Zebrafish in Biomedical Research and Drug Discovery

Abstract

Many model organisms like yeast (Saccharomyces), Drosophila, zebrafish, mouse, rats, hamsters, rabbits, cat, chicken, monkey etc. are being used in biomedical research. Invertebrate models like yeast, Drosophila etc. are used to study genetic functions. On the other hand vertebrate model systems like mouse, rats, hamsters, rabbits, cat, chicken, monkey preferred models for research in diseased conditions when compared to invertebrate model organisms but vertebrate models are the more complex model systems. Zebrafish though a vertebrate with physiological and anatomical characteristics of higher organism it also provides the ease of use of a lower organisms. Hence zebrafish offers an important model system which can connect development, disease, and toxicological studies.

Keywords

Zebrafish, Cancer, Alzheimer’s, Diabetes, Drug Discovery

Introduction

The zebrafish (Daniorerio) is a freshwater fish found in tropical environment a native of Himalayan region and is commonly kept in aquaria in India. It belongs to the Cyprinidae family and Cypriniformes order. Initially it is studied for vertebrate development and it is the first vertebrate organism to be cloned. Over the period of time large number of zebrafish models has been developed for investigating different human diseases and toxicity studies. Both embryos and the adult zebrafish are widely being used in the research.

The use of zebrafish as an experimental animal model is increasing these days. This model is gaining popularity in the fields of biomedical research and toxicology. The reason behind wide acceptance of zebrafish as animal model is because of exceptional characteristics which are discussed below.

Zebrafish have a fully mapped genome with 400 distinct genes and >2000 microsatellite markers which is found significantly homologous to the human genome (about 75% Similar), including noncoding regions which suggests that many genes involved in human diseases can be matched with zebrafish genome. Signaling pathways of both zebrafish and humans are highly conserved with high genomic homology. Gene function assessment can be performed in zebrafish with ease by transgenic development and knockdown experiments making it a handy model for analytical studies [1-6].

Zebrafish possess high fecundity produce large number of embryos. On an average female spawn around 300 eggs per week under optimal conditions. It is found that the hatching of eggs and organogenesis occurs rapidly [7-14]. In contrast to other mammalian models they develop outside uterus which makes it possible to raise them in petri dishes or in multi- well plates containing water. They can be used for larval experiments from 3rd days post fertilization (dpf). The embryos are transparent (Figure 1) upto 7 dfp, and all cells cells can be observed since intial larval stage. In addition to it tissues and organs can also be visualized in vivo the transparency can be extended to up to 9-14 dpf by adding melanocyte inhibitor like phenylthiourea. Moreover, recently transparent adult zebrafish like the Casper line (Figure 2) is produced which provides new imaging possibilities. Additionally the use of sophisticated fluorescent technologies to indicate signaling proteins and cellular entities help in making time-lapse imaging of biological processes and diseases possible (Figure 3).

Figure 1: Figure showing different stages of transparent zebrafish embryo.

Figure 2: Figure showing transparent zebrafish (Casper line).

Figure 3: Genetically modified zebrafish with circulatory system glowing with a green fluorescence to study the development of circulatory system.

Drugs can be administered systemically to zebrafish just by adding it to the water in the aquarium on the other hand if embryos are used, then the test compound is added to water in the petri plates that holds the embryo [16-20]. Hence these models can be preferred to test scarce or expensive compounds. Drugs can also be locally delivered into the tissues with the use of surgical implants or electrophoresis. Animal breeding, developing and maintaining animal house facilities involve high cost whereas zebrafish with their smaller size, high fecundity, simple and rapid lifecycle and developemental stages make them ideal for reliable rapid and economic screenings during pre-regulatory phases and toxicity studies [21-31].

Zebrafish are even used in high-throughput screening (HTS) of drug libraries. Zebrafish embryos or larvae, in the same development stage, are loaded into multi well plates, and are then screened with chemical compounds at different concentrations. Robotics and automated fluid handling systems are also used in HTS [32-35].

Zebra Fish As Disease Models

Alzheimer’s Disease

Though rodents are more closely related to human physiology than fish, Zebrafish whose embryos are easily manipulable because of their large size, their ready availability and the ease of gene manipulation even in their development for assay of particular gene activities make zebrafish embryos a felicitous vertebrate system to examine the cellular and molecular functions of genes implicated in Alzheimer’s Disease [36-40].

Depression

Because of neuroanatomical, neuroendocrine, neurochemical and genetic homology to mammals, chemical genetic screens, zebrafish offers ideal experimental models of depression helps in discovering novel therapeutics. Behavioral testing models like–cognitive, avoidance and social paradigms are available in zebrafish and can be used to identify depression in zebrafish by exposing them to physiological, environmental, genetic, and/or psychopharmacological alterations. Moreover they are highly sensitive to commonly used psychotropic drugs [41-45].

