An insight on established retinal injury mechanisms and prevalent retinal stem cell activation pathways in vertebrate models

Abstract Implementing different tools and injury mechanisms in multiple animal models of retina regeneration, researchers have discovered the existence of retinal stem/progenitor cells. Although they appear to be distributed uniformly across the vertebrate lineage, the reparative potential of the retina is mainly restricted to lower vertebrates. Regenerative repair post‐injury requires the creation of a proliferative niche, vital for proper stem cell activation, propagation, and lineage differentiation. This seems to be lacking in mammals. Hence, in this review, we first discuss the many forms of retinal injuries that have been generated using animal models. Next, we discuss how they are utilized to stimulate regeneration and mimic eye disease pathologies. The key to driving stem cell activation in mammals relies on the information we can gather from these models. Lastly, we present a brief update about the genes, growth factors, and signaling pathways that have been brought to light using these models.

we discuss the different developed injury mechanisms and then talk about underlying genes, growth factors and signaling pathways involved in retinal stem cell activation in different animal models.

| MECHANIS MS OF RE TINAL INJ URY
The injury mechanism of the retina has shown a wide range of variations ranging from whole-cell retinal injury to exploiting each layer individually viz. photoreceptor, [9][10][11] ganglionic layer, 12 etc, using light, chemicals, transgenic lines, and genetic ablations. Various injury methods were applied with the following aims: 1. To determine where the regeneration capacity lies within the retina.
2. To study regenerative response during different modes of injury and be able to mediate whole-cell as well as targeted injuries, thereby mimicking diverse retinal diseases.

| Mechanical injury
Successful experiments with autoplastic eye implantation in salamander larvae and restoration of optic nerve severing in newt 13 were among the first significant research that provided evidence for the regenerative capabilities of the eye. Following these experiments, numerous amphibian and teleost fish models have been exploited using the mechanical mode of injury. 14,15 Mechanical injury of the retina is achieved through surgical procedures such as incisions, poke as exemplified in Figure 1A, or removal of a small part of the retina. The past two decades have witnessed various methods of retinal injuries such as transscleral injuries, poke injuries, [16][17][18][19] and retinal detachments 20,21 as a result of the experiments being performed on different animal models.
Transscleral injury involves using a microknife to excise a small flap of the retina, where local excision of the retina with all the layers is done and used for the study of neuroretina regeneration. 22 In poke or stab injury, eyeballs of the animal model are tilted with forceps and stabbed on the edges with a syringe, thus inflicting damage to all layers. Retinal incisions followed by subretinal injections of saline and hyaluronic acid help create retinal detachment, that is, separation of the neural retina from the underlying retinal pigment epithelium (RPE). 20,21 This injury method helps study changes in photoreceptor outer segment apoptosis and regeneration. 21,23 The use of mechanical injury for retina regeneration is one of the oldest yet most feasible injury mechanisms. Moreover, it is the best option for someone aiming to study whole-cell retinal injury because it achieves uniform damage to the retina. 16,17,24 F I G U R E 1 An illustrative representation of mechanical, light, and chemical induced injury models of the retina. A, In mechanical poke injury, a needle gauge/ syringe is used to poke a hole in the retina that causes uniform damage to all layers. B, In light-induced injury, disruption of the light/dark cycle is done by exposing model organisms to high-intensity light for varied periods. C, In chemically induced injuries such as ouabain toxicity, tissues of model organisms injected with ouabain show cellular apoptosis due to ouabain-mediated blocking of sodium-potassium ATPase pump, which causes an increase in intracellular sodium ion concentration that in turn inhibits the function of the sodium-calcium exchanger (NCX)

