Embryonic patterning of pigmentation in Danio rerio is a highly investigated area of study because of the many implications it has on the disorders and coloring of upper level metazoans. In fact, melanin pigmentation abnormalities have been associated with inflammation and cancer as well as visual, endocrine, auditory, and platelet defects . Attempts to discover genes of cellular proliferation and tumor suppression have led to the discovery of a zebrafish pigmentation gene and its human homolog. 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 .
The number, size, and density of melanosomes, or the pigmented organelles of melanocytes, have been associated with variations in human pigmentation. The greater the abundance of melanosomes, the darker and richer the skin pigmentation. In humans, the protein product of SLC24A5, solute carrier family 24 member 5, is also known as sodium/potasium/calcium exchanger 5 (NCKX5) . Single nucleotide polymorphisms from alanine to threonine in this sequence have been associated with differences in skin pigmentation . Because the change occurs at the 111th amino acid, it was abbreviated “A111T”. The paper discussed below, “SLC24A5, a Putative Cation Exchanger, Affects Pigmentation in Zebrafish and Humans” links mutations in the SLC24A5 gene to golden mutants– lightly pigmented zebrafish and human European populations– while associating the conserved ancestral allele with African and East Asian populations.
The Zebrafish golden Phenotype
A number of techniques were utilized in analysis of the golden mutant phenotype. Transmission electron microscopy (TEM) was previously used to determine the timing and cellular basis of characteristic hypopigmentation. Hypopigmentation is the loss of skin color caused by melanocyte, melanin, or tyrosine depletion. The amino acid tyrosine is used by melanocytes in melanin synthesis, so its absence results in the reduction of pigmentation. TEM was used to detect pigmentation in both skin melanophores and retinal pigment epithelium (RPE) in wild-type and mutant cells in the hours following fertilization.
The Zebrafish golden Genotype
To determine the gene(s) responsible for the golden phenotype, positional cloning, shotgun sequencing, contig assembly, morpholino knockdown, DNA and RNA rescue, and expression analyses were used. The specific mutation in the golb1 allele was determined through comparison of complementary cDNA libraries and genomic sequences from wild-type and golb1 embryos. A linkage analysis of the 1,126 homozygous golden mutant (golb1) embryos was used to estimate recombination frequency. Following γ-radiation-induced deletion, Polymerase Chain Reaction (PCR) was performed for functional analysis of the mutant allele. The zebrafish genomic library had been screened by previous studies, allowing for microinjection of a clone (PAC215f11) containing key markers. Cheng’s lab predicted partial rescue of the wild-type pigmentation following injection of the PAC215f11 clone.
Results and Conclusion
The Zebrafish golden Phenotype
golb1, the recessive golden mutation, was shown to cause hypopigmentation of skin melanophores (Figure 1). The golb1 mutation was the first of its kind studied in D. rerio. Using TEM during the hours postfertilization (hpf), the golden phenotype was characterized. The authors noted that delayed reduced development of melanin pigmentation occurred in comparison to the wild-type. Melanin pigmentation in melanophores and RPE is apparent in wild-type embryos after 48 hpf (Fig. 2A) but is not visible in the golden mutant (Fig. 2B). It is not until 72 hpf that the mutated melanophores begin to develop pigmentation (Fig. 2F and 2G). Even then, the color is sparse and light in comparison to the wild-type (Fig. 2D and 2E). Even the melanophore-rich stripes of the adult zebrafish are much darker in the wild-type than in the lighter golden mutant (Fig. 1A-B). Figure 1A and 1B also show that areas of low melanophore density reveal melanin-poor cells in golden adults when compared to the wild-type.
