EMG1 is essential for mouse pre-implantation embryo development

Background  

Essential for mitotic growth 1 (EMG1) is a highly conserved nucleolar protein identified in yeast to have a critical function in ribosome biogenesis. A mutation in the human EMG1 homolog causes Bowen-Conradi syndrome (BCS), a developmental disorder characterized by severe growth failure and psychomotor retardation leading to death in early childhood. To begin to understand the role of EMG1 in mammalian development, and how its deficiency could lead to Bowen-Conradi syndrome, we have used mouse as a model. The expression of Emg1 during mouse development was examined and mice carrying a null mutation for Emg1 were generated and characterized.     

Ubiquinone is necessary for mouse embryonic development but is not essential for mitochondrial respiration

Ubiquinone (UQ) is a lipid found in most biological membranes and is a co-factor in many redox processes including the mitochondrial respiratory chain. UQ has been implicated in protection from oxidative stress and in the aging process. Consequently, it is used as a dietary supplement and to treat mitochondrial diseases. Mutants of the clk-1 gene of the nematode Caenorhabditis elegans are fertile and have an increased life span, although they do not produce UQ but instead accumulate a biosynthetic intermediate, demethoxyubiquinone (DMQ). DMQ appears capable to partially replace UQ for respiration in vivo and in vitro. We have produced a vertebrate model of cells and tissues devoid of UQ by generating a knockout mutation of the murine orthologue of clk-1 (mclk1). We find that mclk1-/- embryonic stem cells and embryos accumulate DMQ instead of UQ. As in the nematode mutant, the activity of the mitochondrial respiratory chain of -/- embryonic stem cells is only mildly affected (65% of wild-type oxygen consumption). However, mclk1-/- embryos arrest development at midgestation, although earlier developmental stages appear normal. These findings indicate that UQ is necessary for vertebrate embryonic development but suggest that mitochondrial respiration is not the function for which UQ is essential when DMQ is present.     

Beta-catenin is essential for lamination but not neurogenesis in mouse retinal development

During vertebrate retinal development, the seven retinal cell types differentiate sequentially from a single population of retinal progenitor cells (RPCs) and organize themselves into a distinct laminar structure. The purpose of this study was to determine whether beta-catenin, which functions both as a nuclear effector for the canonical Wnt signaling pathway and as a regulator of cell adhesion, is required for retinal neurogenesis or lamination. We used the Cre-loxP system to either eliminate beta-catenin or to express a constitutively active form during retinal neurogenesis. Eliminating beta-catenin did not affect cell differentiation, but did result in the loss of the radial arrangement of RPCs and caused abnormal migration of differentiated neurons. As a result, the laminar structure was massively disrupted in beta-catenin-null retinas, although all retinal cell types still formed. In contrast to other neural tissues, eliminating beta-catenin did not significantly reduce the proliferation rate of RPCs; likewise, activating beta-catenin ectopically in RPCs did not result in overproliferation, but loss of neural retinal identity. These results indicate that beta-catenin is essential during retinal neurogenesis as a regulator of cell adhesion but not as a nuclear effector of the canonical Wnt signaling pathway. The results further imply that retinal lamination and retinal cell differentiation are genetically separable processes.     

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.     

Vitamin E is essential for mouse placentation but not for embryonic development itself

Vitamin E (alpha-tocopherol) was discovered 80 years ago to be an indispensable nutrient for reproduction in the female. However, it has not been clarified when or where vitamin E is required during pregnancy. We examined the role of alpha-tocopherol in pregnancy using alpha-tocopherol transfer protein (Ttpa)-deficient mice fed specific alpha-tocopherol diets that led to daily, measurable change in plasma alpha-tocopherol levels from nearly normal to almost undetectable levels. A dietary supplement of alpha-tocopherol to pregnant Ttpa-/- (homozygous null) mice was shown to be essential for maintenance of pregnancy from 6.5 to 13.5 days postcoitum but found not to be crucial before or after this time span, which corresponds to initial development and maturation of the placenta. In addition, exposure to a low alpha-tocopherol environment after initiation of placental formation might result in necrosis of placental syncytiotrophoblast cells, followed by necrosis of fetal blood vessel endothelial cells. When Ttpa(-/-)-fertilized eggs were transferred into Ttpa+/+ (wild-type) recipients, plasma alpha-tocopherol concentrations in the Ttpa-/- fetuses were below the detection limit but the fetuses grew normally. These results indicate that alpha-tocopherol is indispensable for the proliferation and/or function of the placenta but not necessary for development of the embryo itself.     

Mouse paraxial protocadherin is expressed in trunk mesoderm and is not essential for mouse development

Paraxial protocadherin (PAPC) is a cell adhesion molecule that marks cells undergoing convergence-extension cell movements in Xenopus and zebrafish gastrulating embryos. Here a mouse homologue (mpapc) was identified and characterized. During early- to mid-gastrulation, mpapc is expressed in the primitive streak as the trunk mesoderm undergoes morphogenetic cell movements. At head-fold stage mpapc expression becomes localized to paraxial regions in which somites are formed in the segmental plate. At later stages, mpapc displays a complex expression pattern in cerebral cortex, olfactory bulb, inferior colliculus, and in longitudinal stripes in hindbrain. To analyze the effect of the loss of PAPC function during mouse development, a null allele of the mouse papc gene was generated. Homozygous animals show no defects in their skeleton and are viable and fertile.     

