Characteristics of the expression of t12 mutation in mouse embryos with structural aberrations of chromosome 17

Developmental profiles of mouse embryos with deletions, duplications of nullisomy for the proximal part AB of chromosome 17, including genes of the T-t complex, were studied in mice with marker translocations Rb (16.17)7Bnr or T(16;17)43H and heterozygous for lethal t12 mutation. The embryos t12t12 and t12t12-(Dp17CDE; Dl16) were shown to be eliminated at the morula stage; embryos t12+, t12+ + or t12t12+ survive during preimplantation and early postimplantation stages: t12 embryos (hemizygous for all genes of the 17AB region, including all t-alleles) have quite normal cleavage, blastulation and implantation, but die soon thereafter. The embryos with nullisomy 17AB combined with deletion 17CDE survive up to the morula stage. These data are in line with previously proposed hypothetical mechanism for mutual activation of homologous chromosomes and their segments during initial stages of embryogenesis in mice. The system of marker chromosomes Rb7Bnr and T43H in combination with various alleles of the T complex might be recommended as a useful tool in analysis of primary developmental effects of different t-alleles in mice.     

Chromosomal control of early embryonic development in mice I. Experiments on embryos with autosomal monosomy

The effects of autosomal monosomy on early embryonic development were studied in mice with Robertsonian and reciprocal (non-Robertsonian) translocations. It was found that monosomy for autosomes 1, 2, 3, 5, 6, 16, 17 and 19 had substantially different effects on preimplantation development and survival of mouse embryos. Monosomy for autosomes 1, 3, 6, 16 and 19 does not affect cleavage, compaction and blastulation and in some cases is compatible with implantation. Most of these embryos, however, die as early blastocysts (Ms 3, 6 or 19) and some of them are eliminated at early postimplantation stages (Ms 1 or 16). The embryos with monosomy for autosomes 2, 5 or 17 can be identified during cleavage owing to the reduced blastomere number and pathological changes in the nuclei. Most of these monosomies do not survive beyond the morula stage. The results indicate that differential genetic activity of autosomes in mice becomes already evident in very early embryonic development. A hypothetical mechanism for homologous autosome activation at the onset of embryonic development in mice is suggested.     

Detrimental effects of two active X chromosomes on early mouse development

Matings between female mice carrying Searle's translocation, T(X;16)16H, and normal males give rise to chromosomally unbalanced zygotes with two complete sets of autosomes, one normal X chromosome and one X16 translocation chromosome (XnX16 embryos). Since X chromosome inactivation does not occur in these embryos, probably due to the lack of the inactivation center on X16, XnX16 embryos are functionally disomic for the proximal 63% of the X chromosome and trisomic for the distal segment of chromosome 16. Developmental abnormalities found in XnX16 embryos include: (1) growth retardation detected as early as stage 9, (2) continual loss of embryonic ectoderm cells either by death or by expulsion into the proamniotic cavity, (3) underdevelopment of the ectoplacental cone throughout the course of development, (4) very limited, if any, mesoderm formation, (5) failure in early organogenesis including the embryo, amnion, chorion and yolk sac. Death occurred at 10 days p.c. Since the combination of XO and trisomy 16 does not severely affect early mouse development, it is likely that regulatory mechanisms essential for early embryogenesis do not function correctly in XnX16 embryos due to activity of the extra X chromosome segment of X16.     

The Tribolium castaneum ortholog of Sex combs reduced controls dorsal ridge development

Several constitutional chromosomal rearrangements occur on human chromosome 17. Patients who carry constitutional deletions of 17q21.3-q24 exhibit distinct phenotypic features. Within the deletion interval, there is a genomic segment that is bounded by the myeloperoxidase and homeobox B1 genes. This genomic segment is syntenically conserved on mouse chromosome 11 and is bounded by the mouse homologs of the same genes (Mpo and HoxB1). To attain functional information about this syntenic segment in mice, we have generated a 6.9-Mb deletion [Df(11)18], the reciprocal duplication [Dp(11)18] between Mpo and Chad (the chondroadherin gene), and a 1.8-Mb deletion between Chad and HoxB1. Phenotypic analyses of the mutant mouse lines showed that the Dp(11)18/Dp(11)18 genotype was responsible for embryonic or adolescent lethality, whereas the Df(11)18/+ genotype was responsible for heart defects. The cardiovascular phenotype of the Df(11)18/+ fetuses was similar to those of patients who carried the deletions of 17q21.3-q24. Since heart defects were not detectable in Df(11)18/Dp(11)18 mice, the haplo-insufficiency of one or more genes located between Mpo and Chad may be responsible for the abnormal cardiovascular phenotype. Therefore, we have identified a new dosage-sensitive genomic region that may be critical for normal heart development in both mice and humans.     

