Nuclear transfer reprogramming does not improve the low developmental potency of embryonic stem cells induced by long-term culture.

Epigenetic states of embryonic stem (ES) cells are easily altered by long-term cultivation and lose their developmental potential. To rescue this reduced developmental capacity, nuclear transfer (NT) of ES cells was carried out, and original ES and ES cells from cloned blastocysts (ntES) cells established after NT were compared with in vitro differentiation ability and developmental potential by embryoid body formation and tetraploid aggregation respectively. In the establishment of ntES cell lines, the oocytes fused with the ES cell were activated, and further cultured to cloned blastocysts. When in vitro differentiation ability was examined between original and ntES cell lines derived from ES cells with extensive passages (ES-ep), the day of appearance of simple embryoid body, cystic embryoid body, and spontaneous beating was almost similar. The developmental rates of ES-ep cells, that aggregated with tetraploid embryos to term, ranged from 3 to 6%. Moreover, the majority of live pups died soon after birth. In the ntES cell lines derived from ES-ep cells, developmental rates ranged from 0 to 5%. Those pups also died soon after birth, similar to the ES-ep-derived pups. These results suggest that profound epigenetic modifications of ES cells were retained in the re-established cell lines by NT.

Mice lacking histidine decarboxylase exhibit abnormal mast cells.

Histidine decarboxylase (HDC) synthesizes histamine from histidine in mammals. To evaluate the role of histamine, we generated HDC-deficient mice using a gene targeting method. The mice showed a histamine deficiency and lacked histamine-synthesizing activity from histidine. These HDC-deficient mice are viable and fertile but exhibit a decrease in the numbers of mast cells while the remaining mast cells show an altered morphology and reduced granular content. The amounts of mast cell granular proteases were tremendously reduced. The HDC-deficient mice provide a unique and promising model for studying the role of histamine in a broad range of normal and disease processes.

Placental cell fates are regulated in vivo by HIF-mediated hypoxia responses.

Placental development is profoundly influenced by oxygen (O(2)) tension. Human cytotrophoblasts proliferate in vitro under low O(2) conditions but differentiate at higher O(2) levels, mimicking the developmental transition they undergo as they invade the placental bed to establish the maternal-fetal circulation in vivo. Hypoxia-inducible factor-1 (HIF-1), consisting of HIF-1alpha and ARNT subunits, activates many genes involved in the cellular and organismal response to O(2) deprivation. Analysis of Arnt(-/-) placentas reveals an aberrant cellular architecture due to altered cell fate determination of Arnt(-/-) trophoblasts. Specifically, Arnt(-/-) placentas show greatly reduced labyrinthine and spongiotrophoblast layers, and increased numbers of giant cells. We further show that hypoxia promotes the in vitro differentiation of trophoblast stem cells into spongiotrophoblasts as opposed to giant cells. Our results clearly establish that O(2) levels regulate cell fate determination in vivo and that HIF is essential for mammalian placentation. The unique placental phenotype of Arnt(-/-) animals also provides an important tool for studying the disease of preeclampsia. Interestingly, aggregation of Arnt(-/-) embryonic stem (ES) cells with tetraploid wild-type embryos rescues their placental defects; however, these embryos still die from yolk sac vascular and cardiac defects.

Early exclusion of hand1-deficient cells from distinct regions of the left ventricular myocardium in chimeric mouse embryos.

The basic helix-loop-helix transcription factor gene Hand1 has been implicated in development of the heart. However, the early lethality of Hand1-null mutant mouse embryos has precluded a precise understanding of its function. In this study, we have generated Hand1 homozygous mutant ES cells and performed in vitro differentiation experiments and chimeric analysis to study the role of Hand1 function during cardiac development. Hand1-null ES cells were able to differentiate into beating cardiomyocytes in vitro that expressed cardiac myosin and several cardiac-specific transcripts including Nkx2-5, alpha-cardiac actin, and the myofilament genes myosin light chain 2a and 2v. In chimeras derived from Hand1-null ES cells and ROSA26 embryos, mutant cells were underrepresented in the left caudal region of the linear heart tube at E8.0. By E9.5, after cardiac looping, mutant cells were underrepresented in the anterior region of the outer curvature of the left ventricular myocardium, but did contribute to other parts of the left ventricle and to other cardiac chambers. These results imply that Hand1 is not essential for differentiation of ventricular cardiomyocytes. Hand1-null cells were also underrepresented in several other regions of later embryos, including the rhombencephalic neural tube that was associated with a deficiency of mutant cells in the neural crest cell-derived cardiac outflow tract and first branchial arch. In summary, Hand1 has cell-autonomous functions during cardiac morphogenesis in both mesodermal and neural crest derivatives.

