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Biological Models to Assess Copper Homeostasis in Response to Deficit and Excess |
1 From the Center for Genomics of the Cell, Facultad de Ciencias. Universidad de Chile, Santiago, Chile
2 Presented at the symposium "Molecular Biomarkers of Copper Homeostasis," held in Viña del Mar, Chile, September 26–29, 2007.
3 Supported by the Millennium Science Initiative (ICM P06-039-F) and the International Copper Association (MLA).
4 Reprints not available. Address correspondence to ML Allende, Departamento de Biologia, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile. E-mail: allende{at}uchile.cl.
ABSTRACT
Unicellular eukaryotes and cultured cells from several animal species were invaluable in discovering the mechanisms that govern incorporation, handling, and excretion of copper at the cellular level. However, understanding the systemic regulation of copper availability and distribution among the different tissues of an intact multicellular organism has proven to be more challenging. This analysis is made even more difficult if the genetic variability among organisms is taken into account. The zebrafish has long been considered a powerful animal model because of its tractable genetics and embryology, but it has more recently become a player in environmental studies, pharmaceutical screening, and physiologic analysis. In particular, the use of the larvae, small enough to fit into a microtiter well, but developed enough to have full organ functionality, represents a convenient alternative for studies that aim to establish effects of environmental agents on the intact, living organism. Studies by our group and others have characterized absorption and tissue distribution of copper and have described the acute effects of the metal on larvae in terms of survival, organ stress, and functionality of sensory organs. A large body of work has shown that there is strong conservation in mechanisms and genes between fish and mammals, opening the possibility for genetic or small molecule screens or for generating fish models of human diseases related to copper metabolism. The variability within humans in terms of tolerance to copper excess or deficiency requires a genetic approach to be taken to understand the behavior of populations, because markers and vulnerabilities need to be identified. The zebrafish could represent a unique tool to move in this direction.
INTRODUCTION
The opposing effects of copper in the organism, essential and toxic, pose an unparalleled challenge to cells such that internalization, homeostasis, and excretion have to be finely balanced. Perturbations of this balance, through diet or disease, can lead to a number of disorders. To date, a handful of experimental systems have been used to describe the basic mechanisms of copper handling in the eukaryotic cell, most notably yeast cells and mammalian cells in culture. However, we still lack suitable whole-animal models that can be used to define mechanisms, risk factors, and genetic predisposition to copper-related metabolic disease in humans. Solving some of the uncertainty may be reached by using the fruit fly Drosophila melanogaster or the nematode Caenorhabditis elegans, 2 powerful invertebrate systems amenable to genetic screens that have provided valuable information related to copper homeostasis (1, 2). However, there are likely to be vertebrate-specific mechanisms that will escape analysis in these systems. The zebrafish (Danio rerio) is a small freshwater teleost favored by developmental biologists for the optical transparency of its embryos and rapid development. A sequenced genome and large-scale mutagenesis screens have contributed to establish the zebrafish as a bona fide genetic system and have provided numerous examples of the structural and functional conservation of genes across all vertebrates. In consequence, this organism is also becoming valued for studies in basic physiology because, despite its small size, analysis at the level of the whole organ, tissue, or the intact organism is possible (3). The real power of the fish though may be that during the larval stages, 2–5 d after fertilization, zebrafish are small and can thus be easily manipulated in large numbers, while possessing fully functional organs, not unlike those present in an adult animal. The nervous system, heart, vasculature, muscles, digestive tract, liver, pancreas, and immune system, among others, are differentiated and support the animals' physiology at these stages. Importantly, the larvae do not require food until after the fifth day of life; therefore they can be analyzed with complete disregard to diet (other than the nutrients provided by the yolk sac). Moreover, habitation in an aqueous environment allows the addition of compounds directly to the water or growth medium, facilitating dosage of test substances and timing of delivery. These features have now made the zebrafish of value to the pharmaceutical industry, which sees the opportunity to carry out large-scale drug discovery screens at a fraction of the cost and time compared with other vertebrate models (4).
Here, we review current progress on our understanding of copper metabolism in the zebrafish and on the identification of the relevant genes. We explore some of the strategies being used to generate new knowledge that can be of use to clinicians and nutritionists in terms of markers and potential targets.