Chromosomal control of early mammalian development

A review of recent studies on mammalian embryos, mostly mice, with chromosomal aberrations. Morphological, biochemical and cytological studies on mice with polyploidy, aneuploidy and some structural aberrations are discussed. Some types of chromosomal aberrations, especially monosomy for individual chromosomes (2, 5, 7, or 17), are already evident during early cleavage and are inevitably lethal by the morula stage. A direct relationship exists between the duration of survival and chromosome aberrations (trisomy and monosomy) for every chromosome. Differential gene activity of the mouse autosomes becomes evident already at the very early developmental stages. Some feasible causes of the early death of embryos with autosomal monosomy are discussed and a hypothetical mechanism for the activation of homologous autosomes at the early developmental stages is proposed. Perspectives of future studies in cytogenetics of mammalian development are outlined.     

Overlapping deletions define novel embryonic lethal loci in the mouse t complex

Summary: The t complex region of mouse chromosome 17 contains genetic information critical for embryonic development. To identify and map loci required for normal embryogenesis, a set of overlapping deletions (D17Aus9(df10J), D17Aus9(df12J), and D17Aus9(df13J)) surrounding the D17Aus9 locus and one encompassing the T locus, Del(17)T(7J), were bred in various combinations and the consequences of nullizygosity in overlapping regions were examined. The results indicated that there are at least two functional units within 1 cM of D17Aus9. l17J1 is a peri-implantation lethal mutation within the region deleted in D17Aus9(df13J), whereas l17J2 is a later-acting lethal defined by the region of overlap between Del(17)T(7J) and D17Aus9(df12J). Del(17)T(7J)/D17Aus9(df12J) embryos die around 10.5 dpc. The development of the mutant embryos is characterized by lack of axial rotation, an abnormal notochord structure, and a ballooning pericardium. These studies demonstrate the value of overlapping deletion complexes, as opposed to individual deletion complexes, for the identification, mapping, and analysis of genes required for embryonic development.     

Chromosomal mapping of four different integration sites of Moloney murine leukemia virus including the locus for alpha 1(I) collagen in mouse

Infection of mouse embryos with Moloney murine leukemia virus (M-MuLV) has yielded several mouse substrains with stable germ line integration of retroviral DNA at distinct chromosomal loci (Mov loci; Jaenisch et al., 1981). There is evidence that flanking DNA sequences can have an effect on virus expression and, conversely, inserted viral DNA may affect the expression of adjacent host genes. As part of our studies on the interaction of inserted M-MuLV with the mouse genome, we have chromosomally mapped four different Mov loci by hybridizing single-copy mouse sequences, flanking the proviral DNA, to interspecies somatic cell hybrids. Furthermore, these sequences were assigned regionally by in situ hybridization to mouse metaphase chromosomes. In Mov-13 mice, M-MuLV had inserted into the alpha 1(I) collagen gene leading to early embryonic death in homozygotes. We have assigned this locus to the distal region of chromosome 11. Thus, the alpha 1(I) collagen gene is part of an evolutionarily conserved linkage group with the homologous genes on human chromosome 17. Three other proviral integration sites were mapped to chromosome 1, bands BC (Mov-7), chromosome 11, bands BC (Mov-9), and chromosome 3, bands FG (Mov-10). The Mov-10-specific probe detects an EcoRI-specific restriction fragment length polymorphism, which can make this probe a useful genetic marker.     

Studying Early Lethality of 45,XO (Turner's Syndrome) Embryos Using Human Embryonic Stem Cells