Parental origin-specific expression of Mash2 is established at the time of implantation with its imprinting mechanism highly resistant to genome-wide demethylation.

The Mash2 gene encodes a basic helix-loop-helix transcription factor, which is highly expressed in diploid trophoblast cells of the postimplantation mouse embryo and is required for development of the spongiotrophoblast in order to form a functional placenta. Genomic imprinting of Mash2 has been previously reported; transcriptional inactivation of the paternal wild-type allele in heterozygotes carrying a maternal null allele results in a null-equivalent embryonic lethal phenotype. In order to study the Mash2 imprinting mechanism, we have created a new allele at this locus carrying a targeted insertion of an IRES (internal ribosome entry site)-lacZ cassette within the 3' untranslated region of the gene (referred to as "Mash2-lacZ"). This new allele has made it feasible to monitor paternal Mash2 expression in a wild-type-equivalent background. Our data suggest that parental origin-specific expression of Mash2 begins in the early postimplantation conceptus (5.5 dpc) at the time when trophoblast-specific expression is observed. We also show that the paternal allele is continuously repressed up to 9.5 dpc in the developing ectoplacental cone (EPC) and early chorio-allantoic placenta, with some cells escaping paternal repression. When maternally inherited, lacZ expression from this allele reflects the expression pattern of endogenous Mash2 transcripts up to 8.5 dpc. Furthermore, we have addressed the question of a requirement for DNA methylation for the Mash2 imprinting mechanism by crossing our Mash2-lacZ mice with mice mutant for Dnmt1 (DNA-methyltransferase1). Our results show a partial loss of transcriptional repression of the paternal allele in Dnmt1 deficient background. Interestingly, however, this is not sufficient to eliminate the highly biased parental allele-specific expression of Mash2. Thus, the preferential maternal expression of the gene is still maintained in Dnmt1 null mutant embryos, although methylation analyses demonstrate that the Mash2 locus is highly demethylated in Dnmt1 null mutant embryos. The locus is also highly demythyled in wild-type EPCs. Our results suggest the possibility that a mechanism other than DNA methylation, such as allele-specific chromatin conformation, may be involved in maintenance of parental origin-specific expression of Mash2.

Multiple developmental roles of VEGF suggested by a LacZ-tagged allele.

Vascular endothelial growth factor (VEGF) is an angiogenic factor and a potent stimulator of microvascular permeability. It is a mitogen specific for endothelial cells. The expression of VEGF and its two receptors, Flk-1 and Flt-1, is pivotal for the proper formation of blood vessels in embryogenesis as shown by gene-targeting experiments. Interestingly, the loss of even a single allele of VEGF led to embryonic lethality between day E9.5 and day E10.5 in the mouse. To assess the role of VEGF during embryonic development we decided to tag VEGF expression with LacZ, by inserting an IRES (internal ribosome entry site)-LacZ reporter cassette into the 3' untranslated region of the gene. This alteration enabled us to monitor VEGF expression throughout embryonic development at single-cell resolution. beta-Galactosidase expression from the altered VEGF locus was first observed prior to gastrulation and was detectable at all stages of vascular development in the embryo. Later, the specific cellular distribution and the level of VEGF expression indicated its pleiotropic role in development. High expression levels seemed to be associated with vasculogenesis and permeability, whereas lower levels were associated with angiogenesis and cell migration. In addition, we found VEGF expression in a subtype of endothelial cells present in the endocardium. We believe that the LacZ-tagged allele we have generated offers a precise means of detecting VEGF expression under a variety of physiological and pathological conditions.

Z/AP, a double reporter for cre-mediated recombination.

The Cre/loxP site-specific recombination system combined with embryonic stem cell-mediated technologies has greatly expanded our capability to address normal and disease development in mammals using genetic approaches. The success of this emerging technology hinges on the production of Cre-expressing transgenic lines that provide cell type-, tissue-, or developmental stage-specific recombination between loxP sites placed in the genome. Here we describe and characterize the production of a double-reporter mouse line that provides a convenient and reliable readout of Cre recombinase activity. Throughout all embryonic and adult stages, the transgenic animal expresses the lacZ reporter gene before Cre-mediated excision occurs. Cre excision, however, removes the lacZ gene, allowing expression of the second reporter, the human alkaline phosphatase gene. This double-reporter transgenic line is able to indicate the occurrence of Cre excision in an extremely widespread manner from early embryonic to adult lineages. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system.

