Chapter 14 Why Do We Use Simple Organisms to Model Complex Human Diseases?

In: Complexity and Simplicity
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Marta Migocka-Patrzałek
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Abstract

A model organism is a species, different from the human one, which we investigate intensively to gain insights about specific biological processes, hoping to grasp the more widely applicable, general laws that regulate the functioning of organisms. Animal models are also used to more comprehensively understand human diseases. Such a strategy is efficient given the common origins of life on Earth and with this the shared developmental and metabolic pathways that owe their existence to the retention of features of the genetic code throughout evolution. One can, therefore, certainly ask why it is that we make use of simple organisms, such as worms, fruit flies, or fish, in our research? Can they really give us answers regarding complex human diseases? At the very least, it is surely worth knowing more about how such research is designed and conducted to simply evaluate its potential benefits.

1 Introduction

Animals have been used not only to study the basic principles of life, but also to better understand the anatomy, and physiology of living organisms. Moreover, animals play a central role in education, pharmacology and research. The ability to conduct experiments in controlled situations, and to mimic the biological conditions of human disease in animals led to the development of scientific methods and originated in the concept of animal models of human diseases. The model organism is a species, different from humans, which we intensively investigate to gain knowledge about specific biological processes and to understand wider, general laws that rule over organisms functioning. Although the use of animals in research has been questioned by the scientific community due to ethical concerns, the foundations of our basic knowledge of human physiology and anatomy are based on preclinical research with the use of animals. This is confirmed by the fact that nearly 90% of research awarded with the Nobel Prize in Physiology and Medicine, was conducted using animals in experimental investigations. It is also important to note that animal studies allow researchers to carry out experiments that would be impractical or ethically prohibited with humans (Andersen & Winter, 2019; Burggren & Warburton, 2007; Dubińska-Magiera et al., 2022; Plantié et al., 2015).

2 Why Does a Particular Animal Become a Research Model?

The main reason is availability and the ease of maintenance. For example, mice, chickens, frogs and fruit flies (Drosophila melanogaster) (Figure 14.1) are widely used just because they are easily accessible and simply cheap. The anecdote regarding one of the frog species, called the African clawed frog (Xenopus), is a nice example showing why a particular animal becomes a research model. Once upon a time, scientists discovered that the urine of pregnant women, containing hormones, can induce the development of Xenopus ovaries. Therefore, between the 1940s and 1960s thousands of frogs started to act as living pregnancy tests all around the world. The Xenopus, abundant in laboratories and easy to maintain, can be utilized in other kinds of experiments, trials and research, and as a consequence became a useful animal model. In general, the amphibians turned out to be great models in a variety of research and took a profound place in scientific areas such physiology of musculoskeletal, cardiovascular, renal, respiratory, reproductive, and sensory systems (Burggren & Warburton, 2007; Kiełbwna & Migocka-Patrzałek, 2017).

To be used as a model, the species must meet certain criteria that enable the feasibility of experimental procedures. From the researcher’s point of view, the high rate of reproductivity and relatively short life cycle (the time between being born and the ability to reproduce) is a very valuable feature. In the case of mice, having an accelerated lifespan, we can assume that one mouse year equals about 30 human years. Therefore, their whole life cycle could be examined within only two or three years. It is beneficial, especially in genetic research, in which crossing individuals to obtain several, subsequent generations in a relatively short time, is crucial. The financial feasibility and previous history of research with the use of a particular species are also useful. Such experimental data allows for research outcome comparison with the other researchers’ results, therefore not only enabling but also greatly facilitating the conclusions drawing process. The known genetic code of animals is crucial for genetic research since the possibility of genetic manipulation is nowadays one of the most important research techniques.

FIGURE 14.1
FIGURE 14.1

Fruit fly (Drosophila melanogaster). Fruit flies are one of the most popular model organisms. With the low cost of husbandry, short generation time, and variety of molecular tools for genetic engineering, flies become a useful model in biomedical experiments

Choosing an animal model for a particular purpose is a complex decision, involving scientific and practical considerations. Many species are used in biomedical research, such as yeasts, insects, nematodes (Caenorhabditis elegans), fish (Danio rerio – zebrafish), frogs and many mammals, such as mice, rats, dogs, cats, pigs and even monkeys. Several other issues should be also taken into account during experiment planning, such as the availability of adequate techniques and methods, including equipment, and molecular tools (such as specific antibodies, etc.) (Andersen & Winter, 2019; Dubińska-Magiera et al., 2016; Ericsson et al., 2013; Robinson et al., 2019).

