Ординатура / Офтальмология / Английские материалы / Retinal Development_Sernagor, Eglen, Harris, Wong_2006

Weaver, D. R. and Reppert, S. M. (1995). Definition of the developmental transition from dopaminergic to photic regulation of c-fos gene expression in the rat suprachiasmatic nucleus. Brain Res. Mol. Brain Res., 33, 136–48.

Young, R. W. (1984). Cell death during differentiation of the retina in the mouse. J. Comp. Neurol., 229, 362–73.

New perspectives

(Stone and Farthing, 1942). They were even able to exchange eyes between adult animals, with recovery of function. Studies in the 1960s and 1970s applied the technologies of electron microscopy and 3H-thymidine autoradiography to characterize the regeneration process further (Hendrickson, 1964; Keefe, 1973a,b,c,d). These studies confirmed that both the CMZ and the RPE contributed to the regenerated retina, the former to the anterior retina and the latter to the posterior retina. Moreover, by this time it was recognized that the CMZ represents a zone of continued neurogenesis in adult amphibians and fish (Gaze and Watson, 1968; Hollyfield, 1968), and thus the regeneration from this zone represents an increase in normal retinal growth.

Studies of retinal regeneration in fish have shown that there is a source for genesis of new retinal neurons within the adult retina (Raymond and Hitchcock, 1997), in addition to the CMZ and the RPE. When small regions of gold fish retina are excised, or more widespread damage is induced by neurotoxins, new retinal neurons are generated. The regenerated neurons do not come from either the CMZ or the RPE, but rather arise from a stem-like cell within the retinal parenchyma. Recent work in the post-hatch chick retina is also consistent with an intrinsic source for retinal regeneration (Fischer and Reh, 2001; see Section 15.5). Taken together, the studies indicate that there are many different sources of retinal regeneration in various species (Figure 15.1). All of these sources share several common features:

(1) although they are very different tissues histologically, they are all derived from the neural tube; (2) they all have the capacity to re-enter the cell cycle, while the retinal neurons are incapable of proliferation; and (3) they all express several genes typically present in retinal progenitors at some stage during the regeneration process. In this chapter, we will highlight the common features of the process with an eye towards understanding the essential elements of retinal regeneration.

15.3Regeneration from the ciliary margin zone

As noted above, the CMZ was initially thought to be the only source for retinal regeneration. In addition, this is the one source of neurogenesis in the adult retina common to fish, amphibians and birds. The CMZ is in many ways similar to other regions of persistent progenitors in the CNS, like the subventricular zone and the hippocampal progenitor zone. The CMZ of fish and amphibians allows the retinal growth to keep pace with the overall growth of the eye and the animal. The CMZ of some fish and urodele amphibians continues to generate new retina throughout life, while neurogenesis at the CMZ of anuran amphibians slows considerably at metamorphosis.

The CMZ contributes to the regeneration of the anterior retina in cold-blooded vertebrates. In adult urodeles and larval anurans, proliferation of CMZ cells is up-regulated after retinal damage. Following retinal destruction through devascularization or surgical removal in newts, the CMZ generates a considerably greater amount of new retina than it would in the undamaged eye, and, as described above, early investigators believed that all of the regeneration of the retina was derived from the CMZ. In newts, Keefe reported that the

Figure 15.1 There are many different sources of retinal regeneration in various species. In amphibians, the retinal pigmented epithelium (RPE) is the primary source of new retinal progenitors or stem cells. The proliferating retinal progenitors of the ciliary margin zone (CMZ) generate new neurons in response to damage of the anterior retina in amphibians, fish and birds to a limited extent. The primary source for retinal regeneration in fish is an intraretinal source, likely either the rod progenitor or an intrinsic stem cell; however, in both fish and birds, there is evidence for M¨uller glial-mediated regeneration of retinal neurons. The various parts of the anterior eye, including both the pigmented and non-pigmented epithelia of the ciliary body, and the iris, can generate neurons of various types in mammals and birds, though this has not been shown in fish and amphibians.

number of 3H-thymidine-labelled cells in the CMZ (pars ciliaris retinae) increased approximately tenfold by 20 days after retinal damage (Keefe, 1973a). Similarly, in Rana tadpoles, kainic acid neurotoxic lesions of the retina caused the number of 3H-thymidine-labelled cells to more than double within two or three weeks following damage (Reh, 1987).

