Totipotency and high plasticity in an embryo with a stereotyped, invariant cleavage program

Animal embryos begin as totipotent zygotes, which undergo cell divisions and produce progeny with restricted fate potentials over time. 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. In 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 investigate the embryos of the acoel Hofstenia miamia, which represents an understudied phylum (Xenacoelomorpha) that is distantly related to well-studied developmental systems. Given the invariant duet cleavage program observed in H. miamia embryos, we found unexpected robustness in this species. Isolated 4-cell stage macromeres, the products of an asymmetric, fate specifying cleavage, were totipotent, forming whole organisms upon isolation. Notably, these isolated macromeres produced pharyngeal and neuronal tissues, which they do not produce during normal development. This assay is highly reproducible and can be done at high throughput in H. miamia, making this species an ideal system to investigate the causes of totipotency after specification. Photoconversion-based lineage tracing revealed that rescued cell types are not merely replaced by neoblasts, the adult pluripotent stem cells in H. miamia, suggesting that the macromeres totipotency is the result of changes in the fate potentials of early embryonic cells. Remarkably, all blastomeres at the 8-cell stage were capable of reprogramming their fates in embryo reconstitution assays. By assembling different subsets of 8-cell stage blastomeres, none of which are totipotent on their own, we determined that a minimal unit of two blastomeres, one macromere that produces gut and neoblasts and one micromere that is specified to produce muscle and epidermis, was sufficient to develop into a hatchling worm. Future studies of this system could identify the precise mechanisms that can enable tremendous plasticity, including post-zygotic totipotency, in an embryo with well-defined cellular lineages.