Cold Spring Harb Protoc 2019 Jan 02;20191:. doi: 10.1101/pdb.prot097303.

Cleavage Blastomere Explant Culture in Xenopus.

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The individual blastomeres of Xenopus two- to 32-cell embryos have been fate mapped. This work identified the precursors of most of the embryonic cell types, tissues and organs; however, the maps do not reveal the cell interactions or signaling pathways that are required for establishing cell fates. This protocol describes an explant culture approach for culturing blastomeres in isolation to test whether a cell's fate has been determined. Cleavage blastomeres can be cultured in a simple salt medium without added factors because they contain intracellular yolk platelets, which provide an intrinsic energy source. This method allows one to test whether an isolated blastomere can produce specific cell types or express tissue-specific genes independent of interactions with other cells or specific signaling pathways. The role of cell-cell interactions can be revealed by co-culturing different sets of blastomeres. One can identify the molecules that are required for those cell fates by applying knockdown approaches to the isolated cell. One also can determine the developmental time at which cell fates are committed by explanting blastomere lineages at different stages.

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