Turner''s syndrome (caused by monosomy of chromosome X) is one of the most common chromosomal abnormalities in females. Although 3% of all pregnancies start with XO embryos, 99% of these pregnancies terminate spontaneously during the first trimester. The common genetic explanation for the early lethality of monosomy X embryos, as well as the phenotype of surviving individuals is haploinsufficiency of pseudoautosomal genes on the X chromosome. Another possible mechanism is null expression of imprinted genes on the X chromosome due to the loss of the expressed allele. In contrast to humans, XO mice are viable, and fertile. Thus, neither cells from patients nor mouse models can be used in order to study the cause of early lethality in XO embryos. Human embryonic stem cells (HESCs) can differentiate in culture into cells from the three embryonic germ layers as well as into extraembryonic cells. These cells have been shown to have great value in modeling human developmental genetic disorders. In order to study the reasons for the early lethality of 45,XO embryos we have isolated HESCs that have spontaneously lost one of their sex chromosomes. To examine the possibility that imprinted genes on the X chromosome play a role in the phenotype of XO embryos, we have identified genes that were no longer expressed in the mutant cells. None of these genes showed a monoallelic expression in XX cells, implying that imprinting is not playing a major role in the phenotype of XO embryos. To suggest an explanation for the embryonic lethality caused by monosomy X, we have differentiated the XO HESCs in vitro an in vivo. DNA microarray analysis of the differentiated cells enabled us to compare the expression of tissue specific genes in XO and XX cells. The tissue that showed the most significant differences between the clones was the placenta. Many placental genes are expressed at much higher levels in XX cells in compare to XO cells. Thus, we suggest that abnormal placental differentiation as a result of haploinsufficiency of X-linked pseudoautosomal genes causes the early lethality in XO human embryos.     

Modeling del(17)(p11.2p11.2) and dup(17)(p11.2p11.2) contiguous gene syndromes by chromosome engineering in mice: phenotypic consequences of gene dosage imbalance

Contiguous gene syndromes (CGS) are a group of disorders associated with chromosomal rearrangements of which the phenotype is thought to result from altered copy numbers of physically linked dosage-sensitive genes. Smith-Magenis syndrome (SMS) is a CGS associated with a deletion within band p11.2 of chromosome 17. Recently, patients harboring the predicted reciprocal duplication product [dup(17)(p11.2p11.2)] have been described as having a relatively mild phenotype. By chromosomal engineering, we created rearranged chromosomes carrying the deletion [Df(11)17] or duplication [Dp(11)17] of the syntenic region on mouse chromosome 11 that spans the genomic interval commonly deleted in SMS patients. Df(11)17/+ mice exhibit craniofacial abnormalities, seizures, marked obesity, and male-specific reduced fertility. Dp(11)17/+ animals are underweight and do not have seizures, craniofacial abnormalities, or reduced fertility. Examination of Df(11)17/Dp(11)17 animals suggests that most of the observed phenotypes result from gene dosage effects. Our murine models represent a powerful tool to analyze the consequences of gene dosage imbalance in this genomic interval and to investigate the molecular genetic bases of both SMS and dup(17)(p11.2p11.2).     

FGF-16 is required for embryonic heart development

Fibroblast growth factor 16 (FGF-16) expression has previously been detected in mouse heart at mid-gestation in the endocardium and epicardium, suggesting a role in embryonic heart development. More specifically, exogenously applied FGF-16 has been shown to stimulate growth of embryonic myocardial cells in tissue explants. We have generated mice lacking FGF-16 by targeting the Fgf16 locus on the X chromosome. Elimination of Fgf16 expression resulted in embryonic death as early as day 11.5 (E11.5). External abnormalities, including hemorrhage in the heart and ventral body region as well as facial defects, began to appear in null embryos from E11.5. Morphological analysis of FGF-16 null hearts revealed cardiac defects including chamber dilation, thinning of the atrial and ventricular walls, and poor trabeculation, which were visible at E10.5 and more pronounced at E11.5. These findings indicate FGF-16 is required for embryonic heart development in mid-gestation through its positive effect on myocardial growth.     

Cyclin D and cdk4 are required for normal development beyond the blastula stage in sea urchin embryos

cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development. In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases. Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis. An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA. Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo. In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae. These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle.     

Ts65Dn -- localization of the translocation breakpoint and trisomic gene content in a mouse model for Down syndrome

Fluorescent in situ hybridization (FISH) -- using mouse chromosome paints, probes for the mouse major centromeric satellite DNA, and probes for genes on chromosomes (Chr) 16 and 17 -- was employed to locate the breakpoint in a translocation used to produce a mouse model for Down syndrome. The Ts65Dn trisomy is derived from the reciprocal translocation T(16;17)65Dn. The Ts65Dn mouse carries a marker chromosome containing the distal segment of Chr 16, a region that shows linkage conservation with human Chr 21, and the proximal end of Chr 17. This chromosome confers trisomy for most of the genes in the Chr 16 segment and Ts65Dn mice show many of the phenotypic features characteristic of Down syndrome. We used FISH on metaphase chromosomes from translocation T65Dn/+ heterozygotes and Ts65Dn mice to show that the Chr 17 breakpoint is distal to the heterochromatin of Chr 17, that the Ts65Dn marker chromosome contains a small portion of Chr 17 euchromatin, that the Chr 16 breakpoint lies between the Ncam2 and Gabpa/App genes, and that the Ts65Dn chromosome contains >80% of the human Chr 21 homologs. The significance of this finding is discussed in terms of the utility of this mouse model.     

