Essential role of eIF5A-1 and deoxyhypusine synthase in mouse embryonic development

The eukaryotic initiation factor 5A (eIF5A) contains a polyamine-derived amino acid, hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed post-translationally by the addition of the 4-aminobutyl moiety from the polyamine spermidine to a specific lysine residue, catalyzed by deoxyhypusine synthase (DHPS), and subsequent hydroxylation by deoxyhypusine hydroxylase (DOHH). The eIF5A precursor protein and both of its modifying enzymes are highly conserved, suggesting a vital cellular function for eIF5A and its hypusine modification. To address the functions of eIF5A and the first modification enzyme, DHPS, in mammalian development, we knocked out the Eif5a or the Dhps gene in mice. Eif5a heterozygous knockout mice and Dhps heterozygous knockout mice were viable and fertile. However, homozygous Eif5a1 (gt/gt) embryos and Dhps (gt/gt) embryos died early in embryonic development, between E3.5 and E7.5. Upon transfer to in vitro culture, homozygous Eif5a (gt/gt) or Dhps (gt/gt) blastocysts at E3.5 showed growth defects when compared to heterozygous or wild type blastocysts. Thus, the knockout of either the eIF5A-1 gene (Eif5a) or of the deoxyhypusine synthase gene (Dhps) caused early embryonic lethality in mice, indicating the essential nature of both eIF5A-1 and deoxyhypusine synthase in mammalian development.     

Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality

Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2beta(-/-) morula development stopped after the blastocyst stage. Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2beta is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2beta. Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution.     

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.     

Extracellular domain of CD98hc is required for early murine development

Background

The multifunctional protein CD98 heavy chain (CD98hc, Slc3a2) associates with integrin β1 through its cytoplasmic and transmembrane domains and the CD98hc-mediated integrin signaling is required for maintenance of ES cell proliferation. CD98hc-null mice exhibit early post-implantation lethality similar to integrin β1-null mice, supporting the importance of its interaction with integrin β1. On the other hand, the extracellular domain of CD98hc interacts with L-type amino acid transporters (LATs) and is essential for appropriate cell surface distribution of LATs. LATs mediate the transport of amino acids and other molecules such as thyroid hormone. In this respect, CD98hc may also affect development via these transporters.

Results

In this study, mice were generated from embryonic stem (ES) cell line (PST080) harboring a mutant CD98hc allele (CD98hc^Δ/+). Expression of the CD98hc mutant allele results in ΔCD98hc-β geo fusion protein where extracellular C-terminal 102 amino acids of CD98hc are replaced with β geo. Analyses of PST080 ES cells as well as reconstituted frog oocytes demonstrated that ΔCD98hc-β geo fusion protein preserved its ability to interact with integrin β1 although this mutant protein was hardly localized on the cell surface. These findings suggest that ΔCD98hc-β geo protein can mediate integrin signaling but cannot support amino acid transport through LATs. CD98hc^Δ/+ mice were normal. Although some of the implantation sites lacked embryonic component at E9.5, all the implantation sites contained embryonic component at E7.5. Thus, CD98hc^Δ/Δ embryos are likely to die between E7.5 and E9.5.

Conclusions

Considering that CD98hc complete knockout (CD98hc^-/-) embryos are reported to die shortly after implantation, our findings suggest potential stage-specific roles of CD98hc in murine embryonic development. CD98hc may be essential for early post-implantation development by regulating integrin-dependent signaling, while the other function of CD98hc as a component of amino acid transporters may be required for embryonic development at later stages.     

N-myristoyltransferase 1 is essential in early mouse development

N-Myristoyltransferase (NMT) transfers myristate to an amino-terminal glycine of many eukaryotic proteins. In yeast, worms, and flies, this enzyme is essential for viability of the organism. Humans and mice possess two distinct but structurally similar enzymes, NMT1 and NMT2. These two enzymes have similar peptide specificities, but no one has examined the functional importance of the enzymes in vivo. To address this issue, we performed both genetic and biochemical studies. Northern blots with RNA from adult mice and in situ hybridization studies of day 13.5 embryos revealed widespread expression of both Nmt1 and Nmt2. To determine whether the two enzymes are functionally redundant, we generated Nmt1-deficient mice carrying a beta-galactosidase marker gene. beta-Galactosidase staining of tissues from heterozygous Nmt1-deficient (Nmt1+/-) mice and embryos confirmed widespread expression of Nmt1. Intercrosses of Nmt1+/- mice yielded no viable homozygotes (Nmt1-/-), and heterozygotes were born at a less than predicted frequency. Nmt1-/- embryos died between embryonic days 3.5 and 7.5. Northern blots revealed lower levels of Nmt2 expression in early development than at later time points, a potential explanation for the demise of Nmt1-/- embryos. To explore this concept, we generated Nmt1-/- embryonic stem (ES) cells. The Nmt2 mRNA could be detected in Nmt1-/- ES cells, but the total NMT activity levels were reduced by approximately 95%, suggesting that Nmt2 contributes little to total enzyme activity levels in these early embryo cells. The Nmt1-/- ES cells were functionally abnormal; they yielded small embryoid bodies in in vitro differentiation experiments and did not contribute normally to organogenesis in chimeric mice. We conclude that Nmt1 is not essential for the viability of mammalian cells but is required for development, likely because it is the principal N-myristoyltransferase in early embryogenesis.     

