ZEBRAFISH : AS A MODEL ORGANISM.

A presentation on Zebrafish's history, taxonomy , genetics, life cycle and future prospects of zebrafish and some of its medical implications in human life. Most importantly the major interest is to investigate those particular gene that are responsible for regenerating the heart in zebrafish so that they can be applied to human heart and help im regenerating human heart without the formation of any scar.

1. ZEBRAFISH: AS A MODEL ORGANISM PRESENTED BY GUIDED BY Namrata Singh Prof. ( Mrs ) Pravati Kumari Mahapatra M.Sc. Semester III P.G. Department of Zoology Roll number -15 ZOO 016 Utkal University, Vani Vihar Bhubaneswar- 751 004. 1

2. CONTENTS Introduction Taxonomy History Genetics of Zebrafish Uses as a model organism Future prospects Conclusion References. 2

3. INTRODUCTION Danio rerio , commomly known as Zebrafish is a tropical fresh water fish belonging to the minnow family. It has become another popular model organism to study fundamental biological questions. It is a small 1–1.5 inches fish that grows easily in aquaria. Fig. 01 Zebrafish. 3

4. TAXONOMY Kingdom: Animalia Phylum: Chordata Class: Actinopterygii Order: Cypriniformes Family: Cyprinidae Genus: Danio Species: rerio . 4

5. The Zebrafish is named for the five uniform, pigmented, horizontal, blue stripes on the side of the body, which are reminiscent of a Zebra's stripes, and which extend to the end of the caudal fin. It is laterally compressed with its mouth directed upwards. DESCRIPTION Fig.02 A Zebrafish showing arrangement of stripes. 5

6. The Zebrafish is native to the streams of the South-Eastern Himalayan region. The species arose in the Ganges region in eastern India and commonly inhabits streams, canals, ditches, ponds and slow-moving or stagnant  water bodies. Zebrafish have been introduced to parts of the United States. DISTRIBUTION 6

7. HISTORY The use of Zebrafish as a model organism was pioneered at the University of Oregon,U.S.A . by George Streisinger in 1970. He is the “Founder Father” of Zebrafish Developmental and Genetic Research. Photograph 01 George Streisinger . 7

8. In 1990, the first large scale mutagenesis of Zebrafish were conducted by Christiane Nusslein Volhard in Oxford University, United Kingdom to identify developmental mutations . Photograph 02 Christiane Nusslein Volhard . 8

9. Thomas Look of Dana-Farber Cancer Institute, Boston uses the translucent Zebrafish to study how cancer behaves in 1995. Photograph 03 Thomas Look. 9

10. FLOWN INTO SPACE On 22 nd July, 1976, the Space Station, Salyut 5 was launched in which Zebrafish was one of the crew members. Fig. 03 Salyut 5. 10

11. Fig.04 Anatomy of male Zebrafish. 11

12. Zygote Blastula Gastrula Hatching Larva Juvenile Adult Cleavage Segmentation Fig.05 Developmental stages of Zebrafish. 12

13. MODEL ORGANISM A model organism is a non-human species that has been widely studied, usually because it is easy to maintain and breed in a laboratory setting and has particular experimental advantages. They are used in the laboratory to help scientists understand biological processes. 13

14. ADVANTAGES Advantages of Zebrafish as a model organism: 1. Optically translucent embryos 2. Rapid hatching of eggs 3. Maintenance cost is significantly lower than those for mammals. 4. Amenable for molecular and genetic analysis. 5. As Zebrafish eggs are fertilized and develop outside the mother’s body it is an ideal model organism for studying early development. 14

15. GENETICS 70% of protein-coding human genes are related to genes found in the Zebrafish. 84% of genes known to be associated with human disease have a Zebrafish counterpart. They can be cloned from somatic cells. They can be made transgenic. As a vertebrate, the Zebrafish has the same major organs and tissues as humans. Their muscle, blood, kidney and eyes share many features with human systems. 15

16. ZEBRAFISH AS A MODEL IS USED TO STUDY Regeneration of heart Tail regeneration Retinal regeneration Human pigmentation Cancer research Autism. 16

17. HEART REGENERATION Mammals respond to a myocardial infarction by irreversible scar formation. By contrast, the Zebrafish are able to resolve the scar and to regenerate functional cardiac muscle. The reparative and regenerative process is achieved through Smad3-dependent TGF β signaling. 17

18. 7. During a heart attack, heart muscle cells are deprived of oxygen and they die, leaving scar tissue 8. Scientists are working to find out the specific factors involved in this process to see if this will help us to develop ways of repairing the heart in humans with heart failure or who have suffered heart attacks. 18

19. MECHANISM Zebrafish heart regeneration can be sub-divided into three overlapping phases: Inflammatory phase Reparative phase Regenerative phase. 19

