Mrj encodes a DnaJ-related co-chaperone that is essential for murine placental development.
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We have identified a novel gene in a gene trap screen that encodes a protein related to the DnaJ co-chaperone in E. coli. The gene, named Mrj (mammalian relative of DnaJ) was expressed throughout development in both the embryo and placenta. Within the placenta, expression was particularly high in trophoblast giant cells but moderate levels were also observed in trophoblast cells of the chorion at embryonic day 8.5, and later in the labyrinth which arises from the attachment of the chorion to the allantois (a process called chorioallantoic fusion). Insertion of the ROSAbetageo gene trap vector into the Mrj gene created a null allele. Homozygous Mrj mutants died at mid-gestation due to a failure of chorioallantoic fusion at embryonic day 8.5, which precluded formation of the mature placenta. At embryonic day 8.5, the chorion in mutants was morphologically normal and expressed the cell adhesion molecule beta4 integrin that is known to be required for chorioallantoic fusion. However, expression of the chorionic trophoblast-specific transcription factor genes Err2 and Gcm1 was significantly reduced. The mutants showed no abnormal phenotypes in other trophoblast cell types or in the embryo proper. This study indicates a previously unsuspected role for chaperone proteins in placental development and represents the first genetic analysis of DnaJ-related protein function in higher eukaryotes. Based on a survey of EST databases representing different mouse tissues and embryonic stages, there are 40 or more DnaJ-related genes in mammals. In addition to Mrj, at least two of these genes are also expressed in the developing mouse placenta. The specificity of the developmental defect in Mrj mutants suggests that each of these genes may have unique tissue and cellular activities. (+info)
The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development.
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Csx/Nkx2.5 is a vertebrate homeobox gene with a sequence homology to the Drosophila tinman, which is required for the dorsal mesoderm specification. Recently, heterozygous mutations of this gene were found to cause human congenital heart disease (Schott, J.-J., Benson, D. W., Basson, C. T., Pease, W., Silberbach, G. M., Moak, J. P., Maron, B. J., Seidman, C. E. and Seidman, J. G. (1998) Science 281, 108-111). To investigate the functions of Csx/Nkx2.5 in cardiac and extracardiac development in the vertebrate, we have generated and analyzed mutant mice completely null for Csx/Nkx2.5. Homozygous null embryos showed arrest of cardiac development after looping and poor development of blood vessels. Moreover, there were severe defects in vascular formation and hematopoiesis in the mutant yolk sac. Interestingly, TUNEL staining and PCNA staining showed neither enhanced apoptosis nor reduced cell proliferation in the mutant myocardium. In situ hybridization studies demonstrated that, among 20 candidate genes examined, expression of ANF, BNP, MLC2V, N-myc, MEF2C, HAND1 and Msx2 was disturbed in the mutant heart. Moreover, in the heart of adult chimeric mice generated from Csx/Nkx2.5 null ES cells, there were almost no ES cell-derived cardiac myocytes, while there were substantial contributions of Csx /Nkx2.5-deficient cells in other organs. Whole-mount &bgr;-gal staining of chimeric embryos showed that more than 20% contribution of Csx/Nkx2. 5-deficient cells in the heart arrested cardiac development. These results indicate that (1) the complete null mutation of Csx/Nkx2.5 did not abolish initial heart looping, (2) there was no enhanced apoptosis or defective cell cycle entry in Csx/Nkx2.5 null cardiac myocytes, (3) Csx/Nkx2.5 regulates expression of several essential transcription factors in the developing heart, (4) Csx/Nkx2.5 is required for later differentiation of cardiac myocytes, (5) Csx/Nkx2. 5 null cells exert dominant interfering effects on cardiac development, and (6) there were severe defects in yolk sac angiogenesis and hematopoiesis in the Csx/Nkx2.5 null embryos. (+info)
Accelerated accumulation of somatic mutations in mice deficient in the nucleotide excision repair gene XPA.
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Inheritable mutations in nucleotide excision repair (NER) genes cause cancer-prone human disorders, such as xeroderma pigmentosum, which are also characterized by symptoms of accelerated ageing. To study the impact of NER deficiency on mutation accumulation in vivo, mutant frequencies have been determined in liver and brain of 2-16 month old NER deficient XPA-/-, lacZ hybrid mice. While mutant frequencies in liver of 2-month old XPA-/-, lacZ mice were comparable to XPA+/-, lacZ and the lacZ parental strain animals, by 4 months of age mutant frequencies in the XPA-deficient mice were significantly increased by a factor of two and increased further until the age of 16 months. In brain, mutant frequencies were not found to increase with age. These results show that a deficiency in the NER gene XPA causes an accelerated accumulation of somatic mutations in liver but not in brain. This is in keeping with a higher incidence of spontaneous liver tumors reported earlier for XPA-/- mice after about 15 months of age. (+info)
Ectopic expression of the transforming growth factor beta type II receptor disrupts mesoderm organisation during mouse gastrulation.
