Novel cell states arise in embryonic cells devoid of key reprogramming factors.

The capacity for embryonic cells to differentiate relies on a large-scale reprogramming of the oocyte and sperm nucleus into a transient totipotent state. In zebrafish, this reprogramming step is achieved by the pioneer factors Nanog, Pou5f3, and Sox19b (NPS). Yet, it remains unclear whether cells lacking this reprogramming step are directed towards wild type states or towards novel developmental canals in the Waddington landscape of embryonic development. Here we investigate the developmental fate of embryonic cells mutant for NPS by analyzing their single-cell gene expression profiles. We find that cells lacking the first developmental reprogramming steps can acquire distinct cell states. These states are manifested by gene expression modules that result from a failure of nuclear reprogramming, the persistence of the maternal program, and the activation of somatic compensatory programs. As a result, most mutant cells follow new developmental canals and acquire new mixed cell states in development. In contrast, a group of mutant cells acquire primordial germ cell-like states, suggesting that NPS-dependent reprogramming is dispensable for these cell states. Together, these results demonstrate that developmental reprogramming after fertilization is required to differentiate most canonical developmental programs, and loss of the transient totipotent state canalizes embryonic cells into new developmental states .