A fundamental problem in developmental biology concerns how multipotent precursors choose specific fates. Neural crest cells (NCCs) are multipotent, yet the mechanisms driving specific fate choices remain incompletely understood. Sox10 is required for specification of neural cells and melanocytes from NCCs. Like sox10 mutants, zebrafish shady mutants lack iridophores; we have proposed that sox10 and shady are required for iridophore specification from NCCs. We show using diverse approaches that shady encodes zebrafish leukocyte tyrosine kinase (Ltk). Cell transplantation studies show that Ltk acts cell-autonomously within the iridophore lineage. Consistent with this, ltk is expressed in a subset of NCCs, before becoming restricted to the iridophore lineage. Marker analysis reveals a primary defect in iridophore specification in ltk mutants. We saw no evidence for a fate-shift of neural crest cells into other pigment cell fates and some NCCs were subsequently lost by apoptosis. These features are also characteristic of the neural crest cell phenotype in sox10 mutants, leading us to examine iridophores in sox10 mutants. As expected, sox10 mutants largely lacked iridophore markers at late stages. In addition, sox10 mutants unexpectedly showed more ltk-expressing cells than wild-type siblings. These cells remained in a premigratory position and expressed sox10 but not the earliest neural crest markers and may represent multipotent, but partially-restricted, progenitors. In summary, we have discovered a novel signalling pathway in NCC development and demonstrate fate specification of iridophores as the first identified role for Ltk.
Stem and other multipotent cells generate diverse cell-types, but our understanding of how they make these decisions, which is important for their therapeutic use, is incomplete. Neural crest cells are an important class of multipotent cells and generate multiple stem cell types. We have looked at how pigment cells are made from the neural crest in the zebrafish. The silver shine familiar in so many fish is due to specialised mirror-like pigment cells, called iridophores. We show that these cells are missing in zebrafish shady mutants. We identify the shady gene as encoding a cell signalling receptor, leukocyte tyrosine kinase (Ltk), that has recently been associated with human auto-immune disease. We show that in zebrafish this gene is most likely required to make iridophores from neural crest cells. Thus, we identify a novel pathway required for diversification of these multipotent cells. Our work defines the first role for Ltk in a vertebrate. It provides a mutant resource that will allow us to discover the full breadth of roles for this important gene. Furthermore, the loss of iridophores forms a simple visual screen for inhibition of LTK function and might well have implications in drug discovery.