EMBRYONIC LETHALITY OF VARIOUS MOUSE STRAINS AS A CONSEQUENCE OF X-IRRADIATION-INDUCED INJURIES OF THE FATHER'S SPERMATOZOA.
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Animals↗
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Results for “embryonic lethality”
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In vivo and in vitro studies on the early embryonic lethal oligosyndactylism (Os) in the mouse.
The development of mouse embryos homozygous for oligosyndactylism (Os) is arrested during implantation. Histological investigations confirm a previous report that cells become blocked in mitosis, and air-dried spreads of the mutant embryos reveal that large numbers of cells accumulate in metaphase. Trophoblastic giant cells appear unaffected by the action of the mutant gene both in utero and during culture over the lethal phase. It is proposed that the form of endoreduplication undergone by giant cells renders them refractory to the metaphase block.
Animals↗
Oligosyndactylism, an early embryonic lethal in the mouse.
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Animals↗
In vivo and in vitro development of mouse embryos homozygous for the embryonic lethal velvet coat (Ve) mutation.
Embryos homozygous for the velvet coat mutation, Ve/Ve, were recognized at 6.5 days post coitum by the reduced size of the ectodermal portions of the egg cylinder and the loose, columnar nature of the overlying endoderm. Later in development ectoderm tissues were sometimes entirely absent. Abnormalities appeared in the ectoplacental cone at 8.5 days but trophoblast giant cells and parietal endoderm appeared unaffected. Homozygotes could not be unequivocally identified at 5.5 days nor at the blastocyst stage but were recognized in blastocyst outgrowths by poor development of the inner cell mass derivatives. It has previously been suggested that Ve may exert its action at the blastocyst stage by reducing the size of the inner cell mass, but no evidence for such a reduction was found. Most of the observations on Ve/Ve homozygotes are, however, consistent with the hypothesis that Ve exerts its action primarily on later primitive ectoderm development.
Animals↗
Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation.
The Mre11/Rad50 protein complex functions in diverse aspects of the cellular response to double-strand breaks (DSBs), including the detection of DNA damage, the activation of cell cycle checkpoints, and DSB repair. Whereas genetic analyses in Saccharomyces cerevisiae have provided insight regarding DSB repair functions of this highly conserved complex, the implication of the human complex in Nijmegen breakage syndrome reveals its role in cell cycle checkpoint functions. We established mRad50 mutant mice to examine the role of the mammalian Mre11/Rad50 protein complex in the DNA damage response. Early embryonic cells deficient in mRad50 are hypersensitive to ionizing radiation, consistent with a role for this complex in the repair of ionizing radiation-induced DSBs. However, the null mrad50 mutation is lethal in cultured embryonic stem cells and in early developing embryos, indicating that the mammalian Mre11/Rad50 protein complex mediates functions in normally growing cells that are essential for viability.
Animals↗
Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development.
The mammalian target of rapamycin (mTOR) is a key component of a signaling pathway which integrates inputs from nutrients and growth factors to regulate cell growth. Recent studies demonstrated that mice harboring an ethylnitrosourea-induced mutation in the gene encoding mTOR die at embryonic day 12.5 (E12.5). However, others have shown that the treatment of E4.5 blastocysts with rapamycin blocks trophoblast outgrowth, suggesting that the absence of mTOR should lead to embryonic lethality at an earlier stage. To resolve this discrepancy, we set out to disrupt the mTOR gene and analyze the outcome in both heterozygous and homozygous settings. Heterozygous mTOR (mTOR(+/-)) mice do not display any overt phenotype, although mouse embryonic fibroblasts derived from these mice show a 50% reduction in mTOR protein levels and phosphorylation of S6 kinase 1 T389, a site whose phosphorylation is directly mediated by mTOR. However, S6 phosphorylation, raptor levels, cell size, and cell cycle transit times are not diminished in these cells. In contrast to the situation in mTOR(+/-) mice, embryonic development of homozygous mTOR(-/-) mice appears to be arrested at E5.5; such embryos are severely runted and display an aberrant developmental phenotype. The ability of these embryos to implant corresponds to a limited level of trophoblast outgrowth in vitro, reflecting a maternal mRNA contribution, which has been shown to persist during preimplantation development. Moreover, mTOR(-/-) embryos display a lesion in inner cell mass proliferation, consistent with the inability to establish embryonic stem cells from mTOR(-/-) embryos.
Alleles↗
Chimeric analysis of Notch2 function: a role for Notch2 in the development of the roof plate of the mouse brain.