Goldfish and zebrafish also display an excellent capacity for retinal regeneration after different types of damage. However, most studies have focused on regeneration from a source within the retinal parenchyma. Nevertheless, evidence indicates that the CMZ survives neurotoxic damage, like ouabain, and that the anterior retina is regenerated from the CMZ in goldfish (Stenkamp et al., 2001). In addition, neurotoxic doses of 6-hydroxydopamine increase the width of the CMZ in fish by 50% (Negishi et al., 1988).

Does the CMZ contribute to retinal regeneration in birds and mammals? In the past few years, our lab has found evidence for a CMZ in the retina of post-hatch chicks and quails up to one year of age (Fischer and Reh, 2000; Kubota et al., 2002). The chicken CMZ can be stimulated to regenerate new retinal neurons after damage as the fish and amphibian CMZs do, but only if exogenous growth factors are injected into the vitreous after neurotoxin treatment (Fischer et al., 2002; Fischer and Reh, 2002). While the normal mammalian retina does not continue to generate new neurons at the retinal margin in adult animals, a recent analysis of a mouse mutant has provided evidence that the mammalian retina may be capable of regeneration (Fischer et al., 2002; Fischer and Reh, 2002) at the margin (Figure 15.2). In mice with a single functioning allele of the Shh receptor, patched, a CMZ-like zone forms at the retinal margin (Moshiri and Reh, 2004). Cells labelled with 5 -Bromo-2 -deoxy-uridine (BrdU) can be found for weeks after the normal cessation of retinal neurogenesis. Moreover, the proliferation of the progenitors at the retinal marginal zone is increased significantly when the patched mutant mouse is crossed onto a retinal degeneration background.

The CMZ of persistent progenitors or retinal stem cells that exists in cold-blooded vertebrates is thus a key source of retinal regeneration that has progressively receded in homeothermic vertebrates like chickens and mice. While the CMZ can regenerate (and generate) all types of retinal neurons in frogs and fish, only a few cell types have been demonstrated to be regenerated from this zone in birds (Fischer and Reh, 2000; Fischer et al., 2002), and even fewer have been found in mice (Moshiri and Reh, 2004). Thus, there is a limitation in both the quantity of neurogenesis at the retinal margin in warm-blooded vertebrates, as well as in the regeneration potential of the proliferating precursor cells and/or their local microenvironment. Regeneration from zones of persistent progenitors in other areas of the CNS, like the subventricular zone or the hippocampal progenitor zone, is similarly limited, with primarily granule neurons generated from both regions (Doetsch et al, 1999). Pyramidal cells in the hippocampus are not replaced by the hippocampal progenitors after lesions (Nakatomi et al., 2002), and, in the song bird, only those neurons normally generated in the adult are capable of being regenerated (Scharff et al., 2000). It should also be emphasized that, even though nearly all animals examined demonstrate some capacity for regeneration from the stem/progenitor cells at the retinal margin, and it is possible to stimulate proliferation and regeneration from these cells with intraocular growth factor injections, the regeneration is relatively local and confined to a few hundred microns of the marginal zone. There is no evidence for long-distance migration from this region to lesions in the central retina.

