Heterozygous Embryos Research Articles

Mosaic expression of Med12 in female mice leads to exencephaly, spina bifida, and craniorachischisis

A precise temporal and spatial regulation of gene expression is necessary to achieve neural tube closure. Med12, a subunit of the mediator complex, can bind transcription factors and modulate expression of their target genes. Med12 is essential during early mouse development and is important for neural tube closure. We have made use of a mouse line carrying a conditional null allele of the X-linked Med12 gene to generate heterozygous female embryos that express Med12 in a mosaic fashion thus allowing the study of the role of Med12 during neural tube closure. Mosaic expression of Med12 causes a wide variety of embryonic phenotypes. Some embryos were unable to complete turning and were found with arrested development at embryonic day (ED) 9.5. Others were able to pass ED 12.5 and displayed defects in neural tube closure. These defects included exencephaly, spina bifida, craniorachischisis, split face, and curly tail. Histologic and skeletal analyses of these mutant females show that the neural plate is unable to elevate and is completely flat in the regions of the body axis where neural tube closure fails. We report examples of all known neural tube defects implying Med12 in the full process of neural tube closure along the complete body axis. Our work points to Med12 being an essential coregulator of transcription factors controlling neural tube closure.

The effect of naked neck gene and ambient temperature and their interaction on reproductive traits of heavy broiler dams

High ambient temperature is a major factor for diminishing reproductive performance of broiler parent stocks. Homozygous naked neck (NaNa) broilers, which possess a higher adaptation to heat due to a reduction of feather coverage, exhibited higher growth rates and meat yield. This study was conducted to investigate the influence of genotype x temperature interactions on the reproduction traits of heavy broiler dams caused by different feathering genotypes induced by naked neck gene (Na). In an additional experiment, the effect of Na gene on embryonic activity using oxygen uptake was examined. Normal-feathered (nana) and NaNa hens were maintained in separate cages under high (30 degrees C) and moderate (19 degrees C) temperatures, with RH of 55% from the 18th to 72nd week of age. Egg production, fertility, hatchability, and the time of embryonic mortality were recorded. In comparison with the NaNa genotype, the nana hens showed clear performance depressions under thermal stress with respect to egg production (63%), fertility (20%), hatchability, and number of chicks (72%). Under temperate ambient temperature, there were no differences in laying performance and fertility between both genotypes, with the exception of hatchability due to an increase in embryonic mortality as a result of the Na gene and consequently in the complex trait number of chicks. The early embryonic mortality of eggs laid by nana hens exposed to heat stress is clearly higher than of eggs by nana hens kept under temperate ambient temperature. In contrast, there were no significant differences in early embryonic mortality of eggs of NaNa birds kept under high and temperate environmental temperatures, demonstrating that heat stress leads to reduced early embryonic livability. Embryonic mortality in the late developing stage is significantly increased, and the homozygous genotype is much more affected than the heterozygous embryo. The possible reason for the Na gene-induced embryonic mortality is still not clear. In this investigation, it is shown that the phenomenon cannot be explained by the oxygen consumption.

Acyl-CoA binding protein gene ablation induces pre-implantation embryonic lethality in mice.

Unique among the intracellular lipid binding proteins, acyl-CoA binding protein (ACBP) exclusively binds long-chain fatty acyl-CoAs (LCFA-CoAs). To test if ACBP is an essential protein in mammals, the ACBP gene was ablated by homologous recombination in mice. While ACBP heterozygotes appeared phenotypically normal, intercrossing of the heterozygotes did not produce any live homozygous deficient (null) ACBP((-/-)) pups. Heterozygous and wild type embryos were detected at all post-implantation stages, but no homozygous ACBP-null embryos were obtained-suggesting that an embryonic lethality occurred at a pre-implantation stage of development, or that embryos never formed. While ACBP-null embryos were not detected at any blastocyst stage, ACBP-null embryos were detected at the morula (8-cell), cleavage (2-cell), and zygote (1-cell) pre-implantation stages. Two other LCFA-CoA binding proteins, sterol carrier protein-2 (SCP-2) and sterol carrier protein-x (SCP-x) were significantly upregulated at these stages. These findings demonstrate for the first time that ACBP is an essential protein required for embryonic development and its loss of function may be initially compensated by concomitant upregulation of two other LCFA-CoA binding proteins, but only at the earliest pre-implantation stages. The fact that ACBP is the first known intracellular lipid binding protein whose deletion results in embryonic lethality suggests its vital importance in mammals.