Notch proteins are transmembrane receptors involved in cell-fate determination throughout development. Targeted disruption of either the Notch1 or Notch2 gene in mice results in embryonic lethality around embryonic day (E) 10.5 with widespread cell death. Although Notch1-deficient mice show disorganized somitogenesis, Notch2 mutants did not show definitive abnormalities in any tissue expressing high levels of the Notch2 gene, including the central nervous system. To study Notch2 function in development beyond the embryonic lethal stage, we performed chimeric analysis between Notch2 mutant and wild-type mouse embryos. Chimeric embryos developed normally and homozygous Notch2 mutant-specific cell death was not observed. Although chimeric embryos showed normal mosaicism until E9.5 in all tissues studied to date, Notch2 homozygous mutant cells failed to contribute to formation of the roof plate of the diencephalon and mesencephalon at later developmental stages, when Notch2 is normally expressed at high levels at there. Furthermore, Notch2 heterozygous mutant cells were also excluded from the roof plate of the chimera, however, Notch2 heterozygous mutant mice developed normally. We also showed that Wnt-1 and Mash1 expression patterns at the roof plate were disorganized in Notch2 homozygous mutant embryos. These results indicate that Notch2 plays an important role in development of the roof plate of the diencephalon and mesencephalon, and suggest that cellular rearrangement is involved in this process.
Animals↗
Genetic identification, sequence, and alternative splicing of the Caenorhabditis elegans alpha 2(IV) collagen gene.
The nematode Caenorhabditis elegans has two type IV collagen genes homologous to the mammalian alpha 1(IV) and alpha 2(IV) collagen genes. We demonstrate by transgenic rescue of mutant animals that the genetic locus encoding the C. elegans alpha 2(IV) collagen gene is let-2 on the X chromosome. The most severe effect of mutations in let-2 is temperature-sensitive embryonic lethality. The embryonic lethal phenotype is similar to that seen in animals with mutations in the alpha 1(IV) collagen gene, emb-9. The sequence of the entire C. elegans alpha 2(IV) collagen gene is presented. Comparisons with mammalian type IV collagen sequences show high amino acid sequence conservation in the C-terminal NCl domain and of crosslinking residues (Cys and Lys) in the N-terminal 7S domain. RT-PCR analysis shows that transcripts of the C. elegans alpha 2(IV) collagen gene are alternatively spliced. Transcripts contain one of two mutually exclusive exons, exon 9 or 10. These exons encode very similar products, differing primarily in the sequence of a 9-10 amino acid Gly-X-Y interruption. The expression of these alternatively spliced alpha 2(IV) collagen transcripts is developmentally regulated. In embryos over 90% of the alpha 2(IV) collagen mRNA contains exon 9, while larval and adult RNAs contain 80-90% exon 10. This shift in expression of alternative alpha 2(IV) collagen transcripts suggests that C. elegans embryos may require a different form of alpha 2(IV) collagen than do larvae and adults.
Alternative Splicing↗
Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi.
Genome-wide analysis of gene function is essential for the post-genome era, and development of efficient and economical technology suitable for it has been in demand. Here we report a large-scale inactivation of the expressed genes in the nematode Caenorhabditis elegans. For this purpose, we have established a high-throughput "RNAi-by-soaking" methodology by modifying the conventional RNAi method [1, 2]. A set of tag-sequenced, nonredundant cDNAs corresponding to approximately 10,000 genes [3] (representing half of the predicted genes [4]) was used for the systematic RNAi analysis. We have processed approximately 2500 genes to date. In development, 27% of them showed detectable phenotypes, such as embryonic lethality, post-embryonic lethality, sterility, and morphological abnormality. Of these, we analyzed the phenotypes of F1 sterility in detail, and we have identified 24 genes that might play important roles in germline development. Combined with the ongoing analysis of expression patterns of these cDNAs [3, 5], the functional information obtained in this work will provide a starting point for the further analysis of each gene. Another finding from this screening is that the incidence of essential genes is significantly lower in the X chromosome than in the autosomes.
Animals↗
Telomere-associated protein TIN2 is essential for early embryonic development through a telomerase-independent pathway.
TIN2 is a negative regulator of telomere elongation that interacts with telomeric DNA repeat binding factor 1 (TRF1) and affects telomere length by a telomerase-dependent mechanism. Here we show that inactivation of the mouse TRF1-interacting protein 2 (TIN2) gene results in early embryonic lethality. We further observed that the embryonic lethality of TIN2 mutant mice was not affected by inactivation of the telomerase reverse transcriptase gene, indicating that embryonic lethality is not the result of telomerase-dependent changes in telomere length or function. Our findings suggest that TIN2 has a role independent of telomere length regulation that is essential for embryonic development and cell viability.
Animals↗
Mouse embryogenesis requires the tissue factor extracellular domain but not the cytoplasmic domain.
