Rescue of embryonic lethality in hepatocyte growth factor/scatter factor knockout mice

While the targeted disruption of a gene is a powerful tool for investigating the physiological functions of that gene, disruption of a gene essential for embryogenesis leads to embryonic death. Rescue of the defect(s) causing embryonic death should promote survival, thus permitting further evaluation of the roles that the gene plays later in the developmental process. Disruption of the gene for mouse hepatocyte growth factor/scatter factor (HGF/SF) leads to middle-stage embryonic lethality because of a defect in placental development. Here we report that a single injection of HGF/SF at embryonic day 9.5 (E9.5) into the amniotic cavity of HGF/SF(-/-) embryos rescued the placental defect and resulted in the survival of the embryos until term. Histological analysis suggested that HGF/SF is also required at the late stage of development for tissue organogenesis. Thus, injection of a secreted factor can be a useful method to rescue the defects causing embryonic lethality.     

Rhodopsin signaling and organization in heterozygote rhodopsin knockout mice

Rhodopsin (Rho) resides within internal membrane structures called disc membranes that are found in the rod outer segments (ROS) of photoreceptors in the retina. Rho expression is essential for formation of ROS, which are absent in knockout Rho-/- mice. ROS of mice heterozygous for the Rho gene deletion (Rho+/-) may have a lower Rho density than wild type (WT) membranes, or the ROS structure may be reduced in size due to lower Rho expression. Here, we present evidence that the smaller volume of ROS from heterozygous mice is most likely responsible for observed electrophysiological response differences. In Rho+/- mice as compared with age-matched WT mice, the length of ROS was shorter by 30-40%, and the average diameter of ROS was reduced by approximately 20%, as demonstrated by transmission and scanning electron microscopy. Together, the reduction of the volume of ROS was approximately 60% in Rho+/- mice. Rho content in the eyes was reduced by approximately 43% and 11-cis-retinal content in the eye was reduced by approximately 38%, as determined by UV-visible spectroscopy and retinoid analysis, respectively. Transmission electron microscopy of negatively stained disc membranes from Rho+/- mice indicated a typical morphology apart from the reduced size of disc diameter. Power spectra calculated from disc membrane regions on such electron micrographs displayed a diffuse ring at approximately 4.5 nm(-1), indicating paracrystallinity of Rho. Atomic force microscopy of WT and Rho+/- disc membranes revealed, in both cases, Rho organized in paracrystalline and raftlike structures. From these data, we conclude that the differences in physiological responses measured in WT and Rho+/- mice are due to structural changes of the whole ROS and not due to a lower density of Rho.     

Loss of ATM kinase activity leads to embryonic lethality in mice

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.     

Exaggerated effect of fluvoxamine in heterozygote serotonin transporter knockout mice

Clearance rates for serotonin (5-HT) in heterozygote (+/-) and homozygote (-/-) serotonin transporter (5-HTT) knockout (KO) mice have not been determined in vivo. Moreover, the effect of selective serotonin reuptake inhibitors (SSRIs) on 5-HT clearance in these mice has not been examined. In this study, the rate of clearance of exogenously applied 5-HT was measured in the CA3 region of the hippocampus of anesthetized mice using high-speed chronoamperometry. Compared with wild-type mice, the maximal rate of 5-HT clearance from extracellular fluid (ECF) was decreased in heterozygotes and more markedly so in KO mice. Heterozygote mice were more sensitive to the 5-HT uptake inhibitor, fluvoxamine, resulting in longer clearance times for 5-HT than in wild-type mice; as expected, the KO mice were completely unresponsive to fluvoxamine. There were no associated changes in norepinephrine transporter density, nor was there an effect of the norepinephrine uptake inhibitor, desipramine, on 5-HT clearance in any genotype. Thus, adaptive changes in the norepinephrine transport system do not occur in the CA3 region of hippocampus as a consequence of 5-HTT KO. These data highlight the potential of the heterozygote 5-HTT mutant mice to model the dynamic in vivo consequences of the human 5-HTT polymorphism.     

Analysis of spontaneous early embryonic lethality in mice

The incidence of spontaneously occurring deciduomata is considerably higher in T-stock than in (C3H X C57BL)F1 females. The basis for this difference was studied in vivo, by means of embryo transplantation procedure, and in vitro, by means of short-term embryo culture. Both studies indicate that strain differences in the incidence of spontaneous deciduomata may be largely, if not wholly, accounted for by genetic differences between embryos themselves expressed in terms of the rate of development during the preimplantation period and in the ability to survive the preimplantation and early implantation environment.     

Early embryonic lethality of mice lacking POLD2

As a highly conserved DNA polymerase (Pol), Pol δ plays crucial roles in chromosomal DNA synthesis and various DNA repair pathways. However, the function of POLD2, the second small subunit of DNA Pol δ (p50 subunit), has not been characterized in vivo during mammalian development. Here, we report for the first time, the essential role of subunit POLD2 during early murine embryogenesis. Although Pold2 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at gastrulation stages. Outgrowth assays reveal that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Notably, these phenotypes can be recapitulated by small interfering RNA (siRNA)-mediated knockdown, which also exhibit slowed cellular proliferation together with skewed primitive endoderm and epiblast allocation during the second cell lineage specification. In summary, our study demonstrates that POLD2 is essential for the earliest steps of mammalian development, and the retarded proliferation and embryogenesis may also alter the following cell lineage specifications in the mouse blastocyst embryos.     