Cerberus-like is a secreted BMP and nodal antagonist not essential for mouse development

Mouse cerberus-like (cer-l) is a member of the Cerberus/Dan family of secreted factors. As other members of this family of proteins, Cer-l functions in the extracellular space, inhibiting signaling molecules. Here we show that the neural-inducing and mesoderm-inhibiting activities of Cer-l result from specific binding to BMP and Nodal molecules, respectively. These properties resemble the ones from the related factor Xenopus Cerberus. However, Xenopus Cerberus in addition to BMP4 and Nodal also binds to and inhibits Wnt proteins. We show that Cer-l does not directly inhibit Wnt signals. A null allele of the mouse Cer-l gene was generated by targeted inactivation in ES cells. Homozygous embryos show no anterior patterning defects, are born alive, and are fertile. Since mouse Cer-l and Xenopus Cerberus differ in biochemical activities, we propose the existence of additional members of this family of inhibitors, which may compensate for the loss of cer-l.     

Ribosomal protein S1 is not essential for the trans-translation machinery

In eubacteria, ribosome stalling during protein synthesis is rescued by a tmRNA-derived trans-translation system. Because ribosomal protein S1 specifically binds to tmRNA with high affinity, it is considered to be involved in the trans-translation system. However, the role of S1 in trans-translation is still unclear. To study the function of S1 in the trans-translation system, we constructed an S1-free cell-free translation system. We found that trans-translation proceeded even in the absence of S1. Addition of S1 into the S1-free system did not affect trans-translation efficiency. These results suggest that S1 does not play a role in the trans-translation machinery.     

The normal phenotype of Pmm1-deficient mice suggests that Pmm1 is not essential for normal mouse development

Phosphomannomutases (PMMs) are crucial for the glycosylation of glycoproteins. In humans, two highly conserved PMMs exist: PMM1 and PMM2. In vitro both enzymes are able to convert mannose-6-phosphate (mannose-6-P) into mannose-1-P, the key starting compound for glycan biosynthesis. However, only mutations causing a deficiency in PMM2 cause hypoglycosylation, leading to the most frequent type of the congenital disorders of glycosylation (CDG): CDG-Ia. PMM1 is as yet not associated with any disease, and its physiological role has remained unclear. We generated a mouse deficient in Pmm1 activity and documented the expression pattern of murine Pmm1 to unravel its biological role. The expression pattern suggested an involvement of Pmm1 in (neural) development and endocrine regulation. Surprisingly, Pmm1 knockout mice were viable, developed normally, and did not reveal any obvious phenotypic alteration up to adulthood. The macroscopic and microscopic anatomy of all major organs, as well as animal behavior, appeared to be normal. Likewise, lectin histochemistry did not demonstrate an altered glycosylation pattern in tissues. It is especially striking that Pmm1, despite an almost complete overlap of its expression with Pmm2, e.g., in the developing brain, is apparently unable to compensate for deficient Pmm2 activity in CDG-Ia patients. Together, these data point to a (developmental) function independent of mannose-1-P synthesis, whereby the normal knockout phenotype, despite the stringent conservation in phylogeny, could be explained by a critical function under as-yet-unidentified challenge conditions.     

Hypomethylation of paternal DNA in the late mouse zygote is not essential for development

Global demethylation of DNA which marks the onset of development occurs asynchronously in the mouse; paternal DNA is demethylated at the the zygote stage, whereas maternal DNA is demethylated later in development. The biological function of such asymmetry and its underlying mechanisms are currently unknown. To test the hypothesis that the early demethylation of male DNA may be associated with protamine-histone exchange, we ,used round spermatids, whose DNA is still associated with histones, for artificial fertilization (round spermatid injection or ROSI), and compared the level of methylation of metaphase chromosomes in the resulting zygotes with the level of methylation in zygotes obtained after fertilization using mature sperm heads (intracytoplasmic sperm injection or ICSI). In contrast to ICSI-derived zygotes, ROSI-derived zygotes possessed only slightly demethylated paternal DNA. Both types of zygotes developed to term with similar rates which shows that hypomethylation of paternal DNA at the zygotic metaphase is not essential for full development in mice. Incorporation of exogenously expressed histone H2BYFP into paternal pronuclei was significantly higher in ICSI-derived zygotes than in ROSI-derived zygotes. Surprisingly, in the latter the incorporation of histone H2BYFP into the paternal pronucleus was still significantly higher than into the maternal pronucleus, suggesting that some exchange of chromatin-associated proteins occurs not only after ICSI but also after ROSI. This may explain why after ROSI, some transient demethylation of paternal DNA occurs early after fertilization, thus providing support for the hypothesis regarding the link between paternal DNA demethylation and protamine/histone exchange.     