| Light-induced injury
Light is the environmental stimulus that is necessary for vision. However, constant long-term exposures or high-intensity light can be very damaging to the recipient's photoreceptor layer. 25 In the electromagnetic spectrum, the range of light between 400 and 1400 nm is the "retinal hazard region" as it is the range that is allowed to pass to the retina. 26 Although this range consists of only visible and short wavelength infrared light, visual complications may arise depending on the intensity of light and exposure time. Owing to its high exposure rate in humans, the blue light component (415-495 nm) has also been experimented with in animal models and has been shown to inflict damage to the photoreceptors and the retinal pigment epithelium layer as well. [27][28][29] Light-induced injury mechanism usually follows a disruption of the standard 10 hours light-14 hours dark cycle 30 to a long dark cycle followed by exposure to high-intensity visible light 9,31 or ultraviolet (UV) light. 32 Temporal variations for light-induced injury include long or short durations of exposure 27 that are repeated once or several times 10,33 depending on the experimental setup as exemplified by Figure 1B. The light source may vary from tungsten halogen lamps, 10,31,32 to metal halide lamps, 9 and fiber optics, 34 with light intensities at the water interface being as great as 100 000 lux. 32 The 3 modes of light-induced injury include:

| Photomechanical injury
Laser-based irradiation of retinal pigment epithelium (RPE) has the ability to cause 2 distinct modes of cell damage depending on the time of exposure. These are thermal denaturation (exposure duration more than 10 µs) and intracellular cavitation (exposure duration below 10 µs). 35 For exposure duration of up to 10 µs, which falls in the order of the thermal relaxation period of RPE, melanosomes show a very high increase in temperature ranging up to 150°C. 36,37 Thus, cytoplasm contact with these melanosomes undergoes rapid vaporization, thereby creating microcavitation bubbles. 36 Rapid expansion and dissolution of these bubbles cause mechanical damage and induce apoptosis in the underlying RPE cells via disruption of lysozymes. 36

| Photochemical injury
Post light exposure, dissipated energy from excited chromophores such as lipofuscin and flavoprotein may lead to the production of reactive oxygen species (ROS). 26,38 These chemicals are highly damaging to all cell types, and in the retina, they may initiate apoptosis of the light recipient photoreceptor layer. 26

| Photothermal injury
Light in the form of photons is capable of increasing the mean kinetic energy of the recipient molecules. When this energy dissipates, molecular collisions lead to an increase in these molecules' temperature, thereby causing thermal damage to the cells involved. 26,38 This injury mode is observed in laser light photocoagulation and optical coherence tomography (OCT)-guided laser injuries and has been experimented with in many animal models. 39,40

| Chemical injury
Although both mechanical and light injury can be used to target different retinal layers, mechanical injury best portrays whole-retina damage as, for light-induced injury, photoreceptor-specific damage can be well

| Ouabain-mediated chemical injury
The cardiac glycoside ouabain acts by causing the inhibition of Na + /K + ATPase, hence acting as a metabolic poison by increasing intracellular Na + ion concentration that inhibits the sodium-potassium exchanger 41 as shown in Figure 1C. Introduced by Maier and Wolberg in 1979, 42 it can destroy the whole retina 43 when used in high doses, and intravitreal injection of lower doses is efficient in targeting different individual layers such as the inner nuclear layers, 44 amacrine layers, 12 and photoreceptors. 11,42 The injury mechanism usually follows a microknife for incision followed by injection of ouabain in the intravitreal cavity.

| 6-OHDA mediated injury
Dopaminergic neurons are distributed throughout the retina and play an essential role in the growth and survival of retinal cells. 6-OHDA is a neurotoxin that targets noradrenergic and dopaminergic neuron destruction. 45,46 The injury mechanism, similar to ouabain treatment, follows the making of scleral incisions using a microknife and then microsyringe-mediated administration of the chemical. 6-OHDA is quickly converted to its quinone form in solution, thereby generating free radicals. Hence, while using relatively higher doses of 6-OHDA, sodium ascorbate is added to slow down the build-up of these autoxidation products that may cause nonspecific damage. 46