Early embryonic lethality of H ferritin gene deletion in mice

Ferritin molecules play an important role in the control of intracellular iron distribution and in the constitution of long term iron stores. In vitro studies on recombinant ferritin subunits have shown that the ferroxidase activity associated with the H subunit is necessary for iron uptake by the ferritin molecule, whereas the L subunit facilitates iron core formation inside the protein shell. However, plant and bacterial ferritins have only a single type of subunit which probably fulfills both functions. To assess the biological significance of the ferroxidase activity associated with the H subunit, we disrupted the H ferritin gene (Fth) in mice by homologous recombination. Fth(+/-) mice are healthy, fertile, and do not differ significantly from their control littermates. However, Fth(-/-) embryos die between 3.5 and 9.5 days of development, suggesting that there is no functional redundancy between the two ferritin subunits and that, in the absence of H subunits, L ferritin homopolymers are not able to maintain iron in a bioavailable and nontoxic form. The pattern of expression of the wild type Fth gene in 9.5-day embryos is suggestive of an important function of the H ferritin gene in the heart.     

Dynamin-related protein 1 heterozygote knockout mice do not have synaptic and mitochondrial deficiencies

The objective of this study was to elucidate the effect of partial reduction of the mitochondrial fission protein, dynamin-related protein 1 (Drp1) on mitochondrial activity and synaptic viability. Recent knockout studies of Drp1 revealed that homozygote Drp1 knockout mice are embryonic lethal due to reduced mitochondrial fission, and that this reduced fission leads to developmental defects in the brain. In contrast, heterozygote Drp1 knockout mice appear to be normal in terms of lifespan, fertility, and viability, and phenotypically these animals are not different from wild-type mice. However, the effects of partial Drp1 reduction on mitochondrial function and synaptic activity are not well understood. In the present study, we sought to characterize synaptic, dendritic and mitochondrial proteins, and mitochondrial function and GTPase enzymatic activity, in Drp1 heterozygote knockout mice. Interestingly, we found no significant changes in synaptic, dendritic, and mitochondrial proteins in the Drp1 heterozygote knockout mice compared to the wild-type mice. Further, mitochondrial function and GTPase enzymatic activity appeared to be normal. However, H(2)O(2) and lipid peroxidation levels were significantly reduced in the Drp1 heterozygote knockout mice compared to the wild-type mice. These findings suggest that partial Drp1 reduction does not affect mitochondrial and synaptic viability and may have therapeutic use in treating patients with Alzheimer's disease and Huntington's disease.     

Deletion of SNAP-23 results in pre-implantation embryonic lethality in mice

SNARE-mediated membrane fusion is a pivotal event for a wide-variety of biological processes. SNAP-25, a neuron-specific SNARE protein, has been well-characterized and mouse embryos lacking Snap25 are viable. However, the phenotype of mice lacking SNAP-23, the ubiquitously expressed SNAP-25 homolog, remains unknown. To reveal the importance of SNAP-23 function in mouse development, we generated Snap23-null mice by homologous recombination. We were unable to obtain newborn SNAP-23-deficient mice, and analysis of pre-implantation embryos from Snap23(Δ/wt) matings revealed that Snap23-null blastocysts were dying prior to implantation at embryonic day E3.5. Thus these data reveal a critical role for SNAP-23 during embryogenesis.     

Mice lacking connexin45 conditionally in cardiac myocytes display embryonic lethality similar to that of germline knockout mice without endocardial cushion defect

The gap junction protein connexin45-deficient (Cx45-KO) mice die shortly after the hearts begin to beat. In addition to the heart defect, they also show defective vascular development which may be closely related with the cardiac phenotype. Therefore, we created mice whose floxed-Cx45 locus could be removed conditionally. We utilized cardiac alpha-actin-Cre transgenic mice to investigate the specific cardiac muscular function of Cx45 in vivo. The resultant conditional mutants were lethal, showing conduction block similar to that of the Cx45-KO mice. Unlike Cx45-KO, development of the endocardial cushion was not disrupted in the conditional mutants. X-gal staining was detected in the embryonic cardiac myocytes as a hallmark of Cre-loxP mediated floxed-Cx45 deletion. These results reconfirm the requirement of Cx45 for developing cardiac myocytes. These also indicate that establishing the first contractions is a crucial task for the early hearts.     