Zinc finger protein sall2 is not essential for embryonic and kidney development

SALL/Sall is a mammalian homolog of the Drosophila region-specific homeotic gene spalt (sal), and heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. We earlier reported that mice deficient in Sall1 die in the perinatal period and that kidney agenesis or severe dysgenesis are present. We have now generated mice lacking Sall2, another Sall family gene. Although Sall2 is expressed mostly in an overlapping fashion versus that of Sall1, Sall2-deficient mice show no apparent abnormal phenotypes. Morphology and gene expression patterns of the mutant kidney were not affected. Mice lacking both Sall1 and Sall2 show kidney phenotypes comparable to those of Sall1 knockout, thereby demonstrating the dispensable roles of Sall2 in embryonic and kidney development.     

The murine stanniocalcin 1 gene is not essential for growth and development

The stanniocalcin 1 (STC1) gene is expressed in a wide variety of tissues, including the kidney, prostate, thyroid, bone, and ovary. STC1 protein is considered to have roles in many physiological processes, including bone development, reproduction, wound healing, angiogenesis, and modulation of inflammatory response. In fish, STC1 is a hormone that is secreted by the corpuscles of Stannius and is involved in calcium and phosphate homeostasis. To determine the role of STC1 in mammals, we generated Stc1-null mice by gene targeting. The number of Stc1-/- mice obtained was in accordance with Mendelian ratios, and both males and females produced offspring normally. No anatomical or histological abnormalities were detected in any tissues. Our results demonstrated that Stc1 function is not essential for growth or reproduction in the mouse.     

CIB1 is essential for mouse spermatogenesis

CIB1 is a 22-kDa calcium binding, regulatory protein with approximately 50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of effectors, such as integrin alphaIIb, PAK1, and polo-like kinases, in different tissues. However, the in vivo functions of CIB1 are not well understood. To elucidate the function of CIB1 in whole animals, we used homologous recombination in embryonic stem cells to generate Cib1(-/-) mice. Although Cib1(-/-) mice grow normally, the males are sterile due to disruption of the haploid phase of spermatogenesis. This is associated with reduced testis size and numbers of germ cells in seminiferous tubules, increased germ cell apoptosis, and the loss of elongated spermatids and sperm. Cib1(-/-) testes also show increased mRNA and protein expression of the cell cycle regulator Cdc2/Cdk1. In addition, mouse embryonic fibroblasts (MEFs) derived from Cib1(-/-) mice exhibit a much slower growth rate compared to Cib1(+/+) MEFs, suggesting that CIB1 regulates the cell cycle, differentiation of spermatogenic germ cells, and/or differentiation of supporting Sertoli cells.     

The cerberus-related gene, Cerr1, is not essential for mouse head formation

The Xenopus cerberus gene encodes a secreted factor expressed in the Spemann organizer that can cause ectopic head formation when its mRNA is injected into Xenopus embryos. In mouse, the cerberus-related gene, Cerr1, is expressed in the anterior mesendoderm that underlies the presumptive anterior neural plate and its expression is downregulated in Lim1 headless embryos. To determine whether Cerr1 is required for head formation we generated a null mutation in Cerr1 by gene targeting in mouse embryonic stem cells. We found that head formation is normal in Cerr1(-/-) embryos and we detected no obvious phenotypic defects in adult Cerr1(-/-) mice. However, in embryonic tissue layer recombination assays, Cerr1(-/-) presomitic/somitic mesoderm, unlike Cerr1-expressing wild-type presomitic/somitic mesoderm, was unable to maintain expression of the anterior neural marker gene Otx2 in ectoderm explants. These findings suggest that establishment of anterior identity in the mouse may involve the action of multiple functionally redundant factors.     

DnaA protein is not essential for replication of IncFII plasmid NR1.

By transformation of dnaA null mutant host cells that are suppressed either by an rnh mutation or by chromosomal integration of a mini-R1 plasmid, it was shown that replication of miniplasmids composed of the NR1 minimal replicon had no absolute dependence upon DnaA protein. In addition, the suppression of the dnaA null mutation by the integrated mini-R1, which is an IncFII relative of NR1, was found to be sensitive to the expression of IncFII-specific plasmid incompatibility. This suggests that the integrative suppression by mini-R1 is under the control of the normal IncFII plasmid replication circuitry. Although NR1 replication had no absolute requirement for DnaA, the copy numbers of NR1-derived miniplasmids were lower in dnaA null mutants, and the plasmids exhibited a much reduced stability of inheritance during subculture in the absence of selection. This suggests that DnaA protein may participate in IncFII plasmid replication in some auxiliary way, such as by increasing the efficiency of formation of an open initiation complex at the plasmid replication origin. Such an auxiliary role for DnaA in IncFII replication would be different from that for replication of most other plasmids examined, for which DnaA has been found to be either essential or unimportant.