Recent studies indicate that tissue factor (TF) acts in embryogenesis, metastasis, and angiogenesis. Three independent groups showed that targeted disruption of the murine TF (mTF) gene results in 90% lethality of mTF null embryos at embryonic days 9. 5-10.5. We have demonstrated that expression of wild-type human TF (hTF) from a minigene rescues the embryonic lethality of mTF null embryos. To investigate the role of TF in embryogenesis, we made mutant hTF minigenes whose products either bound FVII/VIIa at a reduced level or lacked the cytoplasmic domain. Two independent transgenic lines expressing the hTF extracellular domain mutant failed to rescue the embryonic lethality of mTF null embryos, suggesting that FVII/VIIa binding by TF, proteolytic activity by the TF/FVIIa complex, or both were required for embryogenesis. In contrast, two transgenic lines expressing the hTF cytoplasmic domain mutant rescued the embryonic lethality of mTF null embryos, indicating that the cytoplasmic domain of TF was not required for embryogenesis. We propose that TF/FVIIa-dependent extracellular protease activity is required for embryogenesis.
Animals↗
Ecsit is required for Bmp signaling and mesoderm formation during mouse embryogenesis.
Bone morphogenetic proteins (Bmps) are members of the transforming growth factor beta (TGFbeta) superfamily that play critical roles during mouse embryogenesis. Signaling by Bmp receptors is mediated mainly by Smad proteins. In this study, we show that a targeted null mutation of Ecsit, encoding a signaling intermediate of the Toll pathway, leads to reduced cell proliferation, altered epiblast patterning, impairment of mesoderm formation, and embryonic lethality at embryonic day 7.5 (E7.5), phenotypes that mimic the Bmp receptor type1a (Bmpr1a) null mutant. In addition, specific Bmp target gene expression is abolished in the absence of Ecsit. Biochemical analysis demonstrates that Ecsit associates constitutively with Smad4 and associates with Smad1 in a Bmp-inducible manner. Together with Smad1 and Smad4, Ecsit binds to the promoter of specific Bmp target genes. Finally, knock-down of Ecsit with Ecsit-specific short hairpin RNA inhibits both Bmp and Toll signaling. Therefore, these results show that Ecsit functions as an essential component in two important signal transduction pathways and establishes a novel role for Ecsit as a cofactor for Smad proteins in the Bmp signaling pathway.
Adaptor Proteins, Signal Transducing↗
Distribution of endothelial cell protein C/activated protein C receptor (EPCR) during mouse embryo development.
The endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombomodulin.thrombin complex. Deletion of the EPCR gene in mice has been reported to lead to embryonic lethality before embryonic day 10 (E10.0). To identify potential mechanisms responsible for this lethality, we performed an immunohistological analysis of EPCR distribution during mouse embryogenesis. EPCR was detected in the trophoblast giant cells at the feto-maternal boundary from E7.5 and at later time points in the trophoblasts of the placenta, suggesting a role in the haemostatic regulation of the maternal blood that irrigates these surfaces. In the embryo, EPCR was weakly detected in aortic endothelial cells from E13.5. Thereafter, EPCR levels increased in certain large blood vessels endothelial cells suggesting that the specificity of EPCR to large vessels is conferred in utero. However, not until postnatal day 7 did the intensity and distribution of EPCR staining mimic that observed in adult mice.
Animals↗
Gene targeting in hemostasis. Factor X.
Blood coagulation Factor X (FX) is a vitamin K-dependent serine protease that plays a central role in blood clotting. Additionally, FX may exert other functions beyond coagulation but the precise role of this protein in these processes and the in vivo relevance remains to be further delineated. The development of gene knockout technology has allowed for a direct means to determine the physiological relevance of various proteins in a number of normal and pathological processes. Mice with a total deficiency of FX have been generated by targeted deletion of all exons encoding the mature FX protein. The genotypic distribution indicated that homozygous deficiency results in partial embryonic lethality at embryonic day (E) 11.5-12.5 with signs of massive bleeding but no histologically evident defects in the vasculature of these embryos or their yolk sac. The majority of those that survive to term die within 5 days, most frequently from intraabdominal bleeding. The remainder die between postnatal day (P)5 and P20 with intraabdominal, subcutaneous, or intracranial bleeding or a combination thereof. These observations underline the importance of FX function in embryonic and postnatal survival and confirm that these mice serve as effective models of the bleeding disorders observed in human FX deficiency. While the early FX-/- lethality impedes investigation of potential morphogenetic functions of FX in vivo, such studies may however become feasible through the availability of mice expressing mutant FX proteins, e.g., EPR-1 binding site mutants, or of mice with conditional FX deficiency.
