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Elongin A is a transcription elongation factor that increases the overall rate of mRNA chain elongation by RNA polymerase II. To gain more insight into the physiological functions of Elongin A, we generated Elongin A-deficient mice. Elongin A homozygous mutant (Elongin A(-/-)) embryos demonstrated a severely retarded development and died at between days 10.5 and 12.5 of gestation, most likely due to extensive apoptosis. Moreover, mouse embryonic fibroblasts (MEFs) derived from Elongin A(-/-) embryos exhibited not only increased apoptosis but also senescence-like growth defects accompanied by the activation of p38 MAPK and p53. Knockdown of Elongin A in MEFs by RNA interference also dramatically induced the senescent phenotype. A study using inhibitors of p38 MAPK and p53 and the generation of Elongin A-deficient mice with p53-null background suggests that both the p38 MAPK and p53 pathways are responsible for the induction of senescence-like phenotypes, whereas additional signaling pathways appear to be involved in the mediation of apoptosis in Elongin A(-/-) cells. Taken together, our results suggest that Elongin A is required for the transcription of genes essential for early embryonic development and downregulation of its activity is tightly associated with cellular senescence.

Ovol1 encodes a zinc finger transcriptional repressor that is downstream of the LEF1/beta-catenin complex, nuclear effectors of canonical Wnt signaling. Previous gene knockout studies performed in a 129SvxC57BL/6 mixed genetic background revealed that Ovol1-deficient mice survive to adulthood but display multiple tissue defects. In this study, we describe a C57BL/6 strain-specific reduction in perinatal survival of Ovol1 mutant mice. The perinatal lethality is accompanied by kidney epithelial cysts of embryonic onset and delayed skin barrier acquisition. Genetic analysis suggests a partial functional compensation by Ovol2 for the loss of Ovol1. The expression of Ovol2 was up-regulated in Ovol1-deficient epidermis, and Ovol1 represses the activity of Ovol2 promoter in a DNA binding-dependent manner. Collectively, these studies uncover novel functions of Ovol1 in mouse development and identify Ovol2 as a downstream target of Ovol1.

Objective: Foxc2/MFH-1 is a member of the forkhead family of transcription factors and Foxc2-deficient mice exhibit aortic arch anomalies (type B interruption of the aortic arch). Endothelin receptor type-A (ETA) is one of the two known endothelin receptors that belong to the G-protein-coupled receptor family. ETA-deficient mice show defects in the great arteries, primarily type B interruption of the aortic arch. Based on similar phenotypes in the cardiovascular system of Foxc2- and ETA-deficient mice, we investigated whether Foxc2 and ETA have a close relationship in aortic arch patterning., Methods: The Foxc2 and ETA homozygotes were obtained by crossing the Foxc2 and ETA heterozygotes, respectively. The double Foxc2/ETA homozygotes were obtained by crossing the double Foxc2/ETA heterozygotes., Results: We investigated the expression of ETA in Foxc2-null mice and the expression of Foxc2 in ETA-null mice and found that the absence of either Foxc2 or ETA had no effect on the expression of the other. Next, we analyzed mice lacking both Foxc2 and ETA to examine the relationship between Foxc2 and ETA on aortic arch patterning in vivo. We found that the majority of Foxc2/ETA double-mutant embryos died around 11.5 dpc and that all surviving mice had persistent truncus arteriosus., Conclusions: The results suggest that Foxc2- and ETA-expressing cells additively form the aorticopulmonary septum.

Early mouse development is a complicated process that is controlled by various proteins including transcription factors. Recently, we identified a putative transcription factor, ELYS (embryonic large molecule derived from yolk sac), using a subtraction strategy. To elucidate the role of ELYS in vivo, we generated ELYS-deficient mice by homologous recombination. Although heterozygous mice appeared to be healthy, fertile and normal, embryos homozygous for the ELYS mutation died between embryonic day (E) 3.5 and 5.5. Null mutant blastocysts collected from the uterus at E3.5 were viable and indistinguishable from wild-type littermates. However, when cultured in vitro, they showed impaired proliferation of the inner cells, because of apoptosis. The expression of ELYS mRNA was detected in both the inner cell mass (ICM) and the trophectoderm at the blastocyst stage, and persisted throughout the developing embryo during E4.5 to 6.5. These results indicate that ELYS is a critical factor for early mouse development and is essential for the survival of the inner cells.

The fetoprotein transcription factor (FTF) gene was inactivated in the mouse, with a lacZ gene inserted inframe into exon 4. LacZ staining of FTF+/- embryos shows that the mFTF gene is activated at initial stages of zygotic transcription. FTF gene activity is ubiquitous at the morula and blastocyst stages and then follows expression patterns indicative of multiple FTF functions in fetal development. FTF-/- embryos die at E6.5-7.5, with features typical of visceral endoderm dysfunction. Adult FTF+/- mice are hypocholesterolemic, and express liver FTF at about 40% of the normal level. Overexpression of liver FTF in transgenic mice indicates in vivo that FTF is an activator of CYP7A1. However, CYP7A1 expression is increased in FTF+/- liver. Gene expression profiles indicate that higher CYP7A1 expression is caused by attenuated liver cell stress signaling. Diet experiments support a model where FTF is quenched both by activated c-Jun, and by SHP as a stronger feedback mechanism to repress CYP7A1. A DR4 element is conserved in the FTF gene promoter and activated by LXR-RXR and TR-RXR, qualifying the FTF gene as a direct metabolic sensor. Liver FTF increases in rats treated with thyroid hormone or a high cholesterol diet. The FTF DR4 element tightens functional links between FTF and LXRalpha in cholesterol homeostasis and can explain transient surges of FTF gene activities during development and FTF levels lower than predicted in FTF+/- liver. The FTF-lacZ mouse establishes a central role for FTF in developmental, nutritive, and metabolic functions from early embryogenesis through adulthood.

