The mouse lethal nonagouti (a(x)) mutation deletes the S-adenosylhomocysteine hydrolase (Ahcy) gene.

The lethal nonagouti (a(x)) mutation is a hypomorphic allele of the agouti coat color locus which, when homozygous, also leads to embryonic death around the time of implantation. To understand the molecular basis of these phenotypes, we identified and cloned a deletion breakpoint junction present in the ax chromosome. Long range restriction mapping demonstrated a simple deletion of approximately 100 kb, which does not affect agouti coding sequences, but begins only 4 kb 3' of the last exon, and thus may affect coat color by removing an agouti 3' enhancer. The Ahcy gene, which codes for the enzyme S-adenosylhomocysteine hydrolase (SAHase), is contained within a 20 kb region within the a(x) deletion. SAHase RNA and protein were detectable in early blastocysts and in embryonic stem cells, respectively, and analysis of embryos derived from an a(x)/a x a(x)/a embryo intercross indicated that a(x)/a embryos die between the late blastocyst and early implantation stages. Treatment of cultured embryos with an SAHase inhibitor, 3-deazaaristeromycin, or with metabolites that can result in elevated levels of cellular SAH, resulted in an inhibition of inner cell mass development, suggesting that loss of SAHase activity in a(x)/a(x) embryos is sufficient to explain their death around the time of implantation.     

Chromosome Damage and Early Developmental Arrest Caused by the Rex Element of Drosophila Melanogaster

Rex (Ribosomal exchange) is a genetically identified repeated element within the ribosomal DNA (rDNA) of Drosophila melanogaster. Rex has a semidominant maternal effect that promotes exchange between and within rDNA arrays in the first few embryonic mitoses. Several of Rex's genetic properties suggest that its primary effect is rDNA-specific chromosome breakage that is resolved by recombination. We report here that rDNA crossovers are only a small, surviving minority of Rex-induced events. Cytology of embryos produced by Rex-homozygous females reveals obvious chromosome damage in at least a quarter of the embryos within the first three mitotic divisions. More than half of the embryos produced by Rex females die, and the developmental arrest is among the earliest reported for any maternal-effect lethal. The striking lethal phenotype suggests that embryos with early chromosome damage could be particularly fruitful subjects for analysis of the cell biology of early embryos.     

Mouse zygotes injected with mitochondria develop normally but the exogenous mitochondria are not detectable in the progeny

A microinjection procedure to introduce "paternal" mitochondria from a source other than spermatozoa into fertilized mouse eggs is described. When a mitochondrial suspension isolated from the testes or liver of Mus molossinus mice was microinjected into fertilized eggs of CD1 mice, the microinjected zygotes survived, developed normally, and offspring were produced. Mus molossinus mitochondrial DNA can be distinguished from CD1 mitochondrial DNA by Southern blot analyses using restriction enzymes such as Eco R1, Xba 1, or Spe 1. Although up to 120 viable mitochondria were injected, no exogenous mitochondrial DNA was detected in fetal samples or in the brain, liver, heart, testis, or ovary of the mature progeny. Under the experimental conditions used, similar results were obtained when mitochondria from the testes of New Zealand black mice or from testes of Syrian hamsters were microinjected into fertilized CD1 mouse eggs. Failure to detect the exogenous mitochondrial DNA under our assay conditions suggests that microinjected mitochondria from testis or liver did not selectively replicate during embryonic development. The "foreign" mitochondria appear to have the same fate during early embryogenesis as the mitochondria of the spermatozoon.     