Abstract 3952: CALM deficiency in fit1 embryonic cells alters expression and rate of endocytosis of growth factor receptors

Abstract Background: Gene rearrangements involving the Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) and Mixed-Lineage Leukemia (MLL) or AF10 genes have been identified in aggressive leukemias and lymphomas. Expression of CALM-containing fusion proteins immortalizes murine hematopoietic cells in vitro, correlating with leukemogenesis in vivo. Disruption of normal CALM, MLL or AF10 protein function as a result of these translocations likely contributes to transformation, although the precise mechanisms are unknown. The native CALM protein is involved in clathrin-mediated endocytosis (CME); it localizes to the cytoplasmic side of endocytic vesicles, interacts with both membrane elements and clathrin, and when over- or under-expressed, disrupts endocytosis. To better understand the effects of altered CALM activity, we have focused our studies on mutant fit1 mice that lack CALM expression. Previous studies have shown that these mice have iron deficiency, defective hematopoiesis and ultimately premature death. Here we examine CALM's role in endocytosis and cellular function using cells derived from fit1 mice. Objective: To examine the effect of CALM deficiency on cell surface receptor expression and endocytosis using mouse embryonic fibroblasts (MEFs) and fetal liver cells derived from normal and CALM-deficient mice. Design: Day 14 MEFs were generated from normal, heterozygous, and mutant fit1 embryos. Immortalized MEFs were compared in terms of quantity of cell surface and total cellular receptors (by flow cytometry and Western blotting), receptor mRNA levels, and rate of endocytosis. Receptors were also measured in fetal liver derived from the same embryos. Results: When compared to their normal counterparts, cells lacking full length CALM showed altered cell surface receptor expression: transferrin receptor (TfR) protein levels measured by flow cytometry were increased two-fold, while EGF receptor (EGFR) levels were reduced two-fold. These results correlated with increased total cellular TfR protein and mRNA levels and decreased total EGFR protein levels. Whereas the rate of internalization of TfR was similar, the rate of endocytosis of EGFR was significantly reduced in CALM-deficient compared with wildtype cells. Elevated TfR levels were also identified in CALM-deficient fetal liver cells. Conclusions: CALM deficiency in fit1-derived cells results in increased TfR surface expression; this is consistent with the iron deficiency phenotype of fit1 mice. The reduced surface expression of EGFR seen in fit1 cells may be a compensatory mechanism that counteracts the increased growth factor receptor signaling that is associated with impaired endocytosis. Since CALM haploinsufficiency is a feature of CALM-AF10 and MLL-CALM leukemias, our results suggest that the perturbation of normal growth factor biology may contribute to transformation in these malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3952.

Effects of varying Notch1 signal strength on embryogenesis and vasculogenesis in compound mutant heterozygotes

BackgroundIdentifying developmental processes regulated by Notch1 can be addressed in part by characterizing mice with graded levels of Notch1 signaling strength. Here we examine development in embryos expressing various combinations of Notch1 mutant alleles. Mice homozygous for the hypomorphic Notch112f allele, which removes the single O-fucose glycan in epidermal growth factor-like repeat 12 (EGF12) of the Notch1 ligand binding domain (lbd), exhibit reduced growth after weaning and defective T cell development. Mice homozygous for the inactive Notch1lbd allele express Notch1 missing an ~20 kDa internal segment including the canonical Notch1 ligand binding domain, and die at embryonic day ~E9.5. The embryonic and vascular phenotypes of compound heterozygous Notch112f/lbd embryos were compared with Notch1+/12f, Notch112f/12f, and Notch1lbd/lbd embryos. Embryonic stem (ES) cells derived from these embryos were also examined in Notch signaling assays. While Notch1 signaling was stronger in Notch112f/lbd compound heterozygotes compared to Notch1lbd/lbd embryos and ES cells, Notch1 signaling was even stronger in embryos carrying Notch112f and a null Notch1 allele.ResultsMouse embryos expressing the hypomorphic Notch112f allele, in combination with the inactive Notch1lbd allele which lacks the Notch1 ligand binding domain, died at ~E11.5-12.5. Notch112f/lbd ES cells signaled less well than Notch112f/12f ES cells but more strongly than Notch1lbd/lbd ES cells. However, vascular defects in Notch112f/lbd yolk sac were severe and similar to Notch1lbd/lbd yolk sac. By contrast, vascular disorganization was milder in Notch112f/lbd compared to Notch1lbd/lbd embryos. The expression of Notch1 target genes was low in Notch112f/lbd yolk sac and embryo head, whereas Vegf and Vegfr2 transcripts were increased. The severity of the compound heterozygous Notch112f/lbd yolk sac phenotype suggested that the allelic products may functionally interact. By contrast, compound heterozygotes with Notch112f in combination with a Notch1 null allele (Notch1tm1Con) were capable of surviving to birth.ConclusionsNotch1 signaling in Notch112f/lbd compound heterozygous embryos is more defective than in compound heterozygotes expressing a hypomorphic Notch112f allele and a Notch1 null allele. The data suggest that the gene products Notch1lbd and Notch112f interact to reduce the activity of Notch112f.

