Zebra ish an emerging model for Preclinical Drug Discovery

For understanding the pathogenesis of human disease at cellular and molecular level bio medical research depends on the use of animal models. Maximum animal models used in medical research of human disease are basically performed in murine system. Though there are advantages of using these animals, murine’s have long gestational period, sexual maturation rate and are expensive. These invertebrates lack many structures and organ systems that are involved in human disease pathogenesis. Forward-genetic screens and random mutagenesis based reverse genetics though possible in mouse and are currently underway; they cannot be done on a large scale as they require considerable staff and infrastructure support. Due to these drawbacks other animal models have been developed that provide initial genetic or drug target information. For developmental genetic studies larval zebra ish are used extensively due to their small size, external development, optical transparency, and accessibility in large numbers. Not only are they vertebrate, they also have high fecundity, which can be easily visualised and experimentally manipulated. 70% of all human disease genes have functional homologous in zebra ish. There has been signi icant increase in the use of zebra ish to elucidate the etiology of human disease like cancer, infectious diseases, cardiovascular disease, kidney disease, diabetes, blindness, deafness, digestive diseases, hematopoiesis, muscle disorders and neural disorders.


INTRODUCTION
Maximum animal models used in medical research of human disease are basically performed in murine system. A lengthy gestation period (18-20 days) and rate of sexual maturation (6-8 weeks) when mutualised with high cost of housing and breeding results in signi icant boundaries in spite of the knowledge produced by these murine models (Lieschke and Currie, 2007). Experiments with mice are laborious. Hence, they are not suitable for high-throughpout screening. These limitations have led to the development of other animal models that provide initial genetic or drug target information and will not be expensive (Goldsmith and Jobin, 2012). For studying the developmental biology zebra ish has been considered as an ideal model. Thus, it is a vertebrate akin possessing much similarity to humans compared to any model which consider the invertebrates, with much bene its over other models including vertebrates. As it possesses better rate of reproducibility (a few hundred eggs per spawning) with transparent embryos, and offers visualization of external development, the experimental models can be easily manipulated. The generation time being short (2-3 months) all of these features, previously found mainly in invertebrate models, simplify the genetic and high-throughput functional stud-ies (Kawakami, 2005;Xi et al., 2011;Tavares and Lopes, 2013). River Ganges is the main origin source of Zebra ish. In Latin, the zebra ish is known as Danio rerio and is very common in aquariums around the globe (Lawrence, 2007). The genetic map of zebra ish demonstrates highly conserved similarities with the human genome. Roughly 70% of of the complete human genome has its functional homologs in zebra ish (Santoriello and Zon, 2012). The development of a zebra ish is rapid and most of the organ systems are completely developed 5 days post-fertilization (dpf). Due to the rapid development and especially the smaller small size, the zebra ish is particularly suitable for the testing of chemical compounds that may challenge the developmental pathways (Peterson et al., 2001)and disease mechanisms (Peterson et al., 2004). Zebra ish has become one of the important research tools and is also used for elucidating the etiology of human disease. Zebra ish is the ideal model for the study of various ailments in humans like cancer, infectious diseases, cardiovascular disease, kidney disease, diabetes, blindness, deafness, digestive diseases, hematopoiesis, muscular disorders and neural disorders. Few examples have been highlighted below for the better understanding of why zebra ish is being used to develop a model, explore disease biology and to develop newer principles of therapy (Kar, 2013).

Wound Healing/Restitution
Wound healing is characterised by critical biological response of injured tissues and organs (Richardson et al., 2013). One of the most popular zebra ish wound model is larval tail wounding model, where a section of tail in is resected.
A complete regeneration of the heart is possible in zebra ish, after a 20% resection of the organ, within 2 months (Goldsmith and Jobin, 2012) and hence are used for their ability to regenerate cardiac tissue. Zebra ish larvae expressing a genetically encoded H 2 O 2 sensor showed that H 2 O 2 signals to leukocytes in tissues, in addition to its known antiseptic role indicating the role of H 2 O 2 during the early events of wound responses in Zebra ish (Niethammer et al., 2009).

