The Cell Sorting Process of Xenopus Gastrula Cells Progresses in a Stepwise Fashion Involving Concentrification and Polarization

Ayano Harata, Takashi Matsuzaki, Koichi Ozaki, Setsunosuke Ihara

Faculty of Life and Environmental Science, Department of Biological Science, Shimane University, Shimane, Japan.

DOI: 10.4236/cellbio.2013.22007                

Abstract

Animal pole cells (AC) and vegetal pole cells (VC) dissociated from early Xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. The overall process of cell sorting was found to morphologically consist of two steps, “concentrification” and “polarization”, as designated here. First, AC and VC clusters emerged at random positions in the aggregate, and the individual clusters gradually assembled themselves by 5 hours in culture (5 hC), forming a concentric arrangement, in which the AC cluster was enveloped by the VC cluster. This concentrification step is essentially consistent with the descriptions in earlier studies. As the next step, the AC and VC clusters moved up and down from 7.5 to 12 hC, resulting in the vertical polarization, namely, a serial array just like in vivo. Immunohistochemical analyses showed that AC expressed both C- and E-cadherins, while VC only expressed C-cadherin, as in vivo, suggesting the normal participation of cadherin system. On the other hand, the actin localization showed that the actin bundles accumulated at the edge of the AC cluster until the concentrification was completed, and gradually decreased during the polarization step. Another important finding was that AC cluster could generate cartilage tissues during the long-term (7 days) culture, evidence for a healthy inductive interaction between the AC and VC. Taken together, the present experimental system allows the AC and VC to be viable and grow into an embryo-like organization.

