Mr. Rakesh Sharma. M, M.Sc., in Applied Microbiology, Research Assistant with seven years of experience in Central Inter- Disciplinary Research Facility (CIDRF), SBV - Puducherry has joined as Research Associate in Mahatma Gandhi Medical Preclinical Research Centre (MGMPRC). A talk by him on “Zebrafish as an animal model for biomedical research” is scheduled on 19th November, 2022 (Saturday) at 2.30 pm in A1 conference hall, 1st floor Hospital block, MGMCRI.”

1. Mahatma Gandhi Medical Advanced Research Institute (MGMARI)
Mahatma Gandhi Medical Preclinical Research Center (MGMPRC)
Sri Balaji Vidyapeeth (Deemed to be University), Puducherry, 607402, India.
Tribute to Christiane Nüsslein-Volhard
Noble prize in Physiology

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THE
NOBLE
PRIZE
CHRISTIANE
NÜSSLEIN-VOLHARD
Nobel Prize in Physiology or Medicine 1995
https://youtu.be/d1rQvbahaPA

3. Christiane Nüsslein-Volhard - Genes in a fertilised egg form
an embryo that were responsible for the early
stages of the developmental program (embryogenesis) the
quest to understand nature – is a creative act.
1995 Nobel Prize for Physiology or Medicine
with geneticists Eric F. Wieschaus and Edward B. Lewis
Making of a scientist
nursing course.
Graduate at Frankfurt University, (molecular biology)
University of Tubingen (Biochemistry)
Promoter regions of phages, obtaining a Ph.D. in 1974.
3
(Born October 20, 1942, Magdeburg,
Germany)

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Christiane Nüsslein-Volhard and Eric
Wieschaus in EMBL, ca. 1980
Invented saturation mutagenesis
Mutations in adult fly genes in
order to observe the impact on
offspring.
Identified 20,000 genes in
the chromosomes of fruit flies.
“I immediately loved working with flies. They fascinated me, and followed
me around in my dreams”.

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Drosophila larvae, in a figure from
Christiane Nüsslein-Volhard Nobel
Lecture.
The Drosophila or fruit
fly, is a popular subject
for biological research
because their embryos
develop extremely
rapidly.

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“Creativity is combining facts no one else has connected before”.
Following her award, Nüsslein-Volhard expanded her research
beyond Drosophila to vertebrates.
Zebrafish were ideal subjects for genetic and developmental studies
due to their rudimentary spinal cords, transparent and rapidly
growing embryos and – perhaps most importantly – similarities with
mammals.
Christiane Nüsslein-Volhard with
zebrafish at the Max-Planck
Institute
“Initially, I was just struck by the beauty of the fish, like I had been by the
segmentation pattern of flies: it’s always nicer to work on something you
find beautiful.”

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Christiane Nüsslein-Volhard
at 'Zebrafish development
and genetics meeting', 1996
“When you look at animals like peacocks or birds of paradise, some
evolutionary biologists have argued that they grow such
extraordinary feathers to prove that they're strong enough to carry
them around, or whatever, and of course that's rubbish. It's about
beauty.”
Christiane Nüsslein-Volhard studies the genes
that control the development of embryos.

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Kingdom Animalia
Phylum Chordata
Class Actinopterygii
Order Cypriniformes
Family Cyprinidae
Genus Danio
Species rerio
Taxonomy

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History in Biomedical Research
Francis Hamilton, a surgeon from British East India company, established in West
Bengal, India, in the early XIX century.
Fishes found in the river Ganges and its branches in 1822, (10 copies of the
zebrafish)
Initially it was called Brachydanio rerio until its transfer to the genre Danio rerio.
Development and embryology
In 1934, Charles Creaser - handling of the zebrafish and emphasizing the ease of
studies of this species.

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George Streisinger - zebrafish in the late 1960s.
1981, Streisinger at the University of Oregon, - apply a mutational analysis in
embryonic development, producing haploid embryos from eggs treated with
ultraviolet radiation.
Not only in the development mutagenesis embryonic, as well as molecular genetic
later neuroscience, toxicology, tissue regeneration, among other areas of interest.
Choose the zebrafish as experimental animals - need to move closer to human
models;
In addition, the need for models for genetics that were of small size, rapid
reproduction, and wide progeny (MEUNIER, 2012).