Gastrointestinal Diseases
The gastrointestinal system of a zebra ish is homologous to humans and consists of pancreas, liver, gall bladder. The secretory and absorptive functions are carried out by the intestinal tract similar to humans. This has led to the development of various patho-logical models of the gastrointestinal tract in the zebra ish (Annie et al., 2005;Wallace et al., 2005). For the assessment of prokinetic drugs and GI motility, the zebra ish larva motility model has been very useful (Zhou et al., 2014). Owing to the similarities in the physiology and pathological conditions of the gut, various human diseases can be easily modelled into the zebra ish. To study the pre mammalian gastrointestinal diseases by high throughput screening and genetic manipulation zebra ish is the ideal model (Fleming et al., 2010).

Cancer
Histologically there is high degree of similarity between zebra ish and human tumour. To develop cancer models in zebra ish the employment of chemical treatment, transplantation of mammalian cells, forward genetic screens, and reverse genetic approaches using knockouts and transgenes have been widely accepted (Santoriello and Zon, 2012). Approximately 1:100 high locus mutation rate is produced by gamma ray mutagenesis and hence has been used in screening morphological defects (Haffter et al., 1996). The zebra ish is known to exhibit large deletions and translocation in the genome on introduction to high frequency gamma rays. Thus, chemical mutagenesis is the irst-choice method to induce mutagenesis. The process of insertional mutagenesis involves injection of a plasmid DNA, a mouse pseudo typed retro-virus or using a P-element transposon . These genetic screening helps in elucidation of novel genes and mutants for speci ic organs or processes (Driever et al., 1996).

Cell survival and apoptosis
Major part of the normal development of many organ systems and tissues involves apoptosis. The usefulness of zebra ish in studying early development, and the ability to label apoptotic cells due to recent advancement in technology has made it possible to visualize apoptotic cells in this model system (Cole and Ross, 2001). Apoptosis is known to be modulated in vivo by a plethora of smaller molecules and here lies the current interest of pharmacology. Several investigators studied apoptosis mechanisms in zebra ish embryos due to the high degree of evolutionary conservation of molecular processes governing regulation of apoptosis (Eykelbosh and Kraak, 2010). For example, zebra ish DNA fragmentation in vitro was inhibited by peptidebased caspase inhibitors of both invertebrate and vertebrate homologues of caspases 1, 4, and 5 or caspases 2, 3, and 7. It has also been seen that in early zebra ish development caspase inhibitors prevent nocodazole-induced apoptosis. These data suggest that caspases of the zebra ish share similar substrate-recognition sites with their mammalian homologues and perhaps target the same proteins in vivo. During the development of a zebra ish embryo detection has been done for apoptotic events speci ic to the organs and tissues. For the identi ication and detection of apoptosis in the embryos using high-throughput screening, acridine orange dye is extensively used (Kari et al., 2007).

Angiogenesis and vasculature
An excellent target for cancer therapy is inhibition of angiogenesis since formation of vessels is necessary for tumor growth. Any small molecule affecting the process of angiogenesis can be screened viably by using zebra ish models. Prominent patterning of blood vessel can be visualized microscopically useful in screening for compounds that affect angiogenesis by staining the sub-intestinal vessels (SIVs) (Serbedzija et al., 1999). Vascular system development has been well established in zebra ish embryos (Rubinstein, 2003). Major vessels formation is complete along with beating of heart, ongoing circulation, and angiogenesis in the somites and the anterior portion of the embryos. It is by angiogenic sprouting that blood vessels are formed in many zebra ish and they also seem to require the same proteins that are essential for the growth of blood vessels in mammals. Using endothelial cell-speci ic promoters such as KDR/Flt, transgenic zebra ish have been developed in which green luorescent protein (GFP) expression is directed to endothelial cells. There is no immediate lethal effect due to defective vessel formation because embryos can survive and develop for up to a week without an intact circulatory system. This permits thorough analysis of the effects of pro angiogenic compounds (Kari et al., 2007).

