ABSTRACT
The mouse blastocyst and stem cells derived from its tissue lineages provide a unique genetic system for examining the establishment and loss of pluripotency. The transcription factor Cdx2 plays a central role by repressing pluripotency genes, such as Oct4, and promoting extraembryonic trophoblast fate at the blastocyst stage. However, genetic evidence has suggested that Cdx2 does not work alone in the trophoblast lineage. We have used bioinformatic and functional genomic strategies to identify the transcription factor Gata3 as a trophoblast factor. We show Gata3 to be capable of inducing trophoblast fate in embryonic stem cells and driving trophoblast differentiation in trophoblast stem cells. In addition, Cdx2 is not required for Gata3-induced expression of a subset of trophoblast genes in embryonic stem cells. We show that Gata3 is coexpressed with Cdx2 in the blastocyst, but this does not depend on Cdx2. In the embryo, expression of Gata3, like that of Cdx2, depends on Tead4, and the expression of both factors becomes restricted to trophoblast by a mechanism that does not initially rely on Oct4. These observations suggest that Gata3 and Cdx2 can act in parallel pathways downstream of Tead4 to induce the expression of common and independent targets in the trophoblast lineage, whereas Oct4 is required for continued repression of trophoblast fate in the embryonic lineage.
Subject(s)
DNA-Binding Proteins/physiology , GATA3 Transcription Factor/physiology , Homeodomain Proteins/physiology , Muscle Proteins/physiology , Octamer Transcription Factor-3/physiology , Transcription Factors/physiology , Trophoblasts/cytology , Animals , Blastocyst/cytology , CDX2 Transcription Factor , Cell Differentiation/genetics , Cell Lineage , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryonic Induction/genetics , Embryonic Stem Cells/cytology , Female , Gene Expression Regulation, Developmental , Mice , TEA Domain Transcription FactorsABSTRACT
Autophagy is emerging as a crucial defense mechanism against bacteria, but the host intracellular sensors responsible for inducing autophagy in response to bacterial infection remain unknown. Here we demonstrated that the intracellular sensors Nod1 and Nod2 are critical for the autophagic response to invasive bacteria. By a mechanism independent of the adaptor RIP2 and transcription factor NF-kappaB, Nod1 and Nod2 recruited the autophagy protein ATG16L1 to the plasma membrane at the bacterial entry site. In cells homozygous for the Crohn's disease-associated NOD2 frameshift mutation, mutant Nod2 failed to recruit ATG16L1 to the plasma membrane and wrapping of invading bacteria by autophagosomes was impaired. Our results link bacterial sensing by Nod proteins to the induction of autophagy and provide a functional link between Nod2 and ATG16L1, which are encoded by two of the most important genes associated with Crohn's disease.
Subject(s)
Autophagy , Carrier Proteins/metabolism , Cell Membrane/metabolism , Nod1 Signaling Adaptor Protein/metabolism , Nod2 Signaling Adaptor Protein/metabolism , Animals , Autophagy-Related Proteins , Bacteria/metabolism , Carrier Proteins/genetics , Cell Line , Cell Membrane/microbiology , Cell Membrane/ultrastructure , Cells, Cultured , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Immunoblotting , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Microscopy, Confocal , Microscopy, Electron , Microscopy, Fluorescence , Mutation , Nod1 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/genetics , TransfectionABSTRACT
The Immortal Strand Hypothesis proposes that asymmetrically dividing stem cells cosegregate chromatids to retain ancestral DNA templates. Using both pulse-chase and label retention assays, we show that non-random partitioning of DNA occurs in germline stem cells (GSCs) in the Drosophila ovary as these divide asymmetrically to generate a new GSC and a differentiating cystoblast. This process is disrupted when GSCs are forced to differentiate through the overexpression of Bag of Marbles, a factor that impels the terminal differentiation of cystoblasts. When Decapentaplegic, a ligand which maintains the undifferentiated state of GSCs, is expressed ectopically the non-random partitioning of DNA is similarly disrupted. Our data suggest asymmetric chromatid segregation is coupled to mechanisms specifying cellular differentiation via asymmetric stem cell division.