RESUMEN
The sea urchin embryo is a model for studying cellular interactions that occur in higher organisms because of its availability, transparency, and accessibility to molecular probes. In previous studies, we found that the mannose/glucose-binding lectin Lens culinaris agglutinin entered living sea urchin embryos, bound to specific cell types and caused exogastrulation, when the developing gut (archenteron) falls out of the embryo proper. We have proposed that the lectin bound to sugar-containing ligands, thus preventing attachment of the archenteron to the blastocoel roof, resulting in exogastrulation. Here, we have continued our study of cellular interactions in this model using Lytechinus pictus sea urchin embryos, and have found that inhibitors of glycoprotein/proteoglycan synthesis, tunicamycin and sodium selenate, and the specific glycosidases, beta-amylase, alpha-glucosidase, and alpha-mannosidase, all inhibit archenteron organization, elongation, and attachment to the blastocoel roof in viable swimming embryos. We also show that single cells obtained by disaggregation of 32-h-old sea urchin embryos bind to L. culinaris agglutinin- and concanavalin A-derivatized beads; the binding is blocked by alpha-methyl mannose, but not l-fucose. These cells also bind to beads derivatized with mannan. These results provide evidence for a role of carbohydrate-containing molecules in cellular interactions in sea urchin gastrulation. In a second set of experiments, we found that the supernatant obtained by disaggregation of 24-32-h-old L. pictus embryos in calcium- and magnesium-free sea water contains molecules that cause exogastrulation, archenteron disorganization, inhibition of archenteron elongation and inhibition of archenteron attachment to the blastocoel roof in viable swimming embryos. We propose that the supernatant contains ligands and/or receptors that mediate archenteron development and attachment to the blastocoel roof and are released when embryos are disaggregated into single cells. These studies may lead to a better understanding of the molecular basis of mechanisms that control cellular interactions during development.
Asunto(s)
Comunicación Celular/fisiología , Gástrula/fisiología , Proteoglicanos/metabolismo , Erizos de Mar/embriología , Animales , Adhesión Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Gástrula/citología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glicósido Hidrolasas/metabolismoRESUMEN
Standard histochemical analysis of cells and tissues generally involves procedures that utilize a relatively small number of probes such as dyes, and generally requires hours or days to process. Our laboratory has developed a novel method for histochemical surveys of cell surface properties that utilizes a large number of probes (derivatized agarose beads) and takes seconds or minutes to accomplish. In this study, 4 human cell lines (CCL-255 (LS123) human colon cancer cells that are non-tumorigenic in nude mice; CRL-1459 (CCD-18CO) human colon endothelial cells that are non-malignant; CCL-220 (COLO 320DM) human colon cancer cells that are tumorigenic in nude mice; and HTB-171 (NCI H446) human lung carcinoma cells) were tested for their ability to bind to agarose beads derivatized with 51 different molecules. There were statistically significant differences in binding of the 4 cell types to all of the 51 types of beads, but 15 types of beads showed dramatic differences in binding to one or more of the 4 cell types. For example, only HTB-171 (NCI H446) bound to p-aminophenyl-beta-D-glucopyranoside-derivatized beads and only CCL-220 (COLO 320DM) bound to L-tyrosine-derivatized beads. The specificity of cell-bead binding was examined by performing assays in the presence or absence of exogenously added compounds in hapten-type of inhibition experiments. This assay, that utilizes large numbers of novel probes, may help in the development of new libraries of surface properties of specific cell types, with differing degrees of malignancy, that at this time could not be developed by using other available technologies.