Dominant Negative N-cadherin Research Articles

Neural crest emigration from the neural tube depends on regulated cadherin expression.

During the emergence of neural crest cells from the neural tube, the expression of cadherins dynamically changes. In the chicken embryo, the early neural tube expresses two cadherins, N-cadherin and cadherin-6B (cad6B), in the dorsal-most region where neural crest cells are generated. The expression of these two cadherins is, however, downregulated in the neural crest cells migrating from the neural tube; they instead begin expressing cadherin-7 (cad7). As an attempt to investigate the role of these changes in cadherin expression, we overexpressed various cadherin constructs, including N-cadherin, cad7, and a dominant negative N-cadherin (cN390 ), in neural crest-generating cells. This was achieved by injecting adenoviral expression vectors encoding these molecules into the lumen of the closing neural tube of chicken embryos at stage 14. In neural tubes injected with the viruses, efficient infection was observed at the neural crest-forming area, resulting in the ectopic cadherin expression also in migrating neural crest cells. Notably, the distribution of neural crest cells with the ectopic cadherins changed depending on which constructs were expressed. Many crest cells failed to escape from the neural tube when N-cadherin or cad7 was overexpressed. Moreover, none of the cells with these ectopic cadherins migrated along the dorsolateral (melanocyte) pathway. When these samples were stained for Mitf, an early melanocyte marker, positive cells were found accumulated within the neural tube, suggesting that the failure of their migration was not due to differentiation defects. In contrast to these phenomena, cells expressing non-functional cadherins exhibited a normal migration pattern. Thus, the overexpression of a neuroepithelial cadherin (N-cadherin) and a crest cadherin (cad7) resulted in the same blocking effect on neural crest segregation from neuroepithelial cells, especially for melanocyte precursors. These findings suggest that the regulation of cadherin expression or its activity at the neural crest-forming area plays a critical role in neural crest emigration from the neural tube.

Cadherin Function Is Required for Axon Outgrowth in Retinal Ganglion Cells In Vivo

The cell–cell adhesion molecule N-cadherin strongly promotes neurite outgrowth in cultured retinal neurons. To test whether cadherins regulate process outgrowth in retinal neurons in vivo, we have blocked cadherin function in single cells by expression of a dominant negative N-cadherin mutant. We report that when cadherin function is inhibited, axon and dendrite outgrowth are severely impaired, particularly in retinal ganglion cells. Laminar migration and cell type specification, by contrast, appear unaffected. Further, expression of the catenin-binding domain of N-cadherin, which blocks cadherin-mediated adhesion in early embryos, does not affect axon outgrowth, suggesting that outgrowth and adhesion are mediated by distinct regions of the cytoplasmic domain. These findings indicate that cadherins play an essential role in the initiation and extension of axons from retinal ganglion cells in vivo.

Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development.

When overexpressed in Xenopus embryos, Xwnt-1, -3A, -8 and -8b define a functional class of Wnts (the Wnt-1 class) that promotes duplication of the embryonic axis, whereas Xwnt-5A, -4, and -11 define a distinct class (the Wnt-5A class) that alters morphogenetic movements (Du, S., S. Purcell, J. Christian, L. McGrew, and R. Moon. 1995. Mol. Cell. Biol. 15:2625-2634). Since come embryonic cells may be exposed to signals from both functional classes of Wnt during vertebrate development, this raises the question of how the signaling pathways of these classes of Wnts might interact. To address this issue, we coexpressed various Xwnts and components of the Wnt-1 class signaling pathway in developing Xenopus embryos. Members of the Xwnt-5A class antagonized the ability of ectopic Wnt-1 class to induce goosecoid expression and a secondary axis. Interestingly, the Wnt-5A class did not block goosecoid expression or axis induction in response to overexpression of cytoplasmic components of the Wnt-1 signaling pathway, beta-catenin or a kinase-dead gsk-3, or to the unrelated secreted factor, BVg1. The ability of the Wnt-5A class to block responses to the Wnt-1 class may involve decreases in cell adhesion, since ectopic expression of Xwnt-5A leads to decreased Ca2+-dependent cell adhesion and the activity of Xwnt-5A to block Wnt-1 class signals is mimicked by a dominant negative N-cadherin. These data underscore the importance of cell adhesion in modulating the responses of embryonic cells to signaling molecules and suggest that the Wnt-5A functional class of signaling factors can interact with the Wnt-1 class in an antagonistic manner.