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Transforming growth factor beta (TGFbeta) regulates the cell cycle and extracellular matrix (ECM) deposition of many cells in vitro. We have analysed chimaeric mouse embryos generated from embryonic stem cells with abnormal receptor expression to study the effect of TGFbeta on these processes in vivo and the consequences for normal development. The binding receptor for TGFbeta, TbetaRII, is first detected in the embryo proper around day 8.5 in the heart. Ectopic expression of TbetaRII from the blastocyst stage onward resulted in an embryonic lethal around 9.5 dpc. Analysis of earlier stages revealed that the primitive streak of TbetaRII chimaeras failed to elongate. Furthermore, although cells passed through the streak and initially formed mesoderm, they tended to accumulate within the streak. These defects temporally and spatially paralleled the expression of the TGFbeta type I receptor, which is first expressed in the node and primitive streak. We present evidence that classical TGFbeta-induced growth inhibition was probably the cause of insufficient mesoderm being available for paraxial and axial structures. The results demonstrate that (1) TGFbeta mRNA and protein detected previously in early postimplantation embryos is present as a biologically active ligand; and (2) assuming that ectopic expression of TbetaRII results in no other changes in ES cells, the absence of TbetaRII is the principle reason why the embryo proper is unresponsive to TGFbeta ligand until after gastrulation. (+info)
Comparative analysis of cell distribution in the pigment epithelium and the visual cell layer of chimaeric mice.
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In chimaeras of both rdrdCC in equilibrium ++ cc and rdrdcc in equilibrium ++CC combinations two types of distribution were observed. In a majority of the chimaeras both retinal layers were chimaeric; whereas in a few cases the pigment epithelium was chimaeric but the visual cell layer was made of ++ cells only. No spatial relation was observed in the distribution of the cells in the two layers. The two eyes of the individuals were nearly always identical with regard to occurrence of chimaerism in the two layers. The findings are discussed in the light of the possible site and mode of expression of the rd gene. (+info)
Marmoset species variation in the humoral antibody response: in vivo and in vitro studies.
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A comparison of the in vivo and in vitro antibody response capabilities of two marmoset species, Saguinus fuscicollis and Saguinus oedipus oedipus, revealed the former to be superior in elaborating humoral antibody. In vivo challenges with Escherichia coli lipopolysaccharide (LPS) and Salmonella typhi flagella consistently yielded higher antibody titres in S. fuscicollis; indeed, with LPS antigen, multiple inoculations of S.o. oedipus marmosets led ultimately to a decrease in antibody formation, in contrast to the anamnestic response of S. fuscicollis. This species differential in immune competence was also suggested in the in vitro stimulation of peripheral blood leucocytes (PBL) and spleen cells with sheep red blood cells (RBC). None of 55 S.o. oedipus PBL cultures and 49 of 89 (55%) S. fuscicollis cultures responded to the test antigen. A similar differential in response to sheep RBC was noted with the spleen cells of each species, although this report contrasts the antibody-forming potential of two marmoset species, a comparison of the immunological response profile of marmosets to those of other laboratory animals challenged with similar antigens suggests these primates may be relatively incompetent. The possible relationship between the haemopoietic chimerism of marmosets and a diminished immune competence is discussed. (+info)
Bmp4 is required for the generation of primordial germ cells in the mouse embryo.
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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. (+info)
Interaction of B cells with activated T cells reduces the threshold for CD40-mediated B cell activation.
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CD154-CD40 interactions are of central importance for the induction of antibody responses to T-dependent antigens. Since most anti-CD40 mAb are only weak B cell mitogens, it is believed that under physiological conditions, signals through CD40 synergize with those from other receptors on B cells to induce B cell activation. We show here that the interaction of either normal B cells, or those from CBA/N (xid) mice, with CD3-activated primary T cells in whole spleen cell cultures markedly reduces the threshold for B cell activation via CD40. Hence, these pre-activated cells undergo vigorous proliferation when stimulated with either optimal or suboptimal concentrations of weakly mitogenic anti-CD40 mAb, or with soluble CD40 ligand. Blocking experiments indicate that the establishment of this priming effect requires stimulation via CD40 itself, plus T cell-derived IL-2. In support of this concept, only CD3/CD28-pre-activated, but not CD3-pre-activated T cells induce this effect, unless the co-cultures of B cells with the latter T cells are supplemented with IL-2. Although B cells activated in this fashion do express higher levels of CD40 than naive cells, we believe that this is insufficient to explain the observed dramatic effects on their proliferative capacity. Rather we propose that T cell-dependent B cell activation induces fundamental changes in the signalling machinery invoked by ligation of CD40. It is likely that this amplification loop could play an important role during the initiation of antibody responses to T-dependent antigens, when activated CD4 T cells only express low levels of CD154. (+info)