| Chemically induced hypoxia
Cobalt chloride (CoCl 2 ) prevents iron inclusion in the heme, thereby decreasing hemoglobin that carries oxygen to different parts of the body. 47 This causes hypoxia, leading to the production of hypoxia-inducible factors (HIF). Furthermore, CoCl 2 also inhibits the proteasome-mediated degradation of HIF, thereby causing hypoxic injury. 48 HIF, in turn, stimulates the production of vascular endothelial growth factors that causes neovascularization and aberrant angiogenesis. 49 CoCl 2 -mediated hypoxic injury involves intravitreal administration and has been used to target different retinal layers such as the photoreceptors 11 and ganglionic layers. 50

| NMDA receptor-mediated injury
High doses of NMDA are known to cause NMDA receptor-mediated influx of cations in massive amounts that lead to overexcitation of synapses, causing neuronal death. 51 Based on this principle, NMDA has been used to injure animal models in different retinal layers such as the rods of the photoreceptor layer or retinal ganglionic layer as well as other layers. 52,53

| NTR/Mtz-mediated cell ablation
This injury lies in the ability of the Escherichia coli bacteria's nitroreductase (NTR) enzyme to reduce prodrug metronidazole (Mtz) into a cytotoxic DNA cross-linking agent, the expression of which causes cellular apoptosis. The technique is used to ablate various types of retinal cells such as the ultraviolet cones, 54 blue cones, 55 rods, 56,57 and bipolar cells. 58  are examples of 2 such transgenic lines that express the NTR promoter in the rod cells. 56,59 Chemical injury in the case of CoCl 2 can be best correlated with retinopathy of prematurity in which the retina in the infants is hypoxic because the central retinal vessels cannot reach the periphery, leading to infantile blindness, 60 whereas ouabain, 6-ODHA, NMDA, and NTR/Mtz induced cell ablations create a pathological environment that induces the regeneration process. 12,43,55,59

| Genetic models of retina diseases
With the help of genetic screening tools, scientists have collected numerous data and identified specific genes that are involved in certain retinal diseases. 61 Inherited retinal diseases such as achromatopsia, RP, Leber's congenital amaurosis (LCA), AMD, etc comprise an extensive collection of heterogeneous mutations involving almost 250 genes. 62 Besides, eye diseases such as diabetic retinopathy (DR) and glaucoma also contribute highly to eye diseases worldwide. 63 Experiments for treatment of such diseases would require disease modeling, which is made possible by creation of knockdown, knock-in, or insertion of mutated variants in the model organism.
The science of transgenics, in which foreign genes can be inserted and expressed successfully in some other organism, and other genome editing techniques such as morpholino-based gene silencing, transcription-activator like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR-Cas) enzyme system make possible the imitation of diseases in a model organism. [64][65][66][67][68][69] The creation of such genetic models of a disease hence provides a platform for novel therapeutics. 63 Here we have talked about a few genetic models of RP and DR; furthermore, a few examples from AMD 70,71 and glaucoma 72,73 can be seen in Figure 2.

| Genetic models of RP
The first retina degeneration model discovered as early as 1924 in mice showed mutations in rod phosphodiesterase Pde6b gene and later was shown to be involved in RP. 74 Retinitis pigmentosa is the largest subgroup of inherited retinal diseases and affects more than 1 million people worldwide. 5 Using this information, many Pde6 mutated animal models are created to copy the disease. 5,74,75 For example, in the case of Pde6b rd1/rd1 mouse models, a null mutation creates a loss of rod cells within 2 weeks. 75 Recent RP models include Pde6a V68 M/R562W compound heterozygotes in mice, which is an accurate homolog of human RP. 76 77 It is produced by a cross between Pde6b rd1/rd1 mutants and Prkdc −/− mutants that lack the expression of B cells, T cells, and natural killer T (NKT) cells.
Thus, these double homozygotes help us understand the disease progression from both a genetic and an immunological viewpoint. 77 Rhodopsin mislocalization is often seen in RP patients; utilizing this information, rho Q344ter -dend2 Xenopus laevis transgenic models have been created in which human mutated rhodopsin is fused with Dendra fluorescent protein, and rhodopsin terminal amino acids are expressed in Xenopus to model RP. 78 CRISPR-Cas9 edited rhodopsin in Xenopus is another recent model of RP. 79 In zebrafish, morpholinobased ceramide kinase-like cerkl gene knockdown and expression of an autosomal dominant form of rhodopsin (Q344X) have been used to model RP. 80,81 Apart from the genes mentioned above, approximately 67 genes have been mapped to be involved in heterogenous RP and thus present targets for animal model development. 75

