How do Zebrafish get their stripes?

Q1. Why study pigment cells in Zebrafish??

www.spiritualreformation.com, http://focusonnature.be/gallery/zebrafish

Ans : to solve mystery of our skin color! [2]

While some researchers were studying zebrafish to find cancer genes, they found that pigment cells in a peculiar golden variety of zebrafish looked similar to pigment cells in light-skinned humans. Since then, many researchers have investigated the zebrafish gene responsible for different colors in stripes. In 2005, some studies found the human version of the gene, which affected Europeans differently from Africans and Asians. This gene, SLC24A5, is shared by both humans and zebrafish and makes melanosomes less abundant, less concentrated and smaller in lighter-skinned humans or light-striped zebrafish. Further study in Zebrafish pigment cells is essential, since they provide many opportunities to learn the nature of the human skin color. [2]

Q2. Where do these pigment cells come from?

Pigment cells arise from neural crest cells, which originate at the edge of the neural tube. Neural crest cells migration requires disruption of the basal lamina surrounding the neural tube and loss of adhesion molecules on neural crest cells. Several studies further investigated that some other adult pigment cells are not directly derived from neural crest cells, but from post-embryonic, neural crest-derived stem cells. [1] [4]

Zebrafish display alternating stripes of melanocytes, iridophores and xanthophores. [1]

  1. melanocytes(black)
  2. iridophores(silver)
  3. xanthophores(yellow)

The following video shows how the melanocytes accumulate in embryonic zebrafish.

melanocytes in an embryonic zebrafish

These cells form very differnet patterns during different life cycle phases.

  • Embryonic and Early Larva Stage

http://www.nc3rs.org.uk/downloaddoc.asp?id=527

At this stage, the neural crest cells differentiated into all three cells; the early larvae have stripes of melanocytes and iridophores in a background of xanthophores. [1]


  • Adult

http://zf-health.org/images/zf_adult.jpg

The adult zebrafish have dark stripes of melanocytes and xanthophore stripes in between. [1]

Q3. Which mutants affect pigment patterns in Zebrafish?

There are mutants that affect pigment patterns at both life cycle phases in zebrafish. However, other mutations identify genes that are crucial for the pigment pattern in early larvae or adults, but not both. Therefore, it is important to distinguish mutants at different life cycle phases. [1] [4]

  • Embryonic and Early Larva Stage

major mutants : colourless, nacre, sparse, rose, fms [3] [4]

  • Adult

major mutants : asterix, obelix, leopard, puma, hagoromo [3] [4]

Pigment pattern formation in adult zebrafish was rarely studied in the past, compared to that in embryonic zebrafish. Hence, more recent studies focus on adult zebrafish pigment patterns and find answers to questions like “Which genes are involved in their pigment formation?” and “Do these genes work together?”. For example, one study by Maderspacher and Nusslein-Volhard (2003) found that adult pigment pattern formation in zebrafish requires leopard and obelix dependent cell interactions. [3]

Q4. How do cell interactions affect the pigment formation?

The following is summary of the paper Formation of the adult pigment pattern in zebrafish requires leopard and obelix dependent cell interactions by Maderspacher and Nusslein-Volhard (2003). [3]

  • Introduction

As mentioned already, zebrafish has a pigment pattern consisting of alternating stripes. However, the paper suggests that another class of chromatophores, silvery iridiophores, is irrelevant for stripe formation, because tissues without iridophores can form a stripe pattern. [3]

At the beginning of the adult zebrafish development, the stripe pattern is established through alignment of newly differentiating pigment cells. There are two possible ways in which the cells are organized into these domains. First, the cells could be organized by filling in a prepattern that is set up independent of the pigment cells. Second, pigment cells could interact each other and thus define the striped domains. [3]

In this paper, the researchers used mutants that prevent formation of either melanophores or xanthophores to show that the presence and juxtaposition of both melanophores and xanthophores are necessary and sufficient for stripe formation. Thus, the pattern appears to be largely defined by short-range interactions among pigment cells. [3]

Based on the classification of homo- and heterotypic interactions, the researchers  analyzed the phenotype of two mutants, leopard(leo) and obelix(obe). These mutants alter the adult pigment pattern in a qualitative way. leo and obe play a major role in different subsets of the cell behaviors and form central components of the stripe-forming system. [3]