3 Animal Models of Disease Can Be Divided into Two Categories, Spontaneous and Induced

The use of animals as a model of human diseases had increased dramatically in the twentieth century. The beginnings of detailed biological analysis of particular disorders were possible because animals sometimes suffer from the same kind of diseases as humans. Also, naturally occurring animal genetic variants (mutants) can be utilized as a model of a particular human genetic disease. We call such animals a spontaneous model. There are hundreds of well-characterised animal strains with inherited genetic disorders, allowing us to examine in detail human genetic diseases (see, e.g., the Jackson Laboratory database1). Mice, called sometimes “lifesavers”, because of their usefulness in medical trials, are biologically very similar to humans and get many of the same diseases, for the same genetic reasons. One such example is the so-called, nude mice. Its genome contains a spontaneous mutation in the gene Foxn1nu, leading to two main defects: abnormal hair growth and defective development of the thymic epithelium. As a result, the nude mice do not develop thymus, therefore they lack the T cells, which are crucially important for natural, immunological defence. This model is widely used to induce diverse kinds of cancer by injecting cancer cell lines into nude mice, enabling preclinical testing of anticancer drugs (Cordier & Haumont, 1980; Onaciu et al., 2020; Szadvari et al., 2016).

On the other hand, nowadays, we have learned how to induce particular pathological conditions in animals to create disease models. Such induction could be a combination of physical and chemical factors, or targeting genetic manipulation. The induced models are based on healthy animals, in which the pathological disorder to be examined is experimentally induced. Examples include partial hepatectomy to study liver regeneration, or studies of cow’s milk allergy in mice by immunizing them with tiny doses of cow milk protein (reviewed in: Hau, 2008). Animals can also carry out a mutation in certain, important genes and suffer from genetic diseases, similar to humans. A genetic disease is a disorder caused by an abnormality in an individual’s genome. The genetic disease may be caused by a mutation in a single gene but can be also caused by different factors such as a combination of environmental factors and mutations in multiple genes. The genetic disease could come also from chromosome alterations or mutations in the non-chromosomal DNA of mitochondria (Dubińska-Magiera et al., 2016; Plantié et al., 2015). Mutants are extremely rare in nature, therefore, the acceleration of research conducted on animal models of human diseases took place when mutagenesis techniques became available. Effective mutagenesis tools were needed to make genetic approaches functional and practical. The fact that X-rays could induce mutations in mice was known from 1923. Then in 1980 extensive, chemical mutagenesis studies were continued using the alkylating chemical called ENU (N-ethyl-N-nitrosourea). Next, in 1990, the human genome sequencing project began, aiming for the identification and mapping of all of the genes in the human genome. The project progress was unexpectedly fast and created an urgent need for knowledge regarding particular gene’s functions. Therefore, approximately in the same period, the idea of a large-scale mouse mutagenesis project emerged. The approach is based on large-scale, random mutagenesis using ENU. Then the changes in phenotype, such as disease symptoms, were observed. Finally, the genome of selected individuals was sequenced to find out which gene is responsible for a particular disorder. The similarity between mice and human genes is high enough to let us extrapolate the observations to use mice in diagnosis and medicine. Further progress in molecular methodology and molecular tools for directed mutagenesis (allowing for specific, directed mutation in a particular gene) led to the establishment of the International Mouse Phenotyping Consortium (IMPC) in 2006. Its goal is to fill the gaps in the knowledge by use of the reverse genetics approach. This procedure is called gen-driven mutagenesis, where the target gene is disrupted, and the results are drawn through the phenotypic analysis (reviewed in: Chenouard et al., 2021; Gondo et al., 2009) (Figure 14.2).

FIGURE 14.2
FIGURE 14.2

The key moments in mice mutagenesis. The aim of mutagenesis is to establish an animal model of human genetic diseases. The progress in genetic engineering starts from the discovery that X-rays can induce mutagenesis. Then the chemical compound ENU (N-ethyl-N-nitrosourea) was used to create mutations. With the technique development, many large-scale programs started in 1997, with the culmination point in 2007, when the International Knockout Mouse Consortium (IKMC) was established, with the aim to provide knockout and conditional mouse strains for all mouse genes. Such acceleration was possible because the whole mouse genome was sequenced in 2022. Starting from 2010 new techniques, allowing for precise mutagenesis were discovered. In the beginning, the ZNF s and TALENS were developed. Then, the CRISPR-Cas9 technique was shown to be a real scientific breakthrough, appreciated by the Nobel Prize awarded in 2020 to Emmanuelle Charpentier and Jennifer Doudna (according to: Chenouard et al., 2021; Gondo et al., 2009)

The way from primary, spontaneous animal models of human diseases to directed mutagenesis, allowing for the customization of genetic modifications, was difficult but successful. However, we should keep in mind that the biological interaction between genes and other biological factors is a very complex and multidimensional process. Therefore, detailed studies are needed to understand the whole picture of the genetic disease pathological process.

