Retinal Regeneration

Muller Glia are the most fundamental participants of the retinal regeneration process. In mammals,
muller glia is not so responsive in the retinal regeneration process. But in teleost fish the response of
muller glia is such that it restores all the damaged cell types of retinae which can restore vision. Recent
studies have revealed several important mechanisms underlying muller glial cell reprogramming and
retinal regeneration in the fish that may lead to new strategies for stimulating retina regeneration in
mammals.

The retina of vertebrates is divided into three cellular layers called the outer nuclear layer (ONL), the
inner nuclear layer (INL) and the ganglion cell layer (GCL). The ONL houses photoreceptors which sense
light and transduce this information to GCL through three types of interneurons (bipolar cells, amacrine
cells and horizontal cells) that reside in the INL. Ganglion cells transfer the information to the brain.

Muller Glia arise from multipotent progenitors through a poorly understood process. It is the only cell
type to span all retinal layers and also have contacts with neighboring neurons. These are the well
positioned to monitor retinal homeostasis and contribute to retinal structure and function. But they also
form barriers for a wide range of molecules between retinal cells and compartments. They also support
neurons by releasing trophic factors, controlling ionic balance in the extracellular space, etc. Quite
remarkably, Muller Glia directly contribute to vision by acting as optical fibers to guide light to
photoreceptors. Progenitor characteristics has also been noted in Muller Glia which includes expression
of progenitor like genes, proliferative responses and the ability to generate neurons under special
conditions.

Muller Glia is quite resilient to damage. It responds to retinal damage by changing its morphology,
biochemistry and physiology, which includes change in gene and protein expression.

It shares some characteristics with retinal stem cells. In some species it can regenerate neurons. For
using it in retinal repair, three steps must be followed: reprogramming of Muller Glia to adopt stem cell
characteristics, generation of a proliferating population of multipotent Muller Glial cell-derived
progenitors and progenitor cell cycle exit and neuronal differentiation.

Three animals have dominated to be a model system for studying retina regeneration and the role of
Muller Glia in this process: teleost fish, which naturally regenerates damaged retina; postnatal chicks,
which exhibit a limited regenerative capacity; and mice, which normally do not regenerate but are an
important model for testing mammalian retinal repair.

In rodents and human cell cultures, Muller glia have been observed to generate both neurons and glia.
Studies suggest that human muller glia are capable of generating neurons and can participate in repair.

In mouse retina, NMDA induced retinal damage and EGF treatment is one of the most potent methods
for stimulating proliferation of muller glia. There was also observed a notable lack of expression of Ascl1
gene, which is essential for reprogramming and proliferation of muller glia in zebrafish. Forced
expression of this gene in combination with EGF treatment stimulated the reprogramming and
proliferation of muller glia and generation of bipolar neurons in postnatal mouse.

In teleost fish such as zebrafish, damaged retina can be regenerated. The structures of the retina of
zebrafish differs from a mammalian retina by a ciliary marginal zone in which retinal progenitors reside
and add new neurons and glia, rod precursors in the ONL that selectively generate rods as the retina
 Retinal Regeneration
grows, and muller glia that generate rod progenitors and can be stimulated to generate multipotent
progenitors for retinal repair.

Mechanisms of retinal regeneration in fish was previously experimented on goldfish. Later on muller glia
was identified as the source of the progenitor using transgenic zebrafish in which muller glial cell derived
progenitors were specifically labelled with green fluorescent protein. It was shown that these
progenitors produced all retinal cell types and remained stably integrated into the retinal architecture.

The way in which muller glia act is by sensing injury like chemical injury, mechanical damage and those
caused by intense light. More recent studies suggest that photoreceptor damage is not necessary to
induce a regenerative response, as was earlier thought. Secreted factors such as heparin binding EGF
like growth factor, tumor necrois factor-alpha, WNTS, ADP and ciliary neurotrophic factor have been
reported to promote injury induced muller glial cell reprogramming and progenitor formation in fish.
Two factors that promote muller glial cell proliferation, ADP and TNF-alpha are not only produced in
muller glia but also released by retinal neurons, suggesting that they may have a role as injury signals
that initiate a regenerative response.

