In vitro development of individually cultured whole mouse embryos from blastocyst to early somite stage.

The sequential processes of in vitro development of whole mouse embryos were classified by stages according to the in vivo criteria of E. Witschi (1972, “Biology Data Book,” Part II: “Rat,” L. Altman and D. S. Dittmer, eds., 2nd ed., Vol. 1, pp. 178–180, Federation of American Societies for Experimental Biology, Bethesda, Md.) and K. Theiler (1972, “The House Mouse,” Springer-Verlag, Berlin/New York). The mouse embryos which developed in vitro in each day of culture were then classified into stages according to the characteristics of mouse embryos developed in vivo. A series of 10 blastocysts were inoculated into 35-mm plastic culture dishes (30–50 blastocysts per experiment). Developing embryos were scored on the fourth, sixth, and eighth days and classified into stages. Among the total of 118 blastocysts cultured in three repeated experiments, 100 mouse embryos had attached and developed in culture dishes. Ninety-four percent of the attached mouse embryos developed to the early egg cylinder stage after 4 days of incubation, and 87% grew to the stage of late egg cylinder after 6 days of culture. An average of 62% of the embryos reached the early somite stage with heart beating after 8 days in culture with frequent medium change. In two separate experiments single mouse blastocysts were placed individually in culture dishes in 2 ml of culture medium. The development of each embryo was followed every day. Each of 10 blastocysts had attached in its respective culture dish and had developed to the early egg cylinder stage after 4 days of culture. About 50 to 70% of each of these 20 individually isolated mouse embryos developed in vitro to the early somite stage after 8 days of culture.     

Dll3 pudgy mutation differentially disrupts dynamic expression of somite genes

Mutations in the notch ligand delta-like 3 have been identified in both the pudgy mouse (Dll3(pu); Kusumi et al.: Nat Genet 19:274-278, 1998) and the human disorder spondylocostal dysostosis (SCD; Bulman et al.: Nat Genet 24:438-441, 2000), and a targeted mutation has been generated (Dll3(neo); Dunwoodie et al.: Development 129:1795-1806, 2002). Vertebral and rib malformations deriving from defects in somitic patterning are key features of these disorders. In the mouse, notch pathway genes such as Lfng, Hes1, Hes7, and Hey2 display dynamic patterns of expression in paraxial mesoderm, cycling in synchrony with somite formation (Aulehla and Johnson: Dev Biol 207:49-61, 1999; Forsberg et al.: Curr Biol 8:1027-1030, 1998; Jouve et al.: Development 127:1421-1429, 2000; McGrew et al.: Curr Biol 8:979-982, 1998; Nakagawa et al.: Dev Biol 216:72-84, 1999). We report here that the Dll3(pu) mutation has different effects on the expression of cycling (Lfng and Hes7) and stage-specific genes (Hey3 and Mesp2). This suggests a more complex situation than a single oscillatory mechanism in somitogenesis and provides an explanation for the unique radiological features of the human DLL3-type of SCD.     

Differentiation in vitro of mouse embryos to the stage of early somite

Mouse blastocysts continuously differentiate in vitro to the early somite stage with reconstituted rat tail collagen as the substrate for the attachment. In order for this to occur, it appears that two differentiation barriers must be overcome. The first, the formation of egg cylinders from the inner cell mass, can be overcome by incubating embryos in heat-inactivated fetal calf serum. The second, the formation of the early somite from the presomite stage, can be overcome by replacing fetal calf serum with human cord serum.Mouse blastocysts were initially incubated with calf serum in Eagle's minimum essential medium. After shedding the zona pellucida, the denuded blastocysts lay flat on the surface of the collagen. Soon thereafter, trophoblastic cells invaded the underlying collagen leaving the rounded inner cell mass protruding from the surface of the collagen. By replacing calf serum in the medium with fetal calf serum the inner cell mass differentiated into endoderm and ectoderm to form an egg cylinder.The egg cylinder rapidly became elongated and formed extraembryonic and embryonic regions. However, the embryonic region shrank from this point on in the fetal calf serum, and the resulting yolk sac formation did not contain the embryo proper. When fetal calf serum was replaced with human cord serum at the end of the egg cylinder stage (equivalent to embryos of about 7.5 days gestation) neural tissue, cardiac chambers, and somites were formed.     

