ABSTRACT
Overexpression and inhibitor studies have suggested that the c-Myc target gene for ornithine decarboxylase (ODC), the enzyme which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation, transformation, and apoptosis. To explore the physiological function of ODC in mammalian development, we generated mice harboring a disrupted ODC gene. ODC-heterozygous mice were viable, normal, and fertile. Although zygotic ODC is expressed throughout the embryo prior to implantation, loss of ODC did not block normal development to the blastocyst stage. Embryonic day E3.5 ODC-deficient embryos were capable of uterine implantation and induced maternal decidualization yet failed to develop substantially thereafter. Surprisingly, analysis of ODC-deficient blastocysts suggests that loss of ODC does not affect cell growth per se but rather is required for survival of the pluripotent cells of the inner cell mass. Therefore, ODC plays an essential role in murine development, and proper homeostasis of polyamine pools appears to be required for cell survival prior to gastrulation.
Subject(s)
Apoptosis , Embryo, Mammalian/cytology , Embryonic and Fetal Development , Ornithine Decarboxylase/genetics , Ornithine Decarboxylase/physiology , Animals , Blastocyst/cytology , Cell Survival , Decidua/anatomy & histology , Embryo Implantation , Female , Gene Targeting , Mice , Models, BiologicalABSTRACT
SOCS3 (CIS3/JAB2) is an SH2-containing protein that binds to the activation loop of Janus kinases, inhibiting kinase activity, and thereby suppressing cytokine signaling. During embryonic development, SOCS3 is highly expressed in erythroid lineage cells and is Epo independent. Transgene-mediated expression blocks fetal erythropoiesis, resulting in embryonic lethality. SOCS3 deletion results in an embryonic lethality at 12-16 days associated with marked erythrocytosis. Moreover, the in vitro proliferative capacity of progenitors is greatly increased. SOCS3-deficient fetal liver stem cells can reconstitute hematopoiesis in lethally irradiated adults, indicating that its absence does not disturb bone marrow erythropoiesis. Reconstitution of lymphoid lineages in JAK3-deficient mice also occurs normally. The results demonstrate that SOCS3 is critical in negatively regulating fetal liver hematopoiesis.
Subject(s)
Erythropoiesis/physiology , Gene Expression Regulation, Developmental , Liver/embryology , Proteins/genetics , Proteins/physiology , Repressor Proteins , Transcription Factors , Animals , Dose-Response Relationship, Drug , Flow Cytometry , Hematopoiesis/physiology , In Situ Hybridization , Interleukin-2/pharmacology , Interleukin-4/pharmacology , Liver/physiology , Mice , Mice, Mutant Strains , Models, Genetic , Mutagenesis , Phenotype , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins , Time Factors , TransfectionABSTRACT
We previously identified the ZNF85 (HPF4) KRAB zinc finger gene, a member of the human ZNF91 family. Here, we show that the ZNF85 gene is highly expressed in normal adult testis, in seminomas, and in the NT2/D1 teratocarcinoma cell line. Immunocytochemical localization of a panel of beta-Gal/ZNF85 fusion proteins revealed that ZNF85 contains at least one nuclear localization signal located in the spacer region connecting the KRAB domain with the zinc finger repeats. Bacterially expressed ZNF85 zinc finger domain bound strongly and exclusively to DNA in vitro in a zinc-dependent manner. The KRAB(A) domain of the ZNF85 protein and of several other members of the ZNF91 family exhibited repressing activity when tested in Gal4 fusion protein assays. The repression was significantly enhanced by the addition of the KRAB (B) domain, whereas further addition of other conserved regions had no effect. The ZNF85 KRAB(A) and (B) domains in vitro bound several nuclear proteins that might constitute critical cofactors for repression.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/physiology , Repressor Proteins/physiology , Testis/metabolism , Zinc Fingers/physiology , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Cell Nucleus/metabolism , DNA/metabolism , DNA, Complementary , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Gene Expression , Humans , Kruppel-Like Transcription Factors , Male , Molecular Sequence Data , Protein Binding , Recombinant Fusion Proteins/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Zinc/metabolism , Zinc Fingers/geneticsABSTRACT
Gli-type zinc finger proteins play important regulatory roles in vertebrate and invertebrate embryogenesis. In Xenopus, the Gli-type proteins XGli-3 and XGli-4 are first expressed in earliest stages of mesoderm and neural development. Transient transfection assays reveal that XGli-3 and XGli-4 can function as transcription repressors. Counteracting the Gli-protein repressor activity by ectopic expression of a fusion protein that contains the Gli-zinc finger cluster connected to the E1A activator domain in Xenopus embryos results in specific morphological alterations in the developing somites and in the central nervous system. Altered expression characteristics for a broad set of molecular markers highlighting specific aspects of mesodermal and neural differentiation demonstrate an important role for Gli-type zinc finger proteins in the early mesodermal and neural patterning of Xenopus embryos.