Kinase-dead ATM protein causes genomic instability and early embryonic lethality in mice

Studies on cell division traditionally focus on the mechanisms of chromosome segregation and cytokinesis, yet we know comparatively little about how organelles segregate. Analysis of organelle partitioning in asymmetrically dividing cells has provided insights into the mechanisms through which cells control organelle distribution. Interestingly, these studies have revealed that segregation mechanisms frequently link organelle distribution to organelle growth and formation. Furthermore, in many cases, cells use organelles, such as the endoplasmic reticulum and P granules, as vectors for the segregation of information. Together, these emerging data suggest that the coordination between organelle growth, division, and segregation plays an important role in the control of cell fate inheritance, cellular aging, and rejuvenation, i.e., the resetting of age in immortal lineages.     

Early embryonic lethality of mice lacking the essential protein SNEV

SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.     

Differentiation diversity of mouse parthenogenetic embryonic stem cells in chimeric mice

Recent studies have illustrated multiple differentiation potentials of embryonic stem cells (ESCs), derived from parthenogenetic embryos, to various kinds of cells (all three embryonic germ layers). However, differentiation diversity of the parthenogenetic ESCs (PgESCs) in vivo remains to be elucidated. In the present study, we established mouse PgESC-lines and observed their contribution diversity in vivo by producing chimeric mice using embryos possessing single nucleotide polymorphisms of mitochondrial DNA (mtDNA) as hosts. Based on southern blot analysis using specific probes to detect the SNPs on mtDNA, PgESC-derived mtDNA were contained in many organs such as brain, lung, and heart of the chimeric mouse. We concluded that PgESCs contributed to various internal organs in vivo, and that they were also stably maintained in adult animals.     

Disruption of the phosphatidylserine decarboxylase gene in mice causes embryonic lethality and mitochondrial defects

Most of the phosphatidylethanolamine (PE) in mammalian cells is synthesized by two pathways, the CDP-ethanolamine pathway and the phosphatidylserine (PS) decarboxylation pathway, the final steps of which operate at spatially distinct sites, the endoplasmic reticulum and mitochondria, respectively. We investigated the importance of the mitochondrial pathway for PE synthesis in mice by generating mice lacking PS decarboxylase activity. Disruption of Pisd in mice resulted in lethality between days 8 and 10 of embryonic development. Electron microscopy of Pisd-/- embryos revealed large numbers of aberrantly shaped mitochondria. In addition, fluorescence confocal microscopy of Pisd-/- embryonic fibroblasts showed fragmented mitochondria. PS decarboxylase activity and mRNA levels in Pisd+/- tissues were approximately one-half of those in wild-type mice. However, heterozygous mice appeared normal, exhibited normal vitality, and the phospholipid composition of livers, testes, brains, and of mitochondria isolated from livers, was the same as in wild-type littermates. The amount and activity of a key enzyme of the CDP-ethanolamine pathway for PE synthesis, CTP:phosphoethanolamine cytidylyltransferase, were increased by 35-40 and 100%, respectively, in tissues of Pisd+/- mice, as judged by immunoblotting; PE synthesis from [3H]ethanolamine was correspondingly increased in hepatocytes. We conclude that the CDP-ethanolamine pathway in mice cannot substitute for a lack of PS decarboxylase during development. Moreover, elimination of PE production in mitochondria causes fragmented, misshapen mitochondria, an abnormality that likely contributes to the embryonic lethality.     

Cellular immunity and blocking serum activity in chimeric mice

Mice (9 CBA × T6, 6 T6, and 7 CBA) were irradiated and repopulated with foreign (BALB/c) bone marrow. Lymph node cells from 16 of 18 repopulated mice not showing signs of graft versus host disease (GVH), were cytotoxic to host type fibroblasts but not to BALB/c fibroblasts, and sera from the same mice could block lymphocyte mediated cytotoxicity. No blocking was seen with sera from three mice which had signs of GVH. LNC from the latter three mice were cytotoxic to recipient fibroblasts.It is suggested that the blocking effect detected in vitro may protect against GVH in vivo, but the relative importance of the blocking phenomenon as compared to other mechanisms is not yet settled.     

Placental failure and impaired vasculogenesis result in embryonic lethality for neuropathy target esterase-deficient mice

Age-dependent neurodegeneration resulting from widespread apoptosis of neurons and glia characterize the Drosophila Swiss Cheese (SWS) mutant. Neuropathy target esterase (NTE), the vertebrate homologue of SWS, reacts with organophosphates which initiate a syndrome of axonal degeneration. NTE is expressed in neurons and a variety of nonneuronal cell types in adults and fetal mice. To investigate the physiological functions of NTE, we inactivated its gene by targeted mutagenesis in embryonic stem cells. Heterozygous NTE(+/-) mice displayed a 50% reduction in NTE activity but underwent normal organ development. Complete inactivation of the NTE gene resulted in embryonic lethality, which became evident after gastrulation at embryonic day 9 postcoitum (E9). As early as E7.5, mutant embryos revealed growth retardation which did not reflect impaired cell proliferation but rather resulted from failed placental development; as a consequence, massive apoptosis within the developing embryo preceded its resorption. Histological analysis indicated that NTE is essential for the formation of the labyrinth layer and survival and differentiation of secondary giant cells. Additionally, impairment of vasculogenesis in the yolk sacs and embryos of null mutant conceptuses suggested that NTE is also required for normal blood vessel development.