ABSTRACT
Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity.
Subject(s)
ADAM Proteins/metabolism , Embryo, Nonmammalian/metabolism , Frizzled Receptors/metabolism , Gene Expression Regulation, Developmental , Membrane Proteins/metabolism , Neural Crest/metabolism , Wnt Proteins/metabolism , Xenopus Proteins/metabolism , ADAM Proteins/genetics , Animals , COS Cells , Cell Membrane/metabolism , Cell Movement , Cell Nucleus/metabolism , Cell Proliferation , Cells, Cultured , Chlorocebus aethiops , Embryo, Nonmammalian/cytology , Fluorescent Antibody Technique , Frizzled Receptors/genetics , HEK293 Cells , Humans , Immunoprecipitation , In Situ Hybridization , Membrane Proteins/genetics , Neural Crest/cytology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Wnt Proteins/genetics , Xenopus Proteins/genetics , Xenopus laevis/genetics , Xenopus laevis/growth & development , Xenopus laevis/metabolismABSTRACT
BACKGROUND: Activation of the Wnt signalling cascade is primarily based on the interplay between Wnt ligands, their receptors and extracellular modulators. One prominent family of extracellular modulators is represented by the SFRP (secreted Frizzled-related protein) family. These proteins have significant similarity to the extracellular domain of Frizzled receptors, suggesting that they bind Wnt ligands and inhibit signalling. The SFRP-type protein Fz4-v1, a splice variant of the Frizzled-4 receptor found in humans and Xenopus, was shown to augment Wnt/ß-catenin signalling, and also interacts with those Wnt ligands that act on ß-catenin-independent Wnt pathways. FINDINGS: Here we show that Xenopus Fz4-v1 can activate and inhibit the ß-catenin-dependent Wnt pathway. Gain-of-function experiments revealed that high Wnt/ß-catenin activity is inhibited by low and high concentrations of Fz4-v1. In contrast, signals generated by low amounts of Wnt ligands were enhanced by low concentrations of Fz4-v1 but were repressed by high concentrations. This biphasic activity of Fz4-v1 was not observed in non-canonical Wnt signalling. Fz4-v1 enhanced ß-catenin-independent Wnt signalling triggered by either low or high doses of Wnt11. Antisense morpholino-mediated knock-down experiments demonstrated that in early Xenopus embryos Fz4-v1 is required for the migration of cranial neural crest cells and for the development of the dorsal fin. CONCLUSIONS: For the first time, we show that a splice variant of the Frizzled-4 receptor modulates Wnt signalling in a dose-dependent, biphasic manner. These results also demonstrate that the cystein-rich domain (CRD), which is shared by Fz4-v1 and SFRPs, is sufficient for the biphasic activity of these secreted Wnt modulators.
Subject(s)
Frizzled Receptors/physiology , Wnt Signaling Pathway/physiology , Xenopus Proteins/physiology , Animals , Embryo, Nonmammalian , Embryonic Development/physiology , MAP Kinase Signaling System/physiology , Protein Isoforms , Xenopus laevisABSTRACT
Tissue border formation is an important process that prevents mixing of cells during embryonic development. The establishment of tissue borders is not a trivial problem, particularly in early embryos when cells and tissues are not fully differentiated. An example of an early tissue separation process is the formation of Brachet's cleft in Xenopus. During early gastrulation, this morphologically visible cleft separates mesendoderm and ectoderm. Over the last decade, it was recognized that morphogenetic processes, including tissue separation, can be experimentally uncoupled from embryonic patterning events. In this study, we summarize the data explaining the regulation of Brachet's cleft and introduce the experimental arsenal that was used for this analysis. The formation of Brachet's cleft involves the activity of transcription factors, cell adhesion molecules, and signaling modules, which act in a complex regulatory network. According to the current state of knowledge, Rho signaling seems to be the central player during this process. The mechanisms that regulate Rho during tissue separation and the experimental approaches to monitor Rho activity are discussed.
Subject(s)
Gastrulation , Signal Transduction , Xenopus/embryology , Animals , Xenopus/metabolism , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolismABSTRACT
BACKGROUND: Tissue plasticity and a substantial regeneration capacity based on stem cells are the hallmark of several invertebrate groups such as sponges, cnidarians and Platyhelminthes. Traditionally, Acoela were seen as an early branching clade within the Platyhelminthes, but became recently positioned at the base of the Bilateria. However, little is known on how the stem cell system in this new phylum is organized. In this study, we wanted to examine if Acoela possess a neoblast-like stem cell system that is responsible for development, growth, homeostasis and regeneration. RESULTS: We established enduring laboratory cultures of the acoel Isodiametra pulchra (Acoela, Acoelomorpha) and implemented in situ hybridization and RNA interference (RNAi) for this species. We used BrdU labelling, morphology, ultrastructure and molecular tools to illuminate the morphology, distribution and plasticity of acoel stem cells under different developmental conditions. We demonstrate that neoblasts are the only proliferating cells which are solely mesodermally located within the organism. By means of in situ hybridisation and protein localisation we could demonstrate that the piwi-like gene ipiwi1 is expressed in testes, ovaries as well as in a subpopulation of somatic stem cells. In addition, we show that germ cell progenitors are present in freshly hatched worms, suggesting an embryonic formation of the germline. We identified a potent stem cell system that is responsible for development, homeostasis, regeneration and regrowth upon starvation. CONCLUSIONS: We introduce the acoel Isodiametra pulchra as potential new model organism, suitable to address developmental questions in this understudied phylum. We show that neoblasts in I. pulchra are crucial for tissue homeostasis, development and regeneration. Notably, epidermal cells were found to be renewed exclusively from parenchymally located stem cells, a situation known only from rhabditophoran flatworms so far. For further comparison, it will be important to analyse the stem cell systems of other key-positioned understudied taxa.
