The zebrafish is a useful model for studying organ regeneration due to its ability to regenerate complex tissues like fins, heart muscle, and neurons. It can regrow amputated fins through blastema formation and proliferation. During heart regeneration, cardiomyocytes dedifferentiate and proliferate to replace damaged tissue. In the retina, brain and spinal cord, radial glial cells act as progenitors to regenerate neurons. New genetic techniques allow manipulation of specific genes to better understand regeneration in the zebrafish.

1. Zebrafish as a Model
Organism for Regeneration
Studies
M.Sc. II Biotechnology
Animal Biotechnology
Dariyus Z Kabraji

2. Contents
• Introduction
• Regeneration
• Zebrafish & what makes them special
• Complex Tissue Regeneration:
1. Organ based Regeneration:
Fin regeneration
Heart regeneration
2. Neural Cell based Regeneration:
Retina regeneration
Spinal Cord regeneration
Brain regeneration
• Future Prospects
• References

3. Introduction
• A prosthesis is an artificial device that replaces a
missing body part, which may be lost through trauma,
disease, or congenital conditions.
• Due to the worldwide demand for prosthetics,
stemming from diabetic conditions and organ failure to
amputation due to traumatic accidents in our
neighbourhoods and on the battlefield, we live in a
world where people are dying because their bodies lack
organs that they can’t live without.
• Since we can only provide mechanical appendages to
replace lost limbs, which take months of therapy to get
used to, and our internal organs are mostly replaced by
donors or devices which are extremely complex but still
under development, our best option is to consider the
possibility of complex tissue regeneration

4. Regeneration
• Mammalian tissues achieve remarkable feats of
regeneration. After removal of more than two-thirds of
its mass, the liver rapidly regenerates within several
days by hepatocyte proliferation. Multi-potent
hematopoietic stem cells replenish red and white blood
cells, and skin, muscle, and intestine are repaired by
tissue-specific stem cells
• However, this regenerative capacity is distributed
unequally among mammalian organs: limbs, brain,
spinal cord, and heart display minimal regeneration
after tissue damage or loss
• Certain non-mammalian vertebrates, such as urodele
amphibians and teleost fish, regenerate complex tissues
much more effectively than mammals. Salamanders
have long been the central characters employed in

5. Zebrafish

6. Zebrafish
• The Zebrafish is a derived member of the genus Danio, of
the family Cyprinidae
• The Zebrafish is native to the streams of the south-eastern
Himalayan region, and is found in parts of India, Pakistan,
Bangladesh, Nepal, and Burma. The species arose in the
Ganges region in eastern India, and commonly inhabits
streams, canals, ditches, ponds, and slow moving or
stagnant water bodies, including rice fields
• 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. Its shape is fusiform and laterally
compressed, with its mouth directed upwards.
• The male is torpedo-shaped, with gold stripes between the
blue stripes; the female has a larger, whitish belly and silver
stripes instead of gold. Adult females exhibit a small genital
papilla in front of the anal fin origin.

7. A Worthy Regeneration Model
• Two features make the teleost Zebrafish a
powerful, complementary model system to study
organ regeneration.
• First, they are highly regenerative, equipped to
regrow amputated fins, injured retinae, transected
optic nerves and spinal cord, and resected heart
muscle.
• Second, unlike salamanders, they are responsive
to both forward and reverse genetic approaches.
• As is customary with genetic model systems, a
wide array of community resources is available
for gene discovery and molecular characterization
in Zebrafish, including mutagenesis screens,
transgenesis, microarrays, developmental

8. Organ Regeneration

9. Fin regeneration
• Zebrafish fins are complex appendages that quickly and reliably
regenerate after amputation, restoring both size and shape.
• The key regenerative units are their many rays of dermal bone,
which are segmented and lined by osteoblasts. Rays are cylindrical
and hollowed, with two concave hemirays surrounding an inner
mesenchymal tissue that is innervated, vascularized, and comprised
primarily of fibroblasts.
• 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 (or scleroblasts) proximal to the
amputation plane.
• Blastema formation is only one step in zebrafish fin regeneration,
and fins must then grow to the appropriate size. Regenerative
outgrowth occurs by two processes: maintenance of a proliferative
compartment at the distal end of the regenerate, and differentiation
of more proximal cells.

10. Fin regeneration
• The proliferative compartment is maintained by signaling
interactions between the mesenchyme and basal epidermis. In
addition to regulating blastema formation, Retinoic Acid (RA),
Fibroblast Growth Factor (Fgf) and canonical Wnt signaling
positively regulate blastemal proliferation and outgrowth
• The Wnt signaling pathways are a group of signal transduction
pathways made of proteins that pass signals from outside of a cell
through cell surface receptors to the inside of the cell. Three Wnt
signaling pathways have been characterized: the canonical Wnt
pathway, the noncanonical planar cell polarity pathway, and the
noncanonical Wnt/calcium pathway. All three Wnt signaling
pathways are activated by the binding of a Wnt-protein ligand to a
Frizzled family receptor, which passes the biological signal to the
protein Dishevelled inside the cell.
• The canonical Wnt pathway leads to regulation of gene
transcription, the noncanonical planar cell polarity pathway
regulates the cytoskeleton that is responsible for the shape of the
cell, and the noncanonical Wnt/calcium pathway regulates calcium
inside the cell.

