Loss of Xenopus cadherin-11 leads to increased Wnt/β-catenin signaling and up-regulation of target genes c-myc and cyclin D1 in neural crest

AbstractXenopus cadherin-11 (Xcadherin-11) is an exceptional cadherin family member, which is predominantly expressed in cranial neural crest cells (NCCs). Apart from mediating cell–cell adhesion it promotes cranial NCC migration by initiating filopodia and lamellipodia formation. Here, we demonstrate an unexpected function of Xcadherin-11 in NCC specification by interfering with canonical Wnt/β-catenin signaling. Loss-of-function experiments, using a specific antisense morpholino oligonucleotide against Xcadherin-11, display a nuclear β-catenin localization in cranial NCCs and a broader expression domain of the proto-oncogene cyclin D1 which proceeds c-myc up-regulation. Additionally, we observe an enhanced NCC proliferation and an expansion of specific NCC genes like AP2 and Sox10. Thereby, we could allocate NCC proliferation and specification to different gene functions. To clarify which domain in Xcadherin-11 is required for early NCC development we tested different deletion mutants for their rescue ability in Xcadherin-11 morphants. We identified the cytoplasmic tail, specifically the β-catenin binding domain, to be necessary for proper NCC development. We propose that Xcadherin-11 is necessary for controlled NCC proliferation and early NCC specification in tuning the expression of the canonical Wnt/β-catenin target genes cyclin D1 and c-myc by regulating the concentration of the nuclear pool of β-catenin

TV-based conjugate gradient method and discrete L-curve for few-view CT reconstruction of X-ray in vivo data

High-resolution, three-dimensional (3D) imaging of soft tissues requires the solution of two inverse problems: phase retrieval and the reconstruction of the 3D image from a tomographic stack of two-dimensional (2D) projections. The number of projections per stack should be small to accommodate fast tomography of rapid processes and to constrain X-ray radiation dose to optimal levels to either increase the duration of in vivo time-lapse series at a given goal for spatial resolution and/or the conservation of structure under X-ray irradiation. In pursuing the 3D reconstruction problem in the sense of compressive sampling theory, we propose to reduce the number of projections by applying an advanced algebraic technique subject to the minimisation of the total variation (TV) in the reconstructed slice. This problem is formulated in a Lagrangian multiplier fashion with the parameter value determined by appealing to a discrete L-curve in conjunction with a conjugate gradient method. The usefulness of this reconstruction modality is demonstrated for simulated and in vivo data, the latter acquired in parallel-beam imaging experiments using synchrotron radiation

Quantitating membrane bleb stiffness using AFM force spectroscopy and an optical sideview setup

AFM-based force spectroscopy in combination with optical microscopy is a powerful tool for investigating cell mechanics and adhesion on the single cell level. However, standard setups featuring an AFM mounted on an inverted light microscope only provide a bottom view of cell and AFM cantilever but cannot visualize vertical cell shape changes, for instance occurring during motile membrane blebbing. Here, we have integrated a mirror-based sideview system to monitor cell shape changes resulting from motile bleb behavior of Xenopus cranial neural crest (CNC) cells during AFM elasticity and adhesion measurements. Using the sideview setup, we quantitatively investigate mechanical changes associated with bleb formation and compared cell elasticity values recorded during membrane bleb and non-bleb events. Bleb protrusions displayed significantly lower stiffness compared to the non-blebbing membrane in the same cell. Bleb stiffness values were comparable to values obtained from blebbistatin-treated cells, consistent with the absence of a functional actomyosin network in bleb protrusions. Furthermore, we show that membrane blebs forming within the cell-cell contact zone have a detrimental effect on cell-cell adhesion forces, suggesting that mechanical changes associated with bleb protrusions promote cell-cell detachment or prevent adhesion reinforcement. Incorporating a sideview setup into an AFM platform therefore provides a new tool to correlate changes in cell morphology with results from force spectroscopy experiments

Cadherin-11 localizes to focal adhesions and promotes cell-substrate adhesion

Cadherin receptors have a well-established role in cell–cell adhesion, cell polarization and differentiation. However, some cadherins also promote cell and tissue movement during embryonic development and tumour progression. In particular, cadherin-11 is upregulated during tumour and inflammatory cell invasion, but the mechanisms underlying cadherin-11 stimulated cell migration are still incompletely understood. Here, we show that cadherin-11 localizes to focal adhesions and promotes adhesion to fibronectin in Xenopus neural crest, a highly migratory embryonic cell population. Transfected cadherin-11 also localizes to focal adhesions in different mammalian cell lines, while endogenous cadherin-11 shows focal adhesion localization in primary human fibroblasts. In focal adhesions, cadherin-11 co-localizes with β1-integrin and paxillin and physically interacts with the fibronectin-binding proteoglycan syndecan-4. Adhesion to fibronectin mediated by cadherin-11/syndecan-4 complexes requires both the extracellular domain of syndecan-4, and the transmembrane and cytoplasmic domains of cadherin-11. These results reveal an unexpected role of a classical cadherin in cell–matrix adhesion during cell migration

