LESSONS FROM
VERTEBRATES
D r
R a j e s h
R a m a c h a n d r a n
P o o n a m
S h a r m a
By – Saahil Kumar Limone
Introduction
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Some fishes and amphibians have a remarkable capacity of
regeneration in the CNS.
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They can successfully regenerate their retina
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Mammals on the contrary have a very poor regenerative capacity of
the CNS.
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We aim to look at the different methods of regeneration in various
model organisms like zebrafish, xenopus, chicks, and mice to
understand this process and perhaps apply it to humans
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Layers of Retina
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Over view
W e
w i l l
b e
f o l l o w i n g
l o o k i n g
a t
t h e
p a r a m e t e r s -
Injury paradigms
Regeneration mechanisms
Relating retinal regeneration to cancer and embryonic development
Retinal regeneration and immune response
Molecular basis of retinal regeneration
Epigenetic basis of retinal regeneration
Zebrafish
Mice and
chick
Xenopus
Injury Paradigms
Chemical
Mechanical
Light-Induced
• Oubain
• Poking and stabbing
with a 30G needle
• Retinal detachment
• Removal of small
retinal patch
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NMDA
• 6-OHDA
• Tunicamycin
• MNU
• CoCl2
• Constant exposure
to high intensity
light of range 4001400nm
• Photochemical
damage caused by
oxygen free radicals
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Regeneration mechanisms
Differentiation
Transdifferentiation
Reprogramming
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Differentiation
CMZ – Ciliary Marginal Zone cells are neural stem cells in the
retinal periphery. They contribute to the growth and
regeneration in teleosts and amphibians.
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Zebrafish – CMZ differentiate to form retinal cell types in
the normal sequence
• Xenopus – Most of retina formed during tadpole stage
• Chicks – Only amacrine and bipolar cells in normal
physiological condition
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• Mouse – Only ganglion cells
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T r a n s d i f f e r e n t i a t i o n
R e g e n e r a t i o n
R e t i n a l
o c c u r s
p i g m e n t e d
t h r o u g h -
e p i t h e l i u m ( R P E )
The RPE dedifferentiates into proliferative neurogenic progenitors.
loses pigmentation
changes morphology
detaches from BM
starts expressing progenitor markers like
pax6 and sox2
Note : Transdifferentiation requires interaction between connective tissue and RPE
A rough approximation on the efficiency of
transdifferentiation
• Avians – need growth factor treatments
• Embryonic chicks – nonfunctional retina of reverse polarity
• Xenopus – complete regeneration
• Mammals – cells in-vivo incompetent to regenerate
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AVIANS
EMBRYONIC CHICKS
XENOPUS
MAMMALS
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Re p ro g ra m m i n g
A
p o t e n t i a l
s o u r c e
o f
r e t i n a
r e g e n e r a t i o n
i n
z e b r a f i s h ,
c h i c k s
a n d
m a m m a l s
Muller glia – non neuronal cells with nuclei in the Inner
Nuclear membrane
The chromatin and transcriptome of muller glia ( MG)
allow them to dedifferentiate, enter the cell cycle, and
start expressing early progenitor markers
This Pho by Unknown AuthorThis
is licensed under CC
• Mammalian MG need either growth
factor supplementation, epigenetic
modifications, transgenic approaches
or a combination of all
• On damage, reprogrammed MG are
sustained and express
undifferentiated cell markers
• Without them, mouse MG respond to
damage by expressing progenitors and
markers, but do not enter the cell cycle!
• Even after this, majority MG don’t
produce neurons, probably due to the
non attainment of mature retinal
progenitor cell identity.
• The inherent inability of mice MG to
reprogram could be due to various
factors
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Rod progenitors – known to replace rod
photoreceptors. In a photoreceptor
specific ablation, when a retinal injury
is chronic or not strong enough to
activate MG cells, rod progenitors
respond.
Continuous growth supported by
stem cells in CMZ and by continued
addition of rod photoreceptors.
MG derived progenitors which
migrate to outer nuclear layer
express Crx
In a diabetic model of zebrafish ( pdx1
mutant ) rods and cones regenerate
from slowly dividing neurods.
The photoreceptors originate without
MG activation, which suggests the
involvement of rod progenitors in
replenishing both rods and cones.
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Regeneration – Comparison with
embryonic development and cancer
Various gene regulatory networks
After injury
During regeneration – similar to
Development
In development – multipotent cells
this set of events needs to be
started again
Give rise to specific retinal neuronal cells
A set of gene regulatory events and
Transcription factors ( TF ) control this
Scientific
findings
transition
MG and RPE retain their
capability to dedifferentiate and
transdifferentiate into
progenitors
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MG proliferation – starts with EMT
and ends with MET
Epithelial to mesenchymal transition ( EMT) – essential
during cancer
Mesenchymal to epithelial transition ( MET ) –
Contributes to new cellular phenotype. Similar steps are
needed even during retina regeneration.
SNAIL – facilitates EMT. During zebrafish regn
Decline in SNAIL gene – existence of MET like
conditions
EMT also capable of inducing stem cell like properties
alongside preventing apoptosis and senescence.
Progenitor proliferation in zebrafish depends on TGF –
B signalling.
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Immune Responses
Tissue injury leads to release of
DAMPs ( Damage associated
molecular patterns)
Released extracellularly. Activates
pattern recognition receptors.
Induction of downstream pathways
and production of pro
inflammatory signals
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Inflammation – helps restore tissue homeostasis and
activates healing. Dysregulation of inflammation can lead
to chronic inflammation, which is harmful
Regenerating tissues – inflammatory signals enhance chromatin remodeling for
access to DNA by reprogramming factors and to promote proliferation in
reprogrammed cells
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In mammals – Muller glia undergo reactive gliosis
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Mice- MG quiescence due to TFs SoX5 and nuclear factor 1 ( these factors
downregulated after retinal damage)
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Chick – Injury induced inflammation plays a huge role in postnatal chick retina
regeneration
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Zebrafish – The induction of inflammatory response is necessary for zebrafish
regen, but it needs a timely resolution by immunosuppressants to allow
successful progression.
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