N-cadherin in adult rat cardiomyocytes in culture. I. Functional role of N-cadherin and impairment of cell-cell contact by a truncated N-cadherin mutant.

N-cadherin is a transmembrane Ca(2+)-dependent glycoprotein that is part of adherens junctions. It functions with the cell adhesion N-terminal extracellular domain as a site of homophilic cell-cell contacts. The intracellular C-terminal domain provides via a catenin complex the interaction with the cytoskeleton. Ectopic expression of chicken N-cadherin in adult rat cardiomyocytes (ARC) in culture was obtained after microinjection into non-dividing cardiomyocytes; it was demonstrated that the exogenous protein colocalized with the endogenous N-cadherin at the plasma membrane of the cell and formed contact sites. A dominant negative chicken N-cadherin mutant was constructed by a large deletion of the extracellular domain. This mutant was expressed and inhibited the function of the endogenous rat N-cadherin probably by competing for the catenin complex binding domain, which is essential for the formation of a stable cell-cell contact of ARC. The injected cells lost contact with neighbouring cells and retracted; the connexons of the gap junctions were pulled out as well. This could be avoided by another N-cadherin mutation, which, in addition to the N-terminal truncation, contained a deletion of the catenin binding domain. In the case of the truncated N-cadherin at the N terminus, the sarcomeric structure of the myofibrils of ARC was also affected. Myofibrils were the most vulnerable cytoskeletal structures affected by the overexpressed dominant negative N-cadherin mutation. Similar behaviour was shown when cardiomyocytes separated following Ca2+ depletion and when new cell-cell contacts were formed after Ca2+ replenishment. N-cadherin is thought to be the essential component for establishing new cell-cell contacts which eventually led to a new formation of intercalated disc-like structures in the cardiac cell culture.

Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin.

Cadherins mediate cell adhesion and are essential for normal development. Embryonic stem cells were transfected with a dominant negative N-cadherin mutant (NCAD delta) under the control of promoters active in small intestinal epithelial cells and then introduced into C57BL/6 mouse blastocysts. Analysis of adult chimeric mice revealed that expression of NCAD delta along the entire crypt-villus axis, but not in the villus epithelium alone, produced an inflammatory bowel disease resembling Crohn's disease. NCAD delta perturbed proliferation, migration, and death programs in crypts, which lead to adenomas. This model provides insights about cadherin function in an adult organ and the factors underlying inflammatory bowel disease and intestinal neoplasia.

In vivo analysis of cadherin function in the mouse intestinal epithelium: essential roles in adhesion, maintenance of differentiation, and regulation of programmed cell death.

A model system is described for defining the physiologic functions of mammalian cadherins in vivo. 129/Sv embryonic stem (ES) cells, stably transfected with a dominant negative N-cadherin mutant (NCAD delta) under the control of a promoter that only functions in postmitotic enterocytes during their rapid, orderly, and continuous migration up small intestinal villi, were introduced into normal C57B1/6 (B6) blastocysts. In adult B6<->129/Sv chimeric mice, each villus receives the cellular output of several surrounding monoclonal crypts. A polyclonal villus located at the boundary of 129/Sv- and B6-derived intestinal epithelium contains vertical coherent bands of NCAD delta-producing enterocytes plus adjacent bands of normal B6-derived enterocytes. A comparison of the biological properties of these cell populations established that NCAD delta disrupts cell-cell and cell-matrix contacts, increases the rate of migration of enterocytes along the crypt-villus axis, results in a loss of their differentiated polarized phenotype, and produces precocious entry into a death program. These data indicate that enterocytic cadherins are critical cell survival factors that actively maintain intestinal epithelial function in vivo.

Cadherin-mediated cell interactions are necessary for the activation of MyoD in Xenopus mesoderm.

Muscle progenitors in Xenopus interact in a community of 100 or more cells to activate their myogenic genes and the muscle differentiation pathway. We examine whether the cell adhesion molecule cadherin is involved in this process. Injections of dominant negative N-cadherin RNA into the region of 2- to 4-cell embryos that will give rise to muscle suppress MyoD expression in muscle progenitor cells. By contrast, Xbra expression is unaffected and levels of Xwnt-8 message rise with increasing doses of dominant negative cadherin RNA. MyoD inhibition in embryos injected with the dominant negative cadherin mRNA is rescued by coinjection of full-length cadherin RNA, showing that the inhibition of MyoD occurs through the cadherin pathway. These results show that cadherin-mediated cell interactions play a critical role in the signaling events required for muscle progenitor cells to differentiate, as judged by their stable activation of MyoD.