The Zebrafish golden Genotype
As stated, analytical tools included positional cloning, shotgun sequencing, contig assembly, morpholino knockdown, DNA and RNA rescue, and linkage analysis. Linkage analysis of golb1 embryos showed that a crossover event occurs between golden and microsatellite marker z13836 on chromosome 18. This corresponds to approximately 33 kb in distance between the two. PCR of the golb1 deletion allele showed loss of the z13836 marker and others nearby. When a clone containing these microsatellite markers was inserted into golden embryos, mosaic rescue of wild-type pigmentation in melanophores and REP was achieved (Figure 2H, 2I).
This affirmation of their hypothesis led the Cheng lab to believe a specific golden gene resided in the PAC215f11 clone.
Rescue experiments that called for the injection of slc24a5 transcript into mutant embryos showed partial rescue of wild-type pigmentation (Figure 2J-K). Reversing the experiment, Fig. 2C shows that injection of morpholino targeted to the start site of slc24a5 in a wild-type embryo reproduces the phenotype of the golb1 mutation at 48 hpf in the larval stage. These results solidify the link between the golden phenotype and slc24a5 genotype.
Pigmentation and Tissue-Specific Expression of Slc24a5
The Cheng group switched model organisms to do a comparison of slc24a5 expression in cancerous and noncancerous murine cell lines using quantitative RT-PCR. Specifically, they compared normal mouse tissues to those of the B16 melanoma cell line. Analysis revealed highest relative expression in melanoma, dermal and eye tissues (Figure 3E. Concentrations in skin and eye were 10-fold higher than other tissues. Slc24a5 expression was 100-fold greater in mouse melanoma than normal skin and eye tissues. The researchers were able to conclude that in melanin-producing cells –particularly in the skin– expression of mammalian Slc24a5 and zebrafish golden is greatest.
Having linked mammalian Slc24a5 and zebrafish golden expression with melanin-producing cells, the next step was to determine the role of the SLC24A5 protein in pigmentation. The structure of the protein, with its alternating hydrophobic and hydrophilic segments, suggested membrane localization to previous researchers. Lamason et al. used green fluorescent protein (GFP) and hemagglutinin (HA) to tag the slc24a5, highlight its location and function, and determine if previous researchers were correct. Figure 4A-B show intracellular localization of the tagged gene products in a role separate from the plasma membrane control Fig. 4C. The golden phenotype is unaffected by the addition of the HA tag (Fig. 4D).
The scientists then synthesized established observations about slc24a5 to create a model of its function within the cell. The first role of the SLC24A5 protein is in organellar calcium concentrations. The second observation was that a pH gradient drives calcium uptake in melanosomes. Finally, the positions of multiple V-ATPases and H+/Na+ exchangers are also localized to melanosomes. The summation of these observations was used to devise a model for calcium accumulation in melanosomes (Figure 4E). The genotype, phenotype, and function of SLC24A5 and golden were now known.
Discussion of the Article
The Pennsylvania State University collaboration did well to highlight the significance of model organism study in aiding our understanding of human developoment and pathology. The applicability of zebrafish pigmentation to human pigmentation is made readily apparent several times throughout the article. Unfortunately, the paper launches right into the results of each experiment with very little in terms of methodology. I looked up the manuscript formatting at Science, and was surprised to find that this is the norm. Research articles “are expected…to present a major advance” but Materials and Methods are typically relegated to “supplementary materials”. In essence, the “information needed to support the paper’s conclusions” are often not published with the paper. This is understandable, since the goal of Science is not to publish reproducible experiments, but instead to educate various fields on significant advances. Aside from this explained omission, no other weaknesses or shortcomings were seen in the paper.
 Lamason, RL, MPK Mohideen, JR Mest, KC Cheng, et al. “SLC24A5, a Putative Cation Exchanger, Affects Pigmentation in Zebrafish and Humans.” Science. 310. (2005): 1782-86. Web. 9 Apr. 2012. <http://biolog-e.ls.biu.ac.il/faculty/wides/80-440/skinpigmentation1782.pdf>.
 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. <http://www.msnbc.msn.com/id/10480835/ns/technology_and_science-science>.
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