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Zyxin Regulates Cell Migration and Differentiation in EMT during Chicken AV Valve Morphogenesis

Published online by Cambridge University Press:  07 June 2013

Na Li ,
Richard L. Goodwin  and
Jay D. Potts
Na Li
Affiliation:
Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, SC 29209, USA
Richard L. Goodwin
Affiliation:
Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, SC 29209, USA
Jay D. Potts*
Affiliation:
Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, SC 29209, USA
*
*Corresponding author. E-mail: [email protected]
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Abstract

During heart valve development, epithelial–mesenchymal transformation (EMT) is a key process for valve formation. EMT leads to the generation of mesenchymal cells that will eventually become the interstitial cells (fibroblasts) of the mature valve. During EMT, cell architecture and motility change markedly; significant changes are also observed in various signaling pathways. Here we systematically examined the expression, localization, and function of zyxin, a focal adhesion protein, in EMT during atrioventricular (AV) valve morphogenesis. Expression and localization studies showed that zyxin was expressed in the AV canal region during crucial stages of valve development. An in vitro 3D collagen gel culture system was used to determine zyxin function either after siRNA gene knockdown or after overexpression. Our studies revealed that zyxin overexpression inhibited endocardial cell migration and cell differentiation and also led to a decrease in the number of migrating mesenchymal cells. Moreover, correlative cytoskeletal changes were apparent in response to both overexpression and knockdown treatments. Thus, zyxin appears to play a role as a regulator of cell migration and differentiation during EMT in chicken AV valve formation.

Keywords

EMTAV valve morphogenesiszyxincell migrationdifferentiation
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 4 , August 2013 , pp. 842 - 854
Copyright
Copyright © Microscopy Society of America 2013 