COPPER HANDLING IN THE ZEBRAFISH EMBRYO AND LARVA
Like most freshwater species, adult zebrafish satisfy their dietary need for copper mainly from food sources. In the ovary, maturation of the oocytes is accompanied by accumulation of copper in the yolk, reaching amounts of 3.5 ng by the late vitellogenic stages (5). After fertilization, copper concentrations drop, suggesting that there is excretion and possibly metal exchange with the environment. Elevated copper concentrations in the water can induce accumulation in larvae, indicating that the basic transport mechanisms are in place by these stages (Hernández et al, unpublished observations, 2008).
Zebrafish embryos and larvae can tolerate dissolved copper in the water in the low micromolar range, but lethality increases rapidly with increasing concentrations. Copper can affect hatching at early stages, especially if treatments begin before chorion hardening, which may protect the embryos somewhat (6). Exposure of larvae to a concentration of 250 µmol/L kills all of the fish within a few hours (Hernández et al, unpublished observations, 2008). Although low micromolar concentrations of waterborne copper do not kill zebrafish larvae, deleterious effects were observed in sensory organs, such as the lateral line system (7, 8), and it is likely to affect the olfactory system as it does in other freshwater fish (9). Cells in these sensory systems are directly exposed to the environment; therefore, they escape the protection offered by the integument. The mechanisms of copper ingression into sensory cells are unknown, although there is ubiquitous expression of the high-affinity copper transporter 1 (Ctr-1) beginning early in embryogenesis (10).
When the metal is in excess, cellular stress becomes rapidly evident in the gills and later in the liver, intestine, and nervous system, indicative of a mechanism that restricts distribution of the metal among the different tissues. This was studied with transgenic zebrafish that express green fluorescent protein under the control of a heat-shock promoter, a useful indicator of the stress response (Hernández et al, unpublished observations, 2008; 11). In embryos, a strong response of metallothionein genes is detected, evidence that embryonic cells are capable of inducing expression of copper-handling proteins (12). In adults, a consequence of acute copper toxicity is oxidative stress in the liver and gill and elevated expression of the copper chaperone COX-17 and catalase, although these effects strongly depend on water chemistry (13).
As in other organisms, copper deficiency in zebrafish is also detrimental to embryonic growth and survival. Assays in zebrafish with drugs that chelate copper show that the notochord is extremely sensitive to this deficiency, a phenotype shown to be due to the inactivation of the cuproenzyme lysyl oxidase (14). The essentiality of this micronutrient was also shown genetically, as a mutation in the atp7a transporter, the Menkes disease gene, produces a similar phenotype (15). In addition, in Ctr-1 knockdown experiments (in which translation of Ctr-1 was abrogated by antisense morpholinos), the larvae die at around day 4 after fertilization (10). Therefore, as in mammals, copper absorption and transport into cells are essential for normal development in the zebrafish.
In embryos and larvae, the copper stores present in the yolk are depleted within a few days, and they most likely begin to acquire the metal through food and water sources. Under conditions of low dietary copper, fish absorb copper directly from the water by the gills. Exposure of zebrafish to ion-depleted water (soft water) elicits changes in expression of several key transporters and enzymes related to copper metabolism. Epithelial calcium channel, Na+K+ adenosine triphosphatase, Na+/H+ exchanger, carbonic anhydrase and Ctr-1 show increases in mRNA amounts after a few days of acclimation in soft water (16). Thus, the zebrafish shows an adaptive response at the genomic level to situations of copper deficit, as is the case for excess.
CONSERVATION OF GENES INVOLVED IN COPPER TRANSPORT AND METABOLISM
Many genes previously known from other organisms to be critical for copper homeostasis and transport were identified in the zebrafish, either by in silico examination of the genome or expressed sequence tag databases or by deliberate cloning of homologs. The 20 zebrafish genes described thus far in the literature that are associated with copper regulation are listed in Table 1
. To date, only a handful of these genes has been characterized by mutation, but the zebrafish offers the possibility of analyzing embryonic genes by inhibiting translation with antisense morpholinos, as was the case for the Ctr-1 high-affinity copper transporter (10) or lysyl oxidase (13). Transporters, chaperones, and other regulatory proteins (such as the metal-response transcription factor-1 and metallothionein genes) were studied in embryos and larvae only in terms of their expression (12, 22, 23). Likewise, most proteins that require copper as a cofactor for their activity are found in the fish, and some of these are characterized (see Table 1).