Keywords

Cell Sorting; Xenopus laevis; Concentrification; Polarization; Embryogenesis

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	A. Gierer, S. Berking, H. Bode, C. N. David, K. Flick, G. Hansmann, H. Schaller and E. Trenkner, “Regeneration of Hydra from Reaggregated Cells,” Nature New Biology, Vol. 239, No. 91, 1972, pp. 98-101.
[2]	Y. Takaku, T. Hariyama and T. Fujisawa, “Motility of Endodermal Epithelial Cells Plays a Major Role in Reorganizing the Two Epithelial Layers in Hydra,” Mechanisms of Development, Vol. 122, No. 1, 2005, pp. 109-122. doi:10.1016/j.mod.2004.08.004
[3]	H. Ide, N. Wada and K. Uchiyama, “Sorting out of Cells from Different Parts and Stages of the Chick Limb Bud,” Developmental Biolog, Vol. 162, No. 1, 1994, pp. 71-76. doi:10.1006/dbio.1994.1067
[4]	H. Yajima, S. Yoneitamura, N. Watanabe, K. Tamura and H. Ide, “Role of N-Cadherin in the Sorting-Out of Mesenchymal Cells and in the Positional Identity along the Proximodistal Axis of the Chick Limb Bud,” Developmental Dynamics, Vol. 216, No. 3, 1999, pp. 274-284.
[5]	H. Yajima, K. Hara, H. Ide and K. Tamura, “Cell Adhesiveness and Affinity for Limb Pattern Formation,” The International Journal of Developmental Biology, Vol. 46, No. 7, 2002, pp. 897-904.
[6]	P. L. Townes and J. Holtfreter, “Directed Movements and Selective Adhesion of Embryonic Amphibian Cells,” Journal of Experimental Zoology, Vol. 128, No. 1, 1955, pp. 53-120. doi:10.1002/jez.1401280105
[7]	M. Marsden and D. W. DeSimone, “Integrin-ECM Interactions Regulate Cadherin-Dependent Cell Adhesion and Are Required for Convergent Extension in Xenopus,” Current Biology, Vol. 13, No. 14, 2003, pp. 1182-1191. doi:10.1016/S0960-9822(03)00433-0
[8]	C. M. Niessen and B. M. Gumbiner, “Cadherin-Mediated Cell Sorting Not Determined by Binding or Adhesion Specificity,” Journal of Cell Biology, Vol. 156, No. 2, 2002, pp. 389-399. doi:10.1083/jcb.200108040
[9]	A. Turner, A. M. Snape, C. C. Wylie and J. Heasman, “Regional Identity Is Established before Gastrulation in the Xenopus Embryo,” Journal of Experimental Zoology, Vol. 251, No. 2, 1989, pp. 245-252. doi:10.1002/jez.1402510212
[10]	C. C. Wylie, A. Snape, J. Heasman and J. C. Smith, “Vegetal Pole Cells and Commitment to Form Endoderm in Xenopus laevis,” Developmental Biology, Vol. 119, No. 2, 1987, pp. 496-502. doi:10.1016/0012-1606(87)90052-2
[11]	R. Keller, L. Davidson, A. Edlund, T. Elul, M. Ezin, D. Shook and P. Skoglund, “Mechanisms of Convergence and Extension by Cell Intercalation,” Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 355, No. 1399, 2000, pp. 897-922. doi:10.1098/rstb.2000.0626
[12]	R. Winklbauer and R. E. Keller, “Fibronectin, Mesoderm Migration, and Gastrulation in Xenopus,” Developmental Biology, Vol. 177, No. 2, 1996, pp. 413-426. doi:10.1006/dbio.1996.0174
[13]	Y. S. Choi and B. Gumbiner, “Expression of Cell Adhesion Molecule E-Cadherin in Xenopus Embryos Begins at Gastrulation and Predominates in the Ectoderm,” Journal of Cell Biology, Vol. 108, No. 6, 1989, pp. 2449-2458. doi:10.1083/jcb.108.6.2449
[14]	Y. S. Choi, R. Sehgal, P. McCrea and B. Gumbiner, “A Cadherin-Like Protein in Eggs and Cleaving Embryos of Xenopus laevis Is Expressed in Oocytes in Response to Progesterone,” Journal of Cell Biology, Vol. 110, No. 5, 1990, pp. 1575-1582. doi:10.1083/jcb.110.5.1575
[15]	G. Levi, D. Ginsberg, J. M. Girault, I. Sabanay, J. P. Thiery and B. Geiger, “EP-Cadherin in Muscles and Epithelia of Xenopus laevis Embryos,” Development, Vol. 113, No. 4, 1991, pp. 1335-1344.
[16]	G. Levi, B. Gumbiner and J. P. Thiery, “The Distribution of E-Cadherin during Xenopus laevis Development,” Development, Vol. 111, No. 1, 1991, pp. 159-169.
[17]	Q. Tao, S. Nandadasa, P. D. McCrea, J. Heasman and C. Wylie, “G-Protein-Coupled Signals Control Cortical Actin Assembly by Controlling Cadherin Expression in the early Xenopus Embryo,” Development, Vol. 134, No. 14, 2007, pp. 2551-2561. doi:10.1242/dev.002824
[18]	E. Agius, M. Oelgeschlager, O. Wessely, C. Kemp and E. M. De Robertis, “Endodermal Nodal-Related Signals and Mesodermal Induction in Xenopus,” Development, Vol. 127, No. 6, 2000, pp. 1173-1183.
[19]	P. D. Nieuwkoop, “The Formation of the Mesoderm in Urodelean Amphibians. I. Induction by the Endoderm,” Wilhelm Roux’ Archiv für Entwicklungsmechanik der Organismen, Vol. 162, No. 4, 1969, pp. 334-373. doi:10.1007/BF00578701
[20]	P. D. Nieuwkoop and J. Faber, “Normal Table of Xenopus laevis,” Daudin, North-Holland Publishing Company, Amsterdam, 1956.
[21]	R. M. Harland, “In Situ Hybridization: An Improved Whole Mount Method for Xenopus Embryos,” Methods in Cell Biology, Vol. 36, 1991, pp. 685-695. doi:10.1016/S0091-679X(08)60307-6