Mitochondrial DNA (mtDNA) copy number regulation is altered in several human mtDNA-mutation diseases and it is also important in a variety of normal physiological processes. Mitochondrial transcription factor A (TFAM) is essential for human mtDNA transcription and we demonstrate here that it is also a key regulator of mtDNA copy number. We initially performed in vitro transcription studies and determined that the human TFAM protein is a poor activator of mouse mtDNA transcription, despite its high capacity for unspecific DNA binding. Next, we generated P1 artificial chromosome (PAC) transgenic mice ubiquitously expressing human TFAM. The introduced human TFAM gene was regulated in a similar fashion as the endogenous mouse Tfam gene and expression of the human TFAM protein in the mouse did not result in down-regulation of the endogenous expression. The PAC-TFAM mice thus had a net overexpression of TFAM protein and this resulted in a general increase of mtDNA copy number. We used a combination of mice with TFAM overexpression and TFAM knockout and demonstrated that mtDNA copy number is directly proportional to the total TFAM protein levels also in mouse embryos. Interestingly, the expression of human TFAM in the mouse results in up-regulation of mtDNA copy number without increasing respiratory chain capacity or mitochondrial mass. It is thus possible to experimentally dissociate mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo. In conclusion, our results provide genetic evidence for a novel role for TFAM in direct regulation of mtDNA copy number in mammals.

There are five members of the RFX family of transcription factors in mammals. While RFX5 plays a well-defined role in the immune system, the functions of RFX1 to RFX4 remain largely unknown. We have generated mice with a deletion of the Rfx3 gene. RFX3-deficient mice exhibit frequent left-right (LR) asymmetry defects leading to a high rate of embryonic lethality and situs inversus in surviving adults. In vertebrates, specification of the LR body axis is controlled by monocilia in the embryonic node, and defects in nodal cilia consequently result in abnormal LR patterning. Consistent with this, Rfx3 is expressed in ciliated cells of the node and RFX3-deficient mice exhibit a pronounced defect in nodal cilia. In contrast to the case for wild-type embryos, for which we document for the first time a twofold increase in the length of nodal cilia during development, the cilia are present but remain markedly stunted in mutant embryos. Finally, we show that RFX3 regulates the expression of D2lic, the mouse orthologue of a Caenorhabditis elegans gene that is implicated in intraflagellar transport, a process required for the assembly and maintenance of cilia. In conclusion, RFX3 is essential for the differentiation of nodal monocilia and hence for LR body axis determination.

The mammalian F-box protein Fbw7 and its Caenorhabditis elegans counterpart Sel-10 have been implicated in the ubiquitin-mediated turnover of cyclin E as well as the Notch/Lin-12 family of transcriptional activators. Both unregulated Notch and cyclin E promote tumorigenesis, and inactivating mutations in human Fbw7 suggest that it may be a tumor suppressor. To generate an in vivo system to assess the consequences of such unregulated signaling, we generated mice deficient for Fbw7. Fbw7-null mice die around 10.5 days post coitus because of a combination of deficiencies in hematopoietic and vascular development and heart chamber maturation. The absence of Fbw7 results in elevated levels of cyclin E, concurrent with inappropriate DNA replication in placental giant trophoblast cells. Moreover, the levels of both Notch 1 and Notch 4 intracellular domains were elevated, leading to stimulation of downstream transcriptional pathways involving Hes1, Herp1, and Herp2. These data suggest essential functions for Fbw7 in controlling cyclin E and Notch signaling pathways in the mouse.

Release of E2F1/DP1 heterodimers from repression mediated by the retinoblastoma tumor suppressor (pRB) triggers cell cycle entry into S phase, suggesting that E2F1 and DP1 proteins must act in unison, either to facilitate or to suppress cell-cycle progression. In stark contrast to the milder phenotypes that result from inactivation of E2Fs, we report that loss of Dp1 leads to death in utero because of the failure of extra-embryonic development. Loss of Dp1 compromises the trophectoderm-derived tissues - specifically, the expansion of the ectoplacental cone and chorion, and endoreduplication in trophoblast giant cells. Inactivation of p53 is unable to rescue the Dp1-deficient embryonic lethality. Thus, DP1 is absolutely required for extra-embryonic development and consequently embryonic survival, consistent with E2F/DP1 normally acting to promote growth in vivo.