The albino deletion complex and early postimplantation survival in the mouse

The albino deletion complex in the mouse represents 37 overlapping chromosomal deficiencies that have been arranged into at least twelve complementation groups. Many of the deletions cover regions of chromosome 7 that contain genes necessary for early embryonic development. The work reported here concentrates on two of these deletions (c6H, c11DSD), both of which were known to be lethal around the time of gastrulation when homozygous. A detailed embryological analysis has revealed distinct differences in the lethal phenotype associated with the c6H and c11DSD deletions. c6H homozygous embryos are grossly abnormal at day 7.5 of gestation, whereas c11DSD homozygous embryos appear abnormal at day 8.5 of gestation. There is no development of the extraembryonic ectoderm in c6H homozygotes, whereas extensive development of this tissue type occurs in c11DSD homozygotes. The visceral endoderm is abnormally shaped and the parietal endoderm appears to be overproduced in c6H homozygotes; these structures are not affected in c11DSD homozygotes. The embryonic ectoderm is runted in both types of embryo and it is not possible to obtain homozygous embryo-derived stem-cell lines for either deletion. Mesoderm formation occurs in the c11DSD but not in the c6H homozygotes. The c11DSD deletion chromosome complements the c6H chromosome in that the lethal phenotype of the compound heterozygote is similar to that of the c11DSD homozygote. These results suggest that a gene(s) necessary for normal development of the extraembryonic ectoderm is present in the c11DSD but deficient in the c6H deletion chromosome.     

Structural organization of interspersed repetitive elements present in the DNA of Mus musculus

Two-dimensional displays of the restriction fragments from the DNA of Mus musculus revealed a complex species-specific pattern produced from nonsatellite repetitive sequences. The patterns have been used as a guide in the direct purification of a group of broadly interspersed repeated DNA sequences (characterized by a 1350-bp Eco-Bam fragment) that have been studied by molecular cloning, restriction mapping and genomic Southern blotting. These studies show that the cloned representatives originate from an abundant group of sequences that share homology with about 2% of the mouse genome. The sequences do not appear to share homology with mouse-interspersed-family-1 (MIF-1) nor with the major AT-rich satellite sequences of mouse. They appear to be part of a group of larger repetitive elements that is both broadly interspersed and heavily methylated in normal mouse tissue.     

A simple procedure for maximum yield of high-quality plasmid DNA

We have established a simple procedure for the rapid isolation of high-quality plasmid DNA suitable for various molecular techniques and provided a step-by-step protocol. The DNA samples isolated by this procedure have been used successfully for double-stranded DNA sequencing, restriction enzyme mapping, subcloning, in vitro mutagenesis, generation of deletion clones and so on. The procedure is highly reproducible, and superior quality DNA can be obtained without the use of phenol, chloroform or other organic solvents.     

Meiotic mapping of murine chromosome 17: The string of loci around l(17)-2Pas

We describe a genetic analysis of l(17)-2Pas, an embryonic lethal mutation on murine chromosome 17. Males transmitted the l(17)-2 allele to only 38% of their offspring, whereas females transmitted this allele at 50%. Two-point crosses revealed tight linkage between l(17)-2 and brachyury (T), and deletion mapping placed l(17)-2 outside of the hairpin-tail deletion (T ^hp ). To map this mutation more precisely, we intercrossed hybrid mice that carry distinct alleles at many classical and DNA loci on chromosome 17 and obtained 172 animals recombinant in the T to H-2 region. Strong positive interference was observed over the 14 cM interval from T to H-2K. Thus, a single recombinant can be informative; one such recombinant places l(17)-2 distal of the molecular marker D17Leh66D. Robust genetic maps can be constructed with multilocus crosses that share anchor loci. DNA markers can be interpolated onto these maps retrospectively.     

Effects of 5-Fluorodeoxyuridine and Related Halogenated Pyrimidines on the Sand-Dollar Embryo