Generating green fluorescent mice by germline transmission of green fluorescent ES cells.

Green fluorescent protein (GFP) and its variants currently represent the only non-invasive markers available for labeling mammalian cells in culture or in a multicellular organism through transgenesis. To date this marker gene has been widely used in the study of many organisms, but as yet has not found large-scale application in mammals due to problems encountered with weak fluorescence and instability of the wild-type protein at higher temperatures. Recently, though, several mutants have been made in the wild-type (wt) GFP so as to improve its thermostability and fluorescence. EGFP (enhanced GFP) is one such wtGFP variant. As a first step in assessing the use of EGFP in ES cell-mediated strategies, we have established a mouse embryonic stem (ES) cell lines expressing EGFP, which can be propagated in culture, reintroduced into mice. or induced to differentiate in vitro, while still maintaining ubiquitous EGFP expression. From the results presented we can suggest that: 1) possible improvements in the efficiency of transgenic regimes requiring the germline transmission of ES cells by aggregation chimeras can be made by the preselection chimeric embryos at the blastocyst stage: (2) the expression of a noninvasive marker, driven by a promoter that is active during early postimplantation development, allows access to embryos during a window of embryonic development that has previously been difficult to investigate (3) the behavior of mutant ES cells can be followed with simple microscopic observation of chimeric embryos or adult animals comprising green fluorescent cells/tissues. and (4) intercrosses of F1 mice and subsequent generations of animals show that progeny can be genotyped by UV light, such that mice homozygous for the transgene can be distinguished from hemizygotes due to their increased fluorescence.

Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles.

The N-myc proto-oncogene is expressed in many organs of the mouse embryo, suggesting that it has multiple functions. A null mutation leads to mid-gestation lethality [1-4], obscuring the later roles of the gene in organogenesis. We have generated a multi-purpose gene alteration by combining the potential for homologous and site-specific recombination in a single targeting vector, and using the selectable marker for neomycin-resistance, neo, to downregulate gene activity. This allowed us to create a series of alleles that led to different levels of N-myc expression. The phenotypes revealed a spectrum of developmental problems. The hypomorphic allele produced can be repaired in situ by Cre-recombinase-mediated DNA excision. We show here for the first time the use of a single targeting vector to generate an allelic series. This, and the possibility of subsequent lineage-specific or conditional allele repair in situ, represent new genome modification strategies that can be used to investigate multiple functions of a single gene.

Mash2 acts cell autonomously in mouse spongiotrophoblast development.

The Mash2 gene, which encodes a basic helix-loop-helix transcription factor, is one of the mammalian homologues of the Drosophila achaete-scute genes. It is strongly expressed in diploid trophoblast cells of the postimplantation mouse embryo. Targeted mutagenesis of Mash2 revealed that loss of function results in embryonic lethality at midgestation, due to placental failure associated with a lack of spongiotrophoblast and reduced labyrinthine trophoblast layers. For the further study of Mash2 function in development of the trophoblast cell lineage, we have performed chimeric analysis combining Mash2 mutant and wild-type embryos. We have addressed the question of whether the phenotype of the Mash2 mutant embryo, which affects all of the three trophoblast cell layers, is caused by a cell autonomous or non-autonomous defect and whether Mash2 is required in both spongiotrophoblast and labyrinthine trophoblast development. Our results showed no contribution of Mash2 mutant cells to the spongiotrophoblast layer in chimeric placentae at 10.5 and 12.5 days postcoitum, suggesting that the product of the Mash2 gene is required cell autonomously during the development of the spongiotrophoblast. However, it seems that Mash2 is not required for development of labyrinthine trophoblast or giant cells, since high contributions of Mash2 mutant cells were observed in those trophoblast cell layers in the chimeric placentae analyzed. We can therefore conclude that the primary and cell-autonomous function of Mash2 appears to be an involvement in the development of diploid trophoblast cells in the ectoplacental cone to form the spongiotrophoblast cell layer of the mature chorioallantoic placenta.

Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.

