Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

development, but in a different context. This becomes most obvious when one compares the development of a laminated retina from an unpatterned neuroepithelium to the regeneration of single neuronal cell type from an existing laminated retina. To fully appreciate the differences between these two processes and the mechanisms involved in regeneration, it is important to contrast our understanding of the general events involved in retinal development with our recently acquired knowledge of the processes underlying retinal regeneration.

Embryonic Eye Patterning

Zebrafish Eyes Form from a Single Field in the Anterior Neural Plate

There is a large body of knowledge regarding the genes and genetic pathways that are required for the proper formation of the vertebrate eye. Around 10 h postfertilization (hpf ), several secreted proteins induce the most anterior region of the anterior ectoderm to become the neural plate. Several signaling pathways (wingless (Wnt), fibroblast growth factor, and insulin-like growth factor) then further subdivide the anterior neural plate to produce the presumptive eye field. Some anterior-most cells then begin expressing several homeobox transcription factors, such as the retinal progenitor genes visual system homeobox 2 (vsx2), paired box gene 6 (pax6), and retinal homeobox (rx), which further restrict their fates to be retinal progenitor cells. The midline of the underlying head mesoderm then expresses several secreted, signaling molecules (such as the sonic hedgehog-related protein, sonic-you), which induces the eye field in the anterior neural plate to split into two distinct regions. This yields the first morphological sign of the developing visual system, the bilateral evagination of a single-cell thick epithelium from the anterior end of the neural keel, which will develop into the optic lobe. The optic lobes are morphologically visible by 12 hpf (Figure 1(a)). Each optic lobe expresses diffusible signals that induce the overlying naı¨ve epithelium to commit to form the lens. As the lens placode starts to thicken (18 hpf, Figure 1(b)), it produces soluble signaling molecules that promote the underlying optic lobe to proliferate and invaginate, which results in the formation of a concave neuroepithelium – the optic cup. Expression of the transcription factor genes microphthalmia-associated transcription factor (mitf ) in the ventral optic cup and vsx2 in the dorsal optic cup commits those cells to develop into the retinal pigmented epithelium and neural retina, respectively. The region that lies at the junction of the mitf and vsx2 expressing cells later becomes one region of persistent retinal neurogenesis in the adult retina, the circumferential marginal zone (CMZ, discussed below).

NR

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Figure 1 Zebrafish retinal development. (a) Cells evaginate bilaterally from the anterior neural keel (NK) to form the optic lobes (OL), which are morphologically distinguishable by 10–12 h postfertilization (hpf). (b) By 18 hpf, the proliferating cells of the presumptive neuroretina (PNR) have induced the overlying ectoderm to thicken into the lens placode (LP). The retina then invaginates to form the OC, with the thicker dorsal cells, or presumptive neuroretina, expressing vsx2 and the thinner ventral cells, or presumptive retinal pigmented epithelium (PRPE), expressing mitf. (c) The cells in the neural retina continue to proliferate until a wave of sonic hedgehog (Shh) signaling induces the first retinal ganglion cells (RGCs) in the most basal nuclear layer, the ganglion cell layer (GCL: between the arrowheads), to differentiate around 40 hpf in the center of the retina, whereas cells in the retinal margin remain in an uncommitted state.

(d) Additional waves of Shh signaling produce three nuclear layers, the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL). The ONL is immediately below the retinal pigmented epithelium (RPE), which is now darkened with pigment granules by 3 days postfertilization (dpf). Scale bars represent 20 mm. OC, optic cup; NK, neural keel; NR, neural retina; RPE, retinal pigmented epithelium; CMZ, circumferential marginal zone; GCL, ganglion cell layer; INL, inner nuclear layer; L, lens; LP, lens placode; OL, optic lobe; ONL, outer nuclear layer; PNR, presumptive neuroretina; PRPE, presumptive retinal pigmented epithelium.

The Laminar Structure of the Retina Forms as Cells Exit the Cell Cycle and Differentiate

The number of cells in the NR increases through cell proliferation until the diffusible signaling protein sonic hedgehog (Shh) is produced in a ventronasal patch of the NR. The Shh protein initiates a wave of expression of the basic helix–loop–helix (bHLH) transcription factor atonal homolog 7 (Atoh7) that sweeps radially toward the dorsal retina that induces the differentiation of the retinal

ganglion cells (RGCs) (Figure 1(c)). Shh is then secreted from the newly specified RGCs to sequentially induce the apically located naı¨ve mitotic cells to exit the cell cycle and commit to the other retinal neuronal identities. Later, Shh signaling from the RPE is required for proper photoreceptor differentiation. This process results in the formation of the terminally laminated retina, which is composed of three nuclear layers (ganglion cell layer, inner nuclear layer (INL), and outer nuclear layer) and two synaptic layers (inner plexiform layer and outer plexiform layer) by 72 hpf (Figure 1(d)).