Cardiac Disease
In vivo models provide a very low throughput in exchange for a considerable expense, whereas, the current in vitro assays performed preclinically are biologically simple. On testing of 100 small molecules for their effects on chronotropicity in the heart of zebra ish, it was found that the drugs known for causing QT prolongation in humans consistently caused bradycardia and AV block in the zebra ish. Heritable modi iers of such drug effects can be explored in zebra ish due to their genetic tractability (Milan et al., 2003).
For study of various heart models such as heart development, heart muscle growth, endocardium and valves development, cardiac conduction system, cardiac electro-physiology, heart regeneration zebra ish models haves been used (Poon and Brand, 2013) . They are used for study of a wide variety of cardiovascular disease processes which includes congenital heart defects, arrhythmia, cardiomyopathy, aortic coarctation. The characteristic optical clarity of zebra ish enables it to be used for the studies on valvulogenesis and detailed electrophysiological mapping to screen the conduction system of the heart. Zebra ish larvae oxygenate through diffusion only which is a very unique character. This allows the study of mutations causing severe cardio myopathy phenotypes such as silent heart and pickwick m171 , which mimics titin mutations observed in human dilated cardiomyopathy. The capability of the cardiac tissue in the subject to regenerate allows the formulation of new therapeutic strategies for cardiac injury, including scar formation following myocardial infarction (Bakkers, 2011;Asnani and Peterson, 2014).

Myocardial infarction
Irreversible loss of heart tissue mainly characterized in, myocardial infarction, theand the tissue is replaced with a ibrous scar. In contrast to the partial resection model, cryoinjury heart infarct model causes serious cellular mortality similar to that of the mammalian infarcts mainly observed in 20% of the ventricular heart wall (Chablais et al., 2011).
Cre/lox lineage tracing system have been used to locate the source of the newly formed cardiomyocytes following this injury. It was found that cardiomyocytes which experience partial dedifferentiation gives rise to the regenerated cardiomyocytes characterized by structural changes and expression of cell-cycle progression genes. According to the study, these data suggest that zebra ish heart regeneration is driven by pre-existing cardiomyocytes rather than by progenitors, as previously suggested. A small ubiquitous protein, Thymosin b4 is presumed to be a signal which is known to trigger the formation of new cardiac tissue and blood vessels in ish. These observations enlighten the path towards inding new drugs for therapy of heart injury in zebra ish (Jopling et al., 2010).

Diabetes
In diabetes research the adult zebra ish has become the potential and important model. Genes responsible for the regulation of blood glucose in zebra ish has been identi ied and it has also been found that the mechanism of blood glucose regulation in zebra ish by insulin release is very much similar to that in mammals. Similar to mammalian model adult zebra ish respond to anti-diabetic drugs, by reducing blood glucose levels (Elo et al., 2007;Eames et al., 2010). The working of insulin promoters and insights into diabetes for rodent genes have been investigated. The reports illustrate signi icant dissimilarities between rodents and human promoters and their genes. The similarity of insulin genes in zebra ish and humans authenticates the key importance of the insulin hormone product (Olsen et al., 2012).
It's a complex and stepwise process of development of the vertebrate pancreas. In zebra ish differentiation of the pancreatic cells are mediated by extrinsic signaling molecules which in luence the intrinsic transcriptional programs. Experiments on zebra ish has revealed several signaling molecules responsible for this differentiation process (Kinkel and Prince, 2009).
In addition to hormones, nutrients like lipids and carbohydrates are also responsible for the regulation of transcription of the genes coding for glycolytic and lipogenic enzymes. They execute their functions primariely through the cellular nutrient/energy receptors. Identi ication of such nutrient/energy receptors is done by using fasting blood glucose level as a parameter from the mammalian studies. Zebra ish is an important model system for studying the human metabolic disorder (Craig and Moon, 2011). The mechanism for regulation of gluconeogenesis in both mammals and zebra ish are the same. Expression of phosphoenolpyruvate carboxykinase (PEPCK) regulates insulin levels and glucagon is responsible for the regulation of the rate limiting step of gluconeogenesis. Changes in PEPCK expression can be obtained through real-time PCR analysis of whole larval RNA (Elo et al., 2007).

Lipid research
To study the biological phenomena, Zebra ish are an increasingly popular vertebrate model organism. To study the lipid-related diseases, model that including atherosclerosis, obesity, diabetes and hepatic steatosis Zebra ish is used as an ideal model system (Hölttä-Vuori et al., 2010).
In obesity endocrine signaling and lipid homeostasis is altered by dietary and xenobiotic compounds. Examination of the effects of diet, drugs and environmental contaminants on the white adipose tissues singly or in the combined form can be done by zebra ish obesogenic (ZO) test which is a short-term assay method. Obesogenic and anti-obesogenic compounds and their mixtures provide relevant information by whole organism testing. The ZO test, which provide the information on adipocyte lipid droplet size and adiposity as its endpoints (Tingaud-Sequeira et al., 2011).

