Tiny Fish, Big Discoveries: Zebrafish as a Model for Understanding Human Diseases

Various animal species play vital roles as experimental models in advanced biomedical research. In the late 20th century, with the development of molecular techniques, zebrafish have been used as a model organism in almost all aspects of biology worldwide.

What are Zebrafish?

The zebrafish belong to the Cyprinidae family, which comprises more than 2000 species. Its former scientific name was Brachydanio rerio. However, it was changed to Danio rerio in 1981 because both genera were very similar, from the presence of short dorsal fins and incomplete lateral lines on them.

It is a small tropical freshwater fish originating in the Ganges River and its tributaries in northern India, and it spreads to river basins in Pakistan, Nepal, and Bhutan. Their maturation takes only 2-3 months, and they can produce 200–300 fertilized eggs weekly and complete embryogenesis in 72 hours.

The adults are generally 2.5–4 cm long, and the larval stage is transparent. When the adult stage is reached, a blue stripe develops along with the length of the body. The typical body shape of a male zebrafish is very slender compared to that of the female, and they appear to be more golden or yellowish on the ventral side of their body. On the other hand, females have more distinct characteristics than males because of their enlarged bellies, where eggs are stored, and a silvery blue streak on the lateral side of the body.

Why are Zebrafish considered a suitable animal model?

Zebrafish are an important model in many fields of science. They are used in developmental biology, neurophysiology, and biomedicine, but little is known about natural ecology and behaviour.

In developmental biology, the central aspect is understanding the animal’s fundamental processes. Zebrafish have a unique transparency that allows researchers to observe biological processes under the microscope. Zebrafish embryos develop rapidly and reach their mature stage within a few days. Rapid development enables the researcher to examine desired traits over several generations within a few months. Furthermore, they have high fecundity, providing statistically robust results and reducing ethical concerns about using many animal models. Zebrafish also have a fully sequenced genome, which facilitates further analysis of the genome and determines developmental biology concerning the organism’s genome. Also, zebrafish show distinct similarities with the human genome, which contains approximately 70% of genes. Thus, the zebrafish model is a vital model organism for studying human hereditary developmental functions.

Zebrafish have become an immensely significant animal model in neurophysiology. It acts as an important species for studying the mechanisms of brain functions and dysfunctions. A unique radial glial cell in the adult zebrafish, the telencephalon, possesses the ability of self-renewal to generate new neurons. These unique radial glial cells are also present in the developing mammalian telencephalon, acting as embryonic neural stem cells.

Zebrafish have become a significantly important model animal in biomedicine. Zebrafish intestinal epithelium morphogenesis is a complex process involving growth, lumen formation, and differentiation into distinct segments, making it suitable for studying intestinal diseases. The intestinal epithelium of the zebrafish develops into three distinct segments, with endocrine cells, mucin-containing goblet cells, and the posterior intestine remaining underdeveloped at 126 hours post-fertilization. Furthermore, zebrafish are a good model animal for intestinal inflammation because their gastrointestinal and immune systems are closely related to those of mammals.

There is a wide prevalence of zebrafish as cancer model animals due to their genetic, physiological, and anatomical similarities to humans, their ability to develop various human-like tumors, and their advantages for in vivo studies, including visualization of cancer progression, cost-effectiveness, and suitability for high-throughput screening.

Humans and zebrafish belong to totally different taxonomies. Still, they share several anatomical similarities, including the nervous system, digestive system, circulatory system, skeletal system, and sensory organs, which also have a higher prevalence of developing cancer. According to the studies, it has been shown that zebrafish can develop lymphoma and cancer conditions either by creating gene-targeted mutations and stable transgenes or by creating a fish with temporary overexpression or downregulation by alternating the gene expression pathway.

What are the applications of Zebrafish as a model organism for understanding various human diseases?

In addition to genetic similarity, the presence of conserved organs and organ systems between zebrafish and humans has led to the development of successful models for various human diseases such as Duchenne muscular dystrophy, human melanoma, acute lymphoblastic leukemia, Parkinson’s disease, Huntington’s disease, Alzheimer disease, myocardial infarction, acute kidney injury, obesity, type 2 diabetes mellitus, and atherosclerosis.