Intrinsic commitment and specification of the different retinal cell types also requires the expression of homeobox transcription factors and pro-neural genes of the Notch signaling pathway. Experiments aimed at determining when the different retinal cell types are committed (neuronal birthdating) established a bias of early committing cells to the RGC, amacrine cell, cone, and horizontal cell classes, followed by the bipolar and rod photoreceptor cells, and lastly the Mu¨ller glial cells. The reproducible timing observed with these different cell types suggests that a molecular clock modulates their commitment and differentiation. This model proposes that the commitment of a retinal progenitor cell to a particular neuronal cell type corresponds to when the progenitor cell exits the cell cycle, with early committed cells localizing more basally in the retina. Thus, retinal progenitor cells divide and some daughter cells become ganglion cells, while the remaining daughter cells continue to divide. Some of these retinal progenitors exit the cell cycle and differentiate as amacrine cells and others continue as retinal progenitors. These progenitors continue their asymmetrical cell division to produce some retinal neurons with each round of cell division, until the final retinal progenitors are committed to become Mu¨ller glial cells. This suggests that the Mu¨ller glia is the retinal cell type that is most recently differentiated from the retinal progenitor cell. This model also allows for the presence of external signals to influence the commitment of the cell, which also changes over time. These mechanisms appear to be conserved across species.

Addition of Retinal Cells Throughout the

Life of a Zebrafish

Unlike mammals, the zebrafish eye continuously grows throughout the lifetime of the fish. This growth requires the continual generation of new retinal neurons in a process called persistent neurogenesis. These additional retinal cells are produced from two adult stem cell niches, the CMZ (Figure 1(d) and Figure 2(a)) and an INL stem cell niche (Figure 2(a)). The stem cells within the CMZ continue to express the cell cycle genes and the embryonic retinal progenitor genes, such as orthodenticle homolog 2 otx2, pax6, and rx, throughout the life of the fish. These stem cells proliferate to yield daughter cells that ultimately

differentiate into ganglion cells, amacrine cells, horizontal cells, cone photoreceptors, bipolar neurons, and Mu¨ller glial cells, but not rod photoreceptors. Rods arise from the INL stem cell niche as described below. The addition of these newly differentiated cells to the region adjacent to the CMZ (Figure 2(c) and 2(d)) results in the radial growth of the adult retina.

The INL stem cells have recently been demonstrated to correspond to the Mu¨ller glial cells. Unlike the CMZ stem cells, however, the asymmetric division of the Mu¨ller glia ultimately produce only rod photoreceptors during persistent neurogenesis (Figure 2). Relatively few Mu¨ller glia are actively dividing at any given moment. The asymmetric division of the Mu¨ller glial cell produces neuronal progenitor cells (Figure 2(a)), which continue to proliferate as they migrate to the outer nuclear layer, where they are called rod precursor cells and continue to undergo cell division (Figure 2(b)). Unlike the pluripotent CMZ stem cells, these Mu¨ller glial-derived rod precursor cells are committed to differentiate into only rod photoreceptors during persistent neurogenesis (Figure 2(c)). As the adult eye enlarges, the distance between the originally differentiated rod photoreceptors increases. The Mu¨ller glialderived rods fill in this space. Thus, persistent neurogenesis in the zebrafish retina encompasses both the radial growth of the retina by addition of all retinal cell types by the CMZ and the slow production of additional rod photoreceptors by the Mu¨ller glia (Figure 2(d)).

