Mechanisms associated with cellular desiccation tolerance of Artemia encysted embryos from locations around the world.

Using differential scanning calorimetry we demonstrated the presence of biological glasses and measured the glass transition temperatures (Tg) in dry encysted gastrula embryos (cysts) of the brine shrimp, Artemia, from eleven different locations, two of which provided cysts from parthenogenetic animals. Values for Tg were highest, by far, in Artemia franciscana cysts from the Mekong Delta, Vietnam (VN), these cysts having been produced from previous sequential inoculations into growth ponds of cysts from the San Francisco Bay, California, USA. Tg values for three groups of A. franciscana cysts were significantly higher than those of other cysts (except those of Artemia persimilis) studied here, as well as all other desiccation-tolerant animal systems studied to date. We also measured three stress proteins (hsc70, artemin and p26) in all these cysts as well as the total alcohol soluble carbohydrates (ASC), about 90% of which is the disaccharide trehalose, a known component of biological glasses. We interpret the results in terms of mechanisms involved with desiccation tolerance and, to some extent, with thermal conditions at the sites of cyst collection.

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 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.

The expression of Brachyury (T) during gastrulation in the marsupial frog Gastrotheca riobambae.

Gastrulation in the marsupial frog Gastrotheca riobambae has been analyzed by the distribution of the Brachyury (T) protein. Comparison with other amphibians provides mechanistic insights, since G. riobambae develops slowly and has the most divergent mode of amphibian gastrulation, producing an embryonic disk. The T pattern indicates that the prospective mesoderm is superficial, as in many amphibians. The dorsal blastopore lip could not be identified by the expression of T, or by morphological criteria, thus it is unknown whether Gastrotheca embryos have a dorsal organizer before or after blastopore closure. The circumblastoporal and notochordal expression of T, which are temporally contiguous in Xenopus, are separated in Gastrotheca, implying that distinct regulatory mechanisms may control the expression of T in its two domains. The separation of the T pattern also indicates that involution at the blastopore is separate from notochord formation. In addition, extension of the archenteron and notochord occurs after blastopore closure, suggesting that dorsal convergence and extension have been delayed until after blastopore closure. Therefore, dorsal convergence and extension need not be the cause of blastopore closure in Gastrotheca. The separation of gastrulation events in embryos that have not been experimentally manipulated, such as those of Gastrotheca, helps in understanding the distinct nature of gastrulation processes.