The tetraspanin Cd63 is required for eye morphogenesis in Xenopus.
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ABSTRACT:
SUBMITTER: Kreis J
PROVIDER: S-EPMC7704261 | biostudies-literature | 2020 Nov
REPOSITORIES: biostudies-literature
Publications
The tetraspanin Cd63 is required for eye morphogenesis in <i>Xenopus</i>.
Kreis Jennifer J Bonß Ramona R Vick Philipp P
microPublication biology 20201127
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Project description:Tetraspanins cause the clustering of membrane proteins into a level of organisation essential for cellular function. Given the importance and complicated nature of this mechanism, we attempted a novel approach to identify the function of a single component in a biologically relevant context. A morpholino knockdown strategy was used to investigate the role of cd63, a membrane protein associated with intracellular transport and a melanoma marker, in embryonic zebrafish. By using three separate morpholinos targeting cd63, we were able to identify a specific phenotype. Strikingly, morphant fish failed to hatch due to the lack of secreted proteolytic enzymes required for chorion-softening. The morphology of the hatching gland at both the cellular and intracellular levels was disorganised, suggesting a role for cd63 in the functioning of this organ. This work identifies a specific role for cd63 in the zebrafish embryo and provides evidence for the suitability of zebrafish as a model system for the investigation of tetraspanin enriched microdomains.
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Project description:Desmoplakin (Dsp) is a unique and critical desmosomal protein, that is integral to epidermal development. However, it is unclear whether this protein is required specifically for epidermal morphogenesis. Using morpholinos or Crispr/Cas9 mutagenesis we decreased the function of Dsp in frog embryos to better understand its role during epidermal development. Dsp morphant and mutant embryos had developmental defects such as epidermal fragility that mimicked what has been reported in mammals. Most importantly, we also uncovered a novel function for Dsp in the morphogenesis of the epidermis in X. laevis. In particular, Dsp is required during the process of radial intercalation where basally located cells move into the outer epidermal layer. Once inserted these newly intercalated cells expand their apical surface and then they differentiate into specific epidermal cell types. Decreased levels of Dsp resulted in the failure of the radially intercalating cells to expand their apical surface, thereby reducing the number of differentiated multiciliated and secretory cells. Such defects correlate with changes in E-cadherin levels and actin and microtubule localization which could explain the defects in apical expansion. A mutated form of Dsp that maintains cell-cell adhesion but eliminates the connections to the cytoskeleton results in the same epidermal morphogenesis defect. These results suggest a specific role for Dsp in the apical expansion of cells during radial intercalation. We have developed a novel system, in the frog, to demonstrate for the first time that desmosomes not only protect against mechanical stress but are also critical for epidermal morphogenesis.
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Project description:The tetraspanin CD63 is implicated in pro-metastatic signaling pathways, but so far, it is unclear how CD63 levels affect the tumor cell phenotype. Here, we investigated the effect of CD63 modulation in different metastatic tumor cell lines. In vitro, knock down of CD63 induced a more epithelial-like phenotype concomitant with increased E-cadherin expression, downregulation of its repressors Slug and Zeb1, and decreased N-cadherin. In addition, β-catenin protein was markedly reduced, negatively affecting expression of the target genes MMP-2 and PAI-1. β-catenin inhibitors mimicked the epithelial phenotype induced by CD63 knock down. Inhibition of β-catenin upstream regulators PI3K/AKT or GSK3β could rescue the mesenchymal phenotype underlining the importance of the β-catenin pathway in CD63-regulated cell plasticity. CD63 knock down-induced phenotypical changes correlated with a decrease of experimental metastasis, while CD63 overexpression enhanced the tumor cell-intrinsic metastatic potential. Taken together, our data show that CD63 is a crucial player in the regulation of the tumor cell-intrinsic metastatic potential by affecting cell plasticity. Stable knock down of CD63 was performed in SKOV3ipL ovarian carcinoma cell line using 2 shRNAs lentiviral constructs (sh49 and sh51), and as a negative control, a shNT construct. Parental cells, control shNT and the 2 shCD63 cell lines were seeded 24 hours prior to RNA isolation on a 10 cm dish, labelling and hybridization on microarrays. One color experiment with 2 biological replicates of the 4 experimental conditions: SKOV3ipL, SKOV3ipL_shNT, SKOV3ipL_sh49 and SKOV3ipL_sh51.
2015-01-23 | E-GEOD-61957 | biostudies-arrayexpress
Project description:Hippo signaling is an important regulator of tissue size, but it also has a lesser-known role in tissue morphogenesis. Here we use the Drosophila pupal eye to explore the role of the Hippo effector Yki and its cofactor Mask in morphogenesis. We found that Mask is required for the correct distribution and accumulation of adherens junctions and appropriate organization of the cytoskeleton. Accordingly, disrupting mask expression led to severe mis-patterning and similar defects were observed when yki was reduced or in response to ectopic wts. Further, the patterning defects generated by reducing mask expression were modified by Hippo pathway activity. RNA-sequencing revealed a requirement for Mask for appropriate expression of numerous genes during eye morphogenesis. These included genes implicated in cell adhesion and cytoskeletal organization, a comprehensive set of genes that promote cell survival, and numerous signal transduction genes. To validate our transcriptome analyses, we then considered two loci that were modified by Mask activity: FER and Vinc, which have established roles in regulating adhesion. Modulating the expression of either locus modified mask mis-patterning and adhesion phenotypes. Further, expression of FER and Vinc was modified by Yki. It is well-established that the Hippo pathway is responsive to changes in cell adhesion and the cytoskeleton, but our data indicate that Hippo signaling also regulates these structures.
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Project description:The tetraspanin CD63 is implicated in pro-metastatic signaling pathways, but so far, it is unclear how CD63 levels affect the tumor cell phenotype. Here, we investigated the effect of CD63 modulation in different metastatic tumor cell lines. In vitro, knock down of CD63 induced a more epithelial-like phenotype concomitant with increased E-cadherin expression, downregulation of its repressors Slug and Zeb1, and decreased N-cadherin. In addition, β-catenin protein was markedly reduced, negatively affecting expression of the target genes MMP-2 and PAI-1. β-catenin inhibitors mimicked the epithelial phenotype induced by CD63 knock down. Inhibition of β-catenin upstream regulators PI3K/AKT or GSK3β could rescue the mesenchymal phenotype underlining the importance of the β-catenin pathway in CD63-regulated cell plasticity. CD63 knock down-induced phenotypical changes correlated with a decrease of experimental metastasis, while CD63 overexpression enhanced the tumor cell-intrinsic metastatic potential. Taken together, our data show that CD63 is a crucial player in the regulation of the tumor cell-intrinsic metastatic potential by affecting cell plasticity.
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