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References

Amsellem, V., Kryszke, M.-H., Hervy, M., Subra, F., Athman, R., Leh, H., Brachet-Ducos, C. & Auclair, C. (2005). The actin cytoskeleton-associated protein zyxin acts as a tumor suppressor in Ewing tumor cells. Exp Cell Res 304, 443–456.Google Scholar
Armstrong, E.J. & Bischoff, J. (2004). Heart valve development: Endothelial cell signaling and differentiation. Circ Res 95, 459–470.Google Scholar
Beckerle, M.C. (1986). Identification of a new protein localized at sites of cell-substrate adhesion. J Cell Biol 103, 1679–1687.Google Scholar
Biechler, S.V., Potts, J.D., Yost, M.J., Junor, L., Goodwin, R.L. & Weidner, J.W. (2010). Mathematical modeling of flow-generated forces in an in vitro system of cardiac valve development. Ann Biomed Eng 38, 109–117.Google Scholar
Boyer, A.S., Ayerinskas, I.I., Vincent, E.B., McKinney, L.A., Weeks, D.L. & Runyan, R.B. (1999). TGFbeta2 and TGFbeta3 have separate and sequential activities during epithelial-mesenchymal cell transformation in the embryonic heart. Dev Biol 208, 530–545.Google Scholar
Butcher, J.T. & Markwald, R.R. (2007). Valvulogenesis: The moving target. Philos Trans R Soc Lond, B, Biol Sci 362, 1489–1503.Google Scholar
Camenisch, T.D., Molin, D.G.M., Person, A., Runyan, R.B., Gittenberger-de Groot, A.C., McDonald, J.A. & Klewer, S.E. (2002). Temporal and distinct TGFbeta ligand requirements during mouse and avian endocardial cushion morphogenesis. Dev Biol 248, 170–181.Google Scholar
Cattaruzza, M., Lattrich, C. & Hecker, M. (2004). Focal adhesion protein zyxin is a mechanosensitive modulator of gene expression in vascular smooth muscle cells. Hypertension 43, 726–730.Google Scholar
Chhabra, E.S. & Higgs, H.N. (2007). The many faces of actin: Matching assembly factors with cellular structures. Nat Cell Biol 9, 1110–1121.Google Scholar
Combs, M.D. & Yutzey, K.E. (2009). Heart valve development: Regulatory networks in development and disease. Circ Res 105, 408–421.Google Scholar
Crawford, A.W. & Beckerle, M.C. (1991). Purification and characterization of zyxin, an 82,000-dalton component of adherens junctions. J Biol Chem 266, 5847–5853.Google Scholar
Crawford, A.W., Michelsen, J.W. & Beckerle, M.C. (1992). An interaction between zyxin and alpha-actinin. J Cell Biol 116, 1381–1393.Google Scholar
Délot, E.C. (2003). Control of endocardial cushion and cardiac valve maturation by BMP signaling pathways. Mol Genet Metab 80, 27–35.Google Scholar
Dhawan, R.R., Schoen, T.J. & Beebe, D.C. (1997). Isolation and expression of homeobox genes from the embryonic chicken eye. Mol Vis 3, 7.Google Scholar
Drees, B.E., Andrews, K.M. & Beckerle, M.C. (1999). Molecular dissection of zyxin function reveals its involvement in cell motility. J Cell Biol 147, 1549–1560.Google Scholar
Eisenberg, L.M. & Markwald, R.R. (1995). Molecular regulation of atrioventricular valvuloseptal morphogenesis. Circ Res 77, 1–6.Google Scholar
Goldsmith, E.C., Zhang, X., Watson, J., Hastings, J. & Potts, J.D. (2010). The collagen receptor DDR2 is expressed during early cardiac development. Anat Rec (Hoboken) 293, 762–769.Google Scholar
Hamburger, V. & Hamilton, H.L. (1951). A series of normal stages in the development of the chick embryo. J Morphol 88, 49–92.Google Scholar
Hirata, H., Tatsumi, H. & Sokabe, M. (2008). Mechanical forces facilitate actin polymerization at focal adhesions in a zyxin-dependent manner. J Cell Sci 121, 2795–2804.Google Scholar
Hoffman, J.I. (1990). Congenital heart disease: Incidence and inheritance. Pediatr Clin North Am 37, 25–43.Google Scholar
Hoffman, L.M., Jensen, C.C., Kloeker, S., Wang, C.-L.A., Yoshigi, M. & Beckerle, M.C. (2006). Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling. J Cell Biol 172, 771–782.Google Scholar
