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      Rabconnectin-3a Regulates Vesicle Endocytosis and Canonical Wnt Signaling in Zebrafish Neural Crest Migration

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          A novel role for Rabconnectin-3a in endosome maturation regulates Wnt signaling, migration, and fate specification in zebrafish neural crest cells.


          Cell migration requires dynamic regulation of cell–cell signaling and cell adhesion. Both of these processes involve endocytosis, lysosomal degradation, and recycling of ligand–receptor complexes and cell adhesion molecules from the plasma membrane. Neural crest (NC) cells in vertebrates are highly migratory cells, which undergo an epithelial–mesenchymal transition (EMT) to leave the neural epithelium and migrate throughout the body to give rise to many different derivatives. Here we show that the v-ATPase interacting protein, Rabconnectin-3a (Rbc3a), controls intracellular trafficking events and Wnt signaling during NC migration. In zebrafish embryos deficient in Rbc3a, or its associated v-ATPase subunit Atp6v0a1, many NC cells fail to migrate and misregulate expression of cadherins. Surprisingly, endosomes in Rbc3a- and Atp6v0a1-deficient NC cells remain immature but still acidify. Rbc3a loss-of-function initially downregulates several canonical Wnt targets involved in EMT, but later Frizzled-7 accumulates at NC cell membranes, and nuclear B-catenin levels increase. Presumably due to this later Wnt signaling increase, Rbc3a-deficient NC cells that fail to migrate become pigment progenitors. We propose that Rbc3a and Atp6v0a1 promote endosomal maturation to coordinate Wnt signaling and intracellular trafficking of Wnt receptors and cadherins required for NC migration and cell fate determination. Our results suggest that different v-ATPases and associated proteins may play cell-type-specific functions in intracellular trafficking in many contexts.

          Author Summary

          The neural crest is a highly migratory population of embryonic cells, which requires Wnt signaling at several stages to promote migration and cell fate decisions. Intracellular trafficking of Wnt receptors and associated proteins can affect the timing and intensity of Wnt signaling. An obvious question is whether proton pumps and/or their partner proteins that are associated with intracellular vesicles might have a role in intracellular trafficking, Wnt signaling, and cell migration/adhesion. In this study we demonstrate such a role for Rabconnectin-3a, a protein associated with the vacuolar-ATPase (v-ATPase) proton pump complex. Loss of Rabconnectin-3a in zebrafish embryos disrupts the maturation of endocytic vesicles in neural crest cells, which has two effects: (1) decreasing Wnt signaling in these cells before migration and (2) increasing Wnt signaling after migration. Prior to migration, endosomes that fail to mature reduce Wnt signaling in neural crest cells and disrupt the localization and expression of cadherins, membrane-bound cell adhesion molecules required for these cells to initiate an epithelial-mesenchymal transition. At later stages, however, Wnt receptors accumulate at the membranes of unmigrated neural crest cells due to defective endocytosis, which correlates with high levels of Wnt signaling. Interestingly, Rabconnectin-3a-deficient neural crest cells that fail to migrate become pigment cells, presumably due to elevated Wnt signaling. Rabconnectin-3a may have a conserved role in endosomal maturation, Wnt signaling, and cell migration in many other cell populations.

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          Most cited references 56

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          Stages of embryonic development of the zebrafish.

          We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis--the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts.
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            Epithelial-mesenchymal transitions in tumour progression.

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              The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression.

              The Snail family of transcription factors has previously been implicated in the differentiation of epithelial cells into mesenchymal cells (epithelial-mesenchymal transitions) during embryonic development. Epithelial-mesenchymal transitions are also determinants of the progression of carcinomas, occurring concomitantly with the cellular acquisition of migratory properties following downregulation of expression of the adhesion protein E-cadherin. Here we show that mouse Snail is a strong repressor of transcription of the E-cadherin gene. Epithelial cells that ectopically express Snail adopt a fibroblastoid phenotype and acquire tumorigenic and invasive properties. Endogenous Snail protein is present in invasive mouse and human carcinoma cell lines and tumours in which E-cadherin expression has been lost. Therefore, the same molecules are used to trigger epithelial-mesenchymal transitions during embryonic development and in tumour progression. Snail may thus be considered as a marker for malignancy, opening up new avenues for the design of specific anti-invasive drugs.

                Author and article information

                Role: Academic Editor
                PLoS Biol
                PLoS Biol
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                May 2014
                6 May 2014
                : 12
                : 5
                Department of Developmental and Cell Biology, University of California, Irvine, California, United States of America
                The Wellcome Trust Sanger Institute, United Kingdom
                Author notes

                The authors have declared that no competing interests exist.

                The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: AMT TFS. Performed the experiments: AMT TLH. Analyzed the data: AMT TFS. Contributed reagents/materials/analysis tools: AMT TFS TLH. Wrote the paper: AMT TLH TFS.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Pages: 14
                Funding for this work was provided by NIH grant R01 DE13828 to TFS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Biology and Life Sciences
                Cell Biology
                Molecular Cell Biology
                Developmental Biology

                Life sciences


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