Effect of human β-globin bacterial artificial chromosome transgenesis on embryo cryopreservation in mouse models

The purpose of the present study was to investigate the efficiency of embryo cryopreservation for four transgenic (TG) thalassaemic mouse strains, which is a key element of the ongoing gene banking efforts for these high-value animals. Heterozygous TG embryos were produced by breeding four lines of TG males to wild-type (WT) females (C57BL/6J). Intact two-cell embryos were cryopreserved by vitrification in straws using 35% ethylene glycol. Survival rates of cryopreserved embryos ranged between 91.1% (102/112) and 93.6% (176/188) without significant differences between the lines. In contrast, the paternal line had a significant effect on the development of these embryos to the blastocyst stage, which ranged from 50.6% (92/182) to 77.5% (79/102). This effect was also noted following embryo transfers, with implantation rates varying from 17.3% (19/110) to 78.1% (35/45). The results demonstrate that the in vivo developmental potential is significantly influenced by TG line and reveal a specific line effect on cryosurvival. All bacterial artificial chromosome transgenic fetuses developed from vitrified-warmed embryos showed expression of the human beta-globin transgene. In conclusion, the present study shows a strong TG line effect on developmental competence following cryopreservation and the vitrification method was successful to bank the human beta-globin TG-expressing mouse strains.

Disruption of Sphingosine 1-Phosphate Lyase Confers Resistance to Chemotherapy and Promotes Oncogenesis through Bcl-2/Bcl-xL Upregulation

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in cancer development through stimulation of cell survival, proliferation, migration, and angiogenesis. Irreversible degradation of S1P is catalyzed by S1P lyase (SPL). The human SGPL1 gene that encodes SPL maps to a region often mutated in cancers. To investigate the effect of SPL deficiency on cell survival and transformation, the susceptibility to anticancer drugs of fibroblasts generated from SPL-deficient mouse embryos (Sgpl1(-/-)) was compared with that of cells from heterozygous (Sgpl1(+/-)) or wild-type (Sgpl1(+/+)) embryos. First, loss of SPL caused resistance to the toxic effects of etoposide and doxorubicin. Interestingly, heterozygosity for the Sgpl1 gene resulted in partial resistance to apoptosis. Secondly, doxorubicin-induced apoptotic signaling was strongly inhibited in Sgpl1(-/-) cells (phosphatidylserine externalization, caspase activation, and cytochrome c release). This was accompanied by a strong increase in Bcl-2 and Bcl-xL protein content. Whereas correction of SPL deficiency in Sgpl1(-/-) cells led to downregulation of antiapoptotic proteins, Bcl-2 and Bcl-xL small interfering RNA-mediated knockdown in SPL-deficient cells resulted in increased sensitivity to doxorubicin, suggesting that Bcl-2 upregulation mediates SPL protective effects. Moreover, SPL deficiency led to increased cell proliferation, anchorage-independent cell growth, and formation of tumors in nude mice. Finally, transcriptomic studies showed that SPL expression is downregulated in human melanoma cell lines. Thus, by affecting S1P metabolism and the expression of Bcl-2 members, the loss of SPL enhances cell resistance to anticancer regimens and results in an increased ability of cells to acquire a transformed phenotype and become malignant.