11. 11
Introduction
One of the most popular animal models in research is the
Zebrafish, a tropical fish which is native to the rice paddies of Asia.
Use of animal models within the field of medical research.
“Danio rerio”. striped bodies resemble that of a Zebra.
The stripes are navy and horizontal, running along the length of
the body from the caudal fin to their gills.
First used in medical research by George Streisinger in the 1960s.
Successfully managed to clone a Zebrafish
The Remarkable Life of
George Streisinger (1927–
1984)

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Silvery white - 5 horizontal lines (blue in color) extending to the caudal fin.
Upper (dorsal) yellow in color - Zebrafish to appear more golden.
The dark blue melanophores - blue stripes Iridescent iridophores
Gold xanthophores –pale yellow color of the dorsal and belly
Choi TY, Kim JH, Ko DH, Kim CH, Hwang JS, Ahn S, Kim SY, Kim CD, Lee JH, Yoon TJ. Zebrafish as a new model for
phenotype‐based screening of melanogenic regulatory compounds. Pigment Cell Research. 2007 Apr;20(2):120-7.

13. Zebrafish ecology

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Zebrafish Development: Embryo and Larvae
Embryogenesis of zebrafish can be divided into eight periods:
1. Zygote - Newly fertilized egg in the first zygotic cycle
2. Cleavage - ¾ of an hour, cell cycles 2-7 occur rapidly
3. Blastula 2 ¼ hours, simultaneous cell cycles 8 and 9, lengthening and epibolic movement
begins
4. Gastrula - 5 ¼ hours, morphogenesis, convergence and extension
5. Segmentation - 10 hours, neuromeres develop, organogenesis, tail develops and first
movements
6. Pharyngula - 24 hours, straightening, circulation and pigmentation occur, fins develop
7. Hatching periods - 48 hours, rapid morphogenesis of organs, cartilage develops in the
head
8. Larval stage - 70 hours, swim bladder inflates, food seeking and avoidance behaviors begin

15. Life cycle of Zebrafish
https://youtu.be/7uBWsIUbRqY

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70 % of human genes have at least one zebrafish
orthlogues

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Pharmacology and Toxicology Research
Social Development and Behavioral Research
Learning and Memory Research
Vision and Retina Research
Stress and Anxiety Research
What Types of Research are Zebrafish Useful For?

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The scientists were surprised to find that the intestinal defects in zebrafish were
almost identical to humans
Microvillus inclusion disease model
Dr. Mahendra Sonawane
Tata Institute of Fundamental
Research (TIFR)

22. Heart from this little fish
CCMB - Hox genes (the body’s axis while the embryo is formed).
Dr. Tressa Jacob,
Indian Institute of Science Education and Research (IISER), Pune

23. Zebrafish clue to microplastic health hazard, say scientists 23
Embryos exposed to pollutant show heart defects: Study
Scientists say concentration levels of microplastic would likely determine their biological
effects.

24. Teenage Mutant Zebrafish: Scales Transform the Skin as Fish Grow
Into Adulthood
24
The Skin and Nervous System
Carry Out Touch Sensation
Diagram of the cells required for touch sensation.
The Skin Changes From Birth to
Adulthood
The skin and touch-sensing nervous system change as
newborn animals grow into adults.

25. 25
Scales Promote Changes in the Skin?
Scales send a local signal to transform epidermal cells and touch-sensing
neurons.

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Gnotobiotic zebrafish model for study of host microbiota interaction

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Invertebrate System

28. 28

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30. 30

31. 31

32. Why are Zebrafish a Good Animal Model Organism?
32
Study of human diseases
Surprising number of common features.
Zebrafish have orthologs or counterparts in 80% of human disease-
associated genes.
Genetic disease, early Drug Discovery, and toxicology research in thousands
of studies

33. 33
Zebrafish Research: Everything You Need to Know?
Zebrafish hold a number of advantages over other
organisms in today’s stringent research environment
Zebrafish biology
Leading candidate for multiple fields of study.
Zebrafish Anatomy: What’s so special about zebrafish?
Anatomical similarities with humans
Zebrafish share most major organs with humans

34. 34
Fast Development Process
Produces - hundreds of eggs per spawning - fertilized externally.
Zygotes development - 72 hours - cell division occurring.
Development within 48 to 72 hours, depending on the thickness of the
chorion.
3 days - larval stage - develop anatomical structures.
6 days - swimming, and en start feeding themselves independently.
Lends itself well to research.