Regeneration in the Zebrafish Retina

Zebrafish regenerate all retinal neurons

Persistent neurogenesis involves the continual generation of new neurons in the adult retina, without any prior loss of retinal neurons that must be replaced. It should be noted that the scientific literature often uses retinal regeneration to define the reprojection of axons from viable neuronal soma to replace damaged axons, such as the reprojection of axons from RGCs subsequent to an optic nerve crush or severing. For this discussion, retinal regeneration refers to the replacement of entire neuronal cells that were lost through retinal insult or genetic causes. Zebrafish respond to the loss of retinal neurons by significantly increasing both the number of Mu¨ller glia that reenter the cell cycle and the rate of proliferation in the neuronal progenitor cells, relative to that observed during persistent neurogenesis. This amplified proliferation response appears to be proportional to the amount of damage suffered. In contrast to persistent neurogenesis, these Mu¨ller-glial-derived neuronal progenitors are not committed to become only rod photoreceptors. These neuronal progenitors proliferate, migrate to the retinal layer that contains the missing neurons, and differentiate specifically into the lost neurons. Thus, the damage response alters the persistent neurogenesis program to

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Figure 2 Continued

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significantly increase the number of neuronal progenitors and allow them a greater breadth of cell differentiation potential.

A variety of damage paradigms have been studied in the zebrafish retina. For example, simple surgical lesion induces cells proximal to the cut site to die, which usually results in the death of all the retinal cell types in a small area of the retina. Similarly, heat caused by either a high temperature probe or laser ablation, or high concentrations of ouabain (Na, K-ATPase inhibitor), often causes loss of cells from all three retinal layers. In contrast, other damage models exhibit more restricted cell-type loss. For example, intravitreal injection of low concentrations of ouabain causes the loss of ganglion cells and INL neurons, without the loss of significant numbers of rod and cone photoreceptors. Constant intense light, in contrast, causes apoptosis of only the rod and cone photoreceptors, primarily in the dorsal and central retina, with no detectable cell death in the INL or GCL layers. An advantage of the light damage model is that only two neuronal classes are lost, rod and cone photoreceptors, which limits the complexity of the regeneration response. Furthermore, the light damage extends across a large region of the retina, which induces the participation of a very large number of Mu¨ller glia and neuronal progenitor cells. This results in an amplification of the signals and processes that are required for regeneration, which should increase the likelihood of their identification.

Constant Intense Light Kills Photoreceptors, Which Are then Regenerated by Mu¨ller Glia

Zebrafish rods and cones die by apoptosis upon exposure to prolonged high-intensity light. Apoptosis is a genetically programmed cell death mechanism, in which the dying cell fragments its DNA and the cell body (blebs) to produce easily phagocytosed cell corpses. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), is a standard method to detect this fragmented genomic DNA. Fragmentation of the DNA produces large

numbers of free 30 -hydroxyl ends that can be used by terminal deoxynucleotidyl transferase to add dUTP nucleotides that are covalently modified to a detectable molecule, such as biotin or fluorescein (Figure 3).

The light-induced photoreceptor cell death is rapid, with TUNEL-positive cells first detected in the ONL as early as 3 h after initiating the light treatment. By 16 h of constant light, the photoreceptor cell death is evident based on the reduction in the number of ONL nuclei relative to undamaged retina and is striking by 3 days of constant light (Figure 4(a) and 4(b)). In addition to the strong TUNEL-positive signal that is detected in the ONL, a weaker TUNEL signal is observed throughout the Mu¨ller glial cells in the INL. Strikingly, this INL TUNEL signal is neither restricted to, nor predominantly localized in, the Mu¨ller glia nuclei, which demonstrates that the Mu¨ller glial cells are not apoptotic. Rather, the colocalization of some proteins derived from apoptotic rods with the TUNEL signal in the Mu¨ller glia suggests that the Mu¨ller glia selectively engulf dying rod photoreceptors. These data are consistent with results in the degeneration model of the Tg(Xops:mCFP) transgenic line, which expresses membrane-bound cyan fluorescent protein (mCFP) from the Xenopus rod opsin promoter (Xops) in only zebrafish rods. The Tg(Xops:mCFP) retina exhibits a persistent loss of only rod photoreceptors, even in the absence of any retinal insult, such as constant bright light. Furthermore, the Tg(Xops:mCFP) retina revealed TUNEL labeling in the INL in a morphology similar to Mu¨ller glia. Thus, the engulfment of apoptotic rod photoreceptors by Mu¨ller glia is a common early feature of the damaged zebrafish retina and may be required for regenerative Mu¨ller glial cell stimulation. TUNEL labeling in the Mu¨ller glia can be detected early, within the first 12 h of light treatment, and represents one of the earliest signs of Mu¨ller glial response to apoptotic photoreceptor damage. Robust regeneration of the light-damaged zebrafish retina follows and a normal complement of rod and cone photoreceptors reform within 28 days after terminating the constant light treatment (Figure 4(c)).

