Zebrafish in genome research Can you spot the difference?

What is a zebrafish? Danio rerio Small freshwater fish from South Asia. 4 cm long when fully grown. Common aquarium fish. Very easy to look after. The zebrafish is a small freshwater fish which is no bigger than 4 cm in length. It is naturally found in streams in the South Eastern Himalayan region which includes countries such as India, Pakistan and Bangladesh. It is now a popular aquarium fish and you are very likely to see one in your local garden centre or pet shop. They are popular pets as they are very robust and easy to look after in a small aquarium tank. Due to their robust nature zebrafish are also a key model organism used in scientific research. Image: Wikimedia commons/Marribio2

What is a model organism? Non-human species widely studied to understand human disease. Model organisms are used when experimentation using humans is unfeasible or unethical. Can you think of a model organism? The term model organism is used to describe any non-human species that is used in scientific research to better understand the causes and potential treatments of human diseases. Model organisms are used when experiments using humans would be considered unfeasible or unethical. There are many different model organisms used in scientific research. These include species such as E. coli bacteria, thale cress, Drosophila fruit flies, chickens, mice, rats, takifugu pufferfish and, of course, the zebrafish.

Types of model organism Genetic model organisms Experimental model organisms Genomic model organisms Good candidates for genetic analysis. Good candidates for research into developmental biology. Good candidates for genome research. Breed in large numbers. Have short generation times so large scale crosses can be followed over several generations. Produce robust embryos that can be easily manipulated and studied. Easy to manage genomes e.g. small genome size or limited number of repeats. Genome is similar to a human. Note this slide is animated Broadly speaking there are three main types of model organism: genetic models, experimental models and genomic models. Each has slightly different characteristics and functions. Genetic model organisms are species that are easy to use for genetic analysis studies. They tend to breed quickly and in large numbers. Question to students: Can you think of any organisms (invertebrate or otherwise) that would be good genetic model organisms? Click to reveal answers Examples include Baker’s yeast (Saccharomyces cerevisiae) which is shown in yellow, the fruit fly (Drosophila melanogaster) and a nematode worm, Caenorhabditis elegans. Experimental model organisms are used widely in research into developmental biology. Species used for this purpose tend to produce robust embryos that can be easily manipulated and studied. Question to students: Can you think of any vertebrate organisms that would be good experimental model organisms? Click to reveal A good example of a model organism used in developmental biology is the African clawed frog (Xenopus laevis). Chickens can also be used. Genomic model organisms are used particularly in genome research. Species used for this purpose tend to either have manageable genome sizes and/or have a genome that has similarities with the human genome. When we talk about genomic similarities, remember that 60 per cent of identified human disease genes have similar counterparts in the fly and worm, which tells us that there is a core of about 1,500 gene families that are conserved in all animals. Question to students: Can you think of any vertebrate organisms that would be good genomic model organisms? Good examples of model organisms used in genome research are the zebrafish and mouse. Mice are used in particular for research into genes associated with more evolutionarily advanced features, such as our immune system. This is because these type of gene families are less likely to have direct counterparts in simple animal systems such as invertebrates. Images: Wellcome Trust Sanger Institute

Why use zebrafish? Small size. All major organs present within 5 days post fertilisation. Short generation time (3-4 months). Produces 300-400 eggs every 2 weeks. Translucent embryos. Lots of genome resources available. There are several reasons why the zebrafish is used as a model organism: It is small and therefore does not require large amounts of space. It develops quickly; all of the five major organs are present five days post fertilisation. It has a short generation time with sexual maturity being reached when fish are 3-4 months old. They produce a lot of eggs; a single female can produce over 300 eggs every two weeks. The eggs are fertilised externally and the embryos develop outside of the female (ex utero) making them easy to study. Zebrafish embryos are translucent enabling researchers to watch the tissues develop under a light microscope. There are a lot of zebrafish genome resources available to support studies. This includes the zebrafish reference genome sequence and a database of information on zebrafish mutants called the Zebrafish Model Organism Database or ZFIN. Image: TBC

The zebrafish embryo brain ear muscle block segments swim bladder eye heart notochord This image shows a zebrafish embryo that is five days old. Some of the major tissues, organs and structural features have been labelled. This image shows some of the major morphological features of a zebrafish that is five days old. You can see it has some of the same major organs and body parts as a human, e.g. brain, eyes, ears, heart, muscle. You may wonder why fish have ears? They have ears for the same reasons as we have ears; to hear and control balance. They use their hearing to detect potential predators. The swim bladder is a common feature of fish that is not found in humans. It helps the fish to control its buoyancy, so it can move up and down in the water. Much like a buoyancy control device or BCD is used by SCUBA divers. The notochord is a central support structure in the embryo that later develops into the backbone of the animal. ~3.5 mm

Zebrafish and human disease Zebrafish mutants have been produced to model human diseases such as: Alzheimer's disease congenital heart disease polycystic kidney disease Duchenne muscular dystrophy malignant melanoma leukaemia Zebrafish models have been used to investigate a range of different human diseases and disorders, some inherited and some acquired. Zebrafish can also be used to test candidate drugs for these diseases making them a valuable tool in the development of treatments for human diseases.