4 The Efforts toward Understanding the Disease’s Genetic Source

We, humans, share the vast majority of genes with animals such as mice, zebrafish, and even fruit flies. The similarities between human and mouse genomes allow us to assume that whenever candidate genes for human diseases are found, their homologs usually are present in, e.g., the mouse genome. On the other hand, we can mutate genes with unknown functions and observe the phenotype. Such an approach guides us to the discovery of genes, which disruption can lead to diseases. As an example, we can mention the research results published by (Dickinson et al., 2016) regarding the high-throughput discovery of novel developmental phenotypes. The scientists identified some disorders and changes in phenotypes in different developmental stages, which were the results of mutations in many previously uncharacterized genes (Groza et al., 2023; Muñoz-Fuentes et al., 2018). The effort of several international projects aiming to investigate the function of mouse genes by mutagenesis, and following molecular analysis and phenotyping, greatly expand our knowledge. This approach creates the possibility of using the mouse as a valuable organism to interpret the information coded in the human genome. For example, the phenotypic analysis shows that mutation in the transmembrane protein 132A gene, Tmem132a, leads, among others, to abnormalities in the animal spinal cord structure and narrower limbs (Rosenthal & Brown, 2007). Such information could make the identification of genetic disease sources easier. Also, can help in the therapy and treatment of such diseases, by creating accurate models of human disease for insightful and detailed studies.

5 Ways to Interpret the Myriad of Biological Data

To gain a wide perspective of the particular gene mutation consequences, we have to correlate the information obtained from experimental analysis with clinical data. Particularly taking into account the sequencing data from patient genomes. Such data become more and more available since nowadays whole exome and genome sequencing are often incorporated into personal health care. To achieve this goal, in the USA the Undiagnosed Disease Network (UDN) was established. The UDN brings together clinical and research data to learn more about genetic diseases, especially to help patients affected by unknown conditions. The data from patient genomes provides information regarding gene variants. The gene sequence can differ among populations because genes are mixed during sexual reproduction. Additionally, mutations are also a source of genetic variation. The interpretation of sequencing data, especially in the case of rare genetic variants, is difficult, because we do not know which individual gene or gene variant is responsible for disease. Particular gene variants can potentially lead to disease, and this possibility can be tested experimentally. Therefore, research institutes, such as the Model Organism Screening Center, use model organisms to check the pathogenicity of certain genetic variants identified in patients by the UDN.

Understanding the impact of gene variants would help us to increase the efficiency of diagnosis. To find a “good candidate gene for disease” the scientists have to combine and compare several sets of data, from different resources. This information can be gained from genetic data sets, but also molecular, morphological, and phenotypic data by the use of genetic animal models. The one of approaches called the MARRVEL (Model Organism Aggregated Resources for Rare Variant Exploration2) aims to combine public genetic resources to analyze and choose the gene variants, which most likely are the cause of certain human diseases. Such variants became a candidate for study in model organisms, for in-depth analysis of pathogenic mechanisms. To facilitate this process and gather all the data in one database, MARRVEL extracts data from clinical and scientific public databases and aligns the variants with model organism genetic sequences. Additional biological and experimental data can be gained from public databases regarding particular organisms. For example, FlyBase3 is the most popular database describing fruit fly molecular data, and ZFIN4 contains information concerning zebrafish.

The whole MARRVEL approach starts with a patient, who has a disease symptom with a potential genetic background. The results of Whole Exome Sequencing or Whole Genome Sequencing leads to a long list of candidate gene variants that can cause disease in this particular case. The data from different sources help to assess how likely a particular variant can be a reason for such a genetic disorder. Then the scientist checks if a particular variant has its homolog (counterpart) in the model organism. Sometimes, there is experimental data regarding this variant, which facilitates its assessment. Having such information, scientists are often able to prioritize the variants’ pathogenicity. If there is no data, gene variants can be introduced into the model organism by genetic engineering. The animal genetic model is observed, tested and analyzed with the hope of finding similar phenotypic features, to those observed in the patient. This validation can show if the variant is pathogenic or not. Further, it can help to find appropriate treatment.

