ABSTRACT
Secreted TGFbeta proteins of the Nodal family pattern the vertebrate body axes and induce mesoderm and endoderm . Nodal proteins can act as morphogens , but the mechanisms regulating their activity and signaling range are poorly understood. In particular, it has been unclear how inefficient processing or rapid turnover of the Nodal protein influences autocrine and paracrine signaling properties . Here, we evaluate the role of Nodal processing and stability in tissue culture and zebrafish embryos. Removal of the pro domain potentiates autocrine signaling but reduces Nodal stability and signaling range. Insertion of an N-glycosylation site present in several related TGFbeta proteins increases the stability of mature Nodal. The stabilized form of Nodal acts at a longer range than the wild-type form. These results suggest that increased proteolytic maturation of Nodal potentiates autocrine signaling, whereas increased Nodal stability extends paracrine signaling.
Subject(s)
Autocrine Communication/physiology , Body Patterning/physiology , Paracrine Communication/physiology , Signal Transduction/physiology , Transforming Growth Factor beta/metabolism , Amino Acid Sequence , Animals , COS Cells , Cell Line , Chlorocebus aethiops , DNA Primers , Gene Expression , Humans , Immunoblotting , Molecular Sequence Data , Mutagenesis , Nodal Protein , Protein Structure, Tertiary , Sequence Alignment , Sequence Analysis, DNA , Transfection , ZebrafishABSTRACT
Before implantation in the uterus, mammalian embryos set aside trophoblast stem cells that are maintained in the extraembryonic ectoderm (ExE) during gastrulation to generate the fetal portion of the placenta. Their proliferation depends on diffusible signals from neighboring cells in the epiblast, including fibroblast growth factor 4 (Fgf4). Here, we show that Fgf4 expression is induced by the transforming growth factor beta-related protein Nodal. Together with Fgf4, Nodal also acts directly on neighboring ExE to sustain a microenvironment that inhibits precocious differentiation of trophoblast stem cells. Because the ExE itself produces the proteases Furin and PACE4 to activate Nodal, it represents the first example, to our knowledge, of a stem cell compartment that actively maintains its own microenvironment.
Subject(s)
Fibroblast Growth Factors/genetics , Proto-Oncogene Proteins/genetics , Stem Cells/cytology , Stem Cells/metabolism , Transforming Growth Factor beta/metabolism , Trophoblasts/cytology , Trophoblasts/metabolism , Animals , Cell Differentiation , Female , Fibroblast Growth Factor 4 , Furin/deficiency , Furin/genetics , Furin/metabolism , Gene Expression/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Nodal Protein , Pregnancy , Proprotein Convertases , Protein Processing, Post-Translational , Recombinant Proteins/pharmacology , Serine Endopeptidases/deficiency , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Tissue Culture Techniques , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/pharmacologyABSTRACT
During gastrulation, a cascade of inductive tissue interactions converts pre-existing polarity in the mammalian embryo into antero-posterior pattern. This process is triggered by Nodal, a protein related to transforming growth factor-beta (TFG-beta) that is expressed in the epiblast and visceral endoderm, and its co-receptor Cripto, which is induced downstream of Nodal. Here we show that the proprotein convertases Spc1 and Spc4 (also known as Furin and Pace4, respectively) are expressed in adjacent extraembryonic ectoderm. They stimulate Nodal maturation after its secretion and are required in vivo for Nodal signalling. Embryo explants deprived of extraembryonic ectoderm phenocopy Spc1(-/-); Spc4(-/-) double mutants in that endogenous Nodal fails to induce Cripto. But recombinant mature Nodal, unlike uncleaved precursor, can efficiently rescue Cripto expression. Cripto is also expressed in explants treated with bone morphogenetic protein 4 (BMP4). This indicates that Nodal may induce Cripto through both a signalling pathway in the embryo and induction of Bmp4 in the extraembryonic ectoderm. A lack of Spc1 and Spc4 affects both pathways because these proteases also stimulate induction of Bmp4.