1. Zebrafish as a model for Glucose metabolism

The leading cause of diabetes mellitus in humans is the failure of beta cells in the pancreas to produce insulin, which leads to insulin deficiency. A common pathway for developing type 2 diabetes mellitus was seen between zebrafish and humans. Exposure of zebrafish to high-calorie and fat diets quickly induces obesity and obesity-related disease, activating metabolic pathways similar to human mechanisms.

Generally, if glucose is included in the diet, insulin is produced by the pancreas, and gluconeogenesis is inhibited through the downregulation of genes involved in the pathway. When glucose is absent in the bloodstream, gluconeogenesis is induced by glucagon. Recent research identified that zebrafish immersed in a high-glucose solution for 14 days could increase fructosamine levels by 41% and decrease the amounts of mRNA for insulin receptors in muscle, thus leading to hyperglycemia. When fed a high carbohydrate diet, mutant zebrafish with a knockout in insulin receptor a and b genes showed hyperglycemia, reduced growth hormone signaling, and fatty liver development, similar to human lipodystrophy disease.

2. Zebrafish as a Neurological Model

Another critical research field is curing neurological disorders with zebrafish because they have the same signaling proteins as human brains. Muscular dystrophies and congenital myopathies are inherited diseases related to skeletal muscle. Patients with Duchenne muscular dystrophy have been found to carry mutations in dystrophin, which lead to muscle weakness that gets progressively worse. In both models, the loss of dystrophin gradually leads to necrotic muscle fibers that are replaced by inflammatory cells and abnormally sized muscle fibers.

3. Zebrafish as a model to study Intestinal diseases

Scientists have used zebrafish as a model to study physiology, function, and diseases related to the intestine because the intestinal tract opens six days after post-fertilization. At this stage, the intestine of a zebrafish is easily visible. Its morphology can be observed with the microscope. Generally, zebrafish models are developed to evaluate the intake of bioactive compounds by the intestine. Scientists have concluded that bioactive compounds can cross the intestinal mucosal barriers and pass through the lamina propria to reach the muscle. Recently, a model was developed with zebrafish embryos infected with Salmonella, and according to the results of the research, due to the depletion of the bacterial detector proteins NOD1 and NOD2, the expression of dual oxidase in the intestinal epithelium has been reduced. This finding was considered a good model for human Crohn’s disease.

4. Zebrafish as a model for cancer

Human melanoma has been successfully modeled in zebrafish, and the most identified mutation is a single amino acid change in the gene BRAF. Since a combination of several alterations causes cancer, the knock-in model of zebrafish is used to screen for other potential cancer-causing mutations. It was identified that when gene SETDB1 was added to the BRAF knock-in zebrafish, melanoma rapidly developed; thus, it was confirmed that SETDB1 is an essential gene in melanoma growth.

Limitations of the usage of the Zebrafish model
Even though zebrafish are considered an essential model, they have some limitations. Although sharing similarities, the zebrafish brain is less complex than the human brain, limiting its use for studying higher-order cognitive functions. Differences in reproductive anatomy and physiology restrict the use of zebrafish for studying human fertility and related issues. Considering the genetic level differences between zebrafish and humans, zebrafish have a higher rate of gene duplications than the human genome.
Zebrafish remain an essential research tool due to their distinct advantages. However, knowing their limits is critical for effectively interpreting results and conducting responsible research procedures. To achieve relevant and reliable results, researchers should carefully assess the use of zebrafish as a model organism.

Written by:
Gayashani Colambage and Sandani Iresha

3^rd Year Undergraduates
Immunology and Integrative Molecular Biology

Faculty of Science,

University of Colombo.

References:

1. Bozkurt, Y. (2020). Introductory Chapter: Importance of Zebrafish (Danio rerio) as Model Organism in Biomedical Research. In IntechOpen eBooks. https://doi.org/10.5772/intechopen.91319
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3. Huberman, A. (2019, June 20). Glia Know When to Give Up / Cell, June 20, 2019 (Vol. 178, Issue 1). YouTube. Retrieved February 20, 2024, from https://www.youtube.com/watch?v=YFkmcLmrFn8
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