Estimation of the contribution of thiophosphamide-induced chromosomal aberrations to perimplantation embryonic mortality in mice]

Thiotepa-induced perimplantation mortality in mice was analysed cytologically. The contribution of induced chromosomal aberrations to preimplantation embryonic death was studied. Under the influence of 1.25 mg/kg of thiotepa on late spermatids the stop of cleavage division was found at 2--16 cell stage in 27% of the cases and at 17--22 cell stage in 11.9% (totally in 38.9%) as compared to 6% in the controls. The cytogenetic analysis of embryos consisting of 2--16 cells revealed gross structural chromosomal aberrations in 75% metaphases suitable for analysis.     

Spindle assembly checkpoint regulates mitotic cell cycle progression during preimplantation embryo development

Errors in chromosome segregation or distribution may result in aneuploid embryo formation, which causes implantation failure, spontaneous abortion, genetic diseases, or embryo death. Embryonic aneuploidy occurs when chromosome aberrations are present in gametes or early embryos. To date, it is still unclear whether the spindle assembly checkpoint (SAC) is required for the regulation of mitotic cell cycle progression to ensure mitotic fidelity during preimplantation development. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of SAC components (Bub3, BubR1 and Mad2) in mouse preimplantation embryos. Our data showed that overexpressed SAC components inhibited metaphase-anaphase transition by preventing sister chromatid segregation. Deletion of SAC components by RNAi accelerated the metaphase-anaphase transition during the first cleavage and caused micronuclei formation, chromosome misalignment and aneuploidy, which caused decreased implantation and delayed development. Furthermore, in the presence of the spindle-depolymerizing drug nocodazole, SAC depleted embryos failed to arrest at metaphase. Our results suggest that SAC is essential for the regulation of mitotic cell cycle progression in cleavage stage mouse embryos.     

The X-linked imprinted gene family Fthl17 shows predominantly female expression following the two-cell stage in mouse embryos

Differences between male and female mammals are initiated by embryonic differentiation of the gonad into either a testis or an ovary. However, this may not be the sole determinant. There are reports that embryonic sex differentiation might precede and be independent of gonadal differentiation, but there is little molecular biological evidence for this. To test for sex differences in early-stage embryos, we separated male and female blastocysts using newly developed non-invasive sexing methods for transgenic mice expressing green fluorescent protein and compared the gene-expression patterns. From this screening, we found that the Fthl17 (ferritin, heavy polypeptide-like 17) family of genes was predominantly expressed in female blastocysts. This comprises seven genes that cluster on the X chromosome. Expression analysis based on DNA polymorphisms revealed that these genes are imprinted and expressed from the paternal X chromosome as early as the two-cell stage. Thus, by the time zygotic genome activation starts there are already differences in gene expression between male and female mouse embryos. This discovery will be important for the study of early sex differentiation, as clearly these differences arise before gonadal differentiation.     

Laser microbeam-induced DNA damage inhibits cell division in fertilized eggs and early embryos

DNA double-strand breaks are caused by both intracellular physiological processes and environmental stress. In this study, we used laser microbeam cut (abbreviated microcut or cut), which allows specific DNA damage in the pronucleus of a fertilized egg and in individual blastomere(s) of an early embryo, to investigate the response of early embryos to DNA double-strand breaks. Line type γH2AX foci were detected in the cut region, while Chk2 phosphorylation staining was observed in the whole nuclear region of the cut pronuclei or blastomeres. Zygotes with cut male or female pronucleus showed poor developmental capability: the percentage of cleavage embryos was significantly decreased, and the embryos failed to complete further development to blastocysts. The cut blastomeres in 2-cell, 4-cell, and 8-cell embryos ceased cleavage, and they failed to incorporate into compacted morulae, but instead underwent apoptosis and cell death at the blastocyst stage; the uncut part of embryos could develop to blastocysts, with a reduced percentage or decreased cell number. When both blastomeres of the 2-cell embryos were cut by laser microbeam, cell death occurred 24 h earlier, suggesting important functions of the uncut blastomere in delaying cell death of the cut blastomere. Taken together, we conclude that microbeam-induced DNA damage in early embryos causes compromised development, and that embryos may have their own mechanisms to exclude DNA-damaged blastomeres from participating in further development.