A midline-directed transplant technique reveals long-lived MPCs. (A) A heterochronic cell transplantation scheme, from transgenic or transiently transgenic donors to wild-type zebrafish hosts, maximizes contribution to the MPCs. 25-50 cells are taken from a dye-injected sphere stage donor and placed in the prospective mesendoderm (darker gray band) of a 1000-cell stage host. (B,B′) At 24 h post fertilization (hpf), transplanted cells are observed exclusively in the derivatives of the MPCs. Green, red and blue arrowheads indicate notochord, hypochord and floor plate, respectively. (C) Transplanted cells can remain undifferentiated for an extended time, and can join the notochord, hypochord and floor plate of the neural tube. Arrow marks one cell that divides and differentiates into two notochord cells (arrowheads). t=0 is ∼18 hpf. Fp, floor plate; No, notochord; Hy, hypochord.