Apoptotic nucleus

TdT labeling

In light-damaged zebrafish retinas, the Mu¨ller glia exhibit increased expression of proliferating cell nuclear antigen (Pcna), which is a component of the DNA replication machinery. The expression of this protein in the Mu¨ller glia indicates their reentry into the cell cycle (Figure 4(d)). To confirm that the Mu¨ller glia were proliferating, light-damaged Tg(gfap:EGFP) retinas, which express enhanced green fluorescent protein (EGFP) specifically in the Mu¨ller glial cells from the zebrafish glial fibrillary acidic protein (gfap) promoter, were coimmunolabeled for the expression of the proliferation marker Pcna. The number of actively dividing Mu¨ller glia, coexpressing EGFP and the red fluorescing anti-Pcna antibody, increases through 51 h of constant intense light treatment (Figure 4

(d)), at which point the Mu¨ller glial-derived INL neuronal progenitor cells continue to proliferate to produce clusters of 8–12 progenitor cells associated with a single Mu¨ller glial cell. While this suggests that all the neuronal progenitor cells in a cluster are derived from a single Mu¨ller glial cell and remain associated with that glial cell, this has not been formally demonstrated. This Mu¨ller glial cell-based regeneration response is conserved throughout a number of different damage models. For example, either surgical lesion or ouabain injection results in the death of many different neuronal cell types in the retina, and they both exhibit increased proliferation of the Mu¨ller glial cells (Figure 4(e)–4(h)).

Curiously, not all of the Mu¨ller glial cells proliferate in response to constant intense light treatment. Earlier reports suggested that a threshold of rod cell death was required to induce the Mu¨ller glial proliferation response. However, intravitreal injection of a low concentration of ouabain into the zebrafish retina resulted in massive death of the neurons in both the ganglion cell and INLs, with minimal cell death of photoreceptors (Figure 4(f )). Regeneration of the ouabain-damaged retina takes longer than the light-damaged retina, but still produces a relatively normal retina by 60 days post ouabain injection (Figure 4(g)). This demonstrates that significant rod or cone cell death is not required to induce retinal regeneration from the Mu¨ller glia (Figure 4(h)). Furthermore, this suggested that the number of apoptotic neurons, rather than the type of neuron, was critical for inducing this regeneration response.

To address if only rod photoreceptor cell death was sufficient to induce a Mu¨ller glial-derived regeneration response, the Tg(Xops:mCFP) transgenic line and the phosphodiesterase 6c (pde6c) mutant line were analyzed. The pdge6 mutant line fails to maintain cone cells, in contrast to the rod photoreceptor cell death in the Tg (Xops:mCFP) fish. While no detectable Mu¨ller glial proliferation response was observed in the Tg (Xops:mCFP) line, a small, but significant, Mu¨ller glial proliferation response was detected in the pdge6 mutant line. This suggested that loss of rods requires only increased proliferation of the ONL rod precursor cells

that were derived from the neuronal progenitor cells during persistent neurogenesis, while loss of cones requires the increased proliferation of the pluripotent Mu¨ller glia.

To further test this hypothesis, a Tg (zop:ntr) transgenic line was generated that expresses the bacterial nitroreductase b (ntr) gene from the zebrafish rhodopsin promoter (zop). The NTR enzyme converts the prodrug, metronidazole, into a cell-autonomous toxin within NTR producing cells. Exposing a Tg (zop:ntr) transgenic line, expressing NTR in all rod photoreceptors, to metronidazole results in the death of only rod photoreceptors and the induction of a Mu¨ller glial proliferation response. Addition of metronidazole to a similar transgenic line that expresses NTR in only a subset of rods, however, failed to induce the Mu¨ller glial response. These data suggest that it is the magnitude of the cell death that determines if the Mu¨ller glia exhibit a robust proliferation response. The failure of the Tg (Xops:mCFP) transgenic line to induce the Mu¨ller glial proliferation response may be due to the rod cell death being small and chronic relative to the massive and acute cell death observed in the metronidazole-treated Tg(zop:ntr) transgenic retinas that express NTR protein in all the rod photoreceptors.

