SEK1/MKK4-mediated SAPK/JNK signaling participates in embryonic hepatoblast proliferation via a pathway different from NF-kappaB-induced anti-apoptosis

Mice lacking the stress-signaling kinase SEK1 die from embryonic day 10.5 (E10.5) to E12.5. Although a defect in liver formation is accompanied with the embryonic lethality of sek1(-/-) mice, the mechanism of the liver defect has remained unknown. In the present study, we first produced a monoclonal antibody specifically recognizing murine hepatoblasts for the analysis of liver development and further investigated genetic interaction ofsek1 with tumor necrosis factor-alpha receptor 1 gene (tnfr1) and protooncogene c-jun, which are also responsible for liver formation and cell apoptosis. The defective liver formation in sek1(-/-) embryos was not protected by additionaltnfr1 mutation, which rescues the embryonic lethality of mice lacking NF-kappaB signaling components. There was a progressive increase in the hepatoblast cell numbers of wild-type embryos from E10.5 to E12.5. Instead, impaired hepatoblast proliferation was observed in sek1(-/-) livers from E10.5, though fetal liver-specific gene expression was normal. The impaired phenotype in sek1(-/-) livers was more severe than in c-jun(-/-) embryos, and sek1(-/-) c-jun(-/-) embryos died more rapidly before E8.5. The hepatoblast proliferation required no hematopoiesis, since liver development was not impaired in AML1(-/-) mice that lack hematopoietic functions. Stimulation of stress-activated protein kinase/c-Jun N-terminal kinase by hepatocyte growth factor was attenuated in sek1(-/-) livers. Thus, SEK1 appears to play a crucial role in hepatoblast proliferation and survival in a manner apparently different from NF-kappaB or c-Jun.     

Perturbation of the hematopoietic system during embryonic liver development due to disruption of polyubiquitin gene Ubc in mice

Disruption of the polyubiquitin gene Ubc leads to a defect in fetal liver development, which can be partially rescued by increasing the amount of ubiquitin. However, it is still not known why Ubc is required for fetal liver development and the nature of the defective cell types responsible for embryonic lethality have not been characterized. In this study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We found that Ubc was highly expressed in the embryonic liver, and the proliferation capacity of fetal liver cells was reduced in Ubc(-/-) embryos. Specifically, Ubc was most highly expressed in hematopoietic cells, and the proliferation capacity of hematopoietic cells was significantly impaired in Ubc(-/-) embryos. While hematopoietic cell and hematopoietic stem cell (HSC) frequency was maintained in Ubc(-/-) embryos, the absolute number of these cells was diminished because of reduced total liver cell number in Ubc(-/-) embryos. Transplantations of fetal liver cells into lethally irradiated recipient mice by non-competitive and competitive reconstitution methods indicated that disruption of Ubc does not significantly impair the intrinsic function of fetal liver HSCs. These findings suggest that disruption of Ubc reduces the absolute number of HSCs in embryonic livers, but has no significant effect on the autonomous function of HSCs. Thus, the lethality of Ubc(-/-) embryos is not the result of intrinsic HSC failure.     

Prep1 (pKnox1) Regulates Mouse Embryonic HSC Cycling and Self-Renewal Affecting the Stat1-Sca1 IFN-Dependent Pathway

A hypomorphic Prep1 mutation results in embryonic lethality at late gestation with a pleiotropic embryonic phenotype that includes defects in all hematopoietic lineages. Reduced functionality of the hematopoietic stem cells (HSCs) compartment might be responsible for the hematopoietic phenotype observed at mid-gestation. In this paper we demonstrate that Prep1 regulates the number of HSCs in fetal livers (FLs), their clonogenic potential and their ability to de novo generate the hematopoietic system in ablated hosts. Furthermore, we show that Prep1 controls the self-renewal ability of the FL HSC compartment as demonstrated by serial transplantation experiments. The premature exhaustion of Prep1 mutant HSCs correlates with the reduced quiescent stem cell pool thus suggesting that Prep1 regulates the self-renewal ability by controlling the quiescence/proliferation balance. Finally, we show that in FL HSCs Prep1 absence induces the interferon signaling pathway leading to premature cycling and exhaustion of fetal HSCs.     