Differential expression of TGF beta 1, beta 2 and beta 3 genes during mouse embryogenesis

We have examined by Northern analysis and in situ hybridisation the expression of TGF beta 1, beta 2 and beta 3 during mouse embryogenesis. TGF beta 1 is expressed predominantly in the mesodermal components of the embryo e.g. the hematopoietic cells of both fetal liver and the hemopoietic islands of the yolk sac, the mesenchymal tissues of several internal organs and in ossifying bone tissues. The strongest TGF beta 2 signals were found in early facial mesenchyme and in some endodermal and ectodermal epithelial cell layers e.g., lung and cochlea epithelia. TGF beta 3 was strongest in prevertebral tissue, in some mesothelia and in lung epithelia. All three isoforms were expressed in bone tissues but showed distinct patterns of expression both spatially and temporally. In the root sheath of the whisker follicle, TGF beta 1, beta 2 and beta 3 were expressed simultaneously. We discuss the implication of these results in regard to known regulatory elements of the TGF beta genes and their receptors.     

Comparative FISH mapping of Gab1 and Gab2 genes in human, mouse and rat.

Gab1 and Gab2 are members of the Gab family which act as adapters for transmitting various signals in response to stimuli through cytokine and growth factor receptors, and T- and B-cell antigen receptors. We determined chromosome locations of the two genes in human, mouse and rat by fluorescence in situ hybridization. The Gab1 gene was localized to chromosome 4q31.1 in human, 8C3 in mouse and 19q11.1--> q11.2 in rat, and the Gab2 gene was located on chromosome 11q13.4-->q13.5 in human, 7E2 in mouse and 1q33.2-->q33.3 in rat. All human, mouse and rat Gab1 and Gab2 genes were localized to chromosome regions where conserved homology has been identified among the three species.     

Intermediate filament protein expression in early developmental stages of the mouse

Expression patterns of intermediate filament proteins have been studied during early mouse embryo development. For this purpose, pre-implantation embryos at different stages of development after in vitro fertilization were studied using antibodies to cytokeratins, vimentin and lamins, using the indirect immunofluorescence assay. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies showed that, although the embryos grew in culture, vimentin could not be detected in a filamentous organization. Immunofluorescence for cytokeratins was only positive from the 8-cell stage onwards. In the morula stage an increased level of cytokeratin expression was observed with a transitional staining pattern, combining a filamentous and a diffuse occurrence. In the blastocyst stages profound cytokeratin filaments were seen in trophoblast cells but not in the inner cell mass. When the cytokeratin subtypes were analysed separately, it became apparent that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage and showing a filamentous distribution, were not found as prominently as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected in all developmental stages examined, as a faintly reactive nuclear lamina. In blastocysts both lamin types were detected in trophoblast as well as in inner cell mass.     

Establishment of cell lines from somite stage mouse embryos and expression of major histocompatibility class I genes in these cells

To study the regulation of MHC class I gene expression during embryonic development, we have characterized a number of clonal cell lines derived from somite stage mouse embryos that were established with or without infection by several transforming retroviruses in combination with murine leukemia viruses. Unlike embryonal carcinoma (EC) cells that have been used as a model for early embryos, the cell lines derived from somite stage embryos are negative for stage specific embryonic Ag-1 and do not appear to differentiate after retinoic acid treatment. Morphology varies from clone to clone and is distinct from that of F9 and other EC cells. In agreement with previous findings in in vivo embryos, expression of surface MHC class I antigen in 57 new clones is either undetectable or low (with variability). All of the clones respond to the addition of interferons and express MHC class I antigens at high levels, but the kinetics of mRNA accumulation vary considerably. To examine the basis of the generally low or absent MHC class I gene expression in these cells, we tested promoter activity of a MHC class I gene by CAT assay after transient DNA transfection. Regardless of the basal levels of mRNA or surface Ag, CAT activity directed by various portions of the 5' flanking region of the MHC class I gene was uniformly low. The cells showed neither the negative nor the positive regulation of MHC class I genes that had been noted respectively for EC cells and for cells expressing the Ag constitutively. The pattern seen in the new cell lines suggests that there is an intermediate stage in the developmental regulation of MHC class I gene expression that may operate during the middle to late stage of fetal development.     

