„[…] from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved.“
- this is how Darwin ends in 1859 his great work “On the origin of Species” where he describes how variation and selection can change species and lead to the formation of new ones (1). Pigmentation patterns are very important factors for the evolution of species and the generation of biodiversity, they might be used for camouflage, as warning signals or for intra-species recognition.
Despite their conspicuousness and their general significance for evolution, the question how pigment patterns develop in animals and which genes are responsible for the differences that exist between species is still largely unanswered. Some studies describe evolved genes responsible for the patterns in insects, but very little is known in vertebrates, which are much more difficult to study genetically.
Application of the Nobel Prize for Chemistry awarded CRISPR/Cas9 system
The Tübingen researchers used the relatively new CRISPR/Cas system for genome editing, for which the Nobel Prize for Chemistry was awarded this year, to generate mutations in species that are not usually found in laboratories (3, 4). With elegant genetic tests they succeeded in detecting an evolved gene, which is partly responsible for the differences in colour patterns in closely related species of fish.
During the last 10 million years tropical freshwater fish of the genus Danio, which includes the striped zebrafish (Danio rerio), have split into a number of different species in South-East Asia; these are easily distinguished by their attractive colour patterns (Fig. 1). Despite their close relationship, they develop very diverse patterns such as horizontal stripes, vertical bars or dots in various colours and with different contrasts. The zebrafish has become something like a star for research on vertebrates, to which the genetic studies that have been performed in Christiane Nüsslein-Volhard's laboratory at the Max Planck Institute for Developmental Biology in Tübingen during the last 30 years contributed significantly (5).
Identifying central genetic information from thirty thousand genes
Zebrafish can be kept and bred quite easily in the laboratory. Nowadays, they are used worldwide in many areas of basic biomedical research to better understand human diseases in animal models and to test and develop potential therapies. However, the most prominent feature of the fish is their colour pattern, it consists of precise, horizontal blue and silver stripes along the body and in the anal and caudal fins. Fascinated by the sheer beauty of colour patterns, the Tübingen group is actively investigating their origin and evolution (6, 7).
The stripes in zebrafish are made by the differential distribution of three types of pigment cells in the skin, black melanophores, yellow-orange xanthophores and silvery-blue iridophores. Specific interactions between these cells control their distribution in the skin; a phenomenon known as self-organization. But which of the approximately thirty thousand genes in the fish are involved?
Evidence of central genes and circuit mechanisms for pattern formation
In several complex mutagenesis screens, in which mutations in the genome of the zebrafish were randomly generated, fish with different colour patterns were identified, for example the mutants Obelix - with wider stripes - or Leopard, with spots instead of stripes (8) (Fig. 2).
DNA analysis of these genes revealed that the proteins they code for are involved in cell-cell contacts that control the interactions between the different types of pigment cells in the skin. These genes are essential for the generation of stripes in zebrafish, but are they also required for the different patterns in the other species? Are changes in them maybe even the basis for the different patterns? By simply comparing the DNA sequences of the genes from the different species this question cannot be answered; a myriad of small differences has accumulated over the course of millions of years, most of which are neutral. It can only be shown experimentally which of the genetic differences are actually responsible for the different patterns. An ideal test would be to exchange the gene variants between related species, but so far this is beyond the possibilities of genetic engineering.
Switching central gene functions on and off explains mechanisms of pattern formation
In the ERC (European Research Council) funded project ‘DanioPattern’, the researchers first compared the zebrafish, Danio rerio, with its sister species, Danio aesculapii. Surprisingly, D. aesculapii shows a completely different pattern, namely vertical dark bars (Fig. 1), despite its very close relationship to zebrafish. With the CRISPR/Cas9 system, loss-of-function mutations could now be generated in this fish species, which hasn’t been used in the laboratory before.
It was found that mutations in genes such as Obelix and Leopard, which are essential for stripe formation in D. rerio, also lead to changes in the colour pattern in D. aesculapii, the vertical bars disappear completely (Fig. 2). This means that these genes play important roles in both species, but leading to very different patterns. "We were surprised that the very different patterns evidently need the same genes, but are their activities identical or have they changed during the course of evolution?", asks Prof. Nüsslein-Volhard.
To find evolved genes, the researchers used a critical genetic test, which is based on the fact that the different species of fish studied are so closely related that viable hybrids can be obtained (9). It is also possible to get hybrids between mutants and wild type fish, since (as with almost all genes of diploid organisms) one functional copy of a gene is sufficient to fulfill its role (Fig. 3a,d). This makes it possible to discover differences in gene function: Reciprocal hybrids, in which the gene derived from one of the parental species is not working, only differ in this single gene, everything else is identical. If the phenotypes of these reciprocal hybrids differ, this means that the corresponding gene has evolved; if no differences are found, the function has remained the same. With such hybrids, the researchers were able to identify changes in the Obelix gene: if the functional copy of Obelix comes from D. rerio, then the hybrids show unchanged stripes (Fig. 3b), while those with a working copy only from D. aesculapii form large spots (Fig. 3c). This means that the function of the Obelix gene has changed during evolution of the two species and it contributes to the development of the different patterns. Leopard and two other genes showed no changes in this test.
Evolution of a control gene for numerous cellular processes identified
“Obelix encodes a membrane protein (Kcnj13), which controls interactions between pigment cells. Kcnj13 belongs to the large class of potassium channels that play roles in a variety of cellular contexts; For example, they are important for the function of neurons and for muscle contraction, or for the release of insulin in the pancreas. In humans, a number of diseases such as diabetes or cardiac arrhythmias are associated with defective potassium channels”, reports the project leader Uwe Irion. “In addition to the two sister species we also tested several other Danios, and such hybrids suggest that Obelix plays a role in the creation of different patterns in at least two more species”, adds PhD student Marco Podobnik. "This would make Obelix a hub in the evolution of colour patterns". The researchers now want to further investigate what causes the differences between the gene functions, in particular whether the structure of the potassium channel or the control of gene activity is changed. The study shows that genome editing based on the CRISPR/Cas9 system allows mutations to be generated in animals that were previously inaccessible to genetic analysis. Until now, the “reciprocal hemizygosity test” could be carried out almost exclusively with Drosophila and yeast, two comparatively simple genetic model organisms. Together this means an essential expansion of research methods that will help to decipher the basis for the establishment and evolution of biodiversity, which was previously beyond the possibilities of biological research.
Podobnik, M., Frohnhöfer, H. G., Dooley, C. M., Eskova, A., Nüsslein-Volhard, C., and Irion, U. (2020): Evolution of the potassium channel gene Kcnj13 underlies colour pattern diversification in Danio fish. Nature Communications, DOI: 10.1038/s41467-020-20021-6, https://www.nature.com/articles/s41467-020-20021-6
Dr. Uwe Irion
Max Planck Institute for Developmental Biology
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