Tumor susceptibility gene 101 (Tsg101) was identified in a random mutagenesis screen for potential tumor suppressors in NIH 3T3 cells. Altered transcripts of this gene have been detected in sporadic breast cancers and many other human malignancies. However, the involvement of this gene in neoplastic transformation and tumorigenesis is still elusive. Using gene targeting, we generated genetically engineered mice with a floxed allele of Tsg101. We investigated essential functions of this gene in vivo and examined whether the loss of function of Tsg101 results in tumorigenesis. Conventional knockout mice were generated through Cre-mediated excision of the first coding exon in the germ line of mouse mammary tumor virus (MMTV)-Cre transgenic mice. The complete ablation of Tsg101 in the developing embryo resulted in death around implantation. In contrast, mammary gland-specific knockout mice developed normally but were unable to nurse their young as a result of impaired mammogenesis during late pregnancy. Neither heterozygous null mutants nor somatic knockout mice developed mammary tumors after a latency of 2 years. The Cre-mediated deletion of Tsg101 in primary cells demonstrated that this gene is essential for the growth, proliferation, and survival of mammary epithelial cells. In summary, our results suggest that Tsg101 is required for normal cell function of embryonic and adult tissues but that this gene is not a tumor suppressor for sporadic forms of breast cancer.

The Shh signaling pathway is required in many mammalian tissues for embryonic patterning, cell proliferation and differentiation. In addition, inappropriate activation of the pathway has been implicated in many human tumors. Based on transfection assays and gain-of-function studies in frog and mouse, the transcription factor Gli1 has been proposed to be a major mediator of Shh signaling. To address whether this is the case in mouse, we generated a Gli1 null allele expressing lacZ. Strikingly, Gli1 is not required for mouse development or viability. Of relevance, we show that all transcription of Gli1 in the nervous system and limbs is dependent on Shh and, consequently, Gli1 protein is normally not present to transduce initial Shh signaling. To determine whether Gli1 contributes to the defects seen when the Shh pathway is inappropriately activated and Gli1 transcription is induced, Gli1;Ptc double mutants were generated. We show that Gli1 is not required for the ectopic activation of the Shh signaling pathway or to the early embryonic lethal phenotype in Ptc null mutants. Of significance, we found instead that Gli2 is required for mediating some of the inappropriate Shh signaling in Ptc mutants. Our studies demonstrate that, in mammals, Gli1 is not required for Shh signaling and that Gli2 mediates inappropriate activation of the pathway due to loss of the negative regulator Ptc.

Mice lacking suppressor of cytokine signaling 3 (SOCS3) exhibited embryonic lethality with death occurring between days 11 and 13 of gestation. At this stage, SOCS3(-/-) embryos were slightly smaller than wild type but appeared otherwise normal, and histological analysis failed to detect any anatomical abnormalities responsible for the lethal phenotype. Rather, in all SOCS3(-/-) embryos examined, defects were evident in placental development that would account for their developmental arrest and death. The placental spongiotrophoblast layer was significantly reduced and accompanied by increased numbers of giant trophoblast cells. Delayed branching of the chorioallantois was evident, and, although embryonic blood vessels were present in the labyrinthine layer of SOCS3(-/-) placentas, the network of embryonic vessels and maternal sinuses was poorly developed. Yolk sac erythropoiesis was normal, and, although the SOCS3(-/-) fetal liver was small at day 12.5 of gestation (E12.5), normal frequencies of erythroblasts and hematopoietic progenitor cells, including blast forming unit-erythroid (BFU-E) and, colony forming unit-erythroid (CFU-E) were present at both E11.5 and E12.5. Colony formation for both BFU-E and CFU-E from SOCS3(-/-) mice displayed wild-type quantitative responsiveness to erythropoietin (EPO), in the presence or absence of IL-3 or stem cell factor (SCF). These data suggest that SOCS3 is required for placental development but dispensable for normal hematopoiesis in the mouse embryo.

Holoprosencephaly (HPE) is genetically heterogeneous with four genes, SIX3, SHH, TGIF, and ZIC2 that have been identified to date and that are altered in 12% of patients. To analyze this prevalence in a South American population-based sample (57 HPE cases in 244,511 live and still births or 1 in 4300), we performed a mutational study of these genes in 30 unrelated children (26 newborns and 4 non-newborns) with HPE being ascertained by ECLAMC (Latin American Collaborative Study of Congenital Malformations). We identified three novel mutations: two were missense mutations of the SHH gene (Cys183-->Phe; His140-->Pro); the third mutation was a 2-bp deletion in the zinc-finger region of the ZIC2 gene. These molecular results explained 8% (2/26 newborn samples) of the HPE cases in this South American population-based sample, a proportion similar to our previously published data from a collection of cases.

The murine Foxf1 gene encodes a forkhead transcription factor expressed in extra-embryonic and lateral plate mesoderm and later in splanchnic mesenchyme surrounding the gut and its derivatives. We have disrupted Foxf1 and show that mutant embryos die at midgestation due to defects in mesodermal differentiation and cell adhesion. The embryos do not turn and become deformed by the constraints of a small, inflexible amnion. Extra-embryonic structures exhibit a number of differentiation defects: no vasculogenesis occurs in yolk sac or allantois; chorioallantoic fusion fails; the amnion does not expand with the growth of the embryo, but misexpresses vascular and hematopoietic markers. Separation of the bulk of yolk sac mesoderm from the endodermal layer and adherence between mesoderm of yolk sac and amnion, indicate altered cell adhesion properties and enhanced intramesodermal cohesion. A possible cause of this is misexpression of the cell-adhesion protein VCAM1 in Foxf1-deficient extra-embryonic mesoderm, which leads to co-expression of VCAM with its receptor, alpha(4)-integrin. The expression level of Bmp4 is decreased in the posterior part of the embryo proper. Consistent with this, mesodermal proliferation in the primitive streak is reduced and somite formation is retarded. Expression of Foxf1 and the homeobox gene Irx3 defines the splanchnic and somatic mesodermal layers, respectively. In Foxf1-deficient embryos incomplete separation of splanchnic and somatic mesoderm is accompanied by misexpression of Irx3 in the splanchnopleure, which implicates Foxf1 as a repressor of Irx3 and as a factor involved in coelom formation.