20. INFLAMMATORY PHASE First, myocardial cell death triggers an inflammatory response that is characterized by the infiltration of the infarct with activated leukocytes and fibroblasts-like cells which express TGF β ligands Fig.06 Mechanism of inflammatory phase. 20

21. REPARATIVE PHASE Second as the wound becomes cleared of the dead cells and matrix debris, the reparative phase begins, TGF β stimulates the recruited fibroblast-like cells to synthesize collagen rich ECM. Fig. 07 Mechanism of reparative phase. 21

22. REGENERATIVE PHASE At the onset of scar formation, the regenerative phase begins. While the fibrotic tissue is still maturing TGF β signaling promotes proliferation of the cardiomyocytes located in the vicinity of the post-infarct. Fig.08 Mechanism of regenerative phase. 22

23. Tissue remodeling and invasion of new cardiac muscle to replace scar issue are associated with TGF β dependent expression of Tenascin -C, an extracellular protein with contra-adhesive properties. Thus, the regenerative phase is characterized by two opposing processes: 1. scar deposition in the damaged area 2. scar degradation at the position where new cardiomyocytes invade the post-infarct. 23

24. TAIL FIN REGENERATION 1. Zebrafish fins are complex appendages that quickly and reliably regenerate after amputation, restoring both size and shape. 2. The key regenerative units are their many rays of dermal bone, which are segmented and lined by osteoblasts . 3. An amputated fin ray is covered within the first several hours by epidermis, and within one to two days, a regeneration blastema forms. The blastema is a proliferative mass of morphologically similar cells, formed through disorganization and distal migration of fibroblasts and osteoblasts . 4. Blastema formation is the only one step in zebrafish tail fin regeneration. 5. Wnt signaling positively regulate blastemal proliferation and outgrowth. 24

25. Fig.09 Amputation of Zebrafish tail fin. 25

26. Fig. 10 Blastema formation in Zebrafish tail fin. 26

27. Fig.11 Wnt signaling regulating tail fin regeneration. 27

28. RETINAL REGENERATION Another notable characteristic of the Zebrafish is that it possesses four types of cone cells, with ultraviolet-sensitive cells supplementing the red, green and blue cone cell subtypes found in humans. Zebrafish can thus observe a very wide spectrum of colors. The species is also studied to better understand the development of the retina; in particular, how the cone cells of the retina become arranged into the so-called cone mosaic. 28

29. The researchers studied Müller   glial cells in the eyes of humans aged from 18 months to 91 years, and were able to develop them into all types of retinal neurons. The stem cells of Zebrafish successfully migrated into diseased rats' retinas, and took on the characteristics of the surrounding neurons. The team stated that they intended to develop the same approach in humans. Stem cells from Zebrafish, the staple of genetic research, could regenerate damaged cones in retinas and restore eyesight to people. Fig.12 Regeneration of retina. 29

30. Zebrafish solves the mystery of our skin color. Embryonic patterning of pigmentation in D. rerio is highly investigated area of study. SLC24A5 is shared both by humans and Zebrafish and makes melanosomes less abundant, less concentrated and smaller in lighter skinned humans or light-striped Zebrafish . HUMAN PIGMENTATION 30

31. While some researchers were studying Zebrafish to find cancer genes, they found that pigment cells of Zebrafish looked similar to pigment cells in light-skinned humans. Since then, many researchers have investigated the Zebrafish gene responsible for different colors in stripes. In 2005, some studies found the human version of the gene, which affected Europeans differently from Africans and Asians. Fig.13 Location of pigment cells in Zebrafish embryo. 31

32. At embryonic and early larva stage, the neural crest cells gets differentiated into three cells displaying alternating stripes: 1. Melanocytes (blue) 2. Iridiophores (silver) 3. Xanthophores (yellow). Further study in Zebrafish pigment cells is essential, since they provide many opportunities to learn the nature of the human skin color. Fig.14 Patterns of Zebrafish. 32

33. 1. Zebrafish have been used to make several transgenic models of cancer, including melanoma, leukemia, pancreatic cancer, colon cancer. 2. Researchers have created a model of cancer in Zebrafish that allows them to capture live images of tumors forming and growing. IN CANCER RESEARCH Fig.15 Tumor in Zebrafish. 33

34. STAINING THE CELL In Zebrafish, the endothelial cells are stained with a fluorescent protein. The fluorescent stained tumor cells are highlighted in the transparent Zebrafish embryos and larvae. Now, the process of metasizing tumor cells can be accurately tracked at cellular level. 34