Forward screening for mutants P ENU-treated male +/+ female x F1 +/M +/+ x F2 +/+ (50%) +/M (50%) x This slide can be used to discuss how you generate a zebrafish mutant, if you wish to discuss this with the students. If you do not wish to use this slide, you can right click on the slide and select hide slide, so it does not appear in the presentation. This diagram shows how researchers generate zebrafish mutants using a process known as forward screening. The first stage in the process is to treat adult male zebrafish with a chemical known as ENU (ethylnitrosourea). The fish are immersed in water containing ENU which generates point mutations in the DNA of the zebrafish sperm. The next stage is to mate (or cross) the ENU-treated males with wild type females (+/+). This produces the F1 generation. The F1 generation are heterozygous, shown as +/M in the diagram. These heterozygous fish are then mated with wildtype fish to create the F2 families where 50% of the siblings are heterozygous for a specific mutation (+/M) and the other 50% are wild type (+/+). F2 siblings that are heterozygous for the mutation (+/M) are mated to produce the F3 generation. The resulting clutch contains 25% wildtype fish, 50% heterozygous fish (+/M or M/+) and 25% homozygous fish with a specific mutant phenotype, shown as (M/M). This 25% will show the consequences of the mutation. These can sometimes be obvious physical characteristics such as shorter fins, abnormal development of organs and even different behaviours. Screening for mutations can be aided by the use of green fluorescent protein (GFP) to label proteins so that when they are expressed they glow green and can be easily visualised under a microscope. Forward genetic screens can be used to identify genes associated with human diseases. The phenotypes displayed in the F3 mutants are genetically analysed and disease models can be identified by their relatedness to human conditions. For example, a zebrafish known as pickwick mutant has weakened heart muscles and a dysfunctional heart. This has now been linked to a large gene region known as titin, or TTN, which is associated with familial dilated cardiomyopathy, an inherited heart condition in humans. F3 +/+ (25%) +/M (50%) M/M (25%)

Reverse screening for mutants Potential human disease gene Exciting gene expression pattern Gene of interest Potential new player in developmental pathway Gene knockout Phenotype analysis Note this slide is animated This slide can be used to discuss how you generate a zebrafish mutant using reverse screening, if you wish to discuss this with the students. If you do not wish to use this slide, you can right click on the slide and select hide slide, so it does not appear in the presentation. Reverse screening for mutants is another approach to generating zebrafish mutants. This process differs from forward screening. First it starts with a gene of interest. Click to reveal. The gene could be of interest because it has an exciting gene expression pattern. It may be that it is thought to be associated with a human disease or it may have been identified in an important developmental pathway. Click to reveal Once you have identified a gene of interest you generate a mutant that has had that specific gene “knocked out”. This is then mated to produce heterozygotes and homozygotes of the disease gene. The phenotype of the mutant is then assessed to identify any similarities to the human disease. Reverse genetic screening has been used to generate zebrafish that have mutations in the tumour suppressor gene, P53.

The activity Identify differences between the wildtype zebrafish and mutant zebrafish. A glossary is provided to help you with scientific terms. In this activity you are going to carry out a phenotype analysis. In other words, you are going to spot the difference between a wild type and mutant zebrafish and identify the phenotype, or visual characteristic. You will be given images of embryos, adult fish and areas of tissue. When you have identified the difference, complete the relevant section of your worksheet. You have a glossary to help you diagnose the phenotype and understand any scientific terms you may be unfamiliar with. The glossary may provide clues to what you’re looking for in the phenotype! Image: Rodrigo Young, University College London

Flash cards & worksheets These are the tools you will require to complete this activity. You can either work in pairs or on your own.

Answers

Image 1 What’s the difference? Embryo B has no eye. Image 1 shows an eyeless mutant that is linked to eye development What’s the difference? Embryo B has no eye. Why is this relevant to us? Zebrafish and humans have a network of proteins called the wnt pathway that are associated with embryo formation and development. Eyeless embryos, such as the one shown in the picture, can occur through mutations in genes that encode the proteins in the wnt pathway. Identifying the genes involved in these pathways can aid our understanding of the role of the wnt pathway in early brain and eye development. Embryo B has no eye. Image: Rodrigo Young, University College London

Image 2 What’s the difference? Image 2 show a pigment mutant that is linked to skin colour. What’s the difference? Fish B is a lighter, golden colour compared to fish A. Why is this relevant to us? Lighter skin colour in humans is linked to a reduced number, size and density of melanosomes. These are structures found in skin cells that contain a dark pigment called melanin. These are shown in the boxes. People with darker skin have lots of large and tightly packed melanosomes in their skin cells. Can you see the difference between the melanosomes in the darker striped fish and golden fish in the picture? This difference in colour has been linked to a gene called SLC24A5 , which is present in both zebrafish and humans. The gene encodes a protein that has a major influence on natural skin colour variation. It is also thought to have played a key role in the evolution of light skin in humans of European ancestry. In 2005 a one base pair change in the gene was found to be the major difference between the pale skin colour of Europeans and the darker skin colour of Africans. Fish B is a lighter, golden colour compared to fish A. Image: Keith C. Cheng, Penn State College of Medicine and Wellcome images