The MEN/ELL gene was cloned as a fusion partner of the MLL gene in the t(11;19)(q23;p13.1) translocation, which is found in adult myeloid leukemia. MEN belongs to a family of RNA polymerase II elongation factors and dysregulated production of MEN through the MLL promoter could cause malignant transformation of myeloid cells. To pursue the physiological role and determine the requirement of the MEN gene product in mouse development, we generated knockout mice (MEN-/-) by gene targeting in embryonic stem cells. After intercrossing heterozygous mice to generate homozygous mutants, we identified no homozygotes (MEN-/-) even at E9.5, as well as after birth, by Southern analysis. Moreover, histological examinations revealed degenerative changes in nearly one-fourth of E6.5 embryos, which were gradually resorbed by E8.5. Our findings demonstrated that MEN-/- mice are embryonic lethal, and die before E6.5 and after implantation. MEN should play a nonredundant role in postimplantation development of mice., (Copyright 2000 Academic Press.)

Inherited mutations in the gene BRCA2 predispose carriers to early onset breast cancer, but such mutations account for fewer than 2% of all cases in East Anglia. It is likely that low penetrance alleles explain the greater part of inherited susceptibility to breast cancer; polymorphic variants in strongly predisposing genes, such as BRCA2, are candidates for this role. BRCA2 is thought to be involved in DNA double strand break-repair. Few mice in which Brca2 is truncated survive to birth; of those that do, most are male, smaller than their normal littermates and have high cancer incidence. Here we show that a common human polymorphism (N372H) in exon 10 of BRCA2 confers an increased risk of breast cancer: the HH homozygotes have a 1.31-fold (95% CI, 1.07-1.61) greater risk than the NN group. Moreover, in normal female controls of all ages there is a significant deficiency of homozygotes compared with that expected from Hardy-Weinberg equilibrium, whereas in males there is an excess of homozygotes: the HH group has an estimated fitness of 0.82 in females and 1.38 in males. Therefore, this variant of BRCA2 appears also to affect fetal survival in a sex-dependent manner.

The Ets family of transcription factors have been suggested to function as key regulators of hematopoeisis. Here we describe aberrant hematopoeisis and hemorrhaging in mouse embryos homozygous for a targeted disruption in the Ets family member, Fli1. Mutant embryos are found to hemorrhage from the dorsal aorta to the lumen of the neural tube and ventricles of the brain (hematorrhachis) on embryonic day 11.0 (E11.0) and are dead by E12.5. Histological examinations and in situ hybridization reveal disorganization of columnar epithelium and the presence of hematomas within the neuroepithelium and disruption of the basement membrane lying between this and mesenchymal tissues, both of which express Fli1 at the time of hemorrhaging. Livers from mutant embryos contain few pronormoblasts and basophilic normoblasts and have drastically reduced numbers of colony forming cells. These defects occur with complete penetrance of phenotype regardless of the genetic background (inbred B6, hybrid 129/B6, or outbred CD1) or the targeted embryonic stem cell line used for the generation of knockout lines. Taken together, these results provide in vivo evidence for the role of Fli1 in the regulation of hematopoiesis and hemostasis.

Women who inherit mutations in the BRCA2 cancer susceptibility gene have an 85% chance of developing breast cancer. The function of the BRCA2 gene remains elusive, but there is evidence to support its role in transcriptional transactivation, tumor suppression, and the maintenance of genomic integrity. Individuals with identical BRCA2 mutations display a different distribution of cancers, suggesting that there are low-penetrance genes that can modify disease outcome. We hypothesized that genetic background could influence embryonic survival of a Brca2 mutation in mice. Brca2-null embryos with a 129/SvEv genetic background (129(B2-/-)) died before embryonic day 8. 5. Transfer of this Brca2 mutation onto the BALB/cJ genetic background (BALB/c(B2-/-)) extended survival to embryonic day 10.5. These results indicate that the BALB/c background harbors genetic modifiers that can prolong Brca2-null embryonic survival. The extended survival of BALB/c(B2-/-) embryos enabled us to ask whether transcriptional regulation of the Brca1 and Brca2 genes is interdependent. The interdependence of Brca1 and Brca2 was evaluated by studying Brca2 gene expression in BALB/c(B1-/-) embryos and Brca1 gene expression in BALB/c(B2-/-) embryos. Nonisotopic in situ hybridization demonstrated that Brca2 transcript levels were comparable in BALB/c(B1-/-) embryos and wild-type littermates. Likewise, reverse transcriptase-polymerase chain reactions confirmed Brca1 mRNA expression in embryonic day 8.5 BALB/c(B2-/-) embryos that was comparable to Brca2-heterozygous littermates. Thus, the Brca1 and Brca2 transcripts are expressed independently of one another in Brca1- and Brca2-null embryos. Mol. Carcinog. 28:174-183, 2000., (Copyright 2000 Wiley-Liss, Inc.)