35. XENOTRANSPLANTATION AND METASTASIS During zebrafish development, a cluster of neural cells called the "posterior lateral line primordium " ( PLLp ) mimics the behavior of metastasizing human cancer. The cluster of zebrafish neural cells travels the entire length of the embryo, driven by the same molecular pathways that drive human cancer cells to new sites in the body. So, if we can find drugs that block it then we can also find drugs that potentially block cancer metastasis in humans as well. Fig. 16 Transplantation and metastasis. 35

36. DISCOVERY OF A DRUG Rosuvastatin prevents the spreading of cancerous cells in Zebrafish from one part to another. The same drug was then tested on human cancer cells, and it had the same migration-stopping effect on both leukemia and pancreatic cancer cells. Fig. 17 3D Structure of Rosuvastatin . 36

37. AS A MODEL TO STUDY AUTISM Zebrafish model gives new insight on autism spectrum disorder. A team of researchers at Massachusetts Institute of Technology focused on genes that have been found to be either missing or copied in about 1% of patients with autism. Their study revealed that when they deleted these genes from Zebrafish embryos, nearly all the fish developed brain abnormalities. It is found that estrogens, the primary female sex hormone, could reverse abnormal behavior in Zebrafish carrying a mutation in CNTNAP2, a gene linked to genes of autism in humans,SYNGAP1 and SHANK3. The researchers found that Zebrafish carrying the CNTNAP2 mutation are more prone to seizures than Zebrafish without the mutation. 37

38. Fig.18 Zebraboxes . 38

39. Genome sequencing The  Wellcome Trust Sanger Institute, U.K.   was the first to start the Zebrafish Genome Sequencing Project. In 2009 , Institute of Genomics & Integrative Biology , New Delhi reported sequenced genes in Zebrafish. The paper “ The zebrafish reference genome sequence and its relationship to human genome ” was published in Nature in 17 th April, 2013. Its genome (1.4 x 10 9  base pairs) has been sequenced revealing 26,606 protein-coding genes. The Zebrafish genome has been fully sequenced to a very high quality. This has enabled scientists to create mutation in more than 14,000 genes to study their function. 39

40. GloFish The  GloFish  is a patented  brand of genetically modified fluorescent fish. Different varieties of GloFish are currently available in the market. Zebrafish were the first GloFish available in pet stores. These fish are a valuable tool for examining erythrocyte development and circulation defects in the developing embryo. Fig.19 GloFish in aquarium. 40

41. GENE EXPRESSION Due to their short lifecycles and relatively large clutch sizes, Zebrafish are a useful model for genetic studies. Morpholino  antisense technology reduces gene expression. Morpholino   oligonucleotides (MO) are stable, synthetic macromolecules that contain the same bases as DNA or RNA; by binding to complementary RNA sequences, they can reduce the expression of specific genes or block other processes from occurring on RNA. 41

42. DRUG DISCOVERY As demonstrated through ongoing research programmes , the Zebrafish model enables researchers not only to identify genes that might underlie human disease, but also to develop novel therapeutic agents in drug discovery programmes . 42

43. ENVIRONMENTAL MONITORING The researchers cloned estrogen-sensitive genes and injected them into the fertile eggs of Zebrafish. The modified fish turned green if placed into water that was polluted by estrogen. 43

44. ZEBRAFISH WEBSITES The Zebrafish Server The Fishnet ZFIN- Zebrafish Information Network. 44

45. Zebrafish Labs in India Institute of Genomics and Integrative Biology (New Delhi) Center for Cellular and Molecular Biology (Hyderabad) Indian Institute of Sciences (Bangalore) Tata Institute of Fundamental Research (Mumbai) Institute of Life Sciences (Hyderabad) National Centre for Radio Astrophysics ( Pune ). 45

46. FUTURE PROSPECTS As Zebrafish are now being used in virtually all disciplines, from neurogenesis to oncogenesis , from behavior to genetics, its popularity is steadily increasing. The emergence of Zebrafish Conferences and the increasing presence of Zebrafish studies at large annual meetings are also boosting the use of Zebrafish as an appropriate model organism. 46

47. CONCLUSION It is a tiny fish with a big splash. 47

48. REFERENCES Chablais F, Jazwinska A (2012) The regenerative capacity of the zebrafish heart is dependent on TGF β signaling. Development and Stem Cell, 139(10 ): 1921-1930 . Gilbert SF(2010) Developmental Biology. 9 th Edition. Sinauer Associates, Inc. Sunderland, Massachusetts, USA, pp 323- 332. Hsu CH, Wen ZH, Lin CS, Chakraborty C (2007) The zebrafish model: use in studying cellular mechanism for a spectrum of clinical disease entities. current neurovascular reseasrch , 4(2 ): 111-120 . Slack JMW (2006) Essential Developmental Biology. 2 nd Edition. Blackwell Publishing Ltd., Malden, USA, pp 61-89. 48

49. THANKYOU 49