The embryo of the sand-dollar (Echinarachnius parma) was exposed to various concentrations of fluorinated pyrimidines immediately after fertilization. FUDR (5-fluorodeoxyuridine) was most active, and a concentration of 2 to 4 mγ/10 cc. (0.8 to 1.6 x 10^-6 m.eq./liter) blocked development at the early blastula stage. Larger doses interrupted development at the same stage. This effect was prevented by thymidine (TDR) and thymine (T); and these pyrimidines protected against many times the minimal lethal concentration of FUDR. TDR was active as a protective agent if added just before early blastula formation. The other fluorinated pyrimidines, 5-fluorouracil (FU), 5-fluorouridine (FUR), 5-fluorocytidine (FCR), 5-fluorodeoxycytidine (FCDR), and 5-fluoroorotic acid (FO), were also studied. These drugs produced effects on embryonic development similar to those seen with FUDR. The effective concentrations, however, varied greatly. T and TDR provided protection against these drugs, but in most cases they were not so effective as against FUDR. 5-Bromodeoxyurdine (BrUDR), beginning at the early blastula stage, caused a random pattern of embryonic death up to the pluteus stage. This drug has been shown to be incorporated into bacterial DNA. BrUDR protected embryos against the early lethal effects of FUDR presumably acting as a thymidine substitute, but the embryos died subsequently in a pattern similar to that seen with BrUDR alone. FUDR and BrUDR appear to inhibit the formation and alter the structure of DNA, respectively, distinctive effects whch may provide a means for studying the role of DNA in embryonic development.     

Molecular Genetics of the Brown (B)-Locus Region of Mouse Chromosome 4. I. Origin and Molecular Mapping of Radiation- and Chemical-Induced Lethal Brown Deletions

Over a period of many years, germ-cell mutagenesis experiments using the mouse specific-locus test have generated numerous radiation- and chemical-induced alleles of the brown (b; Tyrp1) locus in mouse chromosome 4. We describe here the origin, maintenance and initial molecular characterization of 28 b mutations that are prenatally lethal when homozygous. Each of these mutations is deleted for Tyrp1 sequences, and each of 25 mutations tested further is deleted for at least one other locus defined by molecular clones previously found to be closely linked to b by interspecific backcross analysis. A panel of DNAs from mice carrying a lethal b mutation and a Mus spretus chromosome 4 was used in the fine structure mapping of these molecularly defined loci. The deletional nature of each of these prenatally lethal mutations is consistent with the hypothesis that the null phenotype at b has an effect only on the quality (color) of eumelanin produced in melanocytes. The resulting deletion map provides a framework on which to build future molecular-genetic and biological analyses of this region of mouse chromosome 4.     

Requirement for the Xrcc1 DNA base excision repair gene during early mouse development

Surveillance and repair of DNA damage are essential for maintaining the integrity of the genetic information that is needed for normal development. Several multienzyme pathways, including the excision repair of damaged or missing bases, carry out DNA repair in mammals. We determined the developmental role of the X-ray cross-complementing (Xrcc)-1 gene, which is central to base excision repair, by generating a targeted mutation in mice. Heterozygous matings produced Xrcc1-/- embryos at early developmental stages, but not Xrcc1-/- late-stage fetuses or pups. Histology showed that mutant (Xrcc1-/-) embryos arrested at embryonic day (E) 6.5 and by E7.5 were morphologically abnormal. The most severe abnormalities observed in mutant embryos were in embryonic tissues, which showed increased cell death in the epiblast and an altered morphology in the visceral embryonic endoderm. Extraembryonic tissues appeared relatively normal at E6.5-7.5. Even without exposure to DNA-damaging agents, mutant embryos showed increased levels of unrepaired DNA strand breaks in the egg cylinder compared with normal embryos. Xrcc1-/- cell lines derived from mutant embryos were hypersensitive to mutagen-induced DNA damage. Xrcc1 mutant embryos that were also made homozygous for a null mutation in Trp53 underwent developmental arrest after only slightly further development, thus revealing a Trp53-independent mechanism of embryo lethality. These results show that an intact base excision repair pathway is essential for normal early postimplantation mouse development and implicate an endogenous source of DNA damage in the lethal phenotype of embryos lacking this repair capacity.     

Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.