The Notch genes encode single-pass transmembrane receptors that transduce the extracellular signals responsible for cell fate determination during several steps of metazoan development. The mechanism by which extracellular signals affect gene transcription and ultimately cell fate decisions is beginning to emerge for the Notch signalling pathway. One paradigm is that ligand binding to Notch triggers a Presenilin1-dependent proteolytic release of the Notch intracellular domain from the membrane, resulting in low amounts of Notch intracellular domain which form a nuclear complex with CBF1/Su(H)/Lag1 to activate transcription of downstream targets. Not all observations clearly support this processing model, and the most rigorous test of it is to block processing in vivo and then determine the ability of unprocessed Notch to signal. Here we report that the phenotypes associated with a single point mutation at the intramembranous processing site of Notch1, Val1,744-->Gly, resemble the null Notch1 phenotype. Our results show that efficient intramembranous processing of Notch1 is indispensable for embryonic viability and proper early embryonic development in vivo.

We previously isolated and identified peroxisome proliferator-activated receptor (PPAR)-binding Protein (PBP) as a coactivator for PPARgamma. PBP has recently been identified as a component of the multiprotein complexes such as TRAP, DRIP, and ARC that appear to play an important role in the transcriptional activation by several transcriptional factors including nuclear receptors. To assess the biological significance of PBP, we disrupted the PBP gene (PBP/PPARBP) in mice by homologous recombination. PBP(+/-) mice are healthy, fertile, and do not differ significantly from PBP(+/+) control littermates. PBP null mutation (PBP(-/-)) is embryonically lethal at embryonic day 11.5, suggesting that PBP is an essential gene for mouse embryogenesis. The embryonic lethality is attributed, in part, to defects in the development of placental vasculature similar to those encountered in PPARgamma mutants. Transient transfection assays using fibroblasts isolated from PBP mutant embryos revealed a decreased capacity for ligand-dependent transcriptional activation of PPARgamma as compared with fibroblasts derived form the wild type embryos. These observations suggest that there is no functional redundancy between PBP and other coactivators such as steroid receptor coactivator-1 and that PBP plays a critical role in the signaling of PPARgamma and other nuclear receptors.

Mice lacking the erythroid Kruppel-like factor (EKLF) die in utero at embryonic day 15 (E15) from severe anemia. EKLF(-/-) embryos display a marked deficit in beta-globin gene expression. To test whether beta-globin deficiency was solely responsible for the anemia and intrauterine death, we corrected the globin chain imbalance in EKLF(-/-) embryos by breeding with a strain of mice that express high levels of human gamma-globin. Despite efficient production of hybrid malpha(2)-hgamma(2) hemoglobin in the fetal livers of EKLF(-/-) animals, hemolysis was not corrected and survival was not prolonged. We concluded that deficiency of nonglobin EKLF target genes is a major contributor to the definitive red blood cell abnormalities and prenatal death in EKLF(-/-) embryos. These results suggest that strategies designed to antagonize EKLF function in adults with hemoglobinopathy, in an attempt to reactivate gamma-globin gene expression, may adversely affect other essential aspects of red blood cell physiology. (Blood. 2000;95:1827-1833)

Genetic and molecular evidence indicates that visceral endoderm, an extraembryonic cell lineage, is required for gastrulation, early anterior neural patterning, cell death and specification of posterior mesodermal cell fates. We show that variant Hepatocyte Nuclear Factor 1 (vHNF1), a homeodomain-containing transcription factor first expressed in the primitive endoderm, is required for the specification of visceral endoderm. vHnf1-deficient mouse embryos develop normally to the blastocyst stage, start implantation, but die soon afterwards, with abnormal or absent extraembryonic region, poorly organised ectoderm and no discernible visceral or parietal endoderm. However, immunostaining analysis of E5.5 nullizygous mutant embryos revealed the presence of parietal endoderm-like cells lying on an abnormal basal membrane. Homozygous mutant blastocyst outgrowths or differentiated embryonic stem cells do not express early or late visceral endoderm markers. In addition, in vHnf1 null embryoid bodies there is no activation of the transcription factors HNF-4alpha1, HNF1alpha and HNF-3gamma. Aggregation of vHnf1-deficient embryonic stem cells with wild-type tetraploid embryos, which contribute exclusively to extraembryonic tissues, rescues periimplantation lethality and allows development to progress to early organogenesis. Our results place vHNF1 in a preeminent position in the regulatory network that specifies the visceral endoderm and highlight the importance of this cell lineage for proper growth and differentiation of primitive ectoderm in pregastrulating embryos.

vHNF1/HNF1beta, a member of the divergent HNF1/vHNF1 homeoprotein family, is expressed in polarized epithelia of several adult organs and may participate in controlling the transcription of specific genes. In addition to this late requirement, vHNF1 may play earlier roles during development, as it is first expressed in the visceral endoderm at the onset of gastrulation. In order to shed light on its function during embryogenesis, we have inactivated the murine gene by homologous recombination. The homozygous mutation results in embryonic lethality by day 7.5 of development and vHNF1(-)(/)(-) embryos display a disorganized visceral endoderm and a significantly reduced size. Studies of ES cell differentiation and aggregation with tetraploid morulae establish that vHNF1 expression is essential for visceral endoderm differentiation, both in vitro and in vivo. Analysis of differentiation markers confirms that vHNF1 is part of a genetic network that directs the expression of HNF4 and downstream endodermal genes. Furthermore, the complementation of the mutant embryos with wild-type visceral endoderm rescues the day 7.5 lethality and reveals an additional phenotype linked to vHNF1 later expression. The examination of chimeric embryos suggests that vHNF1 expression might be cell-autonomously required in the gut for the proper morphogenesis of the embryo.

