ABSTRACT
Cadherin shedding affects migration and occurs in development and cancer progression. By examining the in vivo biological function of the extracellular cadherin domain (CEC1-5) independently of the shedding process itself, we identified a novel function for cadherins in convergent extension (CE) movements in Xenopus. CEC1-5 interfered with CE movements during gastrulation. Unexpectedly, CEC1-5 did not alter cell aggregation or adhesion to cadherin substrates. Instead, gastrulation defects were rescued by a membrane-anchored cadherin cytoplasmic domain, the polarity protein atypical PKC (aPKC) or constitutive active Rac, indicating that CEC1-5 modulates a cadherin-dependent signalling pathway. We found that the cadherin interacts with aPKC and, more importantly, that the extracellular domain alters this association as well as the phosphorylation status of aPKC. This suggests that CE movements require a dynamic regulation of cadherin-aPKC interaction. Our results show that cadherins play a dual role in CE movements: a previously identified adhesive activity and an adhesion-independent function that requires aPKC and Rac, thereby directly connecting cadherins with polarity. Our results also suggest that increased cadherin shedding, often observed in cancer progression, can regulate migration and invasion by modulating polarity protein activity.
Subject(s)
Cadherins/metabolism , Cell Movement , Protein Kinase C/metabolism , Xenopus laevis/metabolism , Animals , CHO Cells , Cadherins/genetics , Cell Adhesion , Cell Line, Tumor , Cricetinae , Cricetulus , Dogs , Gastrulation , Humans , Morphogenesis , Phosphorylation , Protein Kinase C/genetics , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Signal Transduction , Tissue Culture Techniques , Transfection , Xenopus Proteins/metabolism , Xenopus laevis/embryology , Xenopus laevis/genetics , rac GTP-Binding Proteins/metabolismABSTRACT
The lifelong self-renewal of the epidermis is driven by a progenitor cell population with high proliferative potential. To date, the upstream signals that determine this potential have remained largely elusive. Here, we find that insulin and insulin-like growth factor receptors (IR and IGF-1R) determine epidermal proliferative potential and cooperatively regulate interfollicular epidermal morphogenesis in a cell autonomous manner. Epidermal deletion of either IR or IGF-1R or both in mice progressively decreased epidermal thickness without affecting differentiation or apoptosis. Proliferation was temporarily reduced at E17.5 in the absence of IGF-1R but not IR. In contrast, clonogenic capacity was impaired in both IR- and IGF-1R-deficient primary keratinocytes, concomitant with an in vivo loss of keratin 15. Together with a reduction in label-retaining cells in the interfollicular epidermis, this suggests that IR/IGF-1R regulate progenitor cells. The expression of dominant active Rac rescued clonogenic potential of IR/IGF-1R-negative keratinocytes and reversed epidermal thinning in vivo. Our results identify the small GTPase Rac as a key target of epidermal IR/IGF-1R signalling crucial for proliferative potential and interfollicular morphogenesis.
Subject(s)
Cell Proliferation , Epidermis/physiology , Receptor, IGF Type 1/physiology , Receptor, Insulin/physiology , rac GTP-Binding Proteins/physiology , Animals , Animals, Newborn , Apoptosis/physiology , Cell Differentiation , Cells, Cultured , Epidermal Cells , Epidermis/embryology , Insulin/metabolism , Insulin-Like Growth Factor I/metabolism , Keratin-15/metabolism , Keratinocytes/cytology , Keratinocytes/physiology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/physiology , Mice , Morphogenesis , Signal TransductionABSTRACT
In order to facilitate in situ detection of biomolecules in large sample series the processing of whole-mount specimens has been automated. A freely programmable liquid handling system is described by which embryos or similar biological materials are processed. Possible applications include in situ hybridization (ISH), immunocytochemistry (ICC) or reporter gene assays. Process times required for the preparation of whole-mount in situ hybridizations in Drosophila, Xenopus, Gallus and in hydroids were - in part - significantly reduced as compared with manual processing. Application of automated in situ detection (AISD) in random screening is demonstrated in hydroids. Potential further applications are discussed.