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SEARCHING FOR NEW RELEVANT GENES AND MARKERS
The potential of the zebrafish as a discovery tool in copper research has yet to be realized because the identification of new participants and markers is only beginning to be explored. Pioneering work was recently carried out by Mendelsohn et al (15), who have performed large-scale screens with small molecules to generate a copper-deficiency model in the zebrafish. In a screen carried out in larvae, which relies on function of the tyrosinase enzyme for pigment synthesis, several candidate molecules that can effectively limit copper availability to the organism were identified (15). The deficiency phenotype included defects in notochord formation, neural cell survival, cartilage formation, and hematopoiesis, indicating that certain tissues are differentially sensitive to the lack of this micronutrient. They also identified a mutation in atp7a that shows an identical phenotype to that produced by exposure to the identified drugs (15). Obviously, this study paves the way for further small molecule screens because these models for human diseases can be used as testing assays for a desired biological activity, eg, recovery of cuproenzyme function in the mutant background.
Mutations can be efficiently recovered in fish with chemicals that induce single nucleotide substitutions, the traditional way for mutagenizing the genome on a large scale. In addition, a significant collection of insertional mutants now have the advantage of simpler access to the mutated gene (avoiding the laborious mapping of the mutations implicit with chemical mutagenesis). Many genes of interest to the copper biology community are present in these collections.
Various types of genetic screens aimed at finding copper metabolism genes can be envisioned, such as resistance to deficiency or to toxicity. The possibility of looking through large numbers of mutagenized animals under controlled conditions (eg, a specific concentration of copper in the water in which larvae are raised) could identify new genes and potential therapeutic targets as well. To our knowledge, this type of forward genetic approach has been used before only in Drosophila among multicellular animals (1). The robust lethality values we have for zebrafish larvae should provide a suitable assay for even minor deviations from the average population. Likewise, quantitative trait locus analysis could be performed to assist in the identification of genes that confer resistance or susceptibility after classic genetic selection; the large numbers of organisms and rapid generation time of the zebrafish should be instrumental in this type of approach.
The availability of genomic expression tools such as microarrays or other expression-profiling methods (serial analysis of gene expression, etc) were crucial developments for the emerging fields of toxicogenomics or nutrigenomics. The zebrafish has already been providing relevant information in these fields, and copper-related research stands a chance for significant advancement. Besides the obvious acute-effect screens for changing patterns of gene expression, it should be straightforward to carry out screens in fish that combine copper status with other influencing variables such as water hardness, pH, or the presence of interacting ions or metals such as zinc or iron. In addition, it should be possible to carry out long-term studies in adults, in which the effects of a slight excess or deficiency of copper over a significant portion of the animals' life span can be analyzed. Reliable methods for assaying copper status in humans suffer from a lack of adequate markers, an aspect that could clearly benefit from new candidates identified with nonbiased approaches (35).
Expression profiling can provide new markers and players, but it has the disadvantage of potentially generating many false-positive genes. Candidates will have to be tested for relevance at least in terms of expression, which, again, is amenable to large format processing in the fish through in situ hybridization in larvae. Large-scale functional analysis will be more challenging though, because a reverse genetic approach to study known genes by gene targeting ("knockouts") is still unavailable in fish. A strategy of targeting induced local lesions in genomes (TILLING) has been developed (36) and could become a useful alternative if the desired mutants are not part of the currently available collections. At least as far as embryonic and early larval stages go, essentiality and function of candidate genes may be tested by knock-down with antisense morpholinos.
In summary, the zebrafish shows strengths in specific areas that can be useful for gene and marker discovery and for unraveling some of the more complex aspects of the genetics of copper metabolism in vertebrates.
ACKNOWLEDGMENTS
We thank Natalia Mackenzie, Mónica Brito, Viviana Gallardo, Cristian Undurraga, and Ariel Reyes who have participated in different aspects of copper-related research in our laboratory.
The author's responsibilities were as follows—PPH: researched the literature and performed the database searches for the genes listed in Table 1
; MLA: did the writing; and both authors revised the submitted manuscript. None of the authors had a personal or financial conflict of interest.
REFERENCES
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