| Genetic models of DR
Diabetic retinopathy is an associated complication of diabetes mellitus and is shown in one-third of the diseased patients. 63 There are 5 genetic models of DR in mice; of these, leptin receptor deficient mouse model Lepr db dates back to the 1990s and shows association with type 2 diabetes. 83 Ins2 Akit , on the other hand, mimics type-1 diabetes associated DR and happens as a result of Insulin 2 gene missense mutation. 63,84 The amenability of forward and reverse genetics makes zebrafish another valuable model for genetic manipulation.
To mimic DR in zebrafish, mutations in the von Hippel-Lindau tumor suppressor gene are created providing us with vhl −/− zebrafish 85,86 that phenotypically mimic detachment of the retina, vascular leakage, and macular edema. 87 Another novel model for DR is well exemplified by INS C94Y transgenic pig model made via expression of mutation insulin gene. 88

| AC TIVATI ON OF RE TINAL S TEM CELL S
As the name suggests, retinal stem cells (RSC) are progenitor cells specific to the retina and are induced exclusively to form the retinal cells. They are present throughout the vertebrate lineage 3 but, in comparison with lower vertebrates higher vertebrates such as mammals, are incapable of employing the regenerative property as opposed to their counterparts. 8 This biased ability to regenerate may be due to the presence of stem cells alone not conferring the capability to regenerate but instead requiring a highly coordinated activity between the stem cell and its niche, 6 which seems to be lacking in the case of mammals. These so-called niches are microenvironmental compartments that sustain neural stem cell and endow them the properties of self-renewal and lineage differentiation. 89 Certain conditions needed for creation of this proliferative niche include proper mode of stimulation in the form of injury, followed by F I G U R E 2 Genetic models of retinal diseases. A, An ophthalmoscopic view of the eye during AMD as identified by yellow extracellular membrane accumulation/drusen deposits: [1] CXCR1 is a CX3CR1/fractalkine chemokine receptor and has shown low transcript levels and SNPs during AMD; CCL2 is CC chemokine thought to play an immunoregulatory role in AMD CXC3cr1 −/− Ccl2 −/− are thus homozygous mice created to model AMD. [2] The nonhuman primate Rhesus monkeys have been used to model AMD, DR, etc via AAV Vegf expression which controls neovascularization. (CXCR1: C-X-C motif chemokine receptor; CCL2: C-C motif ligand 2 chemokine; AAV-Vegf: adeno-associated viral vector mediated vascular endothelial growth factor expression). B, An ophthalmoscopic view of the eye during DR as identified by neovascularization and leaky blood vessels: [1] Leptin receptors are essential regulators of obesity; hence, mouse