Discovery and Analysis of Candidate Genes

Involved in Retinal Regeneration

Several groups have performed microarray analyses of mRNA expression patterns in different retinal damage models to determine what genes might change their expression during specific points of regeneration. In both the surgical-lesioned and light-damaged models, signal transducer and activator of transcription 3 (stat3) and its negative regulator suppressor of cytokine signaling 3 (socs3) exhibited increased expression shortly after the retinal insult (e.g., within 16 h of starting the constant intense light treatment). The increased stat3 expression suggests that Gp130 receptor signaling is involved in the early damage response. Gp130 is a promiscuous receptor that binds a number of different extracellular signaling molecules, such as cytokines, to activate the Stat3 transcription factor in neuronal niches to promote cell proliferation in the adult vertebrate brain. The socs3 gene, which is transcriptionally activated by the Stat3 protein, encodes a protein that binds the activated receptor to prevent further Stat3 activation. Increased stat3 expression is also known to occur in RGCs following optic nerve crush, further supporting Stat3’s role in the damage response. Microarrays also revealed that both the achaetescute complex-like 1a (ascl1a) pro-neural gene and the notch pathway genes were upregulated in the surgicallesioned and light-damaged retinal models. By contrast, retinal progenitor genes, such as rx and vsx2, whose expression are maintained in the CMZ throughout life, are not significantly increased in expression in the damaged retina.

Hallmarks

Genes expressed

Histology

This suggests that Mu¨ller glia exhibit a state of competency that is downstream of the CMZ stem cells, but upstream of retinal neuron differentiation.

The ability to test the function of these various candidate genes in the regenerating retina has been recently advanced by the development of a method to electroporate morpholinos into the adult retina. Morpholinos are modified oligonucleotides that contain a morpholine ring, rather than deoxyribose. The morpholinos, which are complementary to a specific mRNA sequence, can base pair with and transiently block the efficient translation of the target mRNA. Because some protein could still be correctly translated in the presence of a morpholino, these loss-of-function experiments and organisms are termed knockdowns and morphants, respectively. Morpholinos have traditionally been introduced into zebrafish embryos by direct injection into either the relatively large cells of the early embryo (1–32 cell stage) or the yolk. The morpholinos then diffuse into the daughter cells as they divide during embryonic development. This approach has tested the function of numerous proteins in zebrafish development. Recently, morpholinos that are covalently attached to a positively charged fluorochrome, lissamine, have been injected into the vitreous and then electroporated into the adult retina. These morpholinos can disrupt retinal regeneration if they are electroporated into the retina to knockdown the expression of a target protein prior to its role in regeneration.

Proof-of-principle experiments to test the effectiveness of this method were shown for the requirement of Pcna in retinal regeneration. Morpholino-induced knockdown of Pcna expression resulted in the Mu¨ller glia failing to proliferate in the light-damaged retina, which led to the premature death of the stimulated Mu¨ller glial cells due to their inability to proceed through the S phase of the cell cycle. Retinas that were injected and electroporated with anti-Pcna morpholinos also failed to upregulate expression of the retinal progenitor cell marker pax6 in the Mu¨ller glial-derived neuronal progenitor cells. Similar morpholino knockdown studies revealed that Stat3 and Pax6 are also required at different steps in the regeneration process. Microarray analyses of mRNA expression at different time points during regeneration of the surgical-lesioned and light-damaged retinas

have revealed numerous candidate genes for functional study. The electroporation of morpholinos will permit a relatively rapid loss-of-function analysis to elucidate the genes that are required for regeneration and the key steps and processes underlying retinal regeneration.

Events Underlying Regeneration of the

Light-Damaged Retina

Adult Mu¨ller glial cells are characterized by the expression of cell-specific markers (Figure 5(a)), such as glutamine synthetase (Glul) and glial fibrillary acidic protein (Gfap) among others. During persistent retinogenesis, rods are added to the established repeating mosaic of cone and rod photoreceptors from the Mu¨ller glial cell population. It is not clear if there is a signal (arrow) to stimulate Mu¨ller glia proliferation or if a small subset of Mu¨ller glia remain in a slow cell cycle (Figure 5(b)). This limited number of proliferating Mu¨ller glia can be detected by Pcna and Pax6 expression (Figure 5(c)). The small number of dividing Mu¨ller glial cells produce a daughter Mu¨ller glial cell (Figure 5(d), brown) and a neuronal progenitor cell (green), which continues to express genes important in the cell cycle (Pcna and cyclins) and genes required for cell specification signaling (notch, delta, and hes). The neuronal progenitor continues to proliferate and migrates to the ONL. As the neuronal progenitor reaches the ONL (where it is now called a rod precursor), it continues dividing or begins to differentiate, expressing rod specification genes (neurogenic differentiation (neurod ), nuclear receptor subfamily 2e3 nr2e3, crx, and rx1) and giving rise only to rod photoreceptors (Figure 5(f ), green).

