J Exp Zool 1997 Feb 15;2773:181-97. doi: 10.1002/(sici)1097-010x(19970215)277:3<181::aid-jez1>3.0.co;2-k.

Evidence for the presence and participation of 85-75 KDa extracellular matrix components in cell interactions of Bufo arenarum gastrulation.

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We studied the presence and distribution of the extracellular materials (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 which comprised 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.

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Species referenced: Xenopus laevis
Genes referenced: bcr