Planarian dorsoventral Netrins organize a muscle midline signaling center and regulate blastema formation

Integration of positional information across body axes is likely critical for whole-body regeneration to define the territories of missing tissue in three dimensions with fidelity. The body-wall musculature in planarians expresses patterning factors regulating the anteroposterior, dorsoventral, and mediolateral axes, but how this information coordinates is not fully understood. We identify a previously described factor specifically expressed in dorsal midline muscle as a BMP/Activin decoy receptor bambi-2. Analysis of scRNAseq indicates bambi-2+ cells coexpress midline-specifying transcription factor pitx and longitudinal muscle-specifying factor myoD, and production of bambi-2+ cells requires these factors. In laterally amputated animals regenerating an entirely new midline, bambi-2+ cells are initially formed at the wound site, then dynamically spread, and ultimately reset to restore bilateral symmetry. We further identify a system of dorsoventral Netrin and Netrin receptor signals expressed from body-wall muscle that control midline identity and blastema morphology. Ventral and laterally expressed netrins -1, -4, and -5 signal via dorsally-enriched netrin repulsion receptors unc5-C, unc5-E, and dcc-2, which together limit mediolateral spread of bambi-2+ dorsal midline muscle and influence the architecture of the muscle system. Our results suggest a model in which ventral determinants dictate mediolateral information important for blastema morphology.