Image 3 What’s the difference? Image 3 shows a nebulin mutation which is linked to congenital nemaline myopathy. What’s the difference? The picture shows two young zebrafish, known as fry. The body of fry B is curved. If you look closely you’ll also see that its mouth is open. This is because it is unable to fully close its mouth as its muscles are too weak. Why is this relevant to us? Zebrafish fry B is showing symptoms of a condition called nemaline myopathy. This is a genetic disease where muscles fibres do not form and function properly. This leads to poor muscle tone and weak muscles. Children with this condition usually have problems with breathing and feeding as their muscles are not strong enough to support the body. This can also lead to skeletal problems such as curvature of the spine (scoliosis). Genes that are associated with this condition are present in both zebrafish and human genomes. Observing the physical and genetic changes in zebrafish with nemaline myopathy can help us understand more about the human condition and potentially lead to new and better treatments for the disease.   The body of fry B is curved. If you look closely you’ll also see that its mouth is open. This is because it is unable to fully close its mouth as its muscles are too weak. Image: Elisabeth Busch, Wellcome Trust Sanger Institute and Lehtokari et al 2008, European Journal of Human Genetics

Image 4 What’s the difference? Image 4 shows mutants that cannot form red blood cells. What’s the difference The zebrafish embryos in picture B look paler and are not stained red. These embryos have been stained for the presence of haemoglobin, the protein found inside red blood cells that carries oxygen. Picture A shows normal embryos containing red blood cells with haemoglobin. The embryos in picture B have been injected with a substance that blocks a gene important for red blood cell formation. As a result these embryos are exhibiting severe anaemia with very few mature red blood cells. This is why they do not stain red for haemoglobin. Why is this relevant ? Red blood cell formation and iron uptake has been found to be linked to a gene called ARGHGEF3 that is found in both humans and zebrafish. This gene encodes an exchange factor which activates other genes along a cell signaling pathway. When the function of this gene and its target genes is blocked it leads to a severe decrease in mature red blood cells. This type of research can give us a better understanding of the function of the genes and the roles they play in iron metabolism and the development of red blood cells and blood vessels.   The zebrafish embryos in picture B look paler and are not stained red. Image: Ana Cvejic, Wellcome Trust Sanger Institute

Image 5 What’s the difference? Image 5 shows a section of muscle block exhibiting Nebulin deficiency What’s the difference? There are bright green blobs in picture B. These pictures show a close up of muscle fibres in two zebrafish. The muscle fibres attach to the skeleton of the fish via cartilage-like structures called myotendinous junctions. In both pictures the muscle has been stained green to highlight a muscle protein called actin. Picture A shows normal muscle fibre arrangement so this fish would be able to swim normally. Picture B shows a build up of clumps of actin in the areas where the muscle attaches to tendons. This fish would not be able to swim well as its muscle would be a lot weaker and unable to function properly. Why is this relevant The build-up of actin in the muscle block is one of the features of a zebrafish that lacks nebulin, a protein that binds to actin. Nebulin deficiency is associated with the genetic condition nemaline myopathy, a disease that leads to muscle weakness. Understanding more about the genetic changes involved in nemaline myopathy may eventually lead to better treatment of the disease in humans. There are bright green blobs in picture B. Image: Elisabeth Busch, Wellcome Trust Sanger Institute

Image 6 What’s the difference? Image 6 shows neutrophil migration (wound inflammatory response) in a cut zebrafish fin What’s the difference? Embryo A has more blue dots than embryo B. The image shows a close up of the fins of two zebrafish embryos. Both embryos have had a small nick cut into their fin. The blue dots are neutrophils (a type of white blood cell involved in the response to injury) that have been stained with a dye. The wound on embryo A (wildtype) has a lot more neutrophils than the wound in embryo B (mutant). This is because the WASp gene has been “knocked down” in embryo B. This means gene expression was inhibited by a chemical and the protein product Wiskott-Aldrich syndrome protein (WASp) was not produced. The result of this gene knock down is reduced movement of neutrophils to the wound site. This suggests the WASp gene and its protein product play a role in the navigation of neutrophils towards chemical cues coming from wound sites or sites of infection. This is an essential part of the body’s immune response. Why is this relevant to us? The Wiskott-Aldrich syndrome protein (WASp) is associated with the X-linked immunodeficiency disease, Wiskott-Aldrich syndrome. This is a severe human disorder which is characterised by regular infections, eczema and a low platelet count. This condition can cause death through severe infections or haemorrhaging. Understanding the mechanisms of the WASp gene can help researchers understand more about the body’s immune response and identify the best treatments for Wiskott-Aldrich syndrome as well other immune and inflammatory diseases. Embryo A has more blue dots than embryo B. The blue dots are stained neutrophils moving towards a wound on the zebrafish fin. Image: Ana Cvejic, Wellcome Trust Sanger Institute