Heterochronically expressed midline netrin was recruited to guide mesoderm migration in epibolic gastrulation of the leech

Netrin is a remarkably conserved midline landmark, serving as a chemotactic factor that organizes the bilateral neural architecture in the post-gastrula bilaterian embryos. Netrin signal also guides cell migration in many other neural and non-neural organogenesis events in later developmental stages, but it has never been before found to participate in gastrulation - the earliest cell migration in metazoan embryogenesis. Here, we found that netrin signaling molecules and their receptors are expressed during gastrulation of the leech Helobdella. Intriguigly, Hau-netrin-1 was expressed in the N lineage, the precursor of ventral ectoderm, at the onset of gastrulation. We demonstrated that the N lineage is required for the entrance of mesoderm into the germinal band and that misexpression of Hau-netrin-1 in early gastrulation prevented mesoderm from entering the germinal band. Together, these results suggested that Hau-netrin-1 secreted by the N lineage guides mesoderm migration during germinal band assembly. Furthermore, ectopic expression of Hau-netrin-1 after the completion of germinal band assembly disrupted the epibolic migration of the germinal bands in a later stage of gastrulation. Thus, Hau-netrin-1 is likely involved in two distinct events in sequential stages of leech gastrulation: the assembly of germinal bands in early gastrulation and their epibolic migration in mid-gastrulation. This mode of gastrulation as observed in the leech is apomorphic for clitellate annelids. We postulated that a heterochronic shift of netrin gene expression in the clitellate ancestor might have facilitated the evolutionary emergence of a novel form of gastrulation in this lineage.