Molecular background of marble pigment pattern formation in trout
Since 2006 we have been working on identification and characterization of genes involved in pigmentation of Salmo trout.
The initial analysis started in a frame of Slovenian national project (J4-93909)
representing a PhD thesis of Urška Sivka. To explore the genetic background of color pattern formation in salmonid fishes
marble trout (Salmo marmoratus) was selected as a model organism due to its particular marble (or labyrinthine) color pattern
(Figure 1 ) and endemism in the Adriatic Sea basin. Marble or labyrinthine pattern is also typical of a population of brown trout (Salmo trutta)
inhabiting the River Otra, Norway (Skaala & Solberg, 1997), and has been observed in some hybrids between species of different salmonid genera (e.g. S. trutta X Salvelinus fontinalis (Miyazawa et al., 2010))
, as well as in some zebrafish mutants (jaguar/obelix (Iwashita et al., 2006) and leopard (Watanabe et al., 2006)) (Figure 1).
Figure 1: Examples of labyrinthine colour pattern in fish. A) Salmo marmoratus from Zadlaščica river, Slovenia; B) Salmo trutta from river Otra, Norway; C) tiger trout
(S. trutta X Salvelinus fontinalis) (from Red's Fishing Report and Blog); D) zebrafish obelix mutant (from Iwashita et al., 2006) and E) zebrafish leopard mutant (from Asai et al., 1999)
For identification and characterization of candidate genes involved in marble colour pattern formation in marble trout we used subtractive cDNA library and cDNA cGRASP 32K microarrays.
Using SSH, we identified two differentially expressed genes, KITA and EIF3EA, which play an important role in survival of melanophores and maintaining the colour pattern.
Microarray analysis of transcripts isolated from the skin of marble and brown trout and F3 hybrids with marble colour pattern, revealed five differentially expressed genes, HDAC1, VPS18,
GNAQ, DCT and SCGII involved in biological process of pigmentation in animals. Out of five, three (HDAC1, GNAQ and DCT) are directly or indirectly involved in Wnt signaling pathway (Sivka et al., 2013).
Given the phenotypic similarity between marble and labyrinthine pattern, which is predicted by reaction-diffusion mathematical model (Miyazawa et al., 2010), and the involvement of differentially expressed genes
in Wnt signaling pathway, marble pattern formation depends on Wnt signaling pathway and is based on reaction-diffusion mechanism.
Since the formation of colour pattern depends on the type and number of pigment cells, we extended our field of research to histology, more specifically to morphology, density and distribution of melanophores in marble trout skin (Figure 2).
Histological analysis revealed a slightly thicker epidermis in marble trout than in brown trout. Melanophores are larger in marble trout than in brown trout. Their average density was more or less constant across all age classes and was three times
lower when compared to brown trout (Sivka et al., 2012).
Figure 2: A) Skin from gill cover of S. marmoratus with light and dark areas. B) Dermis of gill cover, section perpendicular to the surface, border between light and dark areas in S. marmoratus
(from Sivka et al., 2012).
In order to see how the melanophores progress during the marble colour pattern formation, Urška photographed the same region of the gill cover of one individual marble trout aged 2+ once a month in period from February to November, 2009.
Click on the link to see an interesting movie about pattern formation.
After a short standstill the project of characterization of molecular background for marble colour pattern formation continues from December, 2012, when Ida Djurdjevič started with her PhD program.
In the framework of her thesis and hopefully also some additional future projects we intend to apply the technique of laser capture micro-dissection (LCM) in cooperation with Slovenian Forestry Institute,
to gather cell populations of each pigment cell type from skin of marble and brown trout, and to determine their expression profiles with next-generation sequencing (NGS).
We envision that results from these two coupled techniques will create a broader view that will help identify candidate genes involved in the process of pigmentation
and enable us to draw conclusions or hypotheses about cellular processes involved in the formation of color pattern (e.g. intercellular contacts and communication, exo- and endocytosis, etc.).
Furthermore, we intend to analyze the structure of the skin, especially the dermis and the pigment cells, by transmission electron microscopy, in cooperation with
Institute of Cell Biology from the Faculty of Medicine. Through this method we will be able to determine the shape of individual pigment cell types, as well as their ultrastructure,
interactions and location in the dermis.
We are looking forward to continuing collaboration with Pattern formation research group from Osaka University, Japan, that has begun recently.
This group has for years been one of the leading groups in pigment pattern formation research in Danio rerio.
Together we aim to elucidate the molecular and evolutionary mechanisms underlying the amazing diversity of animal color patterns. We will focus on two phylogenetically distant teleost groups
(Salmonidae and Tetraodontidae) that share mysterious and spectacular color pattern variations, among them the labyrinthine one being the most interesting for both groups.
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