[22]	H. L. Sive, R. M. Grainger and R. M. Harland, “Early Development of Xenopus laevis: A laboratory Manual,” Cold Spring Harbor Laboratory Press, New York, 1998.
[23]	R. A. Foty, C. M. Pfleger, G. Forgacs and M. S. Steinberg, “Surface Tensions of Embryonic Tissues Predict Their Mutual Envelopment Behavior,” Development, Vol. 122, No. 5, 1996, pp. 1611-1620.
[24]	J. L. Stubbs, L. Davidson, R. Keller and C. Kintner, “Radial Intercalation of Ciliated Cells during Xenopus Skin Development,” Development, Vol. 133, No. 13, 2006, pp. 2507-2515. doi:10.1242/dev.02417
[25]	A. Nose, A. Nagafuchi and M. Takeichi, “Expressed Recombinant Cadherins Mediate Cell Sorting in Model Systems,” Cell, Vol. 54, No. 7, 1988, pp. 933-1001. doi:10.1016/0092-8674(88)90114-6
[26]	M. Takeichi, “The Cadherins: Cell-Cell Adhesion Molecules Controlling Animal Morphogenesis,” Development, Vol. 102, No. 4, 1988, pp. 639-655.
[27]	R. A. Foty and M. S. Steinberg, “The Differential Adhesion Hypothesis: A Direct Evaluation,” Developmental Biology, Vol. 278, No. 1, 2005, pp. 255-263. doi:10.1016/j.ydbio.2004.11.012
[28]	M. S. Steinberg, “Differential Adhesion in Morphogenesis: A Modern View,” Current Opinion in Genetics & Development, Vol. 17, No. 4, 2007, pp. 281-286. doi:10.1016/j.gde.2007.05.002
[29]	S. Nandadasa, Q. Tao, N. R. Menon, J. Heasman and C. Wylie, “N- and E-Cadherins in Xenopus Are Specifically Required in the Neural and Non-Neural Ectoderm, Respectively, for F-Actin Assembly and Morphogenetic Movements,” Development, Vol. 136, No. 8, 2009, pp. 1327-1338. doi:10.1242/dev.031203
[30]	R. A. Foty and M. S. Steinberg, “Cadherin-Mediated Cell-Cell Adhesion and Tissue Segregation in Relation to Malignancy,” The International Journal of Developmental Biology, Vol. 48, No. 5-6, 2000, pp. 397-409. doi:10.1387/ijdb.041810rf
[31]	T. Lecuit and P. F. Lenne, “Cell Surface Mechanics and the Control of Cell Shape, Tissue Patterns and Morphogenesis,” Nature Reviews Molecular Cell Biology, Vol. 8, No. 8, 2007, pp. 633-644. doi:10.1038/nrm2222
[32]	R. Winklbauer, M. Nagel, A. Selchow and S. Wacker, “Mesoderm Migration in the Xenopus Gastrula,” International Journal of Developmental Biology, Vol. 40, No. 1, 1996, pp. 305-311.
[33]	L. A. Davidson, R. Keller and D. W. DeSimone, “Assembly and Remodeling of the Fibrillar Fibronectin Extracellular Matrix during Gastrulation and Neurulation in Xenopus laevis,” Developmental Dynamics, Vol. 231, No. 4, 2004, pp. 888-895. doi:10.1002/dvdy.20217
[34]	M. Krieg, Y. Arboleda-Estudillo, P. H. Puech, J. Kafer, F. Graner, D. J. Muller and C. P. Heisenberg, “Tensile Forces Govern Germ-Layer Organization in Zebrafish,” Nature Cell Biology, Vol. 10, No. 4, 2008, pp. 429-436. doi:10.1038/ncb1705
[35]	E. M. Schotz, R. D. Burdine,F. Julicher, M. S. Steinberg, C. P. Heisenberg and R. A. Foty, “Quantitative Differences in Tissue Surface Tension Influence Zebrafish Germ Layer Positioning,” HFSP Journal, Vol. 2, No. 1, 2008, pp. 42-56. doi:10.2976/1.2834817
[36]	G. S. Davis, H. M. Phillips and M. S. Steinberg, “Germ-Layer Surface Tensions and ‘Tissue Affinities’ in Rana pipiens Gastrulae: Quantitative Measurements,” Developmental Biology, Vol. 192, No. 2, 1997, pp. 630-644. doi:10.1006/dbio.1997.8741
[37]	A. K. Harris, “Is Cell Sorting Caused by Differences in the Work of Intercellular Adhesion? A Critique of the Steinberg Hypothesis,” Journal of Theoretical Biology, Vol. 61, No. 2, 1976, pp. 267-285. doi:10.1016/0022-5193(76)90019-9
[38]	M. L. Manning, R. A. Foty, M. S. Steinberg and E. M. Schoetz, “Coaction of Intercellular Adhesion and Cortical Tension Specifies Tissue Surface Tension,” Proceedings of the National Academy of Sciences of USA, Vol. 107, No. 23, 2010, pp. 2517-12522. doi:10.1073/pnas.1003743107
[39]	H, Kuroda, H. Sakumoto, K. Kinoshita and M. Asashima, “Changes in the Adhesive Properties of Dissociated and reaggregated Xenopus laevis Embryo Cells,” Current Opinion in Genetics & Development, Vol. 41, No. 3, 1999, pp. 283-291. doi:10.1046/j.1440-169X.1999.413428.x
[40]	K. Okabayashi and M. Asashima, “Tissue Generation from Amphibian Animal Caps,” Current Opinion in Genetics & Development, Vol. 13, No. 5, 2003, pp. 502-507. doi:10.1016/S0959-437X(03)00111-4
[41]	Y. Fukui, M. Furue, Y. Myoishi, J. D. Sato, T. Okamoto and M. Asashima, “Long-Term Culture of Xenopus Presumptive Ectoderm in A Nutrient-Supplemented Culture Medium,” Current Opinion in Genetics & Development, Vol. 45, No. 5-6, 2003, pp. 499-506. doi:10.1111/j.1440-169X.2003.00717.x
[42]	M. Furue, Y. Myoishi, Y. Fukui, T. Ariizumi, T, Okamoto and M. Asashima, “Activin A induces Craniofacial Cartilage from Undifferentiated Xenopus Ectoderm in Vitro,” Proceedings of the National Academy of Sciences of USA, Vol. 99, No. 24, 2002, pp. 15474-15479. doi:10.1073/pnas.242597399