Regeneration Studies in zebra ish
A perfect coordination in the proliferation and pat-terning of mature cells following any severe injury or amputation provide the information regarding complex tissue regeneration process. As compared to mammals certain lower vertebrates have a superior regenerative capacity. Very little information is available regarding the molecular and cellular mechanisms of regeneration. Zebra ish is a suitable model system to overcome this de iciency. The regenerative process refers to perfectly or near perfectly replacing of damaged or lost structures. In mammals, majority of organs heal by scarring. In the process of understanding the regenerative medicine for humans the irst leap is the better understanding of the regenerative process in lower organisms. Zebra ish is a teleost ish that can regenerate multiple structures like ins, optic nerve, scales, heart, and spinal cord which is open to standard molecular and genetic manipulations (Poss et al., 2003;Curado et al., 2007). For investigating the heart regeneration a model developed by using zebra ish embryo/larva provides the greatest of advantages, during irst week of development the embryo/larva oxygenates only through diffusion thus tampering with its cardiac function within that period does not risk the survival of the subject (Stainier, 2001). Regeneration studies in multiple tissues of zebra ish have helped identify new mechanisms and has served as a guide for anticipating regenerative strategies in mammals (Gemberling et al., 2013).

Central nervous system disorders in humans include
Parkinson's disease, schizophrenia, Alzheimer's disease, and depression. Aminergic neurotransmitters are found to be readily involved in the pathogenesis of these disorders. On comparison it was found that the central aminergic system of humans and zebra ish are considerably similar. There is highly similar noradrenergic, serotonergic, and histaminergic system. Similar dopaminergic systems come to exist. A major difference is observed in the mesencephalon of zebra ish where it is lacking dopaminergic neurons. . By alterations of brain dopaminergic systems with MPTP, not only alterations in the dopaminergic system is observed but it also shows abnormal motor behavior. An alteration in the histaminergic neuron networks along with effect on memory and swimming was observed when histamine de iciency was induced chemically. With the help of imaging techniques and behavioral methods zebra ish genetics study reveal how the important behaviors are produced by interaction of modulatory transmitter systems, and in what way they are regulated in disease conditions and modulate pathophysiology (Panula et al., 2006;Bandmann and Burton, 2010). Detection of alterations in basic motor function, changes associated with exteroceptive and interoceptive sensory cues, and alterations in learning and memory performance by using various test methods available, helps in study the altered neurological function. Zebra ish is the ideal model system for study the many of the behavioral disease models which are similar in mammals (Tierney, 2011). Zebra ish provides the most considerable vision into the formation of sensory circuits, and exhibit great potential for explaining the functioning of sensory systems at an organismal level (Guo, 2004).
Proconvulsant activity of PTZ and the combination of PTZ with proconvulsive caffeine was estimated by the latency of seizure produced in zebra ish (Gupta et al., 2014). Locomotor response of nicotine was studied in larval zebra ish (Cousin et al., 2014).

Drug Development process (or discovery)
Testing of drugs in a large scale in mammals requires time and capital thus making the process not so reasonable. Such a method can only be appropriate for application into lower group animals or merely cultured cells. However, when such tests or assays are carried out in lower organisms or cells it portrays a major disadvantage. Such organisms often are not capable of portraying a human disease and even if the disease is induced, the measure of outcome likely to provide relevance for the identi ication of drugs to be applied into the clinical setting is not enough. The smaller size, rapid rate of development, ease of availability of the zebra ish brands them advantageous in the drug discovery processes. Zebra ish screens in melanoma achieved the greatest success, wherein as the result of tremendous research a novel drug has made it as far as upto the clinical setting and has stimulated the sanguinity that similar screens of other diseases will harvest parallel results (White et al., 2011). To study the toxicity much earlier in drug development process, high-throughput zebra ish toxicity assays model was established (Parng, 2005;Zon and Peterson, 2005). The most pertinent distinguishing property of zebra ish embryo assays and also their integrative capacity, it allows the resolve the wide range of effects in a single system. Image-based high-throughput methodologies, due to its developmental characteristics and transparency allow to screening of the large number of compounds in preclinical research purpose (Raldúa and Piña, 2014). These screenings can be performed with reasonable effortlessness, as chemicals can be simply added to the water. A high throughput screening for small molecules from the available libraries a plethora of compounds have been identi ied which can potentially make it to the clinical setting due to their sup-pressing effect on the altered phenotypes (Bassett and Currie, 2004;Zon and Peterson, 2005). Various new compounds are currently under clinical trials which have been screened in the past few years (Wiley et al., 2017).