Developmental aberrations of liver gene expression in bovine fetuses derived from somatic cell nuclear transplantation

Cloning by somatic cell nuclear transfer (NT) has been accomplished. However, the process itself is inefficient since most clones die before birth and survivors often display various anomalies. In an effort to determine global expression profiles of developmentally regulated liver genes in NT bovine fetuses, we employed a custom-made bovine liver complementary DNA (cDNA) microarray. The NT fetuses in early pregnancy were derived from cumulus cells as the nuclear donor cells. Normal fetuses were derived from in vitro fertilization (IVF) and artificial insemination (AI). Gene expression levels in NT, IVF, and AI fetal livers were obtained by comparing individual fetal liver samples with that of adult liver of nonpregnant cycling cows. Statistical analyses of the expression data showed widespread dysregulation of developmentally important genes in the three NT fetuses examined. It was found that the number of dysregulated genes was within a range of 3.5-7.7% of the tested genes in the NT fetal livers. The analyses revealed that one NT fetus was markedly different in liver gene expression profile from the other two NT fetal livers in which the expression profiles were highly correlated. Thus, our findings demonstrate that widespread dysregulation of liver genes occurs in the developing liver of NT bovine fetuses. It is possible that inappropriate genomic reprogramming after NT is a key factor associated with abnormal gene expressions in the livers of NT fetuses, whereas distinct expression patterns between the fellow cloned fetuses likely have resulted from variable epigenetic status of the donor nuclei.     

Effects of endogenous and exogenous glucocorticoids on liver differentiation

The effects of maternal bilateral adrenalectomy on day 1 of gestation and betamethasone treatment on fetal liver development were compared, in terms of biochemical and morphological parameters. For fetuses 20 days old (E20), absence of maternal glucocorticoids during gestation caused an increase in the number of nuclei in whole livers, and a significantly decrease of both body weight and protein content per nucleus, in comparison with the control group (C). Betamethasone injection on days 15, 16 and 17 of gestation into adrenalectomized pregnant rats (ADX + BET) did not completely prevent these effects. The electron microscopic analysis of the ADX fetal liver (E20) showed some hepatocyte lesions such as loss of cytoplasmic organelles, increase in hematopoietic cell number as well as a lower cellular maturation in comparison with the control group. The fetal liver from ADX + BET mothers 20 days after gestation displayed a noticeable involution of the hematopoietic component in spite of its relatively immature stage. However, there was no significant change in the degree of fetal hepatocyte lesions. Therefore, supply of maternal glucocorticoids from the beginning of gestation is essential for maintenance of the integral structure of the rat fetal hepatic parenchyma, for the correct maturation of the blood strains and for the beginning of involution of the hematopoietic tissue at the end of gestation.     

Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity

Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic stem cells and progenitors of Mdm2(-/-) p53(515C/515C) mice were normal in fetal livers but were depleted in postnatal bone marrows. After birth, these mice had elevated reactive oxygen species (ROS) thus activating p53R172P. In the absence of Mdm2, stable p53R172P induced ROS and cell cycle arrest, senescence, and cell death in the hematopoietic compartment. This phenotype was partially rescued with antioxidant treatment and upon culturing of hematopoietic cells in methycellulose at 3% oxygen. p16 was also stabilized because of ROS, and its loss increased cell cycling and partially rescued hematopoiesis and survival. Thus, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis.     

Raf-1 sets the threshold of Fas sensitivity by modulating Rok-alpha signaling

Ablation of the Raf-1 protein causes fetal liver apoptosis, embryonic lethality, and selective hypersensitivity to Fas-induced cell death. Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha. In this study, we show that the kinase-independent modulation of Rok-alpha signaling is also the basis of the antiapoptotic function of Raf-1. Fas activation stimulates the formation of Raf-1-Rok-alpha complexes, and Rok-alpha signaling is up-regulated in Raf-1-deficient cells. This leads to increased clustering and membrane expression of Fas, which is rescued both by kinase-dead Raf-1 and by interfering with Rok-alpha or its substrate ezrin. Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts. Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.     