We characterized a region of the mouse genome disrupted by integration of a gene trap (GT) vector in ES cells. On 5' rapid amplification of cDNA ends analysis of the fusion transcripts containing the GT vector, we identified the eukaryotic protein synthesis initiation factor 4A1 gene (Eif4a1) as a promoter-trapped gene. Plasmid rescue was used to show that the other end of the integrated vector disrupted the murine homolog of the human fragile X mental retardation syndrome-related protein 2 gene (Fxr2h). Structural analysis of P1 clones, isolated from the wild-type mouse genome by PCR with Eif4a1-specific primers, indicated that the integration of the GT vector was accompanied by the deletion of about 35 kb of genomic DNA and that the disrupted region also included three genes, Cd68, Supl15h and Sox15, the latter two of which are transcribed in opposite directions with overlapping 3' ends. These five different genes at least, Eif4a1, Cd68, Supl15h, Sox15 and Fxr2h, are clustered in a 40 kb region. The chromosomal location of this region was mapped by means of interspecific backcross panel DNAs to the central part of mouse chromosome 11, exhibiting a known region of synteny with human chromosome 17.

TREB5 (hXBP-1) is a basic region leucine zipper protein which binds to a CRE-like element in both human T-cell leukemia virus type 1 and MHC class II genes. To study the function(s) of TREB5 in normal development, we have generated TREB5 deficient mice by gene targeting. Heterozygous mutant mice have not exhibited any obvious abnormalities; however, homozygous mutant embryos die between embryonic days 10.5 and 14.5. The major defect responsible for lethality is cellular necrosis of cardiac myocytes located at the atrium and the truncus arteriosus with its following ventricle. Necrotic alteration was not observed in either the endocardial cushion or the conotruncal ridge. These results indicate that TREB5 plays an essential role in maintenance and/or growth of cardiac myocytes during cardiogenesis., (Copyright 1999 Academic Press.)

Hypoxia-inducible factor 1 (HIF-1) is a dimeric transcription factor composed of HIF-1alpha and HIF-1beta subunits that plays an essential role in mammalian O2 homeostasis. In Hif1a-/- knockout mice, complete deficiency of HIF-1alpha resulted in cardiac and vascular malformations and embryonic lethality at E10.5. Between E8. 75 and E9.25 striking vascular regression and abnormal remodeling occurred in the cephalic region concomitant with marked mesenchymal cell death. Similar vascular defects were observed in HIF-1alpha- and VEGF-deficient embryos and VEGF mRNA expression was not induced by hypoxia in Hif1a-/- embryonic stem cells. Surprisingly, Hif1a-/- embryos demonstrated increased VEGF mRNA expression compared to wild-type embryos. In tissue culture cells, VEGF mRNA expression was induced by glucose deprivation independent of HIF-1alpha, providing a mechanism for increased VEGF mRNA expression in Hif1a-/- embryos, in which absence of adequate tissue perfusion resulted in both O2 and glucose deprivation. Rather than being associated with VEGF deficiency, the vascular defects in Hif1a-/- embryos were spatially correlated with cell death, the onset of which preceded vascular regression., (Copyright 1999 Academic Press.)

We have engineered a mouse mutation that specifically deletes the C-terminal 18 amino acid sequence of the RXRalpha protein. This deletion corresponds to the last helical alpha structure (H12) of the ligand-binding domain (LBD), and includes the core of the Activating Domain of the Activation Function 2 (AF-2 AD core) that is thought to be crucial in mediating ligand-dependent transactivation by RXRalpha. The homozygous mutants (RXRalpha af2(o)), which die during the late fetal period or at birth, exhibit a subset of the abnormalities previously observed in RXRalpha -/- mutants, often with incomplete penetrance. In marked contrast, RXRalpha af2(o)/RXRbeta -/- and RXRalpha af2(o)/RXRbeta -/- /RXRgamma -/- compound mutants display a large array of malformations, which nearly recapitulate the full spectrum of the defects that characterize the fetal vitamin A-deficiency (VAD) syndrome and were previously found in RAR single and compound mutants, as well as in RXRalpha/RAR(alpha, beta or gamma) compound mutants. Analysis of RXRalpha af2(o)/RAR(alpha, beta or gamma) compound mutants also revealed that they exhibit many of the defects observed in the corresponding RXR alpha/RAR compound mutants. Together, these results demonstrate the importance of the integrity of RXR AF-2 for the developmental functions mediated by RAR/RXR heterodimers, and hence suggest that RXR ligand-dependent transactivation is instrumental in retinoid signalling during development.

The incidence of translocations involving the 11q23 gene MLL is markedly increased in leukaemias that occur in infants <1 year of age. Epidemiological and molecular data have demonstrated that at least some of these translocations occur in utero. In this report we describe a case of fetal death at 36 weeks of gestation. At autopsy the fetus was found to have widely disseminated acute myelogenous leukaemia (AML), FAB subtype M5. Molecular cytogenetic studies of nuclei recovered from paraffin-embedded tissue sections demonstrated that the leukaemic cells contained an MLL translocation. This is the first detailed report, to our knowledge, of fetal death due to acute leukaemia, and directly demonstrates oncogenesis in utero.

