ABSTRACT
The inside-outside model has been invoked to explain cell-fate specification of the pre-implantation mammalian embryo. Here, we investigate whether cell-cell interaction can influence the fate specification of embryonic blastomeres by sequentially separating the blastomeres in two-cell stage mouse embryos and continuing separation after each cell division throughout pre-implantation development. This procedure eliminates information provided by cell-cell interaction and cell positioning. Gene expression profiles, polarity protein localization and functional tests of these separated blastomeres reveal that cell interactions, through cell position, influence the fate of the blastomere. Blastomeres, in the absence of cell contact and inner-outer positional information, have a unique pattern of gene expression that is characteristic of neither inner cell mass nor trophectoderm, but overall they have a tendency towards a 'trophectoderm-like' gene expression pattern and preferentially contribute to the trophectoderm lineage.
Subject(s)
Blastomeres/cytology , Blastomeres/physiology , Cell Communication , Embryo, Mammalian/cytology , Embryonic Development , Animals , Cell Differentiation , Cell Lineage , Embryo, Mammalian/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Mice , PregnancyABSTRACT
Autosomal recessive cutis laxa (ARCL) describes a group of syndromal disorders that are often associated with a progeroid appearance, lax and wrinkled skin, osteopenia and mental retardation. Homozygosity mapping in several kindreds with ARCL identified a candidate region on chromosome 17q25. By high-throughput sequencing of the entire candidate region, we detected disease-causing mutations in the gene PYCR1. We found that the gene product, an enzyme involved in proline metabolism, localizes to mitochondria. Altered mitochondrial morphology, membrane potential and increased apoptosis rate upon oxidative stress were evident in fibroblasts from affected individuals. Knockdown of the orthologous genes in Xenopus and zebrafish led to epidermal hypoplasia and blistering that was accompanied by a massive increase of apoptosis. Our findings link mutations in PYCR1 to altered mitochondrial function and progeroid changes in connective tissues.
Subject(s)
Cutis Laxa/etiology , Cutis Laxa/genetics , Mutation , Pyrroline Carboxylate Reductases/genetics , Skin/metabolism , Agenesis of Corpus Callosum , Base Sequence , Case-Control Studies , Child, Preschool , Chromosomes, Human, Pair 17 , Consanguinity , Cutis Laxa/metabolism , Female , Fibroblasts/metabolism , Frameshift Mutation , Gene Deletion , Genes, Recessive , Genetic Markers , Homozygote , Humans , Infant , Infant, Newborn , Intellectual Disability/genetics , Male , Molecular Sequence Data , Mutation, Missense , Pedigree , Physical Chromosome Mapping , Polymorphism, Single Nucleotide , Pyrroline Carboxylate Reductases/metabolism , Skin/cytology , Skin/ultrastructure , delta-1-Pyrroline-5-Carboxylate ReductaseABSTRACT
The severe acute respiratory syndrome coronavirus (SARS-CoV) 7a protein, which is not expressed by other known coronaviruses, can induce apoptosis in various cell lines. In this study, we show that the overexpression of Bcl-XL, a prosurvival member of the Bcl-2 family, blocks 7a-induced apoptosis, suggesting that the mechanism for apoptosis induction by 7a is at the level of or upstream from the Bcl-2 family. Coimmunoprecipitation experiments showed that 7a interacts with Bcl-XL and other prosurvival proteins (Bcl-2, Bcl-w, Mcl-1, and A1) but not with the proapoptotic proteins (Bax, Bak, Bad, and Bid). A good correlation between the abilities of 7a deletion mutants to induce apoptosis and to interact with Bcl-XL was observed, suggesting that 7a triggers apoptosis by interfering directly with the prosurvival function of Bcl-XL. Interestingly, amino acids 224 and 225 within the C-terminal transmembrane domain of Bcl-XL are essential for the interaction with the 7a protein, although the BH3 domain of Bcl-XL also contributes to this interaction. In addition, fractionation experiments showed that 7a colocalized with Bcl-XL at the endoplasmic reticulum as well as the mitochondria, suggesting that they may form complexes in different membranous compartments.
Subject(s)
Apoptosis , Viral Matrix Proteins/physiology , Viral Proteins/physiology , bcl-X Protein/metabolism , Amino Acid Sequence , Animals , Chlorocebus aethiops , Gene Deletion , Humans , Immunoprecipitation , Mitochondria/metabolism , Molecular Sequence Data , Mutation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Vero Cells , Viral Matrix Proteins/metabolism , Viral Proteins/metabolismABSTRACT
Here we analyzed the gene expression profile of cells that stably express the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein to determine its effects on host functions. A lung epithelial cell-line, A549, was chosen for this study because the lung is the primary organ infected by SARS-CoV and fatalities resulted mainly from pulmonary complications. Our results showed that the expression of 3a up-regulates the mRNA levels of all three subunits, Aalpha, Bbeta, and gamma, of fibrinogen. Consequently, the intracellular levels as well as the secretion of fibrinogen were increased. We also observed increased fibrinogen levels in SARS-CoV-infected Vero E6 cells.
