The striped pattern of the adult zebrafish arises during a metamorphic period that begins at the age of approximately 3 weeks and lasts for about one month. During this period newly formed pigment cells emerge in the skin and start to cover the fish completely. The cells are distributed in three superimposed monolayers under the skin, bounding the underlying musculature: xanthophores in the top layer, iridophores in a middle layer, and melanophores in the bottom layer. Whereas melanophores are only present in the dark stripes in zebrafish, both xanthophores and iridophores are spread over the entire body, albeit in different shapes and densities in the light and dark stripes.
By imaging fluorescently labelled clones induced by Cre-recombinase under the control of the neural crest specific sox10-promoter in individual fish over several weeks, the dynamics of cell divisions, cell spreading and cell shape changes were monitored during stripe formation in vivo. The pigment cells originate from stem cells located at the segmentally reiterated dorsal root ganglia. These stem cells are multi-potent, giving rise to all three types of pigment cells and to neurons and glia of the peripheral nervous system.
The first light stripe is formed by dense iridophores entering the skin along the horizontal myoseptum, which serves as a morphological pre-pattern. Iridophores divide and spread in the skin, subsequently melanophores appear dorsally and ventrally to form the first two dark stripes. The larval xanthophores contribute directly to the adult pattern by giving rise to adult-specific xanthophores; these begin to proliferate at the onset of metamorphosis. Melanoblasts, the progenitors for melanophores, reach the skin at the presumptive dark stripe regions via the peripheral nerves dorsal and ventral to the first light stripe. In contrast to iridophores and xanthophores, melanophores do not proliferate anymore after they have reached the skin but dramatically expand in size to produce a contiguous sheet of cells forming the dark stripes. Hence, the dark and light stripes are formed by completely different cellular behaviours.
Pigment cells, while populating the skin, constantly communicate with their neighbours in order to regulate their behaviours. Each cell type is dependent on a different signalling system and the analysis of clones induced in mutants lacking iridophores (shady) or xanthophores (pfeffer) indicates that the lack of one cell type does not impair the spreading of the other. The proliferation and spreading is controlled by contact-dependent homotypic competition. On their own, pigment cells of each type can uniformly cover the skin. The stable stripe pattern emerges due to restrictions imposed by heterotypic interactions within and between the three pigment cell types. Our analysis reveals that all three chromatophore types have to communicate and interact to form the final striped pattern. Direct cell-cell contacts involving potassium channels and gap junctions are thought to be the primary mode of communication among different pigment cell types. Genetic screens have identified several components of this communication system. These components directly participate in channel/junction formation, such as Obelix/Kcnj13, leopard/Connexin 41.8 and luchs/Connexin 39.4, or modulate their function, such as spermidine. Although several genes encoding integral membrane proteins have been identified that are involved in zebrafish colour pattern formation, the actual underlying biochemical and molecular events remain highly conjectural.
The arrangements of pigment cells vary in different parts of the body: All three pigment cell types are also present on the dorsum, the dorsal, pectoral and pelvic fins, and on the exposed rim of scales; however in these regions they are intermixed, and formation of stripes is restricted to the flank region, the anal- and tailfins. Stripe formation in the fins of zebrafish does not involve iridophores but requires only melanophores and xanthophores, demonstrating that the mechanisms of pigment cell interactions differ in body and fins. Only the anal and tail fins are striped, whereas the dorsal, pectoral and pelvic fins display a salt and pepper mixture of all three cell types. Other Danio species often display distinct colour motifs in the fins, which may differ from that of the body.
Genetic evidence has pointed to a role of non-pigment cells in shaping the pattern but the responsible tissues have not been identified. In addition to the observational analysis we carry out RNAseq experiments to compare the transcriptomes of striped with non-striped regions. We are creating loss-of-function alleles for identified the candidate genes using the CRISPR/Cas9 system with the aim to find the environmental factors that cause the region-specific rules of communication for the pigment cells.