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The cadherin-catenin complex is necessary for cell adhesion and embryogenesis in Nematostella vectensis - PubMed

  • ️Tue Jan 01 2019

The cadherin-catenin complex is necessary for cell adhesion and embryogenesis in Nematostella vectensis

D Nathaniel Clarke et al. Dev Biol. 2019.

Abstract

The cadherin-catenin complex is a conserved, calcium-dependent cell-cell adhesion module that is necessary for normal development and the maintenance of tissue integrity in bilaterian animals. Despite longstanding evidence of a deep ancestry of calcium-dependent cell adhesion in animals, the requirement of the cadherin-catenin complex to coordinate cell-cell adhesion has not been tested directly in a non-bilaterian organism. Here, we provide the first analysis of classical cadherins and catenins in the Starlet Sea Anemone, Nematostella vectensis. Gene expression, protein localization, siRNA-mediated knockdown of α-catenin, and calcium-dependent cell aggregation assays provide evidence that a bonafide cadherin-catenin complex is present in the early embryo, and that α-catenin is required for normal embryonic development and the formation of cell-cell adhesions between cells dissociated from whole embryos. Together these results support the hypothesis that the cadherin-catenin complex was likely a complete and functional cell-cell adhesion module in the last common cnidarian-bilaterian ancestor. SUMMARY STATEMENT: Embryonic manipulations and ex vivo adhesion assays in the sea anemone, Nematostella vectensis, indicate that the necessity of the cadherin-catenin complex for mediating cell-cell adhesion is deeply conserved in animal evolution.

Keywords: Adhesion; Cadherin; Calcium; Catenin; Cnidaria; Nematostella.

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Figures

Figure 1 –
Figure 1 –. Cadherin/Catenin proteins in the genome of N. vectensis versus other animals

A. (top) schematic representation of the adherens junction (yellow) linking the cortical actomyosin network (red) between neighboring cells; (bottom) illustration of the cadherin-catenin complex within the adherens junction, with calcium-dependent interactions between cadherins (green), are linked by β- (blue) and α-Catenin (yellow) to FActin (red). B. The presence, absence, and abundance of cadherin/catenin components across animal lineages and non-animal relatives, with a phylogenetic tree for reference. C. The cadherin-catenin complement from N. vectensis in comparison to representative vertebrate orthologs.

Figure 2 –
Figure 2 –. N. vectensis α-catenin, β-catenin, and Cadherin 3 are co-expressed in the early embryo and localize to apical cell junctions.

A. Schematic of normal development in N. vectensis; arrows indicate invagination movements at the onset of gastrulation, asterisk indicates the blastopore, and red line indicates the timing of zygotic gene expression. Whole-mount in situ hybridization of α-catenin (Bi - Biii), β-catenin (Ci - Ciii), cadherin 3 (Di - Div), and cadherin 1 (Ei - Evi) from early embryonic through mid-larval stages. Insets in Biii, Ciii, and Diii are blastoporal views of embryos bisected with the blade of a 22-gauge syringe to show internal staining; inset in Eii is a blastoporal view of a whole gastrula-stage embryo. Fi-iv. confocal maximum projection of a 6-hour blastula embryo co-injected with fluorescent fusion mRNAs encoding α-catenin:mNeonGreen (Fi), β-catenin:mTagBFP2(Fii), and Cadherin 1:mScarlet (Fiii); images are of a single embryo, but representative of 3 separate experiments of 20 or more embryos each. Scale bars are 50μm throughout.

Figure 3 –
Figure 3 –. knockdown of Nv α-Catenin produces gastrulation defects that disrupt metamorphosis

A. Embryos injected with α-catenin siRNAs or start codon-targeted morpholino versus control siRNA at 30 h.p.f., stained for DNA (propidium iodide, magenta), and F-Actin (Alexa-Fluor 488 Phalloidin, green). B. RT-qPCR analysis of Nv α-Catenin expression levels over time in embryos injected with a mixture of siRNA 1 and 2. C, E. Embryos injected with a 1:1 mixture of siRNAs versus control embryos across a series of developmental stages during gastrulation (C), and subsequent larval growth (E), stained as in A. D. quantification of phenotypes at 2 days post-fertilization. F. quantification of phenotypes at 10 days post-fertilization. G. In situ hybridization for endoderm (snailA, hnf1), gastrulation (foxA, brachyury), and axial patterning (foxB, dlx, six3/6) markers in treated versus control embryos at early planula stage. Scale bars are 50μm throughout.

Figure 4 –
Figure 4 –. Nv α-catenin is necessary for cell adhesion and the formation of epithelial tissues

A. Schematic of the hanging drop cell adhesion assay. B. quantification of hanging drop assay indicating the number of cells in different size classes of aggregates formed over time. Data is representative of 3 independent assays. C. representative images of cell aggregate formation observed in the hanging drop assay. D. representative images of epithelial cell aggregates formed after 48 hours in a hanging drop. E. quantification of the mean number of aggregates formed per droplet after 48 hours. Fi-iii. Representative images from a 6-hour time series of an embryo treated with calcium-magnesium free media plus 1mM EDTA at the onset of gastrulation. G. 40× confocal image of the apical cell surface of a phalloidin-stained embryo after 1 hour of CMF + EDTA treatment. Arrow indications forming separation between F-actin cortices of adjacent cells. Hi-v. representative images of variable gastrulation phenotypes observed in embryos treated with CMF media without additional EDTA versus untreated control embryos. I. Quantification of the proportion of phenotypes observed in the CMF without EDTA treatments. Scale bars are 50μm in C, D, F, and H, and 5 μm in G.

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