Roles of Histone Acetyltransferase MORF and MOZ in Hematopoiesis.

Abstract 2525Poster Board II-502MOZ (MOnocytic leukemia Zinc finger protein) and MORF (MOz Related Factor), Myst-type histone acetyltransferases, are involved in chromosome translocations associated with FAB-M4/5 subtypes of acute myeloid leukemia. We have reported that MOZ is essential for hematopoietic cell development and self-renewal of hematopoietic stem cells. To explore the possibility MORF also plays important roles in hematopoiesis, we generated Morf-deficient mice with homologous recombination methods. Morf−/− mice were smaller than their wildtype littermates and died within 4 weeks after birth on C57BL/6 background. In MORF−/− fetal liver, Flt3-negative KSL (c-Kit+ Sca-1+ Lineage-) cells containing hematopoietic stem cells were decreased. When fetal liver cells were transplanted into irradiated recipient mice, MORF−/− cells less efficiently reconstituted hematopoiesis than wild-type cells. Additionally, bone marrow cells reconstituted with MORF−/− cells rarely contributed to hematopoiesis in secondary transplants. To reveal relationship between MORF and MOZ in hematopoiesis, we generated double heterozygous (Moz+/− Morf+/−) mouse. Double heterozygous mice were smaller than wild-type littermates and died at least 4 weeks after birth. Numbers of KSL cells, especially Flt3- KSL cells and common myeloid progenitors were decreased in the double heterozygous embryos. The double heterozygous fetal liver cells also displayed less activity to reconstitute hematopoiesis than MOZ+/− or MORF+/− cells. Since MORF−/− mice and MOZ/MORF double heterozygous mice were alive at adult on a mixed C57BL/6/DBA2 genetic background, we investigated adult hematopoiesis in these mice. MORF−/− or MOZ/MORF double heterozygous mice were smaller than their wild-type littermates and had small numbers of thymocytes and splenocytes. However, there were no significant differences in number of bone marrow cells and hematopoietic lineage population in MORF−/− or MOZ/MORF double heterozygous mice. These results suggest that MORF as well as MOZ plays important roles in self-renewal of hematopoietic stem cells. Disclosures:No relevant conflicts of interest to declare.

Characterization of the chromosomal inversion associated with the Koa mutation in the mouse revealed the cause of skeletal abnormalities

BackgroundKoala (Koa) is a dominant mutation in mice causing bushy muzzle and pinna, and is associated with a chromosomal inversion on the distal half of chromosome 15. To identify the gene responsible for the Koa phenotypes, we investigated phenotypes of Koa homozygous mice and determined the breakpoints of the inversion with a genetic method using recombination between two different chromosomal inversions.ResultsSkeletal preparation of Koa homozygotes showed marked deformity of the ribs and a wider skull with extended zygomatic arches, in addition to a general reduction in the lengths of long bones. They also had open eyelids at birth caused by a defect in the extension of eyelid anlagen during the embryonic stages. The proximal and distal breakpoints of the Koa inversion were determined to be 0.8-Mb distal to the Trsps1 gene and to 0.1-Mb distal to the Hoxc4 gene, respectively, as previously reported. The phenotypes of mice with the recombinant inverted chromosomes revealed the localization of the gene responsible the Koa phenotype in the vicinity of the proximal recombinant breakpoint. Expression of the Trsps1 gene in this region was significantly reduced in the Koa homozygous and heterozygous embryos.ConclusionWhile no gene was disrupted by the chromosomal inversion, an association between the Koa phenotype and the proximal recombinant breakpoint, phenotypic similarities with Trps1-deficient mice or human patients with TRSP1 mutations, and the reduced expression of the Trsps1 gene in Koa mice, indicated that the phenotypes of the Koa mice are caused by the altered expression of the Trps1 gene.

