The pregastrula establishment of gene expression pattern in Xenopus embryos: requirements for local cell interactions and for protein synthesis.

Although it has been proposed that mesoderm forms in the marginal zone of the amphibian embryo through inductive signaling from vegetal pole cells, the details of this process remain to be clarified. To determine when marginal zone cells become committed to mesodermal fates, cell contact and protein synthesis requirements for early transcriptional responses were analyzed in Xenopus blastulae. Marginal zone explants were isolated from embryos at different stages and either were cultured untreated, or were dissociated in a medium lacking calcium and magnesium ions, or cultured in the presence of cycloheximide. Whereas many mesoderm-specific transcripts are efficiently induced by activin in dissociated animal pole cells, the same markers were not activated in dissociated marginal zone cells, which were isolated from mid or even late blastulae and cultured in the absence of exogenous inducers. These observations suggest that early specification of mesodermal fates requires cell-cell interactions within the marginal zone and that the marginal zone cells are not committed to express early mesodermal markers until the late blastula stage. Specification of mesodermal fates was also assessed by the ability of marginal zone cells to express early mesodermal markers in the absence of protein synthesis. Induction of Xlim1, 1A11, and, partially, Xbrachyury transcripts in the marginal zone was blocked by cycloheximide treatment through late blastula stages, whereas Goosecoid and Xwnt8 mRNAs were expressed in the absence of protein synthesis, indicating that these sets of markers are activated in vivo through different pathways. These observations demonstrate that determination of mesoderm in the marginal zone is a multistep process which occurs during late blastula stages and depends on cell-cell contact and protein synthesis.