Fusing VE-Cadherin to α-Catenin Impairs Fetal Liver Hematopoiesis and Lymph but Not Blood Vessel Formation

We have recently shown that genetic replacement of VE-cadherin by a VE-cadherin–α-catenin fusion construct strongly impairs opening of endothelial cell contacts during leukocyte extravasation and induction of vascular permeability in adult mice. Here we show that this mutation leads to lethality at midgestation on a clean C57BL/6 background. Investigating the reasons for embryonic lethality, we observed a lack of fetal liver hematopoiesis and severe lymphedema but no detectable defects in blood vessel formation and remodeling. As for the hematopoiesis defect, VE-cadherin–α-catenin affected neither the generation of hematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium nor their differentiation into multiple hematopoietic lineages. Instead, HSPCs accumulated in the fetal circulation, suggesting that their entry into the fetal liver was blocked. Edema formation was caused by disturbed lymphatic vessel development. Lymphatic progenitor cells of VE-cadherin–α-catenin-expressing embryos were able to leave the cardinal vein and migrate to the site of the first lymphatic vessel formation, yet subsequently, these cells failed to form large lumenized lymphatic vessels. Thus, stabilizing endothelial cell contacts by a covalent link between VE-cadherin and α-catenin affects recruitment of hematopoietic progenitors into the fetal liver and the development of lymph but not blood vessels.     

Abnormal development of genetically normal fetal hematopoietic stem cells in steel mutant mouse fetuses

The role and possible transplantability of the early hematopoietic microenvironment was investigated by transplacental inoculation of fetal liver cells from normal donors into steel mutant early fetuses. Donor hematopoietic stem cells were able to lodge in the livers of recipients and to progress to the bone marrow postnatally. However, self-renewal of stem cells and production of differentiated blood cells was very limited in extent and duration after transplantation into mildly anemic steel as compared with Wv/+ heterozygotes. The microenvironmental defect known to exist (albeit undefined) in steel and not in W mutants thus adversely affects proliferation and differentiation of stem cells from the very inception of hepatic hematopoiesis and is not correctable by introducing normal stromal cells under the conditions of the experiment.     

Regulation of rabbit fetal glycogen: effect of in utero fetal decapitation on the metabolism of glycogen in fetal heart, lung, and liver

To understand the control mechanisms involved in the regulation of fetal glycogen, we have studied the effect of in utero fetal decapitations on glycogen metabolism in rabbit fetal heart, lung, and liver. In utero fetal decapitations were performed between days 18 and 21 of gestation. Two to four fetuses on one side of the horn were decapitated. Fetuses were delivered between days 23 and 26 or between days 28 and 30 of gestation. Fetal heart, lungs, and liver were analyzed for DNA, protein, glycogen, glycogen synthase (I and D forms), glycogen phosphorylase (a and b forms), phosphofructokinase, pyruvate kinase, and lactic dehydrogenase. In fetal heart and lung, no difference was observed in any of the above measurements in the intact and decapitated fetuses. In contrast, fetal liver does not appear to develop the glycogen system as indicated by the very low levels of glycogen (0.02 mg/mg DNA) in decapitated fetuses as compared with intact fetuses (0.4 mg/mg DNA). Similarly the levels of glycogen synthase and phosphorylase were two to three times lower in livers from decapitated fetuses as compared with the livers from intact fetuses. The three enzymes phosphofructokinase, pyruvate kinase, and lactic dehydrogenase were not affected by fetal decapitation in all three tissues. These results indicate that the fetal hypothalamic-pituitary-adrenal (thyroid) axis is not required at least after day 18 of gestation for the normal accumulation and subsequent utilization of glycogen in fetal heart and lungs, while it is an absolute requirement for the development of the fetal liver glycogen system.(ABSTRACT TRUNCATED AT 250 WORDS)