Decaying and expanding Erk gradients process memory of skeletal size during zebrafish fin regeneration

Regeneration of amputated salamander limbs or fish fins restores pre-injury size, illustrating positional memory. Although appreciated for centuries, the identity of position-dependent cues and how they control tissue growth are not resolved. Here, we quantify Erk signaling events in populations of osteoblasts during zebrafish fin regeneration. We find osteoblast Erk activity depends on Fgf receptor signaling and organizes into millimeter-long gradients spanning the distal tip to the amputation site. Erk activity scales with amount of tissue amputated, predicts likelihood of osteoblast cycling, and predicts size of regenerated skeletal structures. Mathematical modeling suggests gradients are established by transient deposition of long-lived ligands that are transported by tissue growth. This is supported by the observed scaling of expression of the essential epidermal ligand fgf20a with extents of amputation. Our work provides evidence that localized, scaled expression of pro-regenerative ligands instructs long-range signaling and cycling to control size in regenerating appendages. Overall design: To isolate RNA from the amputation plane, a region spanning one bone segment above and below the amputation plane was collected from fish at 0, 3.5, 7, 14, and 21 dpa. For each time point, tissue samples from 10 animals were pooled for each of 3 biological replicates. Bulk RNAseq libraries were prepared to identify gene expression profiles at the distal tip of regenerates.