Inactivation of Cdc7 kinase in mouse ES cells results in S-phase arrest and p53-dependent cell death

Cdc7-related kinases play essential roles in the initiation of yeast DNA replication. We show that mice lacking murine homologs of Cdc7 (muCdc7) genes die between E3.5 and E6.5. We have established a mutant embryonic stem (ES) cell line lacking the muCdc7 genes in the presence of a loxP-flanked transgene expressing muCdc7 cDNA. Upon removal of the transgene by Cre recombinase, mutant ES cells cease DNA synthesis, arresting growth with S-phase DNA content, and generate nuclear Rad51 foci, followed by cell death with concomitant increase in p53 protein levels. Inhibition of p53 leads to partial rescue of muCdc7(-/-) ES cells from cell death. muCdc7(-/-)p53(-/-) embryos survive up to E8.5, and their blastocysts generate inner cell mass of a significant size in vitro, whereas those of the muCdc7(-/-)p53(+/-) embryos undergoes complete degeneration. These results demonstrate that, in contrast to cell cycle arrest at the G(1)/S boundary observed in yeasts, loss of Cdc7 in ES cells results in rapid cessation of DNA synthesis within S phase, triggering checkpoint responses leading to recombinational repair and p53-dependent cell death.     

Fibrillarin is essential for early development and required for accumulation of an intron-encoded small nucleolar RNA in the mouse

Fibrillarin, a protein component of C/D box small nucleolar ribonucleoproteins (snoRNPs), directs 2'-O-methylation of rRNA and is also involved in other aspects of rRNA processing. A gene trap screen in embryonic stem (ES) cells resulted in an insertion mutation in the fibrillarin gene. This insertion generated a fusion protein that contained the N-terminal 132 amino acids of fibrillarin fused to a beta-galactosidase-neomycin phosphotransferase reporter. As a result, the N-terminal GAR domain was present in the fusion protein but the methyltransferase-like domain was missing. The ES cell line with the targeted fibrillarin allele was transmitted through the mouse germ line, creating heterozygous animals. Western blot analyses showed a reduction in fibrillarin protein levels in the heterozygous knockout animals. Animals homozygous for the mutation were inviable, and massive apoptosis was observed in early Fibrillarin(-/-) embryos, showing that fibrillarin is essential for development. Fibrillarin(+/-) live-born mice displayed no obvious growth defect, but heterozygous intercrosses revealed a reduced ratio of +/- to +/+ mice, showing that some of the Fibrillarin heterozygous embryos die in utero. Analyses of tissue samples and cultured embryonic fibroblasts showed no discernible alteration in pre-rRNA processing or the level of the U3 snoRNA. However, the level of the intron-encoded box C/D snoRNA U76 was clearly reduced. This suggests a high requirement for snoRNA synthesis during an early stage in development.     

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.     

PINCH1 plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes

PINCH1, an adaptor protein composed of five LIM domains, mediates protein-protein interactions and functions as a component of the integrin-integrin-linked kinase (ILK) complex. The integrin-ILK signaling complex plays a pivotal role in cell motility, proliferation, and survival during embryonic development of many animal species. To elucidate the physiological function of PINCH1 in mouse embryonic development, we have deleted the mouse PINCH1 gene by homologous recombination. Mice heterozygous for PINCH1 are viable and indistinguishable from wild-type littermates. However, no viable homozygous offspring were observed from PINCH1+/- intercrosses. Histological analysis of homozygous mutant embryos revealed that they had a disorganized egg cylinder by E5.5, which degenerated by E6.5. Furthermore, E5.5 PINCH1-/- embryos exhibited decreased cell proliferation and excessive cell death. We have also generated and analyzed mice in which PINCH1 has been specifically deleted in ventricular cardiomyocytes. These mice exhibit no basal phenotype, with respect to mouse survival, cardiac histology, or cardiac function as measured by echocardiography. Altogether, these data indicate that PINCH1 plays an essential role in early murine embryonic development but is dispensable in ventricular cardiomyocytes.     

