Periodic Reporting for period 4 - DanioPattern (Development and Evolution of Colour Patterns in Danio species)
Période du rapport: 2021-05-01 au 2022-10-31
Pigment patterns are prominent features of many animals. They are important for thermoregulation, camouflage or intra- and inter-species communication, examples are deterrence of potential enemies or mate choice. As direct targets of natural and sexual selection they are of great evolutionary significance. The zebrafish (Danio rerio) has a very conspicuous pattern of horizontal dark and light stripes on the body and on the anal and tail fins. As a widely used model organism zebrafish are the ideal starting point to study pigment pattern formation in a vertebrate, as well as the evolution of patterning because closely related Danio species show a great variety of different patterns (see example in Fig.1). In all Danio fish the different patterns are formed by the same three classes of pigment cells, black melanophores, orange xanthophores and silvery iridophores. The stripes of zebrafish are formed by a self-organizing process involving interactions among all three kinds of pigment cells, the horizontal orientation depends on an anatomical pre-pattern, the horizontal myoseptum. How the diverse patterns in other Danio fish are formed is largely unknown. Differences in cell-cell interactions must exist, however, to produce the very different patterns and it is a general question how these cellular interactions are regulated on several different levels. The Danio genus provides an ideal system to study these cell-biological questions in vivo by starting from zebrafish and then use the knowledge gained to examine other species.
We studied pigment pattern formation in zebrafish on the cellular level and found that both, xanthophores and iridophores, come in two different forms, specific for the light and dark stripes, respectively (Fig.2). We found that the patterning process is regulated by galanin signalling in the brain, which in turn regulates thyroid hormone release and thus influences the behaviour of pigment cells (Fig.3). Inter-specific complementation tests using hybrids between zebrafish mutants and other Danio fish showed that one gene, kcnj13, is an important genetic hub with functional differences in three species (Fig.4). To further study the role of kcnj13, which encodes an inwardly rectifying potassium channel, we generated mutants in the corresponding gene in Danio aesculapii, the species most closely related to zebrafish. We could show that kcnj13 is also required for patterning in this species, but the function of the gene must have shifted (Fig.5). Analysing the mutant phenotype in zebrafish in more detail, we found that although the gene function is only required in melanophores the shapes of all three types of pigment cells are altered in the mutants ( Fig.6). This underlines the importance of cell-cell interactions in the patterning process. We could show that the functional differences between zebrafish and Danio aesculapii are based on cis-regulatory evolution and we assume that lower expression in the latter species leads to weakened cellular interactions and contributes to the differences in patterns. We have published our results in four papers, so far, with two more in preparation. In addition, the results were presented at several conferences as talks or in poster form.
For the project we established breeding colonies for several additional Danio species, which were not kept in the laboratory previously. This allowed us to use the CRISPR/Cas system to induce specific mutations in Danio aesculapii and thus test genes for their function in vivo. Using these newly induced mutations we could perform reciprocal hemizygosity tests and clearly demonstrate the functional evolution of one gene, kcnj13 (see Fig.5). So far, this type of test is only very rarely used in vertebrates, it is more commonly applied in yeasts and different Drosophila species. As evolution of kcnj13 happened several times in the Danio genus, we assume that this gene is particularly important for pigment pattern evolution. At the same time, we found that several other genes, which are known to regulate cell-cell interactions in zebrafish, are functionally conserved between a number of different Danio species with divergent pigment patterns. This leads to the conclusion that very different patterns, e.g. horizontal stripes and vertical bars, are produced by remarkably similar genetic and cellular pathways.