Development of Diverse Tools for Pigment Cell Research and Drug Screening in Zebrafish

Abstract

The neural crest (NC) is multipotent population of cells that has been very useful in studying several developmental processes including specification, proliferation and differentiation. Its derivatives include amongst others craniofacial cartilage, glial cells of the peripheral nervous system and of particular importance to us the pigment cells. Defects in the NC are responsible for several clinically important diseases. We report here the development of a permanent labelling system for the NC, and of a new zebrafish NC culture system. Plus one gene study of function for id2a and a new method for testing receptor tyrosine kinase inhibitors in vivo.

We have generated a -4725sox10:cre transgenic line that permanently labels the NC and derivatives: migrating NC cells of the head, craniofacial cartilage, cranial ganglia, iridophores, xanthophores, enteric neurons, Schwann cells of the PLLn, of the DRG and of the ventrally and dorsally projecting spinal nerves and sox10+ populations such as oligodendrocytes and the ear epithelium

By testing different attachment substrates, media compositions and methods to obtain NC cells in vitro we have achieved a set of culture conditions that we believe are suitable for zebrafish NC cells and culturing total disaggregated embryos in these culture conditions has allowed us to identify several NC derivatives.

We injected embryos with two different constitutive RTKs that are able to generate ectopic and/or extra iridophores in vivo, using the TAE684 ALK inhibitor, we were able to inhibit this process. Treatment with TAE684 also inhibited production of endogenous Ltk, we used this to our advantage to study iridophore development to distinguish a role for Ltk early in specification and later in proliferation of iridophores.

We characterised the id2a expression pattern in NC in WT and two NC mutants. Using knockdown of id2a we show a role for id2a in NC pigment cell and craniofacial cartilage development.

Date of Award	1 Dec 2010
Original language	English
Awarding Institution	University of Bath
Supervisor	Robert Kelsh (Supervisor)