Anxiety

All the “classic” neurotransmitters present in vertebrates are possessed by Zebrafish and its neuroendocrine system shows different physiological stress responses. Two important methods namely light/dark test and the novel tank test are demonstrated successfully in zebrafish to study anxiety disorders [46-49].

Immune System

Zebrafish possess an innate immune system composed of NK cells, neutrophils, and macrophages/monocyte which starts functioning from 2 dpf and an adaptive immune system that is functioning during 4–6 weeks post fertilization which is highly similar to that of mammalian, with T lymphocytes and B lymphocytes that have Rag-dependent V(D)J recombination which makes zebrafish a suitable animal model for immune system [50,51].

Cancer

Stanton, et al. in 1960s first used Zebrafish in cancer research to test the effects of carcinogens. Though it have a very low rate of spontaneous neoplasia, which account to about only 10% of zebrafish develop a tumour in lifetime, when exposed to carcinogenic agents likes MNNG (N-methyl-N-nitro-N-nitrosoguanidine), DMBA (7,12- dimethylbenz(a)anthracene) and DENA (diethylnitrosamine) they develop cancer. It has been proved to be an ideal model to study the malignancy of many tumours by using tumour transplantation assays. They were found to be robust and have an additional advantage of high fecundity as discussed earlier which provide donor and recipient fish in large numbers. Many types of cancers like melanoma, leukemia, endocrine or liver cancer are studied using zebrafish. Moreover by using xenotransplantation of human tumor cells into zebrafish embryos (xenografts) phenomena like metastasis, tumor cell migration, angiogenesis can be studied. Availability of forward and reverse genetic tools, the non-invasive in vivo imaging technology, and the above characteristics made it an ideal vertebrate model to study cancer [52-54].

Diabetes and Lipid Diseases

Because of accessibility of zebrafish for developmental studies, a complete description of pancreatic de- velopment and morphogenesis is available which led to the understanding of extrinsic signaling molecules, like Shh, retinoic acid and FGF, in influencing intrinsic transcriptional programs. These studies made zebrafish an alternative model to study the onset of diabetes along with its treatment. Hypoglycaemia can be induced in zebrafish by exposing it to high glucose and even retinopathies are developed with prolonged high blood sugar levels. All these makes it suitable model for diabetes.

Zebrafish possess many similarities with mammals in terms of lipid absorption, processing and metabolism, moreover application of new imaging methods with subcellular resolution to whole organism and the use of fluorescent lipid. It is even used in obesity studies [55-63].

Gastrointestinal Disorders

The gastrointestinal system of zebrafish is highly homologous to mammalian counterpart, which contains a liver, gall bladder, pancreas and a linearly segmented intestine with secretory and absorptive functions. The intestinal epithelium possess similar proximal-distal functional specification and many of same epithelial cell lineages like goblet cells, enteroendocrine cells and absorptive enterocytes. With the help of all these similarities zebrafish are used to model numerous gastrointestinal pathologies [64].

Cardio Vascular Diseases

The development of the zebrafish cardiovascular system is thoroughly studied and characterized, which provides great insights in cardiac development, vasculogenesis and angiogenesis. Some outstanding features like external embryological development, its optical clarity of embryo, closed cardiovascular system and similar cardiac cycle to that of humans make the sequential observation of the developing blood vessels and heart possible without invasive technique. Researchers have studied the origins of defects in heart shape, size and function. All these make zebrafish model useful in cardiovascular research [65-69].

Renal Disorders

Zebrafish provides a promising model for studying kidney development it provides many advantages which make it suitable model for genetic research, like the generation of offsprings in large numbers (exutero) with rapid development, low maintenance and ease of genetic modification. Use of genome editing techniques, like TALENs and CRISPR/Cas9 for modeling human genetic disease in zebrafish is making progress moreover zebrafish larvae during 2-3 dpf possess a pronephros which is a simple reflection of human nephron [70-75].

Conclusion

Animal models are being used in medical research since ages among which the most commonly and successful models are that of rodents [76-101]. Though a lot of knowledge is attained from this models few factors like long gestation time of about 2-3 weeks, sexual maturation rate of 6–8 weeks and expensive housing and breeding techniques lead to search for other model organisms. The zebrafish appears as a model organism with large amounts of untapped potential. As it provides comparative anatomy and physiology, genome to that of humans this models can be used to study initial genetic or drug target information before scaling up to expensive systems moreover the transparent, larval zebrafish models can be used to study of human disease, and enables rapid physiologically relevant in vivo screening. The transparency of zebrafish also allows real-time imaging of pathogenesis, which can provide insights into the molecular mechanisms. Furthermore the amenability of this model for high throughput screening and different human disease makes it more helpful to researchers. However the utility of this vertebrate model though cannot replace mammalian models in the drug development mostly in the later stages where regulatory authorities demands mammalian studies and clinical trials it will provide a powerful complement to the murine system.

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