06-P014 High nephron number in betaglycan heterozygous mice

the foregut endoderm and the defective expansion of the midand hindgut at early somite stages. However, there remains unclear whether or not Sox17 is required for the subsequent development, maturation and maintenance of the endoderm derivatives such as livers and pancreas due to the early embryonic lethal of Sox17-null mutant embryos before 10.0 dpc. Recently, we noticed the neonatal lethality of Sox17 heterozygous pups which were back-crossed against C57BL/6 mice. Pathological analyses revealed that a reduced Sox17 activity clearly leads to the defective maintenance of fetal liver development from 16.5 dpc. Molecular marker analyses also demonstrated the ER-stress induction in the hepatocytes, leading the defective maintenance of Hnf4alpha expression the fetal hepatocyte of the Sox17 heterozygous embryos. This is clearly in contrast to their proper vascularization and hematopoiesis in the defective Sox17 heterozygous livers at 16.0–17.5 dpc. In this meeting, we will discuss the possible roles of Sox17 in the maintenance of the fetal liver development/maturation on the basis of the liver transcriptome data revealed by the microarray analyses.

Implication of Interferon Stimulated Gene 15 (ISG15) in the Recruitment of Uterine Natural Killer Cells into the Murine Implantation Site.

Interferon stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that has being implicated in a number of physiological events including innate immune response, interferon signaling and cancer. ISG15 also is induced by pregnancy in the endometrium of rodents, primates and ruminants. Previous reports from our laboratory indicate that expression of ISG15 in the uterus is functionally required for maintenance of pregnancy in mice. Isg15 null females (Isg15-/-) exhibit embryo mortality ranging from 30-70% by 12.5 days post coitum (dpc), in contrast to 4% embryo mortality observed in wild type control females (Isg15+/+). As a consequence, Isg15+/+ mice have larger litters (7.94 ± 0.32 pups/ litter) when compared to Isg15-/- mice (4.2 ± 0.24). Additionally, Isg15-/- females carrying heterozygous embryos showed similar embryonic mortality rates when compared to Isg15-/- females carrying Isg15-/- embryos. This finding suggests that maternal Isg15 genotype contributes to embryonic death. Uterine natural killer cells (uNK) are the most prevalent maternal uterine immune cell and they are found in large number at the feto-maternal interface in mice. They interact closely with the invading placental trophoblast cells, which are responsible for remodeling of spiral arteries. This process is essential to ensure a normal blood supply to the fetus and placenta throughout pregnancy. Uteirne NK cells also produce cytokines (interferon gamma and interleukins 18 and 27), and proliferate within the myometrium at each implantation site forming the mesometrial lymphoid aggregate of pregnancy (MLAp). Uterine NK cells have been linked with failure of pregnancy: studies in mice lacking uNK cells and components of IFN-gamma pathway indicate that uNK cell-derived IFN-gamma is essential for spiral artery remodeling. Because Isg15 is a component of the IFN pathway, we hypothesized that uNK population may be altered in Isg15-/-. To address this question we designed a set of experiments to compare the number and distribution of uNK cells at implantation sites between Isg15+/+ and Isg15-/- mice. Implantation sites of 7.5 dpc and 12.5 dpc (n=5 each genotype and day) were fixed in 4% parformaldehyde and embedded in paraffin. Five μm sections were cut from paraffin-embedded tissues and uNK cells were identified using Dolichos biflorus agglutinin (DBA) lectin histochemistry. The number of uNK was assessed through a computer-assisted image analysis (Image Pro Plus for Windows, Ver 4.0, Media Cybernetics, Silver Spring, MD) at 200 X. A minimum of 3 non-adjacent mid-sagittal sections 40 μm apart were analyzed per implantation site. There was a very conspicuous difference in the density and distribution of uNK cells between Isg15+/+ and Isg15-/- mice. On 7.5 dpc, Isg15-/- mice showed reduced (P<0.001) uNK cell numbers and substantially reduced migration into the mesometrial pole of the decidua when compared to Isg15+/+ mice. By 12.5 dpc, there was a dramatic difference in the distribution of uNK cells in histological sections of Isg15-/- mice when compared with Isg15+/+. In Isg15-/- uNK cells were more concentrated in the decidua basalis in addition to an apparent impaired development of the metrial zone. The present study suggests that ISG15 may play a role in the recruitment of uNK cells in the uterus during early gestation. We suspect that faulty uNK migration and distribution contributes to embryonic mortality observed in ISG15-/- mice. (poster)

Bmp2 and Bmp4 genetically interact to support multiple aspects of mouse development including functional heart development