11. Fin regeneration

12. Heart regeneration
• There is no significant regeneration of adult mammalian cardiac
muscle after experimental injury paradigms. This deficiency is
highly relevant to human disease, given that ischemic myocardial
infarction (MI) and scarring is a primary cause of morbidity and
mortality.
• There are currently several injury models that stimulate heart
regeneration in zebrafish, including surgical resection of the
ventricular apex, cryoinjury, and inducible genetic ablation
• Whereas cryoinjury mimics aspects of MI, genetic ablation
produces massive injuries, removing 60% or more of
cardiomyocytes and inducing signs of end-stage heart failure.
Unlike severe heart failure in humans, these signs regress within
weeks and the animals typically make a full recovery
concomitant with muscle regeneration
• Heart regeneration involves two fundamental components: 1)

13. Heart regeneration
• Genetic fate-mapping experiments in Zebrafish have made
it clear that the regenerative ability of the Zebrafish heart
relies mainly or exclusively on proliferation of existing
cardiomyocytes. These source cardiomyocytes show
characteristics of dedifferentiation, including a reduction in
contractile structure
• Cardiomyocyte proliferation occurs at a low rate in the adult
Zebrafish heart, but is sharply increased in response to
tissue damage
• Injury to the zebrafish heart initiates an organ-wide reaction
detectable as induced expression of raldh2 (a Retinoic Acid-
synthesizing enzyme) as early as 1 hour post-injury in the
endocardium, the endothelial lining of the lumen
• Within a day or two of injury, the epicardium, the outer
lining of the heart, shows an analogous organ-wide response
of raldh2 induction. Then, epicardial cells proliferate and
surround the regenerating muscle, where they release
signals that facilitate cardiomyocyte proliferation.

14. Heart regeneration

15. Neural regeneration
• Neuronal cell loss causes visual, motor, or mental
impairment in humans.
• This neuronal cell death often leads to glial cell
hypertrophy, limited proliferation, and gliotic scarring,
which prevents neuronal regeneration.
• Zebrafish, by contrast, have the capacity to regenerate
neurons within the retina, spinal cord, and brain from
resident radial glial cells.
• New genetic approaches have facilitated the
investigation of commonalities and distinctions in the
pathways necessary for regeneration of different
neuronal tissues and cell types.

16. Retina regeneration
• Because of the relative ease of manipulating the retina,
numerous damage strategies have been employed to
either destroy all or a restricted type of retinal neurons
• All of these damage models induce some Müller glia to
dedifferentiate and re-enter the cell cycle to produce
multi-potent neuronal progenitor cells (NPCs)
• Microarray studies and two-dimensional differential
protein gel analysis identified several candidate genes
and proteins that may be required for aspects of
regeneration. Recently, a technique to electroporate
morpholinos into the adult retina to knockdown the
expression of specific target proteins was developed to
functionally validate the role of these proteins in

17. Retina regeneration

18. Spinal Cord regeneration
• Damage to the human spinal cord results in irreversible loss of
neurons and impaired sensory and motor functions.
• By contrast, Zebrafish possess the ability to regrow new
axonal projections from viable brain neurons across the
severed spinal cord
• In addition to axonal growth, Zebrafish can produce new
neurons and interneurons at the region of damage. Similar to
development, the type of regenerated neuron depends on
dorso-ventral location of its corresponding progenitor radial
glial cell in the spinal cord.
• Axonal regeneration across the lesion site is dependent on
proliferating radial glial cells that infiltrate the site. The
responding glia divide soon after lesion and assume bipolar
morphology. They migrate into the damaged site and connect
the two sides of the lesion guiding the new axons, a process

19. Brain regeneration
• Surgical lesion of the telencephalon causes neuronal cell
death and induces radial glia to proliferate and new neurons
to regenerate.
• Like the spinal cord, regeneration originates from radial glia
that line the ventricles
• These glia proliferate to yield progenitor cells that then
express the transcription factor Eomesa, which regulates
glutamatergic neuron differentiation
• Recent advances using Vivo-Morpholinos to knockdown
protein expression and transgenesis have allowed for
functional studies of specific proteins during brain
regeneration. Vivo-Morpholinos, which contain eight
guanidium residues covalently attached to a trizine residue
in the standard morpholino antisense-oligonucleotides
penetrate into the most proximal cells of the zebrafish
telencephalon ventricle without electroporation or
intracellular injection, after cerebroventricular

20. Future Prospects
• Zebrafish have advantages over other regenerative vertebrate model
systems in regards to the relative ease and diversity by which
potential factors can be manipulated.
• One drawback of the Zebrafish model system has been the inability
to generate conditional loss-of-function alleles.
• Over the past few years, zinc finger nucleases (ZFNs) and, more
recently, transcription activator-like effector nucleases (TALENs)
and the CRISPR-Cas system have aided directed mutagenesis. Very
recently, a system was described for inducing site-specific
homologous recombination in zebrafish embryos utilizing TALENs.
Double-stranded breaks could enable the incorporation of sequences
from co-injected short single-stranded DNA oligos at a low
frequency, in both somatic and germline cells.
• Adapting this technology, one can envision creating conditional
“knock-out” alleles through the insertion of two compatible loxP
sites targeting a gene of interest.
• This technology will enable the study of individual gene products in
a tissue-specific manner during regeneration, and provide potential
upgrades over current dominant-negative, pharmacologic, and
antisense morpholino-based approaches for loss-of-function studies.

21. References
• The Zebrafish as a model for complex tissue
regeneration; Matthew Gemberling, Travis J.
Bailey, David R. Hyde, and Kenneth D. Poss;
Trends Genet .2013 November ; 29(11):
doi:10.1016/ j.tig.2013.07.003.