Totipotency and high plasticity in an embryo with an invariant, fate-specifying cleavage program.

All animal embryos begin as totipotent zygotes that cleave and produce cells with restricted fate potentials. However, the timing of when totipotency is lost and the processes through which embryonic cells acquire fates vary across species. Embryos with invariant cleavage programs, e.g., of nematodes and spiralians, tend to show early restriction of blastomere potency and limited robustness to perturbation, particularly after asymmetric cleavages have occurred. By contrast, embryos with variant cleavage programs, e.g., of vertebrates, tend to specify fates later in development and correspondingly show higher plasticity at early stages. Here, we report unexpected post-zygotic totipotency and subsequent high plasticity of blastomeres in the acoel Hofstenia miamia, whose embryos undergo an invariant cleavage program called "duet cleavage." Blastomere isolation assays revealed that, at the 4-cell stage, single macromeres, which are products of asymmetric, fate-specifying cleavage, were totipotent and formed whole organisms upon isolation. Photoconversion-based lineage tracing showed that pharyngeal and neuronal tissues, which are not produced by macromeres during normal development, are recovered via blastomere transfating. Remarkably, using embryo reconstitution assays, we found that all 8-cell stage blastomeres could be reprogrammed, demonstrating high plasticity in H. miamia embryos. The embryo manipulation assays we developed have high reproducibility and throughput, making the H. miamia embryo an ideal system to investigate the causes for extended totipotency and plasticity.