Upon light-induced retinal damage (or other forms of retinal insult), Mu¨ller glia hypertrophy and transiently increase their expression of Glul and Gfap (Figure 5(h)). If the number of dying rods and cones (Figure 5(h), red) is sufficiently large, the Mu¨ller glial cells increase their expression of the ascl1a and signal transducer (stat3 genes (Figure 5(i)) through unclear mechanisms. Electroporation of morpholinos into the retina prior to inducing retinal damage revealed that both stat3 and ascl1a are independently required for the Mu¨ller glial proliferation response. The reentry of these Mu¨ller glia into the cell cycle is accompanied by the increased expression of the

cell cycle regulatory proteins, the cyclins. The Mu¨ller glial cell divisions produce neuronal progenitor cells, which continue to proliferate (expressing Pcna and the cyclins) and begin to express the retinal progenitor gene pax6 and genes in the Notch and Delta neuronal signaling pathways (Figure 5(j)). As the neuronal progenitor cells migrate toward the ONL, they begin to express genes that are required for the commitment and differentiation of rod and cone photoreceptors (neuroD, nr2e3, and crx ; Figure 5(k)). Within 28 days of ending the constant intense light treatment, the rod and cone photoreceptors have regenerated (Figure 5(l)).

Vestigial Retinal Regeneration Activity in

Mammals

The finding that the Mu¨ller glial cell acts as an adult neuronal stem cell in the regeneration of the damaged zebrafish retina suggests that it potentially could produce a similar regeneration response in the damaged mammalian retina. Recent studies support the hypothesis that mammalian Mu¨ller glia possess some of the features of adult neuronal stem cells. Transplanted rodent Mu¨ller glia into a damaged retina will produce rhodopsin-positive cells. As expected, intraocular injection of neurotoxic doses of N-methyl-D-aspartic acid (NMDA), which is a synthetic amino acid that binds a glutamate receptor, results in the death of many retinal cell types. However, NMDA damage in the rat retina is followed by a small number of Mu¨ller glia proceeding through one cell division to yield a limited number of cells that differentiate into photoreceptors and bipolar cells. This Mu¨ller glial cell proliferation and regeneration response can be slightly enhanced with the addition of various growth factors. Injection of the toxin N-methyl- N-nitrosourea (NMU) into the adult rat eye, which specifically kills photoreceptors, induces Mu¨ller glial hypertrophy and increased expression of GFAP and the neural stem cell marker, nestin, followed by the proliferation of some Mu¨ller glia. While BrdU labeling confirmed that the Mu¨ller glia actively divided in response to the NMU damage, only 42% of the BrdU-labeled cells remained 2 weeks after the NMU injection. Of these surviving BrdU-labeled cells, which were all located in the INL, 58% expressed glutamine synthetase (suggesting they corresponded to Mu¨ller glial cells that initially divided) and only 8% expressed rhodopsin. Thus, there were very few rhodopsin-positive cells produced by this Mu¨ller cell division and the ones that were generated, failed to properly migrate to the ONL. Expression or addition of various growth factors increased the number of proliferating Mu¨ller glia only to a small extent. Activation of various signaling pathways, such as Notch, similarly resulted in only a slight increase in the number of Mu¨ller glial-derived neuronal progenitors in the damaged rodent retina. While retinal damage induced some mammalian Mu¨ller glia to proliferate and various growth factors or

signaling pathways slightly increased that number, there remained an insufficient number of proliferating Mu¨ller glial cells and neuronal progenitor cells to properly regenerate all the neurons lost from the retinal damage. Analyzing the mechanisms underlying the robust Mu¨ller glial proliferation response in the light-damaged zebrafish retina will provide important clues as to why the regenerative capacity of the mammalian retina is so limited.