The zinc finger transcription factor 191 is required for early embryonic development and cell proliferation

Human zinc finger protein 191 (ZNF191/ZNF24) was cloned and characterized as a SCAN family member, which shows 94% identity to its mouse homologue zinc finger protein 191 (Zfp191). ZNF191 can specifically interact with an intronic polymorphic TCAT repeat (HUMTH01) in the tyrosine hydroxylase (TH) gene. Allelic variations of HUMTH01 have been stated to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. Zfp191 is widely expressed during embryonic development and in multiple tissues and organs in adult. To investigate the functions of Zfp191 in vivo, we have used homologous recombination to generate mice that are deficient in Zfp191. Heterozygous Zfp191(+/-) mice are normal and fertile. Homozygous Zfp191(-/-) embryos are severely retarded in development and die at approximately 7.5 days post-fertilization. Unexpectedly, in Zfp191(-/-) and Zfp191(+/-) embryos, TH gene expression is not affected. Blastocyst outgrowth experiments and the RNA interference-mediated knockdown of ZNF191 in cultured cells revealed an essential role for Zfp191 in cell proliferation. In further agreement with this function, no viable Zfp191(-/-) cell lines were obtained by derivation of embryonic stem (ES) cells from blastocysts of Zfp191(+/-) intercrosses or by forced homogenotization of heterozygous ES cells at high concentrations of G418. These data show that Zfp191 is indispensable for early embryonic development and cell proliferation.     

CUL-4A is critical for early embryonic development

Ubiquitin-mediated degradation targets cell cycle regulators for proteolysis. Much of the ubiquitin pathway's substrate specificity is conferred by E3 ubiquitin ligases, and cullins are core components of some E3s. CUL-4A encodes one of six mammalian cullins and is amplified and/or overexpressed in breast cancer, which suggests a role in regulating cell cycle progression. To examine CUL-4A's physiologic function, we generated a CUL-4A deletion mutation in mice. No viable CUL-4A(-/-) pups and no homozygous mutant embryos as early as 7.5 days postcoitum (dpc) were recovered. However, CUL-4A(-/-) blastocysts are viable, hatch, form an inner cell mass and trophectoderm, and implant (roughly 4.5 dpc), indicating that CUL-4A(-/-) embryos die between 4.5 and 7.5 dpc. Despite 87% similarity between the Cul-4A and Cul-4B cullins, the CUL-4A(-/-) lethal phenotype indicates that CUL-4A has one or more distinct function(s). Surprisingly, 44% fewer heterozygous pups were recovered than expected by Mendelian genetics, indicating that many heterozygous embryos also die during gestation due to haploinsufficiency. Taken together, our findings indicate that appropriate CUL-4A expression is critical for early embryonic development.     

The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse.

Homozygosity for a null mutation in the scl gene causes mid-gestational embryonic lethality in the mouse due to failure of development of primitive hematopoiesis. Whilst this observation established the role of the scl gene product in primitive hematopoiesis, the death of the scl null embryos precluded analysis of the role of scl in later hematopoietic development. To address this question, we created embryonic stem cell lines with a homozygous null mutation of the scl gene (scl-/-) and used these lines to derive chimeric mice. Analysis of the chimeric mice demonstrates that the scl-/- embryonic stem cells make a substantial contribution to all non-hematopoietic tissues but do not contribute to any hematopoietic lineage. These observations reveal a crucial role for the scl gene product in definitive hematopoiesis. In addition, in vitro differentiation assays with scl-/- embryonic stem cells showed that the scl gene product was also required for formation of hematopoietic cells in this system.     