6 How Can Fruit Fly Help You?

To visualize the importance of this huge international scientific effort, we can use the true story as an illustration. Let’s imagine a patient, a small girl, with severe disease symptoms, yet undiagnosed. Bristol was born as a healthy baby, but during the first three months, her parents noticed that her development was not as fast as it should be. Her body was floppy and flexible, she was not able to lift her head, and sometimes her left eye turned inwards. She was also delayed in further progress such as crawling, standing and walking. She cannot smile and seems to be not sensitive to pain. For example, she did not cry when she fell or get her vaccine shots. This girl was diagnosed with congenital hypotonia, a symptom that can be caused by different neurological or non-neurological conditions. To find a reason for Bristol hypotonia, for five years she underwent tests checking for cerebral palsy, Down syndrome, autism, muscular dystrophy and many others, all of which were negative. Finally, thanks to analysis within the Undiagnosed Disease Project, the doctors found two more children, a boy and a girl, with similar symptoms, who carried an identical point mutation in the Early B-Cell Factor 3 (EBF3)-coding gene. The EBF3 gene is responsible for the regulation of various other genes’ expression, therefore it is highly possible that its alterations can widely influence nervous and muscle systems. Therefore, the researchers decided to check the consequences of EBF3 gene mutation in the fruit fly model. The experimental outcomes lead to the conclusion that EBF3 is indeed responsible for disease symptoms observed in small patients. The additional 20 patients all around the world have been found to carry the mutation(s) in this gene, leading to the conclusion that it can be responsible for several cognitive or speech disorders, and hypotonia of unknown origin. The knowledge regarding the disease’s origin allows for easier diagnosis in the future saving families from the “diagnosis odyssey”: a long, uncertain and difficult journey through a variety of specialists and medical tests. Additionally, animal models can be used to test potentially therapeutic compounds leading to the discovery of efficient treatment (Dunlap, n.d.).

7 Animal Models Limitations

Animal models have great importance in the life sciences, however, these models are not devoid of disadvantages. There is no such animal model, which could mimic all features of human disease, simply because we are non-identical. The differences between species cause differences in phenotype, morphology, molecular or physiological pathways, etc. Therefore, it is important to choose the best animal to perform particular research to gain as informative outcome as possible. As mentioned before, the homology between human and animal genes is the key issue when you plan to model human genetic diseases. But other features are also important. The zebrafish are a very good model for drug screening, and phenotype-based drug discovery, pharmacology, and toxicology. Fishes are small, and produce a large number of eggs (one couple can produce 100 eggs, twice a week), which can be kept in small vessels. The transparent body at the early stages of development allows for easy, semi-automatic assessment of fish phenotype. Additionally, there is a growing number of available tools for phenotype assessment. However, the drugs or chemical compounds have to be water-soluble, and used in quite large quantities, since we have to immerse fish into the solution. Zebrafish embryos are protected by an envelope, called chorion, which keeps away many chemicals. Therefore, the experimental procedure requires additional steps, among others, special chemical treatment, to remove chorion. Moreover, the zebrafish development is external, which means that embryos do not have a placenta. Additionally, small animals are not good for monitoring biochemical parameters in blood, since taking samples from tiny organisms is very difficult or just not possible. Also, the drug doses tested in animals cannot be directly related to human physiology. Here we talk about zebrafish, which is only one example, but similar considerations have to be taken into account in every animal model and each experiment. Therefore, careful planning is crucial during the experiment designing and result interpretation (Dubińska-Magiera et al., 2021; Hartung, 2008; Migocka-Patrzałek & Elias, 2021; Robinson et al., 2019).

8 Ethical Issues

Experimentation on animals and ethical issues connected with such activity has been a topic of wide-world debate for decades. The necessity of conducting such experiments, which saves human lives, is undoubtful. However, the price is high because it causes animal distress. The discussion focusing on ethical issues leads scientists to establish rules, which allow them to avoid unnecessary experiments, and protect animals from suffering. In 1959 William Russell and Rex Burch described ethical guidelines, called the three R’s (3Rs). The “R” letters relate to the terms Replacement, Reduction, and Refinement – three main, important practices, which should be applied during planning and conducting experiments involving animals (Tannenbaum & Bennett, 2015).

