The role of tight junction protein 1a and leukocyte tyrosine kinase in the development and organisation of pigment cells in the zebrafish

Vertebrates possess a unique structure, the neural crest, which contributes cells to a wide spectrum of tissues and organs, amongst them are pigment cells or chromatophores. In mammals and birds only one type of chromatophore is present – the melanocytes. However, more basal animals – reptilians, amphibians and fish – display an array of specialized chromatophores. Different arrangements of these cells produce an astonishing multitude of colouration patterns, especially in teleost fish. Zebrafish show a highly regular pattern of alternating dark and light stripes composed of three pigment cell types: black melanophores, yellow xanthophores and silvery iridophores. The formation of this pattern requires coordinated cell movements and cell shape changes of pigment cells during metamorphosis. Iridophores play a crucial part in this process by switching between the dense shape of the light stripes and the loose shape of the dark stripes. It has been suggested that homotypic interactions regulate iridophore proliferation and dispersal. Communication with xanthophores and melanophores, on the other hand, regulates a stereotypical sequence of aggregation and loosening, which establishes the stripes along dorso-ventral axis. However, not much is known about the molecular mechanisms that mediate the formation of the adult striped pattern. Analysing mutants obtained in several screens, we describe two novel regulators of iridophore behaviour. In the schachbrett (sbr) mutants the dark stripes are undulating or broken into spots. Using confocal microscopy and transgenic lines, we demonstrate that sbr iridophores are not able to switch from the dense shape to the loose shape in an appropriate spatio-temporal manner. As a result, dense iridophores invade the dark stripe areas, drive away melanophores, which leads to interruptions. We demonstrate that the phenotype is due to truncations of Tight Junction Protein 1a (Tjp1a, ZO-1a). Antibody labelling and chimeric analysis indicate that Tjp1a is expressed in the dense iridophores, but is down-regulated in the loose iridophores. The moonstone mutants display ectopic iridophores inside the trunk throughout development. In the adult fish, ectopic iridophores are present on the scales, in the fins and occasionally on the eyes. We demonstrate, using pharmacological and genetic approaches, that moonstone is a dominant allele of leukocyte tyrosine kinase, which carries a missense mutation in a conserved position of the kinase domain. Loss-of-function zebrafish mutants in ltk lack iridophores. Therefore, it has been suggested that ltk is required for iridophore specification. Using gain- and loss-of-function ltk alleles and inhibitor treatments, we show that mutant Ltk is hyperactive and that Ltk does not only specifically regulate iridophore establishment, but also proliferation and survival. Using chimeric analysis and confocal microscopy, we propose that Ltk facilitates the homotypic competition-based control of iridophore proliferation, but does not affect communication with xanthophores and melanophores. The question of the conversation of functions of the genes controlling pigment pattern formation is of prime importance in other model organisms, such as guppy. With the example of two guppy mutants, golden and blue, we demonstrate the conserved functions of two tyrosine kinases – Kita and Csf1ra.