Disruption of the pelota gene causes early embryonic lethality and defects in cell cycle progression

Mutations in either the Drosophila melanogaster pelota or pelo gene or the Saccharomyces cerevisiae homologous gene, DOM34, cause defects of spermatogenesis and oogenesis in Drosophila, and delay of growth and failure of sporulation in yeast. These phenotypes suggest that pelota is required for normal progression of the mitotic and meiotic cell cycle. To determine the role of the pelota in mouse development and progression of cell cycle, we have established a targeted disruption of the mouse PELO: Heterozygous animals are variable and fertile. Genotyping of the progeny of heterozygous intercrosses shows the absence of Pelo(-/-) pups and suggests an embryo-lethal phenotype. Histological analyses reveal that the homozygous Pelo deficient embryos fail to develop past day 7.5 of embryogenesis (E7.5). The failure of mitotic active inner cell mass of the Pelo(-/-) blastocysts to expand in growth after 4 days in culture and the survival of mitotic inactive trophoplast indicate that the lethality of Pelo-null embryos is due to defects in cell proliferation. Analysis of the cellular DNA content reveals the significant increase of aneuploid cells in Pelo(-/-) embryos at E7.5. Therefore, the percent increase of aneuploid cells at E7.5 may be directly responsible for the arrested development and suggests that Pelo is required for the maintenance of genomic stability.     

G2 arrest and impaired nucleocytoplasmic transport in mouse embryos lacking the proto-oncogene CAN/Nup214.

The vertebrate nucleopore complex (NPC) is a 125 MDa multiprotein assembly that mediates nucleocytoplasmic transport. One of its components, CAN/Nup214, is an FXFG repeat-containing protein known to be involved in myeloid leukemia in humans. We have devised a powerful genetic approach, using maternally derived protein in murine null embryos, to show that CAN/ Nup214 is essential for NPC function in vivo. We demonstrate that CAN-/- mouse embryonic stem (ES) cells are not viable and that CAN-/- embryos die in utero between 4.0 and 4.5 days postcoitum, following the depletion of their CAN from maternal sources. In 3.5-day-old mutant embryos, cultured in vitro, progressive depletion of CAN leads to cell cycle arrest in G2 phase, and eventually to blastocoel collapse, impaired NLS-mediated protein uptake and nuclear accumulation of polyadenylated RNA. Remarkably, these defective CAN-depleted embryos do not display any gross morphological abnormalities in their nuclear envelopes or NPCs. Our data suggest that CAN is critical to cell cycle progression and required for both nuclear protein import and mRNA export.     

The targeted disruption of the CD98 gene results in embryonic lethality

CD98 is one of the important molecules for development, cell differentiation, cell proliferation, and regulation of cellular function. In this study, CD98 heavy chain (HC) knockout mice were produced and analyzed. Five targeted ES clones were obtained and colony frequency was about 2%. One (clone 113) of the five heterozygous ES cell clones had undergone aberrant recombination at the 5' side. The aberrant recombination happened at the site between second intron and 5' arm. All lines from correctly targeted clones could not transmit the mutated allele to spermatozoa. The mutated allele derived from the aberrant targeted clone was transmitted to the progeny. However, none of the F2 mice was homozygous for the CD98 mutation, indicating that the targeted disruption of the CD98 gene results in embryonic lethality. The point of embryonic lethality is considered to be between 3.5 and 9.5 dps. These findings indicate that CD98 molecules are essential for mouse embryogenesis.     

Generation of normal lymphocyte populations by Rb-deficient embryonic stem cells

BACKGROUND: Mice homozygous for a loss-of-function mutation of the recombination-activating gene-2 (RAG 2), which is required for the rearrangement of antigen receptor genes, do not produce mature B and T lymphocytes. But chimeric mice that result from injection of normal embryonic stem (ES) cells into blastocysts from RAG2-deficient mice develop normal mature lymphocyte populations, all of which are derived from the injected ES cells; we have called this process RAG2-deficient blastocyst complementation. Using ES cells with homozygous mutations, RAG-2-deficient blastocyst complementation could provide a physiological assay with which to determine the potential role of almost any gene in the development and/or function of lymphocytes. To test the general utility of this system, we have used it to test the differentiation-potential of ES cells that harbor homozygous loss-of function mutations of their retinoblastoma susceptibility (Rb) gene loci. We chose Rb for this analysis because of its widespread function in the control of the cell cycle and cell differentiation, the adverse effect of homozygous germline mutations of Rb on hematopoiesis in fetal liver, and the embryonic lethality that results when the homozygous Rb mutation is introduced into the germline. RESULTS: Homozygous Rb mutant ES cells can develop into phenotypically normal, mature B and T lymphocytes in the RAG-2-deficient background. Strikingly, Rb-deficient B and T cells do not have major defects in either activation or function. CONCLUSION: We have demonstrated the efficacy of the RAG-2-deficient blastocyst complementation system for evaluating the role of critical genes in lymphocyte development. Our results indicate that Rb expression is not intrinsically required for B-cell or T-cell function, despite the normally high levels of Rb expressed in lymphoid cells.