The placenta and cardiovascular system are the first organ systems to form during mammalian embryogenesis. We show here that a single gene is critical for development of both. The Hand1 gene, previously called Hxt, eHAND and Thing1, encodes a basic helix-loop-helix (bHLH) transcription factor that starts to be expressed during pre-implantation development. After implantation, Hand1 expression is restricted to placental trophoblast cells and later to embryonic cardiac and neural crest cells. We generated Hand1-null mutant mice by gene targetting. Homozygous mutant embryos arrested by embryonic day (E) 7.5 of gestation with defects in trophoblast giant cell differentiation. This early mortality could be rescued by aggregation of mutant embryos with wild-type tetraploid embryos, which contribute wild-type cells to the trophoblast, but not the embryo. By E10.5, however, the Hand1-null fetuses derived from tetraploid chimaeras died due to cardiac failure. Their heart tubes showed abnormal looping and ventricular myocardial differentiation. Therefore, Hand1 is essential for differentiation of both trophoblast and cardiomyocytes, which are embryologically distinct cell lineages.

The basic helix-loop-helix (bHLH) transcription factors, Hand1 and Hand2 (refs 1,2), also called eHand/Hxt/Thing1 and dHand/Hed/Thing2 (refs 3,4), respectively, are expressed in the heart and certain neural-crest derivatives during embryogenesis. In addition, Hand1 is expressed in extraembryonic membranes, whereas Hand2 is expressed in the deciduum. Previous studies have demonstrated that Hand2 is required for formation of the right ventricle of the heart and the aortic arch arteries. We have generated a germline mutation in the mouse Hand1 gene by replacing the first coding exon with a beta-galactosidase reporter gene. Embryos homozygous for the Hand1 null allele died between embryonic days 8.5 and 9.5 and exhibited yolk sac abnormalities due to a deficiency in extraembryonic mesoderm. Heart development was also perturbed and did not progress beyond the cardiac-looping stage. Our results demonstrate important roles for Hand1 in extraembryonic mesodermal and heart development.

The regulation of mitochondrial DNA (mtDNA) expression is crucial for mitochondrial biogenesis during development and differentiation. We have disrupted the mouse gene for mitochondrial transcription factor A (Tfam; formerly known as m-mtTFA) by gene targetting of loxP-sites followed by cre-mediated excision in vivo. Heterozygous knockout mice exhibit reduced mtDNA copy number and respiratory chain deficiency in heart. Homozygous knockout embryos exhibit a severe mtDNA depletion with abolished oxidative phosphorylation. Mutant embryos proceed through implantation and gastrulation, but die prior to embryonic day (E)10.5. Thus, Tfam is the first mammalian protein demonstrated to regulate mtDNA copy number in vivo and is essential for mitochondrial biogenesis and embryonic development.

Objective: The splotch (Sp2h) mutation disrupts the Pax3 gene and is lethal in homozygotes. The aim of the present study was to investigate the cause of lethality., Methods and Results: Using the splotch (Sp2H) mouse mutant, we demonstrated that approximately 60% of Sp2H homozygotes die in utero at 13.5-14.5 days of gestation. All these embryos have cardiac malformations involving partial or complete failure of septation of the outflow tract. Although the cause of death in utero is unknown, the dying embryos are edematous, their superior caval veins are over-expanded, and the fetal liver is enlarged and engorged with blood, all signs of cardiac failure. The remaining Sp2H homozygotes die around the time of birth, and these embryos have grossly normal hearts. All Sp2H homozygotes have neural tube defects, either spina bifida, exencephaly, or both. Although these defects clearly do not cause death in utero, they are very likely responsible for the perinatal death of homozygotes that survive to late gestation. There is no correlation between the presence or absence of a cardiac defect and the type of neural tube defect. On the other hand, there is a striking correlation between presence of a cardiac defect and reduction or absence of dorsal root ganglia, which are derivatives of the neural crest., Conclusions: In this paper, we show that the lethality has a biphasic pattern, and the data strongly suggests that mid-gestation lethality is due to cardiac defects and not the associated neural tube defects. This finding supports the idea that 'conotruncal' cardiac defects involving the ventricular outflow tracts develop as a result of failure of the 'cardiac' neural crest to colonise the developing heart in the mid-gestation embryo, and that the resulting heart defects are solely responsible for the observed mortality.

Acute myeloid leukaemia (AML) is a major haematopoietic malignancy characterized by the proliferation of a malignant clone of myeloid progenitor cells. A reciprocal translocation, t(8;21)(q22;q22), observed in the leukaemic cells of approximately 40% of patients with the M2 subtype of AML disrupts both the AML1 (CBFA2) gene on chromosome 21 and the ETO (MTG8) gene on chromosome 8 (refs 3-5). A chimaeric protein is synthesized from one of the derivative chromosomes that contains the N terminus of the AML1 transcription factor, including its DNA-binding domain, fused to most of ETO, a protein of unknown function. We generated mice that mimic human t(8;21) with a "knock-in' strategy. Mice heterozygous for an AML1-ETO allele (AML1-ETO/+) die in midgestation from haemorrhaging in the central nervous system and exhibit a severe block in fetal liver haematopoiesis. This phenotype is very similar to that resulting from homozygous disruption of the AML1 (Cbfa2) or Cbfb genes, indicating that AML1-ETO blocks normal AML1 function. However, yolk sac cells from AML1-ETO/+ mice differentiated into macrophages in haematopoietic colony forming unit (CFU) assays, unlike Cbfa2-/- or Cbfb-/-cells, which form no colonies in vitro. This indicates that AML1-ETO may have other functions besides blocking wild-type AML1, a property that may be important in leukaemogenesis.