Amino Acid Sequence↗
Inactivation of the nuclear receptor coactivator RAP250 in mice results in placental vascular dysfunction.
Coactivators constitute a diverse group of proteins that are essential for optimal transcriptional activity of nuclear receptors. In the past few years many coactivators have been identified but it is still unclear whether these proteins interact indiscriminately with all nuclear receptors and whether there is some redundancy in their functions. We have previously cloned and characterized RAP250 (ASC-2/PRIP/TRBP/NRC), an LXXLL-containing coactivator for nuclear receptors. In order to study its biological role, Rap250 null mice were generated by gene targeting. Here we show that genetic disruption of Rap250 results in embryonic lethality at embryonic day (E) 13.5. Histological examination of placentas revealed a dramatically reduced spongiotrophoblast layer, a collapse of blood vessels in the region bordering the spongiotrophoblast, and labyrinthine layers in placentas from Rap250(-/-) embryos. These findings suggest that the lethality of Rap250(-/-) embryos is the result of obstructed placental blood circulation. Moreover, the transcriptional activity of PPAR gamma is reduced in fibroblasts derived from Rap250(-/-) embryos, suggesting that RAP250 is an essential coactivator for this nuclear receptor in the placenta. Our results demonstrate that RAP250 is necessary for placental development and thus essential for embryonic development.
Animals↗
Generation of a Bmp2 conditional null allele.
Bone morphogenetic proteins (Bmp's) are known to play many important roles in embryogenesis. In addition, recent data from human genetic studies has revealed that Bmp's also have important functions in maintenance of the adult phenotype and aging. The original Bmp2 germline null allele resulted in lethality at embryonic day 7.0-10.5 due to malformation of the amnion/chorion and cardiac malformations. Because the early embryonic lethality of the Bmp2 germline null allele hinders further investigation into Bmp2 function at later stages, we generated a Bmp2 conditional null allele. Using gene targeting in mouse embryonic stem (ES) cells, we introduced LoxP sites upstream and downstream of Bmp2 exon 3 that encodes the mature peptide. Our results indicate that the Bmp2 conditional null allele is a true conditional null that encodes wildtype activity and reverts to a null allele after cre recombinase-induced recombination.
Alleles↗
Extraembryonic expression of EPCR is essential for embryonic viability.
The endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin-thrombomodulin complex. Deletion of the EPCR gene (Procr) in mice leads to embryonic lethality before embryonic day 10 (E10.0). EPCR is detected in the giant trophoblast cells at the feto-maternal boundary from E7.5 and weakly in embryonic aortic endothelial cells from E13.5, suggesting that extraembryonic EPCR expression may be essential for embryonic viability. Using conditional knock-out strategies, we demonstrate that Procr-deficient embryos with EPCR expression on placenta giant trophoblasts can be carried to term and then develop normally. Conversely, EPCR expression in the embryo, without expression in the giant trophoblast cells, does not rescue the mice. In genetically modified mice with low tissue factor activity, Procr deficiency is not lethal to the embryo. As adults, Procr-deficient mice generate more thrombin and activate less protein C in response to procoagulant stimuli. Spontaneous thrombin formation in the deficient animals increases with age. These findings show that extraembryonic EPCR expression is critical for embryo development.
Aging↗
Rump white inversion in the mouse disrupts dipeptidyl aminopeptidase-like protein 6 and causes dysregulation of Kit expression.
The mouse rump white (Rw) mutation causes a pigmentation defect in heterozygotes and embryonic lethality in homozygotes. At embryonic day (E) 7.5, Rw/Rw embryos are retarded in growth, fail to complete neurulation and die around E 9.5. The Rw mutation is associated with a chromosomal inversion spanning 30 cM of the proximal portion of mouse chromosome 5. The Rw embryonic lethality is complemented by the W19H deletion, which spans the distal boundary of the Rw inversion, suggesting that the Rw lethality is not caused by the disruption of a gene at the distal end of the inversion. Here, we report the molecular characterization of sequences disrupted by both inversion breakpoints. These studies indicate that the distal breakpoint of the inversion is associated with ectopic Kit expression and therefore may be responsible for the dominant pigmentation defect in Rw/+ mice; whereas the recessive lethality of Rw is probably due to the disruption of the gene encoding dipeptidyl aminopeptidase-like protein 6, Dpp6 [Wada, K., Yokotani, N., Hunter, C., Doi, K., Wenthold, R. J. & Shimasaki, S. (1992) Proc. Natl. Acad. Sci. USA 89, 197-201] located at the proximal inversion breakpoint.
Amino Acid Sequence↗