In vivo differentiation of embryonic cells devoid of key reprogramming factors

Embryonic cell differentiation depends on the reprogramming of the oocyte and sperm nucleus into a transient totipotent state. In zebrafish, this coincides with genome activation, which is regulated by the pioneer factors Nanog, Pou5f3, and Sox19b (NPS). Here, we investigate the role of NPS in developmental reprogramming and differentiation by analyzing the fate of NPS mutant cells in a wild-type embryo using single-cell RNA-seq. We find that many cells fail to activate transcription or undergo cell death, while others acquire gene expression profiles that resemble germ cells, neural progenitors, and motoneuron states. These cells achieve intermediate transcriptional states revealing the essential role of NPS in coordinating nuclear and cytoplasmic reprogramming and preventing the premature activation of lineage-specific differentiation programs. These results demonstrate that most developmental programs require developmental reprogramming by NPS, yet some cell can bypass transient totipotency to achieve intermediate developmental states resembling wild type states in vivo. Donor embryos (WT or MZnps mutants) were injected with fluorescent reporters and transplanted into host wild-type embryos at the 1-cell stage. Embryos were dissociated at 11–12 hpf and enriched for donor-derived cells using hCD4 magnetic selection. Single-cell suspensions were loaded onto 10x Genomics Chromium chips (v3 chemistry), and three libraries were sequenced (two MZnps donor replicates, one WT donor).