Hoffman, L.M., Nix, D.A., Benson, B., Boot-Hanford, R., Gustafsson, E., Jamora, C., Menzies, A.S., Goh, K.L., Jensen, C.C., Gertler, F.B., Fuchs, E., Fässler, R. & Beckerle, M.C. (2003). Targeted disruption of the murine zyxin gene. Mol Cell Biol 23, 70–79.Google Scholar
Hove, J.R., Köster, R.W., Forouhar, A.S., Acevedo-Bolton, G., Fraser, S.E. & Gharib, M. (2003). Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis. Nature 421, 172–177.CrossRefGoogle ScholarPubMed
Kokudo, T., Suzuki, Y., Yoshimatsu, Y., Yamazaki, T., Watabe, T. & Miyazono, K. (2008). Snail is required for TGFbeta-induced endothelial-mesenchymal transition of embryonic stem cell-derived endothelial cells. J Cell Sci 121, 3317–3324.CrossRefGoogle ScholarPubMed
Leccia, M.T., van der Gaag, E.J., Jalbert, N.L. & Byers, H.R. (1999). Zyxin redistributes without upregulation in migrating human keratinocytes during wound healing. J Invest Dermatol 113, 651–657.Google Scholar
Lincoln, J., Alfieri, C.M. & Yutzey, K.E. (2004). Development of heart valve leaflets and supporting apparatus in chicken and mouse embryos. Dev Dyn 230, 239–250.Google Scholar
Macalma, T., Otte, J., Hensler, M.E., Bockholt, S.M., Louis, H.A., Kalff-Suske, M., Grzeschik, K.H., von der Ahe, D. & Beckerle, M.C. (1996). Molecular characterization of human zyxin. J Biol Chem 271, 31470–31478.Google Scholar
Markwald, R.R., Krook, J.M., Kitten, G.T. & Runyan, R.B. (1981). Endocardial cushion tissue development: Structural analyses on the attachment of extracellular matrix to migrating mesenchymal cell surfaces. Scan Electron Microsc Pt. 2: 261–274.Google ScholarPubMed
Markwald, R.R., Norris, R.A., Moreno-Rodriguez, R. & Levine, R.A. (2010). Developmental basis of adult cardiovascular diseases: Valvular heart diseases. Ann NY Acad Sci 1188, 177–183.Google Scholar
Mori, M., Nakagami, H., Koibuchi, N., Miura, K., Takami, Y., Koriyama, H., Hayashi, H., Sabe, H., Mochizuki, N., Morishita, R. & Kaneda, Y. (2009). Zyxin mediates actin fiber reorganization in epithelial-mesenchymal transition and contributes to endocardial morphogenesis. Mol Biol Cell 20, 3115–3124.CrossRefGoogle ScholarPubMed
Nix, D.A. & Beckerle, M.C. (1997). Nuclear-cytoplasmic shuttling of the focal contact protein, zyxin: A potential mechanism for communication between sites of cell adhesion and the nucleus. J Cell Biol 138, 1139–1147.Google Scholar
Nix, D.A., Fradelizi, J., Bockholt, S., Menichi, B., Louvard, D., Friederich, E. & Beckerle, M.C. (2001). Targeting of zyxin to sites of actin membrane interaction and to the nucleus. J Biol Chem 276, 34759–34767.Google Scholar
Norris, R.A., Moreno-Rodriguez, R.A., Sugi, Y., Hoffman, S., Amos, J., Hart, M.M., Potts, J.D., Goodwin, R.L. & Markwald, R.R. (2008). Periostin regulates atrioventricular valve maturation. Dev Biol 316, 200–213.Google Scholar
Person, A.D., Klewer, S.E. & Runyan, R.B. (2005). Cell biology of cardiac cushion development. Int Rev Cytol 243, 287–335.Google Scholar
Pfaffl, M.W., Horgan, G.W. & Dempfle, L. (2002). Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30, e36. Google Scholar
Potts, J.D., Dagle, J.M., Walder, J.A., Weeks, D.L. & Runyan, R.B. (1991). Epithelial-mesenchymal transformation of embryonic cardiac endothelial cells is inhibited by a modified antisense oligodeoxynucleotide to transforming growth factor beta 3. Proc Natl Acad Sci USA 88, 1516–1520.Google Scholar
Potts, J.D. & Runyan, R.B. (1989). Epithelial-mesenchymal cell transformation in the embryonic heart can be mediated, in part, by transforming growth factor beta. Dev Biol 134, 392–401.Google Scholar
Potts, J.D., Vincent, E.B., Runyan, R.B. & Weeks, D.L. (1992). Sense and antisense TGF beta 3 mRNA levels correlate with cardiac valve induction. Dev Dyn 193, 340–345.Google Scholar