Bone morphogenetic proteins (BMPs) have multiple roles during embryogenesis. Current data indicate that the dosage of BMPs is tightly regulated for normal development in mice. Since Bmp2 or Bmp4 homozygous mutant mice show early embryonic lethality, we generated compound heterozygous mice for Bmp2 and Bmp4 to explore the impact of lowered dosage of these BMP ligands. Genotyping pups bred between Bmp2 and Bmp4 heterozygous mice revealed that the ratio of adult compound heterozygous mice for Bmp2 and Bmp4 is much lower than expected. During embryogenesis, the compound heterozygous embryos showed several abnormalities, including defects in eye formation, body wall closure defects, and ventricular septal defects (VSD) in the heart. However, the ratio of the compound heterozygous embryos was the same as expected. Caesarean sections at E18.5 revealed that half of the compound heterozygotes died soon after birth, and the majority of the dead individuals exhibited VSD. Survivors were able to grow to adults, but their body weight was significantly lower than control littermates. They demonstrated progressive abnormalities in the heart, eventually showing a branched leaflet in atrioventricular valves. These results suggest that the dosage of both BMP2 and 4 is critical for functional heart formation during embryogenesis and after birth.

Adherens junction domains are split by asymmetric division of embryonic neural stem cells

Investigating the mechanisms controlling the asymmetric division of neocortical progenitors that generate neurones in the mammalian brain is crucial for understanding the abnormalities of cortical development. Partitioning of fate determinants is a key instructive step and components of the apical junctional complex (adherens junctions), including the polarity proteins PAR3 and aPKC as well as adhesion molecules such as N-cadherin, have been proposed to be candidate determinants. In this study, however, we found no correlation between the partitioning of N-cadherin and fate determination. Rather, we show that adherens junctions comprise three membrane domains, and that during asymmetrical division these are split such that both daughters retain the adhesive proteins that control cell position, but only one daughter inherits the polarity proteins along with the apical membrane. This provides a molecular explanation as to how both daughters remain anchored to the ventricular surface after mitosis, while adopting different fates.

Wnt Signals Organize Synaptic Prepattern and Axon Guidance through the Zebrafish unplugged/MuSK Receptor

Early during neuromuscular development, acetylcholine receptors (AChRs) accumulate at the center of muscle fibers, precisely where motor growth cones navigate and synapses eventually form. Here, we show that Wnt11r binds to the zebrafish unplugged/MuSK ectodomain to organize this central muscle zone. In the absence of such a zone, prepatterned AChRs fail to aggregate and, as visualized by live-cell imaging, growth cones stray from their central path. Using inducible unplugged/MuSK transgenes, we show that organization of the central muscle zone is dispensable for the formation of neural synapses, but essential for AChR prepattern and motor growth cone guidance. Finally, we show that blocking noncanonical dishevelled signaling in muscle fibers disrupts AChR prepatterning and growth cone guidance. We propose that Wnt ligands activate unplugged/MuSK signaling in muscle fibers to restrict growth cone guidance and AChR prepatterns to the muscle center through a mechanism reminiscent of the planar cell polarity pathway.

Efficient production of androgenetic embryos by round spermatid injection

Mammalian androgenetic embryos can be produced by pronuclear exchange of fertilized oocytes or by dispermic in vitro fertilization of enucleated oocytes. Here, we report a new technique for producing mouse androgenetic embryos by injection of two round spermatid nuclei into oocytes, followed by female chromosome removal. We found that injection of round spermatids resulted in high rates of oocyte survival (88%). Androgenetic embryos thus produced developed into mid-gestation fetuses at various rates, depending on the mouse strain used. All the fetuses examined maintained paternally specific genomic imprinting memories. This technique also enabled us to produce complete heterozygous F1 embryos by injecting two spermatids from different strains. The best rate of fetal survival (12% per embryos transferred) was obtained with C57BL/6 x DBA/2 androgenetic embryos. We also generated embryonic stem cell lines efficiently with the genotype of Mus musculus domesticus x M. m. molossinus. Thus, injection of two round spermatid nuclei followed by maternal enucleation is an effective alternative method of producing androgenetic embryos that consistently develop into blastocysts and mid-gestation fetuses.