35. Fluorescent and Transparent Zebrafish 35
Fluorescent proteins - processes or reactions -
microscopy research.
Green fluorescent proteins (GFP) - create chimeric
proteins - expressed in cells, tissues, and whole
organisms.
They are transparent - all organs and
musculoskeletal structures six days after fertilization.
Transparent embryos - observe organs or tissues as
they develop.
Dozens of transgenic zebrafish lines - created -
express fluorescent proteins in organs, glands, and
other bodily structures.

36. Zebrafish as an In Vivo Model 36
Zebrafish offer a host of advantages over other in vivo models
greatly increases sample size
Real-time and non-invasive in vivo zebrafish research, in which zebrafish serve as
animal models, have been popular for decades,
oGenetics
oCell biology
oEnvironmental toxicology
oEmbryology
oPharmaceutical toxicology

37. Cost-Effective Solution for Drug Discovery
37
cost efficiency
zebrafish are extremely affordable
zebrafish are well suited to the task of this High-Content Screening.

38. Zebrafish and the 3Rs: Reducing Animal Experiments 38
Zebrafish are also a great option for following the 3Rs of animal research.
The 3Rs—Replacement, Reduction and Refinement—aim to greatly reduce
animal experiments and suffering overall during research.
Zebrafish provide a viable alternative and act as a bridge between in
vivo and in vitro methods.

39. Toxicity Assays for Drug Discovery Using Zebrafish 39
Toxicity assays using zebrafish include:
oDevelopmental / embryo toxicity
oCardiovascular toxicity
oHepatotoxicity
oNeurotoxicity
oNephrotoxicity
oOcular toxicity
oAnd others

40. Efficacy Assays for Drug Discovery using Zebrafish 40
Efficacy assays have been developed in a range of therapeutic areas
Cancer - Angiogenesis Inhibition
Neurodegenerative and Rare Diseases
Tissue Regeneration

41. Zebrafish for Pharma Research - Develop possible therapeutic compounds at a
faster and more cost-effective rate using zebrafish.
Zebrafish for Agrochemical Research - Toxicity research concerning pesticides
such as herbicides, fungicides, bactericides and insecticides.
Zebrafish for Cosmetic Research - Embryonic and early stage zebrafish, again,
offer a viable alternative in this field.
41

42. Zebrafish Facts 42
How Many Cells Does a Zebrafish Embryo Have?
3 hours of fertilization and about 2,000 cells at around 5 hours. Within 48 hours the
embryos are in the prime stages.
Zebrafish Embryo Size
A zebrafish embryo - 0.7 mm in diameter and grows to roughly 3.5 mm by the time of
hatching.
Zebrafish Embryo Injection
To inject test compounds, dyes, plasmids or RNA for transgenic or mutation generations
into zebrafish embryos.

43. Zebrafish Model Organism in Preclinical Research 43
How they can be utilized in Preclinical Research.
Zebrafish have become an ideal candidate for preclinical research.
In Vivo vs In Vitro vs Ex-Vivo
Ex vivo models are:
More complex in cell diversity
Closer to in vivo models
Less reactive to stresses
In vitro models are:
Less complex in cell diversity
More reactive to stresses

44. 44
Phenotypic Screening
Zebrafish have now become a favored model for large scale in vivo phenotypic
screenings
These can be categorized into four major types of assay output:
Morphological,
Therapeutic,
Pathway,
Behavioral.
Drug Delivery
Easily observed in transparent zebrafish embryos.

45. 45
Zebrafish Model Organism in Early Drug Research
Early Drug Discovery.
Different stages where Zebrafish are crucial in Early Drug Discovery.