Gene targeting in embryonic stem (ES) cells has been used to mutate the murine DNA methyltransferase gene. ES cell lines homozygous for the mutation were generated by consecutive targeting of both wild-type alleles; the mutant cells were viable and showed no obvious abnormalities with respect to growth rate or morphology, and had only trace levels of DNA methyltransferase activity. A quantitative end-labeling assay showed that the level of m5C in the DNA of homozygous mutant cells was about one-third that of wild-type cells, and Southern blot analysis after cleavage of the DNA with a methylation-sensitive restriction endonuclease revealed substantial demethylation of endogenous retroviral DNA. The mutation was introduced into the germline of mice and found to cause a recessive lethal phenotype. Homozygous embryos were stunted, delayed in development, and did not survive past mid-gestation. The DNA of homozygous embryos showed a reduction of the level of m5C similar to that of homozygous ES cells. These results indicate that while a 3-fold reduction in levels of genomic m5C has no detectable effect on the viability or proliferation of ES cells in culture, a similar reduction of DNA methylation in embryos causes abnormal development and embryonic lethality.     

Transcriptome Profiling of Embryonic Development Rate in Rainbow Trout Advanced Backcross Introgression Lines

In rainbow trout (Oncorhynchus mykiss) and other fishes, embryonic development rate is an ecologically and evolutionarily important trait that is closely associated with survival and physiological performance later in life. To identify genes differentially regulated in fast and slow-developing embryos of rainbow trout, we examined gene expression across developmental time points in rainbow trout embryos possessing alleles linked to a major quantitative trait loci (QTL) for fast versus slow embryonic development rate. Whole genome expression microarray analyses were conducted using embryos from a fourth generation backcross family, whereby each backcross generation involved the introgression of the fast-developing alleles for a major development rate QTL into a slow-developing clonal line of rainbow trout. Embryos were collected at 15, 19, and 28 days post-fertilization; sex and QTL genotype were determined using molecular markers, and cDNA from 48 embryos were used for microarray analysis. A total of 183 features were identified with significant differences between embryonic development rate genotypes. Genes associated with cell cycle growth, muscle contraction and protein synthesis were expressed significantly higher in embryos with the fast-developing allele (Clearwater) than those with the slow-developing allele (Oregon State University), which may associate with fast growth and early body mass construction in embryo development. Across time points, individuals with the fast-developing QTL allele appeared to have earlier onset of these developmental processes when compared to individuals with the slow development alleles, even as early as 15 days post-fertilization. Differentially expressed candidate genes chosen for linkage mapping were localized primarily to regions outside of the major embryonic development rate QTL, with the exception of a single gene (very low-density lipoprotein receptor precursor).     

Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification‐induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ‐H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage‐related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ‐H2AX foci in zygotes and two‐cell embryos. Trp53bp1 was upregulated in zygotes, two‐cell embryos and four‐cell embryos in the vitrified group, and Brca1 was increased in two‐cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two‐cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4′‐trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ‐H2AX foci in zygotes and two‐cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification‐induced abnormal ROS generation, γ‐H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ‐H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification‐associated γ‐H2AX accumulation is at least partially due to abnormal ROS generation.     

Essential role for the Pak4 protein kinase in extraembryonic tissue development and vessel formation

Pak4 is a member of the group B family of Pak serine/threonine kinases, originally identified as an effector protein for the Rho GTPase Cdc42. Pak4 knockout mice are embryonic lethal and do not survive past embryonic day 11.5. Previous work on Pak4 knockout mice has focused on studying the phenotype of the embryo. Abnormalities in the extraembryonic tissue, however, are common causes of early embryonic death in knockout mice. Extraembryonic tissue associated with the Pak4-null embryos was therefore examined. Abnormalities in both yolk sacs and placentas resulted when Pak4 was deleted. These included a lack of vasculature throughout the extraembryonic tissue, as well as an abnormally formed labyrinthine layer of the placenta. Interestingly, epiblast-specific deletion of Pak4 using a conditional knockout system, did not rescue the embryonic lethality. In fact, it did not even rescue the extraembryonic tissue defects. Our results suggest that the extraembryonic tissue abnormalities are secondary to defects that occur in response to epiblast abnormalities. More detailed analysis suggests that abnormalities in vasculature throughout the extraembryonic tissue and the epiblast may contribute to the death of the Pak4-null embryos.     