The endothelial cell-specific vascular endothelial growth factor (VEGF) and its cellular receptors Flt-1 and Flk-1 have been implicated in the formation of the embryonic vasculature. This is suggested by their colocalized expression during embryogenesis and the impaired vessel formation in Flk-1 and Flt-1 deficient embryos. However, because Flt-1 also binds placental growth factor, a VEGF homologue, the precise role of VEGF was unknown. Here we report that formation of blood vessels was abnormal, but not abolished, in heterozygous VEGF-deficient (VEGF+/-) embryos, generated by aggregation of embryonic stem (ES) cells with tetraploid embryos (T-ES) and even more impaired in homozygous VEGF-deficient (VEGF-/-) T-ES embryos, resulting in death at mid-gestation. Similar phenotypes were observed in F1-VEGF+/- embryos, generated by germline transmission. We believe that this heterozygous lethal phenotype, which differs from the homozygous lethality in VEGF-receptor-deficient embryos, is unprecedented for a targeted autosomal gene inactivation, and is indicative of a tight dose-dependent regulation of embryonic vessel development by VEGF.

Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice.

The receptor tyrosine kinase Flk-1 (ref. 1) is believed to play a pivotal role in endothelial development. Expression of the Flk-1 receptor is restricted to endothelial cells and their embryonic precursors, and is complementary to that of its ligand, vascular endothelial growth factor (VEGF), which is an endothelial-specific mitogen. Highest levels of flk-1 expression are observed during embryonic vasculogenesis and angiogenesis, and during pathological processes associated with neovascularization, such as tumour angiogenesis. Because flk-1 expression can be detected in presumptive mesodermal yolk-sac blood-island progenitors as early as 7.0 days postcoitum, Flk-1 may mark the putative common embryonic endothelial and haematopoietic precursor, the haemangioblast, and thus may also be involved in early haematopoiesis. Here we report the generation of mice deficient in Flk-1 by disruption of the gene using homologous recombination in embryonic stem (ES) cells. Embryos homozygous for this mutation die in utero between 8.5 and 9.5 days post-coitum, as a result of an early defect in the development of haematopoietic and endothelial cells. Yolk-sac blood islands were absent at 7.5 days, organized blood vessels could not be observed in the embryo or yolk sac at any stage, and haematopoietic progenitors were severely reduced. These results indicate that Flk-1 is essential for yolk-sac blood-island formation and vasculogenesis in the mouse embryo.

Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium.

The vascular endothelial growth factor (VEGF) and its high-affinity binding receptors, the tyrosine kinases Flt-1 and Flk-1, are thought to be important for the development of embryonic vasculature. Here we report that Flt-1 is essential for the organization of embryonic vasculature, but is not essential for endothelial cell differentiation. Mouse embryos homozygous for a targeted mutation in the flt-1 locus, flt-1lcz, formed endothelial cells in both embryonic and extra-embryonic regions, but assembled these cells into abnormal vascular channels and died in utero at mid-somite stages. At earlier stages, the blood islands of flt-1lcz homozygotes were abnormal, with angioblasts in the interior as well as on the periphery. We suggest that the Flt-1 signalling pathway may regulate normal endothelial cell-cell or cell-matrix interactions during vascular development.

Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo.

The receptor tyrosine kinases (RTKs) expressed on the surface of endothelial cells are likely to play key roles in initiating the program of endothelial cell growth during development and subsequent vascularization during wound healing and tumorigenesis. Expression of the Tek RTK during mouse development is restricted primarily to endothelial cells and their progenitors, the angioblasts, suggesting that Tek is a key participant in vasculogenesis. To investigate the role that Tek plays within the endothelial cell lineage, we have disrupted the Tek signaling pathway using two different genetic approaches. First, we constructed transgenic mice expressing a dominant-negative form of the Tek receptor. Second, we created a null allele of the tek gene by homologous recombination in embryonic stem (ES) cells. Transgenic mice expressing dominant-negative alleles of Tek or homozygous for a null allele of the tek locus both died in utero with similar defects in the integrity of their endothelium. By crossing transgenic mice that express the lacZ reporter gene under the transcriptional control of the endothelial cell-specific tek promoter, we found that the extraembryonic and embryonic vasculature was patterned correctly. However, homozygous tek embryos had approximately 30% and 75% fewer endothelial cells at day 8.5 and 9.0, respectively. Homozygous null embryos also displayed abnormalities in heart development, consistent with the conclusion that Tek is necessary for endocardial/myocardial interactions during development. On the basis of the analysis of mice carrying either dominant-negative or null mutations of the tek gene, these observations demonstrate that the Tek signaling pathway plays a critical role in the differentiation, proliferation, and survival of endothelial cells in the mouse embryo.