46. Investigating Developmental Mechanisms via Targeted Protein Degradation Shiv Nadar University, Delhi NCR
Evaluation of Embryotoxicity, genotoxicity and anticlastogenic activity of
Swertiamarin in zebrafish embryos and mice.
JSS College of Pharmacy, Mysore
A zebrafish model to characterize VHL-HIF signaling in tumorigenesis SRM Institute of Science and
Technology, Kattankulathur
Deciphering the role of selective N-glycosyl modification in fate biasing of
melanocytes from Neural crest cells
CSIR Institute of Genomics and
Integrative Biology, New Delhi
Investigating the role of temporally regulated endocytosis in developing zebrafish
epidermis
Tata Institute of Fundamental
Research, Mumbai
Zebrafish Ovary: A Model to study Intra-ovarian Regulatory Mechanisms Karnataka University, Dharwad
Ccn2a-FGFR1-SHH Signaling is Necessary for Intervertebral Disc Homeostasis and
Regeneration in Adult Zebrafish
Agharkar Research Institute, Pune,
Maharashtra
Poly (A)-specific ribonuclease deficiency impacts oogenesis in zebrafish NITTE University Centre for Science
Education and Research, Mangaluru,
Karnataka
Cabozantinib-induced edema in zebrafish represents an adverse effect characterized
by defects in lymphatic vasculature and renal function
Dr. Reddy’s Institute of Life Sciences,
University of Hyderabad Campus,
Gachibowli, Hyderabad
Recent trends of zebrafish in Biomedical research in India

47. ZEBRAFISH AS A MODEL FOR CARDIOVASCULAR AND METABOLIC DISEASE.
Tissue-intrinsic pathological processes can be studied for a broad range of human cardiovascular and metabolic
diseases

48. Development of cardiovascular physiology. Different cell types of the vascular system develop progressively;
fully mature blood-brain barrier is not established before 14 dpf.

50. Set Up Fish Facility
Prepare the room allocated for the facility along with support of structural engineer.
700 kg for each system unit or rack and around 1000 kg for a reverse osmosis storage tank.
Animal research is highly regulated by government legislation in relation to setting up and
running an animal facility.
Water source heated to 28° C and comfortable humidity.
26°C is acceptable and makes for a more pleasant temperature to work in.
Maintain a 13/11 hour day/night cycle, and need to install a device to regulate this cycle.
Light Illumination levels maintained, higher light levels increase the growth of algae, keep
lighting low directly above the fish tanks.
Ensure there is plentiful surface space for setting up breeding cages (shelves, tables, or trolleys,
for example) as well as storage cupboards for equipment.
Need a dry bench for injecting and other procedures (and perhaps for a computer)

53. IISER Pune Zebrafish Facility

54. Zebrafish housing RO and water containment Zone
Breeding zone
MGMPRC Zebrafish Facility
Sterio microscope

56. Zebrafish core facility - equipment and techniques
Techniques and equipment at a glance:
Working stations for microinjection
Epi-fluorescent stereomicroscopes
Working stations for behavior analysis
Compound microscope for high throughput screening
Cryopreservation service
BSL2-high containment laboratory
SPF unit for pathogen-free zebrafish

57. BTX™ MicroJect 1000A Microinjection System (Fisher
Scientific International,)
Stereomicroscope System SZX7 (Olympus)
behavior analysis
ZEISS Light sheet 7
Streamline® Class II BSC - E Series

58. SUMMARY AND CONCLUSIONS
Zebrafish have developed at a breathtaking pace towards being a principal animal model
in biomedical research.
Zebrafish provide a powerful complementary platform. With increasing depth of
phenotyping paved by nanotechnology solutions and sophistication of high-throughput and
high content screening efforts.
The zebrafish to further improve its position as a prime preclinical animal model, and to
benefit human health

59. Indian Zebrafish Investigators Meeting (IZIM) 59
The Indian Zebrafish Investigators Meeting (IZIM) 2022, Conference was hosted by The Agharkar Research Institute (ARI) and
Indian Institute of Science Education and Research, (IISER) Pune and was held at IISER Pune I presented Poster presentation
entitled “Evaluation of Anti-inflammatory property of Terminalia arjuna (Wright and Arnott Roxb) leaf extract in zebra fish
model.” in India.