Immuno and In lamation
There is a high similarity between the human immune system and zebra ish immune system (Traver et al., 2003). Complete development of the immune system (innate or adaptive) is followed even in the small zebra ish. The growth and development of immune system was easily observable due to its transparent embryos. using whole mount in situ (WISH) in ixed embryos, or followed in real time in live transgenic ish in which luorochromes have been used to tag or tracked cells. The in lammatory response which follows an injury directs the immune cells of the host system to tackle pathogens and sustain integrity of the tissue in consideration. Variety of signal gradients established the immune cell recruitment to the site of in lammation (Redd et al., 2006). After injury in zebra ish embryo demonstrated that the wounded cells instantaneously start to produce hydrogen peroxide (H 2 O 2 ) which is none other than a signaling molecule for the leukocytes (Pase et al., 2012). Hematopoiesis studies suggest that maximum cells possess the qualities equivalent to that of the zebra ish but exceptions exists (Stachura and Traver, 2011). In both mammals and zebra ish, the thymus is responsible for production of T lymphocytes. The only difference that prevails is in the morphology of the gland. In zebra ish the gland remains as two separate bilateral structures (Lam et al., 2002). The innate immune system: the acute phase response to infection, the interaction of host and pathogen, and the chemotactic response to injury the zebra ish is the ideal model to study. Due to higher similarities of zebra ish to human and other mammals. Therefore, a surge in the immuno models of zebra ish is noted, providing new understandings in the development of the immune system and its functioning (Meeker and Trede, 2008), and also to study leukocyte recruitment and in lammation Zebra ish is an powerful model system (Lieschke and Currie, 2007;Mathias et al., 2009).

Muscle disorders
Zebra ish models for human muscular disorders stands bene icial due to some speci ic qualities. Its reproducibility, easily measured motor behaviors are the qualities that are present from the irst day of the life. Mammalian skeletal muscle is structurally very similar to that of the zebra ish and shares identical histological and molecular features. Such striking similarities includes conservation of the components of the dystrophin-associated glycoprotein complex, contractile apparatus and the coupling machinery for contraction-relaxation. These structures happen to be the most vital muscular structures required to understand any muscle disease pathophysiology (Guyon et al., 2003;Dou et al., 2008). In a growing zebra ish, skeletal muscle is the largest organ and can be easily visualized. As a inal point, the zebra ish models are capable of precisely estimating the severity of clinical condition compared to corresponding rodent models (Berger et al., 2010;Berger and Currie, 2012). Mutations of SepN1, a selenoprotein, and RyR1, the major component of the ryanodine receptor regulated intracellular calcium channel, result in an overlapping spectrum of congenital myopathies. The immediate developmental and molecular roles of SepN and RyR in vivo, loss-of-function effects were analyzed in the zebra ish embryo. It was demonstrated that these two proteins are required for carrying out the normal calcium luxes in the embryo and also for the same cellular differentiation events. The absence of either SepN or ryanodine receptors are known to cause similar diseases in the human muscle and the zebra ish embryo (Jurynec et al., 2008). Mutagenic screens of the zebra ish genome have led to the identi ication of a class of recessive lethal mutations in which muscle differentiation occurs normally, which is charted by tissue-speci ic degeneration reminiscent of human muscular dystrophies. It was seen that, results from mutations within the zebra ish orthologue of the human Duchenne muscular dystrophy (DMD) gene causes sapje (sap) mutation. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology by disrupting the link between the actin cytoskeleton and the extracellular matrix in skeletal muscle cells (Bassett and Currie, 2004;Kawahara et al., 2011).

