Fish pigmentationfunctional and evolutionary characterization of the agouti locus

Resumo

Pigment pattern formation is a classic problem in developmental biology and has been the subject of extensive experimental investigation and mathematical modelling. Vertebrate pigment patterns frequently show a clear distinction between a pale ventral area and a darker dorsal area. Thanks to the work of Greg Barsh’s lab, we have known for some time that in mammals this pattern results from spatially regulated expression of Agouti-signaling protein. The molecular basis of pigment pattern has also been widely studied in fish, but to date the emphasis has been exclusively on the mechanisms controlling striped and spotted patterns in zebrafish and its sister species. However, fish also show a pronounced dorsal-ventral pigment pattern. In this PhD dissertation, we show that the dorsal-ventral patterning in zebrafish is also Agouti-signaling protein-dependent, and that the striping mechanism is largely independently superimposed upon this evolutionarily-conserved dorsal-ventral patterning mechanism. Our observations are, we believe, the first to show a “loss-of-function” mutation of the signaling molecule (Agouti-signaling protein, Asip1) and its receptor (Mc1r) and to demonstrate that disruption of their expression abolishes the dorsal-ventral pigment pattern difference. Combined with the data from mammals, our data strongly suggests that an Agouti-signaling protein-dependent mechanism regulating dorsal-ventral pigment pattern is a conserved feature of vertebrates, so that other patterning mechanisms, whether generating stripes or spots or other patterns, are likely generated by superimposing secondary patterning mechanisms. Our conclusion is particularly interesting because the cellular basis for the patterns in mammals (and birds) and in fish appear very different, with the former depending on a shift in the biochemistry of melanin produced within melanocytes, whereas the latter seems to affect the ratios of black melanocytes and other pigment cell-types, the yellow xanthophores and the silver iridophore. Our data indicates that at the cellular level there is some conservation of the mechanisms, with Asip1 and Mc1r affecting melanin production (total levels, not melanin type), but also likely affecting the ratio of pigment cell types in different body regions. Additionally, the results of this PhD project unquestionably confirmed the pleiotropic nature of the agouti gene. Thus, this reveals the ability of the melanocortin system to simultaneously regulate pigmentation and other physiological processes in fish skin. Finally, our results also provide evidence that the mechanism leading to the dorsal-ventral pigment pattern pre-dates the origin of teleosts. Lastly, we have to say that this work has been a cross-disciplinary and combined cutting-edge approaches from comparative genomics, cell and developmental biology, reverse genetics, and evolutionary biology. Overall, we feel this thesis not only provide basic knowledge on vertebrate pigmentation development, but also provide information relevant to the design of new strategies for intensive fish culture.