Identification of transgene integration site and anatomical properties of fluorescence intensity in a EGFP transgenic chicken line

Transgenic birds are commonly used for time-lapse imaging and fate mapping studies in developmental biology. When researchers use transgenic birds expressing fluorescent protein, they need to understand the integration site of the transgene in the genome and the intensity of fluorescence in the tissues of interest. In this study, we determined the integration site of the transgene and fluorescence property of developing organs in our transgenic chicken line generated by lentivirus infection. The transgene was localized between exons 3 and 4 of MED27. Some homozygotes and heterozygotes appeared to be lethal at early embryonic stages. We performed histological analysis of EGFP expression in transgenic embryos at St. 14, 17, and 24 by immunohistochemistry with anti-GFP antibody on paraffin sections. Next, we cut cryosections and quantified direct EGFP intensity from the transgene in each tissue without performing immunohistochemistry. These results revealed that EGFP intensity in each tissue was unique in developing embryos and changed according to developmental stages. Finally, we demonstrated that EGFP-expressing cells in a micromass culture with co-culturing wild-type cells were clearly distinguishable via live cell imaging. These results provide essential information on the potential of our transgenic line and indicate that these transgenic chicken lines are useful for research associated with developmental biology.     

Conditional knockout of focal adhesion kinase in endothelial cells reveals its role in angiogenesis and vascular development in late embryogenesis

Focal adhesion kinase (FAK) is a critical mediator of signal transduction by integrins and growth factor receptors in a variety of cells including endothelial cells (ECs). Here, we describe EC-specific knockout of FAK using a Cre-loxP approach. In contrast to the total FAK knockout, deletion of FAK specifically in ECs did not affect early embryonic development including normal vasculogenesis. However, in late embryogenesis, FAK deletion in the ECs led to defective angiogenesis in the embryos, yolk sac, and placenta, impaired vasculature and associated hemorrhage, edema, and developmental delay, and late embryonic lethal phenotype. Histologically, ECs and blood vessels in the mutant embryos present a disorganized, detached, and apoptotic appearance. Consistent with these phenotypes, deletion of FAK in ECs isolated from the floxed FAK mice led to reduced tubulogenesis, cell survival, proliferation, and migration in vitro. Together, these results strongly suggest a role of FAK in angiogenesis and vascular development due to its essential function in the regulation of multiple EC activities.     

Successful transfer of biopsied equine embryos

Embryo biopsy has been used to detect inherited disorders and to improve the phenotype by analyzing of linkages between marker loci and the desired characteristics. Unfortunately, early procedures required the removal of a large portion (one-half) of the embryo for analysis, and the transfer of bisected equine embryos has not been particularly successful. Recent discovery of the polymerase chain reaction (PCR) has made possible the detection of specific DNA sequences from only a few cells. We investigated whether the removal of a small biopsy would allow for successful PCR and normal embryonic development. In the study reported here, 14 microbladebiopsied Day 6 to 7 equine embryos were transferred nonsurgically into recipient mares. The sex of each embryo was determined from the biopsy by means of restriction fragment length polymorphism analysis of the ZFY/ZFX loci after PCR amplification. The embryos were sexed as 8 females and 6 males on the basis of PCR assay results. Two embryos were biopsied using a needle aspiration technique, but no PCR amplification products resulted from these attempts. Eight intact control embryos were transferred to recipient mares using the same method. Pregnancy rates were 3 14 and 6 8 for the microblade biopsy and control groups, respectively. All of the microblade biopsy group pregnancies were females. One was aborted for cytogenetic analysis. Two were born after normal gestation. With improved pregnancy rates, this technique could be used for preimplantation diagnostics of equine embryos. As gene mapping advances and associations between particular DNA sequences and inherited traits become established, a rapid PCR technique could be used to select embryos before transfer.     

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.