models of chronic hyperglycemia have been modeled in Lepr db mouse mutant for leptin receptor. [2] Ins2 Akita with missense mutation in insulin 2 gene is another mouse DR model. [3] Von Hippel Lindau protein regulates Hif levels that can induce angiogenesis, vhl −/− biallelically inactivated zebrafish thus shows DR phenotype. [4] Large animal model of DR is the INS C94Y transgenic pig created by mutating the insulin gene. (Lepr: leptin receptor; Ins/INS: insulin; vhl: von Hippel Lindau). C, An ophthalmoscopic view of the eye during RP identified by the presence of intraretinal melanin deposits/bone spicule pigments, a hallmark of RP: [1] Mutations in the Pde6a and Pde6b gene are known to cause RP. The compound heterozygote RP mice model Pde6a V685M/R562W is an exact homolog of human RP. [2] Pde6b functions in the photo cascade transduction, and Prkdc encodes for catalytic subunit of DNA-dependent protein kinase DNA-PK. The Pde6b −/− Prkdc −/− is an immunocompromised mice model of RP that lacks T, B, and NKT cells. [3] This transgenic zebrafish model expresses a mutated human rhodopsin gene construct Rho-Q334X to model RP. [4] Single mutation in some genes such as cerkl has been shown to cause autosomal dominant RP. Thus, morpholino knockdown of cerkl has been used to mimic RP in zebrafish. [5] Rhodopsin mislocalization is one of the observed features of RP based upon this human class 1 rhodopsin mutation Q344ter has been fused to dendra 2 fluorescent protein to mimic RP in frog. [6] CRISPR-Cas9 has also been used in frogs to create point mutation in rhodopsin gene and model RP phenotype in the animal model. (Pde6a/b: phosphodiesterase 6; Prkdc: protein kinase DNA-activated catalytic subunit; Rho: rhodopsin; cerkl: ceramide kinase like). D, An ophthalmoscopic view of the eye during glaucoma characterized by a relatively large optic cup resulting from loss of optic nerve due to high intraocular pressure. [1] The low-density receptor-related lipoprotein mutation at Q413X shows glaucoma-like phenotype in zebrafish. [2] Purinergic receptor P2Y 6 is important in regulating the intraocular pressure, and thus its knockout has been used to model the disease in mice. (P2Y: purinergic receptor) epigenetic chromatin alterations, expression of specific transcription factors, assembly of growth factors and immune cells, cell signaling initiation, and switching of cells from a glycolytic to the oxidative pathway. 6,7,90 In the upcoming section, we have provided a list of retinal stem cells found in different vertebrates and briefly discussed the genes, growth factors, and signaling pathways that, together with these stem cells, help create a proper microenvironment for RSC activation among different vertebrate models. These have also been summarized in Table 1.

