Divergent roles for Eph and Ephrin in Avian Cranial Neural Crest

<p>Abstract</p> <p>Background</p> <p>As in other vertebrates, avian hindbrain neural crest migrates in streams to specific branchial arches. Signalling from Eph receptors and ephrins has been proposed to provide a molecular mechanism that guides the cells restricting them to streams. In mice and frogs, cranial neural crest express a combination of Eph receptors and ephrins that appear to exclude cells from adjacent tissues by forward and reverse signalling. The objective of this study was to provide comparative data on the distribution and function of Eph receptors and ephrins in avian embryos.</p> <p>Results</p> <p>To distinguish neural crest from bordering ectoderm and head mesenchyme, we have co-labelled embryos for Eph or ephrin RNA and a neural crest marker protein. Throughout their migration avian cranial neural crest cells express EphA3, EphA4, EphA7, EphB1, and EphB3 and move along pathways bordered by non-neural crest cells expressing ephrin-B1. In addition, avian cranial neural crest cells express ephrin-B2 and migrate along pathways bordered by non-neural crest cells expressing EphB2. Thus, the distribution of avian Eph receptors and ephrins differs from those reported in other vertebrates. In stripe assays when explanted cranial neural crest were given the choice between FN or FN plus clustered ephrin-B1 or EphB2 fusion protein, the cells strongly localize to lanes containing only FN. This preference is mitigated in the presence of soluble ephrin-B1 or EphB2 fusion protein.</p> <p>Conclusion</p> <p>These findings show that avian cranial neural crest use Eph and ephrin receptors as other vertebrates in guiding migration. However, the Eph receptors are expressed in different combinations by neural crest destined for each branchial arch and ephrin-B1 and ephrin-B2 appear to have opposite roles to those reported to guide cranial neural crest migration in mice. Unlike many of the signalling, specification, and effector pathways of neural crest, the roles of Eph receptors and ephrins have not been rigorously conserved. This suggests diversification of receptor and ligand expression is less constrained, possibly by promiscuous binding and use of common downstream pathways.</p

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction – A model for cross-modulation

<p>Abstract</p> <p>Background</p> <p>A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC.</p> <p>Methods</p> <p>PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome.</p> <p>Results</p> <p>When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk.</p> <p>Conclusion</p> <p>ST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.</p

Chemotactic Cellular Migration: Smooth and Discontinuous Travelling Wave Solutions

A simple model of chemotactic cell migration gives rise to travelling wave solutions. By varying the cellular growth rate and chemoattractant production rate, travelling waves with both smooth and discontinuous fronts are found using phase plane analysis. The phase plane exhibits a curve of singularities whose position relative to the equilibrium points in the phase plane determines the nature of the heteroclinic orbits, where they exist. Smooth solutions have trajectories connecting the steady states lying to one side of the singular curve. Travelling shock waves arise by connecting trajectories passing through a special point in the singular curve and recrossing the singular curve, by way of a iscontinuity. Hyperbolic partial differential equation theory gives the necessary shock condition. Conditions on the parameter values determine when the solutions are smooth travelling waves versus discontinuous travelling wave solutions. These conditions provide bounds on the travelling wave speeds, corresponding to bounds on the chemotactic velocity or bounds on cellular growth rate. This analysis gives rise to the possibility of representing sharp fronts to waves of invading cells through a simple chemotactic term, without introducing a nonlinear diffusion term. This is more appropriate when cell populations are sufficiently dense

Structural and compositional divergencies in the extracellular matrix encountered by neural crest cells in the white mutant axolotl embryo

The skin of the white mutant axolotl larva is pigmented differently from that of the normal dark due to a local inability of the extracellular matrix (ECM) to support subepidermal migration of neural crest-derived pigment cell precursors. In the present study, we have compared the ECM of neural crest migratory pathways of normal dark and white mutant embryos ultrastructurally, immunohistochemically and biochemically to disclose differences in their structure/composition that could be responsible for the restriction of subepidermal neural crest cell migration in the white mutant axolotl. When examined by electron microscopy, in conjunction with computerized image analysis, the structural assembly of interstitial and basement membrane ECMs of the two embryos was found to be largely comparable. At stages of initial neural crest cell migration, however, fixation of the subepidermal ECM in situ with either Karnovsky-ruthenium red or with periodate-lysine-paraformaldehyde followed by ruthenium red-containing fixatives, revealed that fibrils of the dark matrix were significantly more abundant in associated electron-dense granules. This ultrastructural discrepancy of the white axolotl ECM was specific for the subepidermal region and suggested an abnormal proteoglycan distribution. Dark and white matrices of the medioventral migratory route of neural crest cells had a comparable appearance but differed from the corresponding subepidermal ECMs. Immunohistochemistry revealed only minor differences in the distribution of fibronectin, laminin, collagen types I, and IV, whereas collagen type III appeared differentially distributed in the two embryos. Chondroitin- and chondroitin-6-sulfate-rich proteoglycans were more prevalent in the white mutant embryo than in the dark, especially in the subepidermal space. Membrane microcarriers were utilized to explant site-specifically native ECM for biochemical analysis. Two-dimensional gel electrophoresis of these regional matrices revealed a number of differences in their protein content, principally in constituents of apparent molecular masses of 30-90,000. Taken together our observations suggest that local divergences in the concentration/assembly of low and high molecular mass proteins and proteoglycans of the ECM encountered by the moving neural crest cells account for their disparate migratory behavior in the white mutant axolotl

Spatial analysis of multi-species exclusion processes: Application to neural crest cell migration in the embryonic gut

Hindbrain (vagal) neural crest cells become relatively uniformly distributed along the embryonic intestine during the rostral to caudal colonization wave which forms the enteric nervous system (ENS). When vagal neural crest cells are labeled before migration in avian embryos by in ovo electroporation, the distribution of labeled neural crest cells in the ENS varies vastly. In some cases, the labeled neural crest cells appear evenly distributed and interspersed with unlabeled neural crest cells along the entire intestine. However, in most specimens, labeled cells occur in relatively discrete patches of varying position, area, and cell number. To determine reasons for these differences, we use a discrete cellular automata (CA) model incorporating the underlying cellular processes of neural crest cell movement and proliferation on a growing domain, representing the elongation of the intestine during development. We use multi-species CA agents corresponding to labeled and unlabeled neural crest cells. The spatial distributions of the CA agents are quantified in terms of an index. This investigation suggests that (i) the percentage of the initial neural crest cell population that is labeled and (ii) the ratio of cell proliferation to motility are the two key parameters producing the extreme differences in spatial distributions observed in avian embryos.Benjamin J. Binder, Kerry A. Landman, Donald F. Newgreen, Johanna E. Simkin, Yoshiko Takahashi, Dongcheng Zhan

Fibronectin in the area opaca of the young chick embryo. Immunofluorescence and immuno-electron-microscopic study

Distribution of fibronectin-like immunoreactivity was studied in the area opaca of the young chick embryo (stages 4-6 HH) by use of the immunofluorescence and protein A-coupled to colloidal gold techniques. Fibronectin, associated to the basement membrane, formed a fibrillar network, the pattern of which changed from the centre to the periphery of the area opaca. At the ultrastructural level, differences in fibronectin distribution were found between non-moving and moving cells. The epithelial-like cells presented fibronectin staining exclusively on their basal side. Actively migrating cells (edge and mesodermal cells) showed immunoreactive material localized around their entire surface and within the cytoplasm. The fibronectin distribution is discussed in relation to three important phenomena taking place during the early growth of the area opaca: anchorage and migration of the edge cells, modification of cell shape in relation to mechanical tension, and expansion of the area vasculosa