BRCA1 is proposed to be a tumor suppressor gene. To explore the biological function of BRCA1, a partial deletion (amino acids 300-361) of mouse Brca1 exon 11 was introduced into the genome of embryonic stem (ES) cells by homologous recombination. Mice carrying one mutated allele of Brca1 appear normal and are fertile up to 10 months of age without any sign of illness. However, no viable progeny homozygous for the Brca1 mutant allele were obtained. Detailed analysis of large numbers of embryos at different stages of development indicated that the homozygous mutant concepti are severely retarded in growth as early as embryonic day 4.5 (E4.5) and are resorbed completely by E8.5. Although the homozygotes at E5.5-E6.5 are able to synthesize DNA and display distinguishable embryonic and extraembryonic structures, they fail to differentiate and form egg cylinders. Consequently, they were unable to form primitive streaks and undergo gastrulation. Consistent with these in vivo results, blastocysts homozygous for mutated Brca1 alleles are at a considerable disadvantage when grown in vitro. These observations suggest that Brca1 has an important role in the early development of mouse embryos.

The CBFA2 (AML1) gene encodes a DNA-binding subunit of the heterodimeric core-binding factor. The CBFA2 gene is disrupted by the (8;21), (3;21), and (12;21) chromosomal translocations associated with leukemias and myelodysplasias in humans. Mice lacking a CBF alpha 2 protein capable of binding DNA die between embryonic days 11.5 and 12.5 due to hemorrhaging in the central nervous system (CNS), at the nerve/CNS interfaces of cranial and spinal nerves, and in somitic/intersomitic regions along the presumptive spinal cord. Hemorrhaging is preceded by symmetric, bilateral necrosis in these regions. Definitive erythropoiesis and myelopoiesis do not occur in Cbfa2-deficient embryos, and disruption of one copy of the Cbfa2 gene significantly reduces the number of progenitors for erythroid and myeloid cells.

We have generated mouse lines in which the RXR beta gene was disrupted by homologous recombination. Approximately 50% of the RXR beta homozygous mutants died before or at birth, but those that survived appeared normal except that the males were sterile, owing to oligo-astheno-teratozoospermia. Failure of spermatid release occurred within the germinal epithelium, and the epididymis contained very few spermatozoa that, in addition, exhibited abnormal acrosomes and tails. There was a progressive accumulation of lipids within the mutant Sertoli cells, which were histochemically characterized as unsaturated triglycerides. In old mutant males, progressive degeneration of the germinal epithelium occurred, ending with the formation of acellular lipid-filled tubules. The selective expression of RXR beta in Sertoli cells, together with the timing of appearance of the histological abnormalities, suggests that the primary defect resulting from the mutation resides in these cells.

The goosecoid gene encodes a homeodomain-containing protein that has been identified in a number of species and has been implicated in a variety of key developmental processes. Initially suggested to be involved in organizing the embryo during early development, goosecoid has since been demonstrated to be expressed during organogenesis-most notably in the head, the limbs and the ventrolateral body wall. To investigate the role of goosecoid in embryonic development, we have inactivated the gene by gene targeting to generate mice mutant for the goosecoid gene. Mice that are homozygous for the goosecoid mutation do not display a gastrulation phenotype and are born; however, they do not survive more than 24 hours. Analysis of the homozygotes revealed numerous developmental defects affecting those structures in which goosecoid is expressed during its second (late) phase of embryonic expression. Predominantly, these defects involve the lower mandible and its associated musculature including the tongue, the nasal cavity and the nasal pits, as well as the components of the inner ear (malleus, tympanic ring) and the external auditory meatus. Although the observed phenotype is in accordance with the late expression domains of goosecoid in wild-type embryos, we suggest that the lack of an earlier phenotype is the result of functional compensation by other genes.

NF-kappa B, which consists of two polypeptides, p50 (M(r) 50K) and p65/RelA (M(r) 65K), is thought to be a key regulator of genes involved in responses to infection, inflammation and stress. Indeed, although developmentally normal, mice deficient in p50 display functional defects in immune responses. Here we describe the generation of mice deficient in the RelA subunit of NF-kappa B. Disruption of the relA locus leads to embryonic lethality at 15-16 days of gestation, concomitant with a massive degeneration of the liver by programmed cell death or apoptosis. Embryonic fibroblasts from RelA-deficient mice are defective in the tumour necrosis factor (TNF)-mediated induction of messenger RNAs for I kappa B alpha and granulocyte/macrophage colony stimulating factor (GM-CSF), although basal levels of these transcripts are unaltered. These results indicate that RelA controls inducible, but not basal, transcription in NF-kappa B-regulated pathways.

We have used a retroviral gene trap in embryonic stem (ES) cells to derive a recessive embryonic lethal mouse strain, ROSA beta-geo5. Mutant embryos display an enlarged pericardial cavity, bradycardia, a dilated fourth ventricle in the brain, and die between embryonic days 11 and 12. Whereas heart development in the mutant embryos is extensive, the ventricular wall is abnormally thin with a reduced number of trabeculae. Cloning of the trapped gene indicates that proviral insertion creates a null mutation in the transcriptional enhancer factor 1 (TEF-1) gene. Although transcription of a number of muscle-specific genes believed to be TEF-1 targets appears normal, the defect in cardiogenesis is likely attributable to diminished transcription of one or several cardiac-specific genes.