| Retinal stem cells in teleost fishes
The most remarkable ability to regenerate the retina among the vertebrates is displayed by teleost fishes such as the zebrafish, 91 where the growth of retina takes place throughout their life. 3 93,97 This study has shown RPE to follow a Wnt-mediated reparative pathway 93 and has accounted for the presence of macrophages and microglia indispensable for timely progression of proliferation. 98

| Müller glia in zebrafish
The molecular characterization of neuronal stem cells of the brain showed expression of properties identical to radial glia cells, 99,100 therefore indicating that radial glial cells are the progenitors of neurons. Since the retina is also derived from the CNS and possesses its own glial cells, the "Müller glia," these cells were a potential target for identifying stem cell progenitors in the eye. MG in zebrafish is a potent source of neuronal progenitor and post-injury become reactivated via expression of certain pluripotency genes such as pax6b, 101,102 ascl1, 17,33,103 lin28, 17,95 and stat3. 17,104 Ongoing research has been able to add more to this list. Gene expression analysis of sox2 morpholino knockouts has also shown to cause a significant reduction in ascl1a and lin28 levels, which states its importance in reprogramming. 105,106 Myc is another well-known pluripotency inducing transcriptional activator, 107 and in zebrafish it has been found to regulate lin28 positively via ascl1a expression and negatively via Myc histone deacetylase (hdac) inhibition of her4.1, a delta notch effector. 108 Oct4 is another transcription factor that, apart from upregulating regeneration-associated genes, regulates proliferation and cell cycle exit respectively via inhibiting and eventually inducing transforming growth factor β (Tgfβ) signaling. 18 Several cytokines and growth factors are produced post-injury from MG, and some of these are known to cause diseases such as proliferative vitreoretinopathy in humans. 109,110 However, in regeneration-competent organisms, where timely termination of proliferation occurs, some cytokines and growth factors have been shown to positively regulate regeneration. In the case of zebrafish, these include heparin-binding epidermal growth factor (HbEgf), fibroblast growth factor (Fgf), insulin, insulin-like growth factor (Igf), leptin, etc [111][112][113][114][115] Tumor necrosis factor alpha (Tnfα) is another cytokine that regulates regeneration as it works upstream of Ascl1a. 114 Midkine is another addition to the list and facilitates G1-to-S transition. 116 Furthermore, blocking growth factors such as Tgfβ 18 and platelet-derived growth factor 19 also tend to downregulate regeneration-associated transcription factors and are essential for regeneration.
The major signaling pathways already known to be involved in retina regeneration that positively regulates regeneration include Wnt/β catenin pathway, 111,115,117,118 Jak-stat pathway, 119 and Pik3/ Akt signaling pathway. 4 Hedgehog pathway (Shh) is another known pathway involved in retina regeneration and can control both the proliferation and differentiation of MG. 120 Signaling pathways that inhibit regeneration include the Notch pathway, 111,121,122 let-7 mi-croRNA, 103 and Dikkopf (Dkk) pathway. 117 Certain pathways seem to play both proproliferative and antiproliferative roles to drive differentiation depending upon their temporal expression. Insm1adependent pathway is one such pathway and can inhibit ascl1a expression and induce ascl1a-mediated dkk gene repression. 123,124 Furthermore, it can regulate controlled proliferation by inhibiting hb-egfa gene expression. 116 Tgfβ-mediated upregulation of matrix metalloproteinase mmp2 during the earlier phases of injury TA B L E 1 List of endogenous retinal stem cells source across the vertebrate lineage, their characteristics (in vivo), and an updated list of growth factors, cytokines, and signaling pathways discovered using different animal models involved in retina regeneration

Characteristics of endogenous RSC (in vivo) Growth factors and cytokines
Signaling

| Retinal stem cells in amphibians
Amphibians, like the teleost fishes, possess massive regeneration potential. Urodele newt, salamander, and anuran Xenopus are popular models for eye regeneration studies. [125][126][127] Retinal stem cells in amphibians have also been identified in the MG, CMZ, and RPE.

| Müller cells in amphibians
Until very recently, MG in the case of amphibians was considered not to possess any proliferative abilities. However, detection of proliferating cells in newts and Xenopus larvae 128,129 has paved the way for MG to be a probable source. 128,129 While current findings support this idea, a recent experiment has revealed more profound reactivation in cells from older organisms than from younger ones, 130 which opposes the general feature of regeneration; hence, MG cells in amphibians need further investigation.

| CMZ in amphibians
Similar to the fishes, amphibians such as Xenopus also express RSC markers along the peripheral region of CMZ and follow a centripetal pathway of retinogenesis during development and regeneration. [131][132][133][134] Stem cell markers such as pax6 and six3 show high expression at the periphery; this is followed by neurogenic genes such as delta and notch, and finally proneural genes such as neuroD towards the center. 131 In the past decade, protooncogenes such as c-myc and n-myc, which happen to be important cell cycle regulators, have also been shown to have centripetally increasing expression. 132 Signal transduction pathways also work simultaneously for successful development and regeneration and, to name a few, include the canonical Wnt and the Shh pathway. 133,135,136 While the former promotes proliferation, the latter promotes both proliferation and differentiation via Sprf1-mediated Wnt inhibition. 133

