Regulation of Classical Cadherin Membrane Expression and F-Actin Assembly by Alpha-Catenins, during Xenopus Embryogenesis

Alpha (α)-E-catenin is a component of the cadherin complex, and has long been thought to provide a link between cell surface cadherins and the actin skeleton. More recently, it has also been implicated in mechano-sensing, and in the control of tissue size. Here we use the early Xenopus embryos to explore functional differences between two α-catenin family members, α-E- and α-N-catenin, and their interactions with the different classical cadherins that appear as tissues of the embryo become segregated from each other. We show that they play both cadherin-specific and context-specific roles in the emerging tissues of the embryo. α-E-catenin interacts with both C- and E-cadherin. It is specifically required for junctional localization of C-cadherin, but not of E-cadherin or N-cadherin at the neurula stage. α-N-cadherin interacts only with, and is specifically required for junctional localization of, N-cadherin. In addition, α -E-catenin is essential for normal tissue size control in the non-neural ectoderm, but not in the neural ectoderm or the blastula. We also show context specificity in cadherin/ α-catenin interactions. E-cadherin requires α-E-catenin for junctional localization in some tissues, but not in others, during early development. These specific functional cadherin/alpha-catenin interactions may explain the basis of cadherin specificity of actin assembly and morphogenetic movements seen previously in the neural and non-neural ectoderm

ADAMTS9-Mediated Extracellular Matrix Dynamics Regulates Umbilical Cord Vascular Smooth Muscle Differentiation and Rotation

Despite the significance for fetal nourishment in mammals, mechanisms of umbilical cord vascular growth remain poorly understood. Here, the secreted metalloprotease ADAMTS9 is shown to be necessary for murine umbilical cord vascular development. Restricting it to the cell surface using a gene trap allele, Adamts9Gt, impaired umbilical vessel elongation and radial growth via reduced versican proteolysis and accumulation of extracellular matrix (ECM). Both Adamts9Gt and conditional Adamts9 deletion revealed that ADAMTS9 produced by mesenchymal cells acted non-autonomously to regulate smooth muscle cell (SMC) proliferation, differentiation, and orthogonal reorientation during growth of the umbilical vasculature. In Adamts9Gt/Gt, we observed interference with PDGFRβ signaling via the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which regulates cytoskeletal dynamics during SMC rotation. In addition, we observed disrupted Shh signaling and perturbed orientation of the mesenchymal primary cilium. Thus, ECM dynamics is a major influence on umbilical vascular SMC fate, with ADAMTS9 acting as its principal mediator

N- and E-cadherins in Xenopus are specifically required in the neural and non-neural ectoderm, respectively, for F-actin assembly and morphogenetic movements

Transmembrane cadherins are calcium-dependent intercellular adhesion molecules. Recently, they have also been shown to be sites of actin assembly during adhesive contact formation. However, the roles of actin assembly on transmembrane cadherins during development are not fully understood. We show here, using the developing ectoderm of the Xenopus embryo as a model, that F-actin assembly is a primary function of both N-cadherin in the neural ectoderm and E-cadherin in the non-neural (epidermal) ectoderm, and that each cadherin is essential for the characteristic morphogenetic movements of these two tissues. However, depletion of N-cadherin and E-cadherin did not cause dissociation in these tissues at the neurula stage, probably owing to the expression of C-cadherin in each tissue. Depletion of each of these cadherins is not rescued by the other, nor by the expression of C-cadherin, which is expressed in both tissues. One possible reason for this is that each cadherin is expressed in a different domain of the cell membrane. These data indicate the combinatorial nature of cadherin function, the fact that N- and E-cadherin play primary roles in F-actin assembly in addition to roles in cell adhesion, and that this function is specific to individual cadherins. They also show how cell adhesion and motility can be combined in morphogenetic tissue movements that generate the form and shape of the embryonic organs

The C-cadhering catenin binding domain (CBD) is required for cell adhesion . (A–D

<p>) Immunostaining for C-cadherin (upper panels) and αEC (lower panels) in paraffin sections of stage 9 embryos, showing the non-rescue of cell adhesion in the absence of the CBD. (<b>E–H</b>) 3D projections of high magnification, and high resolution confocal images showing the localization of C-cadherin protein. Scale bars for A-D, 50 µM, E-H, 20 µM.</p

Depletion of α-E-catenin and α-N-catenin in the neural ectoderm.

<p>(<b>A</b>) Neural plates containing αEC-depleted clones (green) stained for C-cadherin (Red) and GFP (Green). (<b>B</b>) Neural plates containing αNC-depleted clones (morpholino-injected side marked by *, and uninjected side marked by # ), stained for αNC (Red) and C-cadherin (Green). (<b>C</b>) αEC-depleted neural plate cells (Green, FLDX) stained for N-cadherin (Red). (<b>D</b>) αNC-depleted neural plate cells (Green, FLDX) stained for N-cadherin (Red). Scale bars in <b>A-D</b>, 50 µM.</p

The distribution of α-N-catenin and α-E-catenin mRNAs in the neural and non-neural ectoderm.

<p>(<b>A-C</b>) αNC <i>In-situ</i> staining of st.13–19 embryos (<b>D-F</b>) αEC In-situ staining of st.13–19 embryos. NE (Neural Ectoderm), n-NE (non-Neural Ectoderm).</p

E-cadherin requires α-E-catenin in the non-polarized blastocelic roof cells, but not in the polarized superficial animal cap cells for junctional localization and cell adhesion.

<p>(A-C) Blastocelic roof cells of st. 9 animal caps stained for C-cadherin (red in A and B), or E-cadherin-HA (red in C), αEC (purple), and F-actin (green) in uninjected (A), αEC-depleted (B), or αEC + E-cadherin-HA mRNA injected (C) embryos. (D-E) Superficial cells imaged from the blastocelic cavity in animal caps where all the deeper cells have fallen off, stained for C-cadherin in (D), or E-cadherin (E), in αEC depleted (D), or αEC depleted + E-cadherin-HA mRNA-injected (E) embryos. (F) Western blot data showing the depletion of αEC protein and the expression of C-, and E-, cadherin protein levels in the experiment. Scale bars, 50 µM.</p

Cortical F-actin staining in alpha catenin-depleted neural and non-neural ectodermal cells.

<p>(<b>A</b>) Alexa-488 phalloidin staining for F-actin (green) in uninjected non-neural ectoderm cells. (<b>B</b>) αEC morpholino-injected non-neural ectoderm cells (Red, RLDX) showing the loss of F-actin (green). (<b>C</b>) Mean F-actin pixel intensity quantitation in uninjected and αEC-depleted non-neural ectoderm cells. (<b>D</b>) F-actin staining (green) in αEC-depleted neural ectoderm cells (Red). (<b>E</b>) Mean F-actin pixel intensity quantitation in uninjected and αEC-depleted neural ectoderm cells. (<b>F</b>) F-actin staining (green) in αNC-depleted neural ectoderm cells (Red). (<b>G</b>) Mean F-actin pixel intensity quantitation in uninjected and αNC-depleted neural ectoderm cells. (H) Neural plates stained for F-actin (green) in αEC, and αNC-depleted neural ectoderm cells (red). Scale bars, 50 µM.</p