roboA and anosmin-1 restrict fate boundaries in planarian stem cells via foxA

Stem cells integrate extracellular cues with downstream transcriptional responses to channel their differentiation, but the underlying mechanisms responsible remain unclear. Planarians possess distinct organs embedded in a vast pool of pluripotent stem cells that maintain tissue turnover and drive whole-body regeneration. This unique configuration provides an ideal model for exploring how pluripotent stem cells sense tissue boundaries, guiding appropriate differentiation. Here, we use the planarian pharynx to define the cell lineages that originate from stem cells expressing the pioneer transcription factor foxA. The anatomical restriction of pharynx-specific lineages is disrupted by knockdown of the Roundabout receptor roboA, leading to ectopic pharynx neurons (EPNs) emerging in the brain. An RNAi screen for genes that phenocopy roboA identifies the extracellular protein anosmin-1, a gene responsible for Kallmann syndrome, as a potential partner of roboA. Aberrant induction of EPNs in the brain requires foxA, while knockdown of foxA induces head-specific neurons in the pharynx, revealing a latent plasticity in stem cells to adopt alternative fates. Together, we propose that RoboA and Anos1 act as fate-reinforcing genes (FRGs) that suppress inappropriate cell fates, maintaining organ integrity and enabling stem cell plasticity during regeneration. 10X 3'single-cell RNA sequencing from control and foxA(RNAi) planarian cells.