Injuries in the retina activate multiple signalling cascades. Pharmacological inhibition and genetic
manipulations suggest that glycogen synthase kinase 3beta-beta catenin, notch, Mapk-Erk and Jak-Stat
signalling pathways regulate the retina regeneration in zebrafish.

The mechanism of action of growth factors like HBEGF is poorly understood. Studies suggests that
combinatorial action of cytokines, WNTS and growth factors stimulate Muller glial cell reprogramming in
zebrafish retina.

Retina regeneration is not only driven by activation of signalling pathways but also by suppression of
pathways that drive Muller glial cell differentiation and quiescence. MicroRNA let-7 signalling and DKK
signalling are two such inhibitory pathways. Notch signaling seems to have an inhibitory role in the
zebrafish retina regeneration. But unlike other signalling pathways which are suppressed following
retinal injury, notch signalling components are induced by injury. It acts in such a way that it seems to
help to match the number of injury responsive Muller glia with the extent of retinal damage. HBEGFA
and TNFA are rapidly induced after injury. It acts in autocrine and paracrine manner to stimulate
proliferation. Injury dependent induction of ASCL1a results in the suppression of cellular differentiation
and proliferation. Lin28 is an RNA binding protein, stimulated by the expression of ASCL1a gene, which is
used to reprogramme somatic cells int into induced pluripotent stem cells. Following intense light
damaged retinal injury, Stat3 levels are increased in both quiescent and proliferating Muller glia.
Surprisingly, Stat3 knockdown only reduces progenitor formation by 40% suggesting that this protein
has a modulatory role. Not only does stat 3 knockdown reduce ascl1aexpression, but ascl1a knockdown
also suppresses Stat3 expression. Pax6 is alsonecessary for the proliferation in zebrafish and its
expression is induced just before Muller glia start dividing.

During iPSC formation, pluripotency genes undergo a demethylation event that enables a state which is
permissive for gene expression. DNA demethylation contributes to reprogramming of Muller glia,
whereas DNA methylation may be necessary for the migration and differentiation of Muller glial cell
derived progenitors. The promoters of several regeneration-associated genes, including ascl1a, lin28,
hbegfa, and insm1a, exhibited a low basal level of methylation in Muller glia that remain unchanged in
 Retinal Regeneration
progenitors. DNA methylation reflects the capacity for gene expression, whereas histone modification
can distinguish active from repressed gene.

Reprogrammed Muller glia in zebrafish divide asymmetrically near the ONL which can be managed by
Gsk3beta inhibition. Pax6b controls the earliest division of the first Muller glial cell derived progenitor.
Progenitors are born apically near the ONL and migrate into the INL in an N-cadherin dependent
manner. INSM1a drives cell cycle exit by inhibiting expression of cell cycle associated genes and
increasing the expression of p57, a gene encoding cdk inhibitor.

In zebrafish Muller glial cell derived progenitors can regenerate all major retinal cell types, but this
ability is severely limited in mammals. This may be due to intrinsic differences in gene expression. The
mechanisms that control the differentiation of Muller glial cell derived progenitors in the adult fish
retina are poorly understood. In the photoreceptor damage model of the fish, Fgf signalling and galectin
Dragal1-L2 are necessary for the regeneration of the rod photoreceptors but not cone photoreceptors.
In mechanical injury model, Notch signalling seem to affect the differentiation of all cell types.

The variety of injury paradigms that stimulate zebrafish retina regeneration include Mapk-Erk,
Gsk3beta-beta-catenin and Jak-Stat signaling is remarkable.and crucial for retinal regeneration.

The goal of scientists studying retinal regeneration is to apply regenerative strategies to eye diseases.
The ability to use endogenous stem cells for repair avoids many concerns associated with prosthetic
devices and cell transplants. For all this, fish will continue to remain important model for uncovering the
mechanisms of retina regeneration, and other species such as birds and mice will serve as important
models for testing these mechanisms..