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Development and Evolution of Colour Patterns in Danio species

Periodic Reporting for period 2 - DanioPattern (Development and Evolution of Colour Patterns in Danio species)

Reporting period: 2018-05-01 to 2019-10-31

The project addresses the problem of colour pattern formation in zebrafish and closely related Danio species. The first aim is to understand the cellular, molecular and genetic basis for the observed behaviours of chromatophores during the development of a model organism. The second aim is then to understand the evolution of pattern formation by expanding to other Danio species. These are closely related to zebrafish, they occupy similar habitats and they develop in a very similar fashion, however, their pigmentation patterns are very different from the stripes seen in zebrafish.
The overall objectives are to understand the genetic regulation of chromatophore interactions during stripe morphogenesis in zebrafish and to use this knowledge as a base to decipher the molecular genetics leading to the evolution of diverse patterns in Danio species. In recent years it has become clear that the process of pigment pattern formation is different in various regions of the body of the fish. We are therefore currently comparing the behaviours of pigment cells in the trunk with their behaviours in the striped fins (anal and caudal) to find out what are the commonalities and where are the differences. In combination with the molecular and computational analysis of the genome sequences and gene expression patterns of the different species we address fundamental questions not only in the field of patterning, but also for biodiversity and evolution.
To obtain a more comprehensive picture of the mechanisms responsible for pigment pattern formation in zebrafish we started the analysis of stripe formation in the anal and tail fins. Whereas in the body iridophores appear to lead in the patterning process, careful microscopic analysis and follow-up studies show that in the anal fin xanthophores seem to fulfil this role; they expand from the body into the fins along the fin rays and condense to delineate the light stripe areas (Figure 1), the dark stripes are subsequently populated by melanophores, which are derived from approx. four stem cells.
In addition to the observational analysis we carried out RNAseq experiments to compare the transcriptomes of striped (anal and caudal) with non-striped (dorsal) fins. We identified 44 transcripts that show significant differences in their expression levels and are therefore good candidates for regulators of patterning (Figure 2). We are now in the process of creating loss-of-function alleles for all of them using the CRISPR/Cas9 system.
To better understand the role of the tissue environment in the patterning process we started to analyse the mutant npm, where pattern formation is disrupted (Figure 3), however all the pigment cells are capable of producing wild type stripes if placed in the correct environment by transplantations. We identified a mutation in the gene coding for Galanin receptor 1A (galr1A) in npm mutants and are currently working to confirm causality and to elucidate the mechanism by which galr1A influences stripe formation in zebrafish.
For the study of other Danio species we have analysed exome-sequencing data from Danio aesculapii, the closest known relative to zebrafish, Danio albolineatus and Danio choprae (Figure 4). Overall we find a very high degree of similarity in the sequences from the different species. To test the role of genes known to be involved in patterning in zebrafish we have started to create loss-of-function alleles of several genes in Danio aesculapii and Danio albolineatus and we are now in the process of establishing the first mutant stocks. In addition we carried out transplantations of wild type cells from other species into zebrafish, so far we could show that xanthophores from several non-striped species can contribute to stripe formation when put into a zebrafish environment (Figure 5). This underscores the high degree of similarity in the patterning process despite the very different outcomes.
Colour patterns are prominent features of most animals, as targets of natural as well as sexual selection they are rapidly evolving. We use the zebrafish as a model organism and paradigm for the development and evolution of adult patterns.
The comparative gene expression analysis of striped vs non-striped fins by RNAseq has given us a first list of candidates for factors that regulate pigment cell behaviour in different regions of the body of zebrafish. The thorough analysis of these candidates and the extension of the approach to other regions of the fish, which also show differences in pattering, e.g. the flank vs. dorsum, will allow us to gain a more complete picture of the different patterning mechanisms that are active in zebrafish. As the same types of pigment cells are present in many other species of fish and amphibians we expect that the principles learned from zebrafish will be widespread and can therefore be the basis for the understanding of pigment pattering in many animals, thus allowing us to better understand the evolutionary processes, which finally lead to the biodiversity we observe in the world.
As a very first step into this direction we have started the analysis of patterning mechanisms in other Danio species. With the help of blastomere transplantations between different species we investigate the commonalities and differences that lead to the vastly different patterns. A key point will be the establishment of lines in other species that harbour mutations in genes known from zebrafish to influence the behaviours of pigment cells during pattering. These lines can then be used to generate inter-species hybrids for reciprocal heterozygosity tests as a means to assess their roles in evolution. Combined with genome and transcriptome data this will lay the foundation to understand the genetic basis of colour pattern formation in different Danio species and the evolution of colour patterns in vertebrates.
Diagram showing the number of differentially expressed genes between striped and non-striped fins
A clone of labeled chromatophores during stripe formation in the anal fin
Danio species analyzed in the project
Transplantations showing the contribution of xanthophores from different species to stripe formation
The phenotype of npm mutants