Maternal enzyme masks the phenotype of mouse embryos lacking dihydrolipoamide dehydrogenase

During early embryogenesis, the phenotype reflecting the embryonic genotype emerges only as maternal proteins are replaced by embryonically encoded forms, a process known as the maternal-to-embryonic transition (MET). Little is understood about MET for most proteins. This study investigates how complete deficiency of the murine dihydrolipoamide dehydrogenase gene (Dld), a gene that encodes an enzyme of mitochondrial energy metabolism, affects the phenotype of the early embryo and how the MET of the DLD protein affects the phenotype. Dld-deficient (-/-) embryos were found to develop similarly to wild-type (+/+) or heterozygous (+/-) embryos throughout the preimplantation period. These three genotypic classes also have comparable rates of glucose uptake (4.9-5.0 pmoles/embryo/h) and lactate production (0.97-1.0 pmoles/embryo/h). Dld-deficient embryos at the end of the preimplantation stage have 44% of DLD enzyme present in oocytes, a proportion similar to that found in +/+ or +/- embryos. This study demonstrates that Dld-deficient preimplantation embryos are phenotypically normal, as the MET for the DLD enzyme is only partially complete by the end of the preimplantation period. These findings have implications for phenotype- or enzyme-based approaches to identify mutations in Dld and other genes that encode proteins with similar MET kinetic profiles.

Bmp2 is required for cephalic neural tube closure in the mouse

BMPs have been shown to play a role in neural tube development particularly as dorsalizing factors. To explore the possibility that BMP2 could play a role in the developing neural tube (NT) beyond the lethality of Bmp2 null embryos, we created Bmp2 chimeras from Bmp2 null ES cells and WT blastocysts. Analysis of Bmp2 chimeras reveals NT defects at day 9.5 (E9.5). We found that exclusion of Bmp2 null ES cells from the dorsal NT did not always prevent defects. For further comparison, we used a Bmp2 mutant line in a mixed background. Phenotypes observed were similar to chimeras including open NT defects, postneurulation defects, and abnormal neural ectoderm in heterozygous and homozygous null embryos demonstrating a pattern of dose-dependent signaling. Our data exposes BMP2 as a unique player in the developing NT for dorsal patterning and identity, and normal cephalic neural tube closure in a dose-dependent manner.

A Transgenic Mouse Model for Congenital Dyserythropoietic Anemia Type I

The Congenital Dyseyrthropoetic Anemias (CDA) are a heterogeneous group of inborn defects characterized by anemia of varying severity and morphological abnormalities of bone marrow erythroblasts. Studying their pathology has the potential to contribute to the understanding of the normal process of erythropoiesis. In CDA type I, the phenotype is largely limited to the red cell lineage with erythroblasts showing characteristic spongy nuclear chromatin on electron microscopy. Its genetic basis is due to missense mutations in the CDAN1 gene (chromosome 15q15), but the biological function of the highly conserved and non-redundant codanin-1 protein remains entirely unknown.We have produced a mouse model for CDA type I by generating transgenic mice from an ES cell line carrying a gene-trap (splice acceptor/β-galactosidase gene/neomycin resistance gene) in intron 25 of the murine CDAN1 gene. Blood and bone marrow from CDAN1gt/wt heterozygotes is morphologically and quantitatively indistinguishable from wild type animals. Northern blot analysis and RT-qPCR of codanin-1 RNA expression confirms a broad pattern of expression with the highest levels seen in erythroid tissue. Flow-cytometric analysis of β-galactosidase activity in erythroid cells from phenylhydrazine treated adult spleens shows the highest levels in the CD71high/Ter119low early erythroblasts, declining with further erythroid maturation. Furthermore, indirect immunofluorescence localizes the codanin-1/β-galactosidase fusion product in the nucleus of the CD71high/Ter119low early erythroblasts, particularly at the interface between euchromatin and heterochromatin. The presence of an in-frame β-gal in the gene trap insert, therefore, provides a method to analyze gene and protein expression from the CDAN1 promoter, and additional distribution data from heterozygous embryos and adults will be presented. Heterozygote crosses produced no homozygotes among liveborn progeny, suggesting embryonic lethality of this state. Analysis of embryos at different stages suggests development of homozygotes ceased at ~6.5d of gestation, prior to the onset of erythropoiesis.These results from the first transgenic mouse model for CDA type I, highlight a non-erythroid role for codanin-1 in early embryonic development, in addition to its role in adult human erythropoiesis. The embryonic lethality of the mouse model is consistent with the absence of homozygote null mutations in the CDAN1 genes analyzed so far in human patients. Creation of mice with the CDA type I phenotype may have to await a knock-in transgenic containing a human mutation, which is currently in preparation. The model described here should be valuable for further studies of the intracellular localization of codanin-1 and the identification of any relevant protein binding partners.