| Retinal pigment epithelium in amphibians
The other important mode of retina regeneration in amphibians is "transdifferentiation" of the RPE seen in urodeles 137,138 and more recently has also been shown in anurans. 126 This is a mechanism where the cell dedifferentiates and returns to a point where it can switch lineages and hence redifferentiate into another cell type. 139 Investigating the molecular mechanism underlying RPEmediated retinogenesis has shown that RPE goes into a brief, unique multipotent state, and unlike mammals where RPE proliferation is associated with PVR, amphibian RPE is well reprogrammed for recovery. [140][141][142] This reprogramming involves the expression of pluripotency factors such as C-myc, Klf4, and Sox2 post injury which takes cells to a dedifferentiated state. 143 This follows the expression of pax6 and mift genes that accounts for redifferentiation. These RPE cells then undergo cell cycle entry initiated by Fgfs and Igf1, which in turn induce MEK-Erk and heparin-susceptible pathways. 141 In the case of newts, apart from the expression of EFTFs of which pax6 shows 2 variants, 143 multipotency state regulates via expression of pluripotency factors C-myc, Klf4, and Sox2. 140 In the case of Xenopus, RPE-mediated regeneration depends on Fgf-dependent MAPK pathway. 144,145

| Retinal stem cells in birds
In the case of birds, the chick is used as a popular model organism

| CMZ in chick
The expression of multipotent progenitor associated transcription factors and incorporation of BrdU at the peripheral margins of retina in chick have shown the capability of cells in CMZ to undergo regeneration. 137 However, unlike their cold-blooded counterparts, CMZ in chick possesses restricted capabilities and can only produce bipolar and amacrine cells. 137 Signaling pathways such as sonic hedgehog alongside FGF signaling 149  to the list include the following: Jak/stat pathway, which promotes MG progenitor cell proliferation but has been shown to be not as efficient in neuronal differentiation 163 ; another mammalian target of rapamycin (mTOR) pathway has also been shown to promote MG reprogramming as its inhibition has a significant effect on PAX6 levels. Stimulatory pathways such as hedgehog and WNT are also blocked on mTOR inhibition. 164 Activation of retinoic acid signaling pathway 165 and growth hormone treatment 166 have also surfaced as recent findings in chick MG regeneration. 144,145 Moreover, the existence of crosstalk between FGF and notch 139 and FGF and hedgehog, 141 and its capability to activate the Jak-stat pathway 142 and mTOR signaling, 143 makes it a focal point in the case of chick regeneration.

| Retinal stem cells in mammals
The regenerative potential of mammalian retina has long been questioned owing to their incapability to "self-heal" upon injury or pathological conditions. However, this "incapability" has proved to be a myth owing to the success of experiments that have shown the presence of RSC existing in the quiescent form in mammals in different locations within the retina. These include the ciliary body (CB), IPE, RPE, and MG. 127 The knowledge gathered from regenerationcompetent animal models and its application in mammals has helped unravel certain intrinsic and extrinsic factors and pathways that induce retinal stem cell activation.

| The ciliary body in mammals
The CB or retinal margin in higher vertebrates represents a location of stem cells that corresponds to the ciliary marginal zone in the case of lower vertebrates. [167][168][169] In vitro analysis of the CB in both mice and humans shows that they are capable of forming neurospheres that express RSC markers and generate most retinal cell types, including photoreceptors. 170