Kidney Disorder
Polycystic kidney disease (PKD) is a common genetic disorder which is characterized by formation of multiple cysts in the kidneys. These cysts are thought to be the result of excessive proliferation of epithelial cells. Similar conditions can be developed by a zebra ish leading to its signi icance in the research revolving around the disease (Sun, 2004).
Zebra ish embryonic kidney is structurally simple which enables it to become a popular model for the study of renal organogenesis and the search for new therapeutic strategies. Studies using zebra ish has shown signi icant advancement in the discovery of the nature and disease severity for Acute Kidney Injury (AKI) and ciliopathies of the kidney (Swanhart et al., 2011).
Gene regulatory networks in the control of kidney development if explained would provide an insight on the origin of birth defects related to the renal system, which will further aid in the clinical interventions for such conditions. Nephrons are the basic microscopical and functional unit of kidney. Podocytes are specialized endothelial cells located on the nephrons that houses the blood ilter, otherwise known as the glomerulus. Any dysfunction of the kidney is bound to arise due to damage to these podocytes. Interaction of podocytes with the vasculature produces sieve like structures that collects and ilters the luid. Filtrate enters the nephron which changes it to urine. Podocytes are also responsible for the protection of nephrons as it prevents the entry of high molecular weight proteins and larger cells. Thus, any damage to podocytes can lead to renal diseases like chronic kidney disease. In zebra ish the genetic intervention required for the development of podocyte if understood, is likely to be applicable to a wide range of vertebrates and mammals (Wingert and Kroeger, 2014).
As a response of various renal diseases there is either partial or complete loss of kidney functions. In mammals the damaged nephrons can be repaired only partially and new nephrons cannot be produced (Humphreys et al., 2008). Whereas, a zebra ish can produce nephrons throughout their complete lifespan and can even regenerate them following an injury. Thus, a model if developed can provide an insight on how can mammalian regeneration of nephrons can be activated (Reimschuessel, 2001;Zhou et al., 2010). These data when integrated concludes that the zebra ish kidney is likely to contain nephron stem cells. If these cells could be identi ied by any means then mammalian cells with similar properties could be isolated or developed which would provide a gateway to therapeutic regeneration of the nephrons in mammals .
Homeostasis in the body is primely maintained by the renal system. Nephrons are responsible for various homeostatic functions like removing metabolic waste from the body, luid regulation, and maintaining the electrolytes balance. AKI is liable to cause the loss of these activities and AKI can occur in response to toxins and ischemia mediated organ damage. The ability to regenerate or repair damaged nephrons following AKI in humans is not completely understood. However, researchers studying AKI in vertebrate animal models such as mammals, and more recently the zebra ish, have documented robust regeneration within the nephron blood ilter and tubule following injury.
Anatomical simplicity of their kidneys has made zebra ish a popular model for studying renal diseases. Zebra ish as a model has been established for polycystic kidney disease (PKD), nephronophthisis, acute kidney injury (AKI), various other ciliopathies (Swanhart et al., 2011).

Leukemia
A high degree of genetic and morphological similarity in hematopoiesis between the zebra ish and human indicates that zebra ish can provide valuable knowledge about the mechanisms behind pathogenesis of leukemia (Teittinen et al., 2012).
About 60% of cases, mutated NOTCH 1 gene found to be responsible for T-cell acute lymphoblastic leukemia (T-ALL). To study the molecular level mechanism of human NOTCH1-induced Tcell leukemia transgenic zebra ish model have been used. The purpose of this model is to detect a strong interaction between NOTCH1 and bcl2 suggests that genetic modi ier screens have a high likelihood of revealing other genes that can cooperate with NOTCH1 to induce T-ALL. Genetic modi ier screens using transgenic zebra ish prone to NOTCH1-induced T-cell leukemia may ultimately reveal suppressors or enhancers of Notch pathway in T-ALLs. These modi ier genes could be the targets for the development of new therapies (Chen et al., 2007).
To study the mechanism of tumorigenesis, Zebra ish is valuable research model. The model organism is induced to develop T-cell leukemias resembling those in humans and is amenable to large-scale forward-genetic screens that can be used to uncover novel cancer pathways (Langenau, 2003).
Comparisons of zebra ish and mammalian hematopoiesis and lymphopoiesis indicate that the genetic programs underlying vertebrate blood development have been highly conserved through evolution (Thisse, 2002).
Acute lymphoblastic leukemia (ALL), a disease with relatively homogeneous morphology and immunophenotype, was another type of cancer found in zebra ish but with great heterogeneity at the genetic level, which can lead to distinct responses to therapy. One of the main causes of ALL is the TEL-AML1 fusion, associated with B-lymphocytes. Transgenic ish were generated from this mutation in all cell lineages, and developed lymphoblastic leukemia, which phenocopied the childhood CD10+, pre-B ALL. Creation of a transgenic line for the oncogene Myc led to the development of T-cell mediated ALL. Other mechanisms responsible for ALL have also been addressed with zebra ish, such as Notch1-induced T-ALL (Tavares and Lopes, 2013).