The C-Terminus of the CBFβ-SMMHC Fusion Protein Is Required for the Myeloblastic Transformation of inv16 Leukemia

Inv(16)(p13q22) is found in almost all human acute myeloid leukemia (AML) subtype M4Eo cases and forms a fusion gene CBFB-MYH11, which encodes a fusion protein CBFβ–SMMHC. CBFβ forms a heterodimeric transcription factor with RUNX1 and both are required for embryonic hematopoiesis, while SMMHC is the smooth muscle myosin heavy chain. Using knock-in mouse strategy, we previously demonstrated that Cbfb+/MYH11 F1 embryos have severe blockage of definitive hematopoiesis and die from CNS hemorrhage. The phenotype was similar to that of embryos with homozygous deletion of Cbfb or Runx1, suggesting that Cbfb-MYH11 dominantly represses Runx1/CBFb function. We further demonstrated that Cbfb-MYH11 is necessary but not sufficient for leukemia to develop, as the Cbfb+/MYH11 mice required additional mutations for leukemogenesis. Several hypotheses have been proposed, based on in vitro studies, to explain how CBFβ-SMMHC dominantly inhibits RUNX1/CBFβ. The C-terminal region of CBFβ-SMMHC is responsible for multimerization and also interacts with corepressors; thus this region might be critical for RUNX1 repression. To determine the importance of this multimerization domain in vivo, we generated knock-in mice expressing CBFβ-SMMHC with a 95 aa C-terminal deletion (CBFβ-SMMHCdC95), which truncates this multimerization/repression domain. CBFβ-SMMHCdC95 expressing F1 heterozygous embryos (Cbfb+/MYH11dC95) developed normally with no hematopoietic defects and no hemorrhage. Hematopoiesis was normal in the adult Cbfb+/MYH11dC95 mice except for mild increase of mature neutrophils and minor T cell developmental defects in the first year. However, the mice proceeded to a lethal myeloproliferative disorder (MPD) during their second year of life. There was a significant increase of Mac1/Gr1 double positive cells in the peripheral blood and the spleen, which were negative for the stem cell/progenitor cell marker, c-kit. Morphologically the erythrocytes and neutrophils were dysplastic, and the mice developed severe splenomegaly. ENU treatment of the Cbfb+/MYH11dC95 mice accelerated the development of the MPD phenotype but could not induce overt, transplantable, myeloid leukemia. These data suggest that the multimerizatin domain of SMMHC is important for both hematopoiesis blockage and leukemogenesis, especially the blastic transformation of inv16 leukemia.

Dnm3os, a non‐coding RNA, is required for normal growth and skeletal development in mice

Dnm3os, a gene that is transcribed into a non-coding RNA (ncRNA), contains three micro RNAs (miRNAs), miR-199a, miR-199a*, and miR-214, whose functions remain unknown in mammals. In this study, we introduced the lacZ gene into the Dnm3os locus to recapitulate its expression pattern and disrupt its function. Dnm3os(+/lacZ) heterozygous embryos showed beta-galactosidase activity, which reflected the authentic expression pattern of Dnm3os RNA. Most of the Dnm3os(lacZ/lacZ) homozygous pups died within one month of birth. After birth, Dnm3os(lacZ/lacZ) mice exhibited several skeletal abnormalities, including craniofacial hypoplasia, defects in dorsal neural arches and spinous processes of the vertebrae, and osteopenia. Importantly, the expression of miR-199a, miR-199a*, and miR-214 was significantly down-regulated in Dnm3os(lacZ/lacZ) embryos, supporting the assumption that Dnm3os serves as a precursor of these three miRNAs. Thus, Dnm3os has emerged as an miRNA-encoding gene that is indispensable for normal skeletal development and body growth in mammals.