THE JOURNAL OF EXPERIMENTAL ZOOLOGY 277:181–197 (1997) © 1997 WILEY-LISS, INC. JEZ 738 Evidence for the Presence and Participation of 85–75 KDa Extracellular Matrix Components in Cell Interactions of Bufo arenarum Gastrulation SUSANA B. GENTA, MANUEL J. AYBAR, MARCELA A. PERALTA, AND SARA S. SÁNCHEZ* Departamento de Biología del Desarrollo, Instituto Superior de Investigaciones Biológicas (INSIBIO), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) y Universidad Nacional de Tucumán (UNT), Chacabuco 461, 4000-San Miguel de Tucumán, Tucumán, Argentina ABSTRACT We studied the presence and distribution of the extracellular materials (ECM), obtained by mild embryonic dissociation through nondenaturing and denaturing PAGE, immuno- blotting and immunocytochemical wholemount in the gastrulation of anuran amphibian Bufo arenarum. The SDS-PAGE, under reducing conditions, revealed the protein profile of the ECM which com- prised six bands. The Western immunoblotting effected with antibodies against fibronectins (FN) of Xenopus laevis, Ambystoma mexicanum and Bufo arenarum revealed that the 210 and 190 KDa bands (EP1–EP2) present in the ECM were identified as FN. Polyclonal antibodies against the 85–75 KDa polypeptides (EP3–EP4) were obtained and used throughout this study. The distribution of FN and EP3–EP4 was comparatively studied in the blastocoelic roof (BCR) of stage 10.5 Bufo arenarum, Xenopus laevis and Ambystoma mexicanum embryos. In the anurans, FN appeared as a network of fine fibrils apparently oriented at random, while in Ambystoma, FN appeared as a complex anastomosing network of oriented fibrils. EP3– EP4 were found in Bufo and in Xenopus both in the intercellular contact zones and in the cellular periphery. No linear arrangements of these proteins were observed. Few, if any, EP3–EP4 were found on the BCR of Ambystoma mexicanum. At stage 11, EP3–EP4, which showed a dramatic increase at the chordomesoderm-neuroectoderm junction in Bufo arenarum embryos, appeared as an amorphous material. For the purpose of analyzing the role of EP3–EP4 during Bufo arenarum gastrulation, anti- EP3–EP4 antibodies and anti-EP3–EP4 Fab´ fragments were microinjected into the blastocoel cavity of stage 9 embryos, an event that cause severe alterations in the gastrulation process. Convergent extension of the dorsal marginal zone and the epiboly of the BCR were the most strongly affected events. Results show that EP3–EP4 are required for normal Bufo arenarum gastrulation. J. Exp. Zool. 277:181–197, 1997. © 1997 Wiley-Liss, Inc. The role played by the extracellular matrix in cell interactions is not limited to the structural organization of cells into tissues. Matrix compo- nents such as fibronectin (FN), laminin (LM) and collagens (COL) exert an influence on the adhe- siveness of cells as well as on their ability to mi- grate and become differentiated. As these events occur extensively in early embryos, it is not sur- prising that the extracellular matrix should play an important role in the morphogenetic process. Embryonic development appears to require the precise and sequential functioning of specific mac- romolecules. The developmentally regulated ap- pearance and disappearance of certain molecules has often been correlated with a possible devel- opmental role (Edelman et al., ’83; Ekblom et al., ’86; Hay, ’91; Thiery et al., ’82). However, the direct demonstration of causal roles for specific molecules during development is difficult. Treatment with antibodies is the most obvious approach to obtain the specific in- hibition of the function of a molecule. For ex- ample, antibodies to FN as well as integrins (INT) and cell binding peptide fragments of FN blocked gastrulation in the same way when in- *Correspondence to: Sara S. Sánchez, Departamento de Biología del Desarrollo, INSIBIO (CONICET-UNT), Chacabuco 461, 4000-San Miguel de Tucumán, Argentina. Received 20 March 1996; Revision accepted 8 October 1996