural structures. In mice, neural inductive signals are thought to reside in an area of visceral endoderm that expresses the Hex gene. Here, we have conducted a morphological and lineage analysis of visceral endoderm cells spanning pre- and postprimitive streak stages. Our results show that Hex-expressing cells have a tall, columnar epithelial morphology, which distinguishes them from other visceral endoderm cells. This region of visceral endoderm thickening (VET) is found overlying first the distal and then one side of the epiblast at stages between 5.5 and 5.75 days post coitum (d.p.c.). In addition, we show that the epiblast has an anteroposterior-compressed appearance that is aligned with the position of the VET. Intracellular labeling of VET/Hex-expressing cells reveals an anterior and anterolateral shift from their distal epiblast position. VET/Hex-expressing cells are first localized to the anterior side of the epiblast by 5.75 d.p.c. and form a crescent on the anterior half of the embryo at the onset of gastrulation. Subsequently, VET descendants are distributed along the embryonic/extraembryonic boundary by headfold stages at 7.5 d.p.c. The morphological characteristics and position of VET/Hex-expressing cells distinguishes the future anteroposterior axis of the embryo and provide landmarks to stage mouse embryos at preprimitive streak stages. Moreover, the morphological characteristics of pregastrulation mouse embryos together with the stereotyped shift in the position of visceral endoderm cells reveal similarities among amniote embryos that suggest an evolutionary conservation of the mechanisms that pattern the rostral neurectoderm at pregastrula stages.

Preimplantation mouse embryos were examined for intercellular permeability to molecules of different molecular weights. Using immunosurgery followed by immunofluorescence, none of the eight-cell embryos, approximately 10% of the early morulae, 50% of the late morulae, and 90% of the early blastocysts were found to block nonspecific anti-mouse thymocyte serum from diffusing into intercellular spaces. Diffusion of horseradish peroxidase and microperoxidase into intercellular spaces of viable embryos was also impeded by some morulae and by all early blastocysts maintained on ice. Peroxidase tracers found within the blastocoel cavity of some early blastocysts examined at 37°C appear to be a result of pinocytosis and transcellular movement. Intercellular diffusion of lanthanum into glutaraldehyde-fixed embryos was impeded only by early blastocysts. These results suggest that a permeability seal is established between external cells of the early mouse embryo prior to blastocoel formation. In addition, freeze-fracture electron microscopy revealed a correlated change in zonula occludens junction organization, indicating that formation of an intercellular permeability barrier and subsequent blastocoel formation may depend upon completion of assembly of these junctions.

Concanavalin A (Con A), a plant lectin which binds to mannose-containing components, has been used to precipitate a specific class of proteins synthesized during preimplantation mouse embryogenesis. Total cellular protein was extracted from labeled embryos with Nonidet P40, reacted with Con A, and then exposed to rabbit antiserum directed against Con A. Immune complexes were precipitated with protein A from Staphylococcus aureus. After washing, Con A-bound proteins were eluted with mannoside which released between 3 and 10% of the total radioactivity from unfertilized and fertilized eggs, 2-cell and 8-to 16-cell embryos, and late blastocysts. In control experiments, in which mannoside was added prior to the Con A, only 0.1–0.5% of the total radioactivity was eluted, indicating little release of proteins nonspecifically bound to the protein A-anti-Con A-Con A complex. When these precipitates were examined by two-dimensional gel electrophoresis, approximately 30–70 peptides in the defined periods of synthesis were detected in autoradiograms. To precipitate cell surface proteins, intact embryos were incubated in Con A and anti-Con A prior to extraction with Nonidet P40. Less than 1.0% of the total radioactivity was eluted with mannoside. A complex of three pairs of peptides was present in 2-cell and 8- to 16-cell embryos. The qualitative changes in two-dimensional gel patterns of Con A-binding proteins has provided a program of synthesis occurring during normal development for a specific class of proteins.

The mutation oligosyndactyly results in syndactyly, abnormal fusion and insertion of certain limb muscles, and diabetes insipidus in heterozygous mice. When homozygous the mutation is lethal; beginning at the blastocyst stage, the homozygous cells arrest in metaphase with intact spindles. The mutant phenotype cannot be corrected by forming aggregation chimeras with wild-type cells, suggesting that the mutation results in a cell autonomous lethal condition. Short-term rescue of the homozygous-induced mitotic arrest can be achieved, however, by cytoplasmic injection of polyadenylated RNA obtained from a rapidly dividing embryo-derived stem cell line.

Spindle-pole organization during early mouse development was examined using a variety of immunological reagents that recognize centrosomal components. Spindle poles of unfertilized eggs and blastocysts were found to react positively with two antisera (centrin and NRS-01), whereas poles of activated eggs and early cleavage-stage embryos were negative when treated with the same sera. In contrast, a third antiserum (5051) showed positive spindle-pole staining throughout the preimplantation stages of development. Two monoclonal antibodies (MPM-1 and MPM-2) that are known to react with mitotic phosphoproteins were also used in this study. Both antibodies stained the cytoplasm of mitotic cells with extremely high intensity. In addition, MPM-2 was found to stain spindle poles. These results suggest that organizational changes in the spindle pole are occurring during early mouse development. Embryos homozygous for a recessive lethal mutation known as oligosyndactyly (Os) were also treated with the reagents described above. This mutation results in a metaphase arrest at the blastocyst stage with intact spindles being present. Spindle poles were observed in Os homozygous mutants stained with centrin, NRS-01, and 5051. However, when Os mutants were stained with the MPM monoclonal antibodies, about half of the mitotic cells completely lacked the dramatic cytoplasmic staining. This observation is in contrast to that observed for wild-type embryos, where greater than 95% of mitotic cells showed positive cytoplasmic staining.