Developmentally regulated expression of the mouse homologues of the potassium channel encoding genes m-erg1, m-erg2 and m-erg3

Deciphering the expression pattern of K+ channel encoding genes during development can help in the understanding of the establishment of cellular excitability and unravel the molecular mechanisms of neuromuscular diseases. We focused our attention on genes belonging to the erg family, which is deeply involved in the control of neuromuscular excitability in Drosophila flies and possibly other organisms. Both in situ hybridisation and RNase Protection Assay experiments were used to study the expression pattern of mouse (m)erg1, m-erg2 and m-erg3 genes during mouse embryo development, to allow the pattern to be compared with their expression in the adult. M-erg1 is first expressed in the heart and in the central nervous system (CNS) of embryonic day 9.5 (E9.5) embryos; the gene appears in ganglia of the peripheral nervous system (PNS) (dorsal root (DRG) and sympathetic (SCG) ganglia, mioenteric plexus), in the neural layer of retina, skeletal muscles, gonads and gut at E13.5. In the adult m-erg1 is expressed in the heart, various structures of the CNS, DRG and retina. M-erg2 is first expressed at E9.5 in the CNS, thereafter (E13.5) in the neural layer of retina, DRG, SCG, and in the atrium. In the adult the gene is present in some restricted areas of the CNS, retina and DRG. M-erg3 displayed an expression pattern partially overlapping that of m-erg1, with a transitory expression in the developing heart as well. A detailed study of the mouse adult brain showed a peculiar expression pattern of the three genes, sometimes overlapping in different encephalic areas.     

Geminin escapes degradation in G1 of mouse pluripotent cells and mediates the expression of Oct4, Sox2, and Nanog

Geminin is an essential cell-cycle protein that is only present from S phase to early mitosis in metazoan somatic cells. Genetic ablation of geminin in the mouse results in preimplantation embryonic lethality because pluripotent cells fail to form and all cells differentiate to trophoblast. Here we show that geminin is present in G1 phase of mouse pluripotent cells in contrast to somatic cells, where anaphase-promoting complex/cyclosome (APC/C)-mediated proteasomal destruction removes geminin in G1. Silencing geminin directly or by depleting the APC/C inhibitor Emi1 causes loss of stem cell identity and trophoblast differentiation of mouse embryonal carcinoma and embryonic stem cells. Depletion of cyclins A2 or B1 does not induce this effect, even though both of these APC/C substrates are also present during G1 of pluripotent cells. Crucially, geminin antagonizes the chromatin-remodeling protein Brg1 to maintain expression of Oct4, Sox2, and Nanog. Our results define a pluripotency pathway by which suppressed APC/C activity protects geminin from degradation in G1, allowing sustained expression of core pluripotency factors. Collectively, these findings link the cell cycle to the pluripotent state but also raise an unexplained paradox: How is cell-cycle progression possible in pluripotent cells when oscillations of key regulatory proteins are lost?     

Differential expression of the ARF GAP genes GIT1 and GIT2 in mouse tissues.

GIT1 and GIT2 belong to the family of ADP-ribosylation factor GTPase-activating proteins (ARF-GAP) and have been implicated in the regulation of G protein-coupled receptor sequestration, cell migration, T-cell activation, neuronal spine formation, and aggregate formation in Huntington's disease. Examination of endogenous GIT protein expression in tissues, however, has been hampered by the lack of GIT2-specific antibodies. To visualize GIT1 and GIT2 gene expression in mouse tissues, we created mice with beta-galactosidase (beta-Gal) reporters inserted into the two GIT genes. beta-Gal staining confirmed the broad tissue distribution of GIT1 and GIT2 in the mouse but also revealed striking differences. GIT2 is expressed in most cells of the body, whereas GIT1 is restricted to only a subset of cells. For example, GIT2 is uniformly expressed throughout lung and liver, whereas GIT1 is restricted to cells lining blood vessels, bronchi, and bile ducts. Expression of GIT1 and GIT2 is mutually exclusive in the testes, where a developmental expression shift occurs, with GIT2 present in spermatogonia but GIT1 in mature spermatids. In conclusion, analysis of endogenous GIT expression revealed a nearly ubiquitous distribution of GIT2, whereas GIT1 is restricted to specific cell types even in tissues with apparently high GIT1 expression and is entirely absent from some tissues.