The first “R” says that, if it is possible the researcher should replace the experiment on animals with another technique. For example, one can use cell cultures (in vitro experiments) for the initial assessment of particular chemical toxicity. Another example is replacing animals with the so-called “artificial skin” (derived from in vitro cultures) in cosmetics testing. The alternative to using animals is the growing potential of bioinformatics analysis. Such in silico analysis allows for the virtual modelling of certain biological processes before testing them with the use of animals. For instance, one can use the prediction of protein-protein interaction to choose the best experimental conditions and parameters.

The second “R” obliged the investigator to reduce the number of animals used in the experiment to a minimum. This approach can be achieved by careful planning, also by using computational tools to calculate the minimal number of individuals in tested groups. Another example of following the “reduction” rule, is obtaining as much data from tested animals as possible. Let’s imagine that we treat fish with a potentially therapeutic substance to observe overall phenotype. We can assess the changes using a variety of tests, for example, record a movie showing the swimming speed and pattern, make a photo of live fish to show its length and shape, and finally use the animal muscles for biochemical tests. In this way, we save at least two or three experimental groups. Additionally, we can use animal tissues, which were not used in experiments, for didactic purposes or other kinds of investigations and research.

The third “R” regards animal experiment refinement to avoid or ameliorate the animal suffering, distress and pain. This is an especially subtle issue because it is really hard to assess if any human intervention in animal natural life rhythms is harmless. Even tests taken as harmless, such as microscopic observations of live animal behaviour, introduce into their lives some additional stimuli such as different light, additional sounds or physical vibrations. All procedures, which could be painful, are carried out on appropriately anaesthetized animals (anaesthetic agents cause loss of feeling in all or part of the body, with or without loss of consciousness). But there are also several other ways to ameliorate the distress, that is. by maintaining stable, proper conditions in the breeding facility. It is also worth noting here, that the animals for research purposes are bred and maintained in proper, strictly controlled conditions. The animal facilities are obliged to keep rigid conditions such as, in the case of zebrafish, a certain quality of water and food, stable temperature, adequate aquatic systems, enough space and take into account many other important factors.

The 3Rs were stated to serve as a base for the development of future alternatives to the use of animals in research. Indeed, nowadays there are expanding range of options to replace experimentations on animals such as 3D cell cultures, computer modelling and bioinformatic tools (Andersen & Winter, 2019; Balls, 2020; Díaz et al., 2020; Petetta & Ciccocioppo, 2021; Robinson et al., 2019).

9 Conclusions

Simple organisms such as bacteria, yeasts, worms, and fruit flies are indispensable to gaining knowledge regarding human organisms and health. The model means something simpler than the original, which we used as an example. The fast development of alternative experimental systems will lead us to use in the future other than animal disease models, at least in some cases (Benam et al., 2015). Nevertheless, the use of animals as a model of the human body is of utmost importance in drug testing and medicine, simply because we, as mankind, are not skilled enough to imitate nature in a sufficient way. The simplest organism created by nature is far too complex to replace it fully with any artificial creation.

Abbreviations

ZNF s

zinc finger nucleases

TALEN s

transcription activator-like effector nucleases

CRISPR

clustered regularly interspaced short palindromic repeats

Cas9

caspase 9

Notes

2

https://marrvel.org/; retrieved October 6, 2021

3

https://flybase.org/; accessed June 24, 2024

4

https://zfin.org/; accessed June 24, 2024

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clustered regularly interspaced short palindromic repeatsClose
Cas9caspase 9Close
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  • Andersen, M. L., & Winter, L. M. F. (2019). Animal models in biological and biomedical research – experimental and ethical concerns. Anais Da Academia Brasileira de Ciencias, 91(suppl 1), e20170238.

    • Search Google Scholar
    • Export Citation
  • Balls, M. (2020). It’s time to reconsider the principles of humane experimental technique. Alternatives to Laboratory Animals: ATLA, 48(1), 4046.

    • Search Google Scholar
    • Export Citation
  • Benam, K. H., Dauth, S., Hassell, B., Herland, A., Jain, A., Jang, K.-J., Karalis, K., Kim, H. J., MacQueen, L., Mahmoodian, R., Musah, S., Torisawa, Y.-S., van der Meer, A. D., Villenave, R., Yadid, M., Parker, K. K., & Ingber, D. E. (2015). Engineered in vitro disease models. Annual Review of Pathology, 10, 195262.

    • Search Google Scholar
    • Export Citation
  • Burggren, W. W., & Warburton, S. (2007). Amphibians as animal models for laboratory research in physiology. ILAR Journal/National Research Council, Institute of Laboratory Animal Resources, 48(3), 260269.

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