Alzheimer's Disease
One of the most signi icant neurodegenerative disorder is Alzheimer's disease.
After extensive research our understanding of the disease still remains incomplete. Both an effective treatment and the validity of the existing disease models are still under question. The zebra ish (and, in particular, its embryos) is a malleable and accessible model possessing a vertebrate neural structure and genome. Zebra ish genes orthologous to those mutated in human familial Alzheimer's disease have been de ined (Newman et al., 2011). The characteristics of these genes are dif icult to observe in any model but in zebra ish some characteristic discoveries have been possible. Zebra ish possess genes orthologous to all the genes known to be involved in Alzheimer's disease (Chen et al., 2007).
Although zebra ish lacks the complexity of advanced cognitive behaviors evident in rodent models, they have proven to be a very informative model for the study of human diseases. Zebra ish possess genes orthologous to those mutated in familial Alzheimer's disease and research using zebra ish has revealed unique characteristics of these genes that have been dif icult to observe in rodent models (Newman et al., 2014).
The presence of various neurotransmitters, both excitatory and inhibitory, is reported in the central system of zebra ish. A tight junction-based blood brain barrier with macromolecular permeability is also found in Zebra ish which is useful to study about the novel AD drugs (neuroprotective drugs).

Future perspectives
Zebra ish is believed to have a strong hold in the study of diseased genes and identifying newer therapeutic strategies. This notion has been picked up due to the unique and very versatile features of zebra ish.

Interfacing with human genetics and genomics
The potential genes causing disease in humans have been identi ied mostly by complete genome screening and the genome association studies. Zebra ish is well known for the functional depiction of such genes and further can be used to investigate coding and noncoding function in vivo. Several studies are conducted to verify the association of genetic variants in the pathogenesis of many diseases in the zebra ish by implying to the genetic approach (Santoriello and Zon, 2012).

Transplantation of tissues
Cell and tissue transplantation techniques have been used to investigate the mutant embryos with defective tissues. Model for tumor transplantation assay using zebra ish is quite reliable. Tumor cell reprogramming, migration, metastasis, and effects on vasculogenesis can be studied by cancer cell lines which can be the derivatives of different tissues or species (Santoriello and Zon, 2012).
Gene function involved in physiological processes is quite similar in ish and humans. Zebra ish models are a much convenient means for the investigation and identi ication of the genetic markers which can either suppress or enhance the disease conditions (Chakraborty et al., 2009).
Zebra ish models stand unique by being transparent and possess strong potentiality to ful il this important function in the study of human disease, enabling rapid, physiologically relevance in in vivo screening. The transparency of zebra ish also allows for screening of pathophysiology by various imaging methods which has proven to be very useful in understanding numerous disease mechanisms.
The zebra ish models stand on an underdeveloped platform with uncovered potentials. With growing understanding of the genetic mechanisms, anatomy and physiology of zebra ish the importance of this model will grow to become a powerful element of various researches involving the murine system. Extensive research on zebra ish has proved its supremacy as a model for human diseases over other conventional models. The unique features of zebra ish provides as the most appropriate in vivo system for investigating the preliminary stage of any pathology before the observations are translated to other expensive murine systems which will conserve both time and capital (Goldsmith and Jobin, 2012).

CONCLUSIONS
The present investigation mainly focuses on establishing zebra ish as an alternative preclinical drug discovery model. Screening of toxicological pro ile of compounds can be easily done in zebra ish due to its high visibility at developmental stages. Zebra ish also used to investigate the causes and pathologies of human diseases like cardiovascular, neurological, wound, gastrointestinal, diabetes. Because of its highly similar genome with human provided the support to zebra ish as an ideal model system for human diseases.