Reinhard, M., Zumbrunn, J., Jaquemar, D., Kuhn, M., Walter, U. & Trueb, B. (1999). An alpha-actinin binding site of zyxin is essential for subcellular zyxin localization and alpha-actinin recruitment. J Biol Chem 274, 13410–13418.Google Scholar
Runyan, R.B. & Markwald, R.R. (1983). Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue. Dev Biol 95, 108–114.Google Scholar
Runyan, R.B., Potts, J.D. & Weeks, D.L. (1992). TGF-beta 3-mediated tissue interaction during embryonic heart development. Mol Reprod Dev 32, 152–159.Google Scholar
Sadler, I., Crawford, A.W., Michelsen, J.W. & Beckerle, M.C. (1992). Zyxin and cCRP: Two interactive LIM domain proteins associated with the cytoskeleton. J Cell Biol 119, 1573–1587.Google Scholar
Serini, G., Bochaton-Piallat, M.L., Ropraz, P., Geinoz, A., Borsi, L., Zardi, L. & Gabbiani, G. (1998). The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1. J Cell Biol 142, 873–881.Google Scholar
Smith, M.A., Blankman, E., Gardel, M.L., Luettjohann, L., Waterman, C.M. & Beckerle, M.C. (2010). A zyxin-mediated mechanism for actin stress fiber maintenance and repair. Dev Cell 19, 365–376.Google Scholar
Sperry, R.B., Bishop, N.H., Bramwell, J.J., Brodeur, M.N., Carter, M.J., Fowler, B.T., Lewis, Z.B., Maxfield, S.D., Staley, D.M., Vellinga, R.M. & Hansen, M.D.H. (2010). Zyxin controls migration in epithelial-mesenchymal transition by mediating actin-membrane linkages at cell-cell junctions. J Cell Physiol 222, 612–624.Google Scholar
Stankunas, K., Ma, G.K., Kuhnert, F.J., Kuo, C.J. & Chang, C.-P. (2010). VEGF signaling has distinct spatiotemporal roles during heart valve development. Dev Biol 347, 325–336.Google Scholar
van der Gaag, E.J., Leccia, M.-T., Dekker, S.K., Jalbert, N.L., Amodeo, D.M. & Byers, H.R. (2002). Role of zyxin in differential cell spreading and proliferation of melanoma cells and melanocytes. J Invest Dermatol 118, 246–254.CrossRefGoogle ScholarPubMed
Vermot, J., Forouhar, A.S., Liebling, M., Wu, D., Plummer, D., Gharib, M. & Fraser, S.E. (2009). Reversing blood flows act through KLF2a to ensure normal valvulogenesis in the developing heart. PLoS Biol 7, e1000246. Google Scholar
Wang, Y. & Gilmore, T.D. (2003). Zyxin and paxillin proteins: Focal adhesion plaque LIM domain proteins go nuclear. Biochim Biophys Acta 1593, 115–120.Google Scholar
Weinberg, E.J., Mack, P.J., Schoen, F.J., Garcia-Cardena, G. & Kaazempur Mofrad, M.R. (2010). Hemodynamic environments from opposing sides of human aortic valve leaflets evoke distinct endothelial phenotypes in vitro. Cardiovasc Eng 10, 5–11.Google Scholar
Wennerberg, K. & Der, C.J. (2004). Rho-family GTPases: It's not only Rac and Rho (and I like it). J Cell Sci 117, 1301–1312.Google Scholar
Wójtowicz, A., Babu, S.S., Li, L., Gretz, N., Hecker, M. & Cattaruzza, M. (2010). Zyxin mediation of stretch-induced gene expression in human endothelial cells. Circ Res 107, 898–902.Google Scholar
Worth, D.C. & Parsons, M. (2008). Adhesion dynamics: Mechanisms and measurements. Int J Biochem Cell Biol 40, 2397–2409.Google Scholar
Wu, H., Liu, T., Wang, R., Tian, S., Liu, M., Li, X. & Tang, H. (2011). MicroRNA-16 targets zyxin and promotes cell motility in human laryngeal carcinoma cell line HEp-2. IUBMB Life 63, 101–108.CrossRefGoogle ScholarPubMed
Yalcin, H.C., Shekhar, A., McQuinn, T.C. & Butcher, J.T. (2011). Hemodynamic patterning of the avian atrioventricular valve. Dev Dyn 240(1), 23–35.Google Scholar
Yamagishi, T., Ando, K. & Nakamura, H. (2009). Roles of TGFbeta and BMP during valvulo-septal endocardial cushion formation. Anat Sci Int 84, 77–87.Google Scholar
Yoshigi, M., Hoffman, L.M., Jensen, C.C., Yost, H.J. & Beckerle, M.C. (2005). Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement. J Cell Biol 171, 209–215.Google Scholar
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