EVIDENCE FOR THE PRESENCE AND PARTICIPATION OF 85-75-KDA EXTRACELLULAR-MATRIX COMPONENTS IN CELL-INTERACTIONS OF BUFO-ARENARUM GASTRULATION

We studied the presence and distribution of the extracellular material s (ECM), obtained by mild embryonic dissociation through nondenaturing and denaturing PAGE, immunoblotting and immunocytochemical wholemount in the gastrulation of anuran amphibian Bufo arenarum. The SDS-PAGE, under reducing conditions, revealed the protein profile of the ECM whi ch comprised six bands. The Western immunoblotting effected with antib odies against fibronectins (FN) of Xenopus laevis, Ambystoma mexicanum and Bufo arenarum revealed that the 210 and 190 KDa bands (EP1-EP2) p resent in the ECM were identified as FN. Polyclonal antibodies against the 85-75 KDa polypeptides (EP3-EP4) were obtained and used throughou t this study. The distribution of FN and EP3-EP4 was comparatively stu died in the blastocoelic roof(BCR) of stage 10.5 Bufo arenarum, Xenopu s laevis and Ambystoma mexicanum embryos. In the anurans, FN appeared as a network of fine fibrils apparently oriented at random, while in A mbystoma, FN appeared as a complex anastomosing network of oriented fi brils. EP3-EP4 were found in Bufo and in Xenopus both in the intercell ular contact zones and in the cellular periphery. No linear arrangemen ts 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, an ti-EP3-EP4 antibodies and anti-EP3-EP4 Fab' fragments were microinject ed into the blastocoel cavity of stage 9 embryos, an event that cause severe alterations in the gastrulation process. Convergent extension o f the dorsal marginal zone and the epiboly of the BCR were the most st rongly affected events. Results show that EP3-EP4 are required for nor mal Bufo arenarum gastrulation. (C) 1997 Wiley-Liss, Inc.