  • Method

In this experiment, the researchers intercrossed homozygous adult zebrafish carriers to generate double mutants between adult specific phenotypes, such as leo and obe, and mutants with an early larval phenotype such as nac and fms. Then, cell-transplantation was performed and tq 270 and td 15 were used for leo and obe. Images of the fish were captured consistently throughout the experiment. [3]

  • Results

http://conlonlab.org/courses/materials/523mats/Maderspacher.pdf

http://conlonlab.org/courses/materials/523mats/Maderspacher.pdf

They first studied the distribution of melanophores, when xanthophores were absent. They used homozygous mutants for kit- related RTK fms for this study because they are responsible for a great reduction of xanthophores during larval stages and for devoid of this cell type in the adult. They found that at around  25 days of age, melanophores started to aggregate. Later on, melanophores failed to be cleared from the corresponding region of the first xanthophores stripe in wild type. [3]

Then, they wanted to figure out the pattern formed by xanthophores, when melanophores were absent. Here, they used mutants for nacre. nac mutants are homozygous viable and devoid of melanophores in larval and adult stages. Most xanthophores were GFP positive (i.e donor derived), while most of the melanophores were normally pigmented( i.e host derived). [3]

This shows that even though melanophores lack fms function, they still can form stripes.   Furthermore, the melanophore pattern in fms mutants is due to the lack of xanthophores. In conclusion, these findings show that juxtaposition of melanophoes and xanthophores is necessary and sufficient for stripe formation. [3]

http://conlonlab.org/courses/materials/523mats/Maderspacher.pdf


The researchers investigated two mutants, leopard (leo) and obelix(obe) that change the adult pigment pattern to find out what controls interactions between the two pigment cell types. From this figure, we can see that heterozygous adults have only two to three melanophore stripes that are wider than in wild type due to dosage dependent effect of obe. In obe homozygous adults, melanophores are located in two broad stripe domains flanking the horizontal myoseptum. From the results, researchers found that obe and leo mutant patterns are not caused by immobility of melanophores and that obe and leo affect stripe integrity and shape respectively. [3]

When double homozygotes for fms and obe are expressed, melanophore clusters are lost and all melanophores rather look evenly scattered.  fms;leo double homozygotes also showed the same effect of loss of melanophore clustering and are not different from fms;obe double mutants. The data indicate that both leo and obe have a similar impact on melanophore behavior, while only leo also influences xanthophores behavior. [3]

In conclusion,  obelix promotes aggregation of melanophores and controls boundary integrity. In contrast, leopard controls boundary shape by regulating homotypic interaction within both melanophores and xanthophores and interaction between the two. The findings from the paper proves that cell-cell interactions among pigment cells are the major driving force for adult pigment pattern formation. One weakness of the paper is that the method section is not very detailed. It is hard to understand how they transplanted cells, as they did not mention much about it. Further study on the relationship between different genes in the pigment formation of zebrafish might help us understand the complex relationships between genes responsible for different human skin colors. [2] [3]

Bibliography

[1] Hanes, Tasha. “Establishment of Spatial Pattern.” Rev. of Pigment Pattern Development in the Zebrafish. 20 Mar. 2002. <tashahanes.ppt>.

[2] Kane, Daniel B. “Fish Help Unlock Mystery of Our Skin Color – Technology & Science – Science – Mysteries of the Universe – Msnbc.com.” Breaking News, Weather, Business, Health, Entertainment, Sports, Politics, Travel, Science, Technology, Local, US & World News- Msnbc.com. 15 Dec. 2005. Web. 03 Apr. 2011. <http://www.msnbc.msn.com/id/10480835/ns/technology_and_science-science>.

[3] Maderspacher, Florian, and Christine Nusslein-Volhard. “Formation of the Adult Pigment Pattern in Zebrafish Requires Leopard and Obelix Dependent Cell Interactions.” Development 130 (2003): 3447-457 <http://conlonlab.org/courses/materials/523mats/Maderspacher.pdf>.

[4] Parichy, David M., Eve M. Mellgren, John F. Rawls, and Et. Al. “Mutational Analysis of Endothelin Receptor B1 (rose) during Neural Crest and Pigment Pattern Development in the Zebrafish Danio Rerio.” <em>Protist.biology. Washington. Edu</em>. Developmental Biolgoy, 2000. Web. 1 Apr. 2011. &lt; <“http://protist.biology.washington.edu/dparichy/DevBiol2000.pdf“>

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