Cadherin-11 Mediates Contact Inhibition of Locomotion during <i>Xenopus</i> Neural Crest Cell Migration

<div><p>Collective cell migration is an essential feature both in embryonic development and cancer progression. The molecular mechanisms of these coordinated directional cell movements still need to be elucidated. The migration of cranial neural crest (CNC) cells during embryogenesis is an excellent model for collective cell migration <i>in vivo</i>. These highly motile and multipotent cells migrate directionally on defined routes throughout the embryo. Interestingly, local cell-cell interactions seem to be the key force for directionality. CNC cells can change their migration direction by a repulsive cell response called contact inhibition of locomotion (CIL). Cell protrusions collapse upon homotypic cell-cell contact and internal repolarization leads to formation of new protrusions toward cell-free regions. Wnt/PCP signaling was shown to mediate activation of small RhoGTPase RhoA and inhibition of cell protrusions at the contact side. However, the mechanism how a cell recognizes the contact is poorly understood. Here, we demonstrate that <i>Xenopus</i> cadherin-11 (Xcad-11) mediated cell-cell adhesion is necessary in CIL for directional and collective migration of CNC cells. Reduction of Xcad-11 adhesive function resulted in higher invasiveness of CNC due to loss of CIL. Additionally, transplantation analyses revealed that CNC migratory behaviour <i>in vivo</i> is non-directional and incomplete when Xcad-11 adhesive function is impaired. Blocking Wnt/PCP signaling led to similar results underlining the importance of Xcad-11 in the mechanism of CIL and directional migration of CNC. </p> </div

Xcad-11 is required for directional CNC migration <i>in vivo</i>.

<p>CNC transplants. First column: Lateral view on transplanted GFP-labelled <i>Xenopus</i> CNC before migration at stage 19. Second column: Lateral view on transplanted GFP-labelled <i>Xenopus</i> CNC after migration at stage 26. Third column: Tracking analysis of six to seven cells by H2B-cherry labelled nuclei during CNC migration (stage 19-26). Anterior is to the left and dorsal to the top. (A) Wildtype grafts showed normal migration. (B) Grafts coinjected with Xcad-11-MO were unable to migrate into the pharyngeal pouches. (C, D) Overexpression by coinjection of dn-Xcad-11 and Dsh(DEP+), respectively, led to disorientated and not directional CNC migration. (E) Schematic illustration of the transplantation assay. Percentage of complete CNC migration given in (F) with n = number of transplanted embryos. Error bar shows standard error. (***) Significance to wildtype with p<0.005 after student’s T-Test. Scale bar, 200 µm.</p

Xcad-11 mediates repulsive response in colliding in single CNC cells <i>in vitro</i>.

<p>Collision assay. First three columns: Single CNC cells before (t-Δ), during (t) and after (t+Δ) mutual contact with tracking. Fourth column: Relative velocity vectors with initial velocity vector (red, n = 10 collisions). (A) Only wildtype CNC cells showed change of direction. (B) Xcad-11-MO treated cells show random distribution. (C) dn-Xcad-11 and (D) Dsh(DEP+) overexpressing CNC cells displayed reduced repulsive response. Scale bar, 50 µm.</p

Depletion of Xcad-11 adhesive function blocks CNC migration <i>in vivo</i>.

<p>Lateral view of <i>Xenopus</i> CNC at stage 26, analysed by whole-mount ISH for the specific CNC marker AP-2α. Left column: Injected side (IS). Right column: Non-injected side (NIS). (A) Wildtype CNC cells migrated in defined streams into the pharyngeal pouches. (B) Xcad-11-MO injected embryos show AP-2α staining only at the dorsal part of the embryo, indicating incomplete CNC migration compared to NIS. (C) Overexpression of dn-Xcad-11 and (D) Dsh(DEP+) both showed incomplete and fused CNC hyoidal and branchial migration streams. Percentage of complete CNC migration given in (E) with n = number of embryos. Error bar shows standard error. (***) Significance to wildtype with p<0.005 after student’s T-Test. Scale bar, 250 µm.</p

Loss of Xcad-11 adhesive function increases CNC invasiveness <i>in vitro</i>.

<p>Confrontation assay. First column: Confronted explants at time point t=0. Second column: Confronted CNC explants at time point of highest invasion Δt. Third column: Morphology of CNC cells. Except for (A) wildtype vs. wildtype, yellow overlapping area increased strongly in (B) Xcad-11 morphant, (C) dn-Xcad-11 and (D) Dsh(DEP+) overexpressing CNC cells reflecting invasiveness of the tissues. Xcad-11 depleted cells displayed blebbing (yellow arrowheads in (B)) in contrast to protrusion formation of CNC cells in other approaches (white arrowheads in (A, C, D)). (E) Schematic illustration of the confrontation assay. Average Overlapping Index (OI) given in (F) with n = number of confrontations (wt: wildtype). Error bar shows standard error. (***) Significance to wildtype vs. wildtype with p<0.001 after student’s T-Test. Scale bar, 50 µm.</p