ABSTRACT
This corrects the article DOI: 10.1038/ncb3628.
ABSTRACT
Apicobasal polarity is known to affect epithelial morphogenesis and cell differentiation, but it remains unknown how these processes are mechanistically orchestrated. We find that ligand-specific EGFR signalling via PI(3)K and Rac1 autonomously modulates apicobasal polarity to enforce the sequential control of morphogenesis and cell differentiation. Initially, EGF controls pancreatic tubulogenesis by negatively regulating apical polarity induction. Subsequently, betacellulin, working via inhibition of atypical protein kinase C (aPKC), causes apical domain constriction within neurogenin3+ endocrine progenitors, which results in reduced Notch signalling, increased neurogenin3 expression, and ß-cell differentiation. Notably, the ligand-specific EGFR output is not driven at the ligand level, but seems to have evolved in response to stage-specific epithelial influences. The EGFR-mediated control of ß-cell differentiation via apical polarity is also conserved in human neurogenin3+ cells. We provide insight into how ligand-specific EGFR signalling coordinates epithelial morphogenesis and cell differentiation via apical polarity dynamics.
Subject(s)
Cell Polarity/physiology , ErbB Receptors/metabolism , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/physiology , Organogenesis/physiology , Signal Transduction/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/physiology , Epithelial Cells/metabolism , Mice , Mice, Knockout , Morphogenesis/physiology , Nerve Tissue Proteins/metabolism , Neuropeptides/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase C/metabolism , rac1 GTP-Binding Protein/metabolismABSTRACT
The production of insulin-producing ß cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny, we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or ß-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state, a principle that could be relevant to other systems. Notably, activation of the key ß-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional ß cells.
Subject(s)
Cell Differentiation/genetics , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression Profiling , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Single-Cell Analysis , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Biomarkers , Cell Lineage/genetics , Computational Biology/methods , Gene Expression Regulation, Developmental , Genes, Reporter , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Immunophenotyping , Models, Biological , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Organogenesis/genetics , Phenotype , TranscriptomeABSTRACT
Canonical Wnt and Nodal signaling are both required for induction of the primitive streak (PS), which guides organization of the early embryo. The Wnt effector ß-catenin is thought to function in these early lineage specification decisions via transcriptional activation of Nodal signaling. Here, we demonstrate a broader role for ß-catenin in PS formation by analyzing its genome-wide binding in a human embryonic stem cell model of PS induction. ß-catenin occupies regulatory regions in numerous PS and neural crest genes, and direct interactions between ß-catenin and the Nodal effectors SMAD2/SMAD3 are required at these regions for PS gene activation. Furthermore, OCT4 binding in proximity to these sites is likewise required for PS induction, suggesting a collaborative interaction between ß-catenin and OCT4. Induction of neural crest genes by ß-catenin is repressed by SMAD2/SMAD3, ensuring proper lineage specification. This study provides mechanistic insight into how Wnt signaling controls early cell lineage decisions.
Subject(s)
Octamer Transcription Factor-3/metabolism , Primitive Streak/metabolism , Smad2 Protein/metabolism , Smad3 Protein/metabolism , beta Catenin/metabolism , Base Sequence , Cell Line , Cell Lineage , Gene Expression Regulation, Developmental , Humans , Models, Biological , Molecular Sequence Data , Neural Crest/cytology , Nodal Protein/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Stem Cells/cytology , Stem Cells/metabolism , Wnt Signaling Pathway/geneticsABSTRACT
Wild-type (dark) and white mutant axolotl (Ambystoma mexicanum) embryos were used to investigate the role of the secreted growth factor bone morphogenetic protein-4 (BMP-4) and its antagonist, Noggin, in dorso-lateral trunk neural crest (NC) migration. Implantation of a BMP-4-coated microbead caused a melanophore-free zone around the bead, reduction of the dorsal fin above the bead, and disappearance of myotome tissue. We established a novel method that allows controlled induction of protein synthesis and release. Xenopus animal cap (XAC) cells injected with heat shock-inducible constructs for BMP-4 and Noggin were implanted into axolotl embryos and protein expression was induced at defined time points. With this approach, we could demonstrate for the first time that Noggin can stimulate melanophore migration in the white mutant. We further showed that implantation of BMP-4 expressing XAC cells alters pigment cell distribution without affecting muscle and dorsal fin development.