Similarities and Differences of Development with Retinal Ontogeny/Genesis

As discussed above, Mu¨ller glia and the neuronal progenitor cells respond to retinal damage by increasing the expression of many of the retinal progenitor genes, such as pax6, that are expressed during embryogenesis and near the CMZ throughout the life of the fish. The inability of the mammalian Mu¨ller glial cells to mount a sufficiently robust proliferative and regenerative response may be due to differences in the signals that stimulate the Mu¨ller glia to respond. All attempts to stimulate sufficient levels of Mu¨ller glial proliferation in the mammalian retina have met with very limited success. Conversely, the failure to robustly proliferate may be due to intrinsic differences between the mammalian and zebrafish Mu¨ller glial cells. For example, transplanted mammalian Mu¨ller glial cells into a damaged retina will differentiate into rhodopsin-positive cells that are predominantly restricted to the INL, rather than repopulating the ONL. Thus, a further understanding of the signals that stimulate the zebrafish Mu¨ller glial and neuronal progenitor cells to proliferate and migrate may reveal insights into how to better manipulate the mammalian retina, and by extension, potentiate human retinal regeneration.

See also: Color Blindness: Inherited; Injury and Repair: Light Damage; Retinal Histogenesis.

Further Reading

Bailey, T. J., El-Hodiri, H., Zhang, L., et al. (2004). Regulation of vertebrate eye development by Rx genes. International Journal of Developmental Biology 48: 761–770.

Bernardos, R. L., Barthel, L. K., Meyers, J. R., and Raymond, P. A. (2007). Late-stage neuronal progenitors in the retina are radial Muller glia that function as retinal stem cells. Journal of Neuroscience 27: 7028–7040.

Fausett, B. V. and Goldman, D. (2006). A role for alpha1 tubulinexpressing Muller glia in regeneration of the injured zebrafish retina.

Journal of Neuroscience 26: 6303–6313.

Fimbel, S. M., Montgomery, J. E., Burket, C. T., and Hyde, D. R. (2007). Regeneration of inner retinal neurons after intravitreal injection

of ouabain in zebrafish. Journal of Neurosciences 27: 1712–1724.

Kassen, S. C., Ramanan, V., Montgomery, J. E., et al. (2007). Time course analysis of gene expression during light-induced

photoreceptor cell death and regeneration in albino zebrafish.

Developmental Neurobiology 67: 1009–1031.

Malicki, J. (2000). Harnessing the power of forward genetics – analysis of neuronal diversity and patterning in the zebrafish retina. Trends in Neurosciences 23: 531–541.

Morris, A. C., Scholz, T. L., Brockerhoff, S. E., and Fadool, J. M. (2008). Genetic dissection reveals two separate pathways for rod and cone regeneration in the teleost retina. Developmental Neurobiology

68: 605–619.

Morris, A. C., Schroeter, E. H., Bilotta, J., Wong, R. O., and Fadool, J. M. (2005). Cone survival despite rod degeneration in XOPS-mCFP transgenic zebrafish. Investigative Ophthalmology and Visual Science

46: 4762–4771.

Otteson, D. C. and Hitchcock, P. F. (2003). Stem cells in the teleost retina: Persistent neurogenesis and injury-induced regeneration.

Vision Research 43: 927–936.

Raymond, P. A., Barthel, L. K., Bernardos, R. L., and Perkowski, J. J. (2006). Molecular characterization of retinal stem cells and their niches in adult zebrafish. BMC Developmental Biology 6: 36.

Raymond, P. A. and Hitchcock, P. F. (2000). How the neural retina regenerates. Results and Problems in Cell Differentiation 31: 197–218.

Thummel, R., Kassen, S. C., Enright, J. M., et al. (2008). Characterization of neuronal progenitors in zebrafish adult retinal regeneration. Experimental Eye Research 87: 433–444.

Thummel, R., Kassen, S. C., Montgomery, J. E., Enright, J. M., and Hyde, D. R. (2007). Inhibition of Muller glial cell division blocks regeneration of the light-damaged zebrafish retina. Developmental Neurobiology 68: 392–408.

Vihtelic, T. S. and Hyde, D. R. (2000). Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina. Journal of Neurobiology 44: 289–307.

Vihtelic, T. S., Soverly, J. E., Kassen, S. C., and Hyde, D. R. (2006). Retinal regional differences in photoreceptor cell death and regeneration in light-lesioned albino zebrafish. Experimental Eye Research 82: 558–575.

Yurco, P. and Cameron, D. A. (2005). Responses of Muller glia to retinal injury in adult zebrafish. Vision Research 45: 991–1002.