| Müller glia in mammals
Unlike fish and amphibian MG, the MG in mammals tends to maintain its quiescence even postembryonically and respond to injury with prolonged reactive gliosis. 179,180 That mammalian MG also possessed the capability to evade this quiescence was first confirmed in a retinal explant culture study of retina taken from NMDA-injured Sprague Dawley rats, which managed to produce bipolar cell rod photoreceptors. 53 Ascl1a overexpression alongside a histone deacetylase inhibitor has been shown to relatively upregulate MG proliferation in mice; however, the effects seem to be transient. 181,182 Findings in a recent paper have shown that the transient nature of proliferation is due to the presence of Nuclear factor 1 (Nf1), a transcription network that inhibits Ascl1a and works to promote reactive gliosis instead of regeneration in mammals. 183 Another gene, Lin28a, a posttranscriptional regulator, can also induce reprogramming as it can control the formation of MG or other neural lineages depending upon its expression or deletion. 184,185 Neurogenin-2, a basic helix-loop-helix (bHLH) transcription factor, is another potential candidate that activates MG regeneration. 186 Taking hints from regenerative models, many growth factors have been identified that help stimulate MG differentiation in mammals. These include retinoic acid, FGF, insulin, epidermal growth factor (EGF), HB-EGF, and progranulin. 158,187,188 Regarding the signaling pathways involved, the most recent inclusion is the "hippo pathway," which is a conserved kinase pathway that works during development. 189 Hippo pathway-mediated phosphorylation of transcriptional cofactor Yes-associated protein (YAP) influences cyclin D1 levels required for the initial burst in MG reprogramming. 190 Apart from these well-known signaling pathways that are already known to us, the WNT and Notch pathway positively regulates MG proliferation, 191,192 with Notch acting as an inhibitor of CDK inhibitor p27 kip . 1193 Intraocular injections of SHH also stimulate MG proliferation and enhance neurogenic potential. 194

| D ISCUSS I ON
In vertebrates, the "regenerative neurogenesis," that is, the ability of postembryonic establishment of functional neuron regeneration, is said to exist, 195 but this feature appears to be compromised in higher vertebrates. Analysis of the above-mentioned endogenous retinal stem cells have helped to unveil species-specific trends and pathways followed during retina regeneration, revealing what appears to be missing in mammals. For instance, while the CMZ shows high regenerative potential in fish and amphibians, chick and mammalian CMZ seems to require the presence of additional mitogens and signaling factors. 196 Similarly, in comparison with the amphibian RPE that transdifferentiates its way to regenerate the retina, mammalian RPE seems to have preserved only a certain fraction of the ability to proliferate. 174 Moreover, the regulatory elements required for induction of transdifferentiation seem to be lacking in mammals. 174,196 Regarding the Müller glia, Ascl1 upregulation appears to be key in facilitating retina regeneration both in fish and birds. MG-specific expression of Ascl1 in combination with a histone deacetylase inhibitor has shown to activate regenerative potential in mammals as well, but the results appear to be transient. 182 Recent experiments have shown that this state of dormancy of mammalian MG is mediated by a dedicated gene regulatory network that upregulates upon injury. 183 The evolutionary aspect of regeneration is far from being understood. However, loss of such recuperative powers in higher vertebrates has been speculated to occur for resolving wounds in the best possible way and to increase the reproductive fitness. 197 In reality, loss of vision in humans has the potential of exacerbating tremendous socioeconomic pressure. Animal models of retina regeneration present us with an opportunity to solve this mystery utilizing an "in vivo" platform that allows for understanding complex interactions happening during the process. These models have allowed the establishment of injury mechanisms that happen to be the first-hand stimulus for RSC activation and hence allow us to explore the underlying molecular mechanisms. Furthermore, they also serve excellently as eye disease models, thereby helping to understand the pathophysiology of the disease and, hence, designing and trial of therapeutic drugs. The findings in one animal model pave way for experimenting with the same in another animal model and allow for a broader understanding of relatedness or heterogeneity of retina regeneration activation among different species. The information thus gathered from these models about the underlying regeneration-associated molecules and pathways can ultimately be used as experimental targets in mammalian models and gradually in humans.

ACK N OWLED G M ENTS
We thank Dr Sujay Ghosh, Dr Arindam Biswas, and Puja Rai for the critical reading of the manuscript. Part of the review is supported by the Ramalingaswami award to SPH and grant received from DBT, Govt of India (BT/HRD/35/02/2006). RDS is recipient of junior research fellowships from CSIR, Govt. of India (09/028(1146)/2020-EMR-I).

CO N FLI C T O F I NTE R E S T
The authors state no conflicts of interests.