15.4 Regeneration from the retinal pigmented epithelium

As noted above, historically, the second cell type recognized as providing a source for retinal regeneration in the newt is the RPE. The RPE is morphologically very distinct from neural retina: it is a monolayer of cuboidal cells without any evidence for neurons in normal animals, and it provides critical functions for the rod and cone photoreceptors, including

Figure 15.2 The ciliary margin zone (CMZ) is a region of retinal progenitors that persists into adulthood in fish, amphibians (A,D) and, to a more limited extent, birds (B,E). (C) Although the CMZ is not present in normal mice, in mice with a single functioning allele of the Sonic hedgehog receptor, patched (F), a CMZ-like zone forms at the retinal margin (modified from Moshiri and Reh, 2004). 5 -Bromo-2 -deoxy-uridine-labelled cells are green in E and F, while the silver grains in D show [3H]-thymidine incorporation. The large arrow points to the retinal margin in D–F. CB, ciliary body. For colour version, see Plate 12.

outer segment phagocytosis and visual pigment regeneration. The common embryological origin of these tissues as the two layers of the optic cup, an evagination of the neural tube, belies their apparent morphological and physiological differences.

Retinal regeneration from the RPE in amphibians has been most extensively studied in newts, salamanders and axolotls, though anuran (frog) tadpoles are also able to regenerate retina from the RPE. The most common experimental design is to remove the retina,

leaving the RPE intact. The RPE subsequently loses pigmentation, proliferates and generates two new epithelial layers, a pigmented layer and a non-pigmented layer. The nonpigmented layer begins to express genes typical of retinal progenitor cells and undergoes extensive cell division to produce sufficient new neurons for the new retina (Reh and Nagy, 1987, 1989). Thus, retinal regeneration occurs in two phases. In the first phase, the RPE cells dedifferentiate to become retinal progenitors. The second phase is much like normal development of the retina and follows a similar time course and developmental programme. In vitro experiments have confirmed that the RPE is the source of neural retinal tissue; RPE cells can dedifferentiate in vitro and generate new retinal neurons (Reh et al., 1987). The demonstration of RPE-cell dedifferentiation has been facilitated by the cells’ pigmentation, which provides an intrinsic marker. The regeneration of retina from the RPE was therefore one of the first well-recognized examples of ‘transdifferentiation’ (Okada, 1981). However, it should be noted that this process involves extensive cell proliferation, and a direct conversion between a RPE cell and a retinal neuron is not typically observed.

The embryonic chick eye is capable of a similar form of RPE transdifferentiation (Coulombre and Coulombre, 1965; Park and Hollenberg, 1989; Pittack et al., 1991). Removal of the retina from a chick embryo within the first three to four days of incubation causes the RPE of the chick to undergo a transdifferentiation into neural retinal progenitors, very similar to that observed in the amphibian. The retina that forms from the RPE is laminated like normal retina, and contains relatively normal ratios of the different retinal cell types. Although the RPE can give rise to new retina in amphibians, embryonic chicks and embryonic mammals, there is an important difference in the process in amphibians that is critical for functional regeneration: the RPE generates normally oriented retina in the amphibian, but generates retina of inverted polarity in embryonic chicks and mammals. The reason for this difference is shown in Figure 15.3. The neural tube is an epithelium, with a basal surface and an apical surface. The involution of the optic vesicle that allows optic cup formation leads to the retinal and pigmented epithelia lying adjacent to one another, but with opposite polarities; i.e. their apical surfaces are adjacent. Ultimately, as the retina develops, the photoreceptor outer segments form at the apical surface of the retinal epithelium, while the RPE microvilli form at the apical surface of the RPE. During retinal regeneration in the amphibian, one of the first stages in the process is when the RPE cells detach from their basement membrane (Bruch’s membrane) and round up (Keefe, 1973a,b,c,d; Reh and Nagy, 1987). The rounded-up RPE cells have apparently lost their polarity, but become repolarized when they make contact with remnants of the vitreal basement membrane. The progenitor cells that are produced by the RPE thus have the normal retinal polarity. By contrast, in embryonic chicks and mammals, there is direct conversion of the RPE cell layer into neural retina, without the cells ever detaching from Bruch’s membrane (Coulombre and Coulombre, 1965; Park and Hollenberg, 1989; Pittack et al., 1991). As a result, the regenerated retina retains the polarity of the original RPE and thus the retina is inverted from its ‘normal’ orientation. In addition to the inverted polarity, the regenerated retina of chicks and mammals has another obvious problem: in regions where the RPE is converted to retina,

Figure 15.3 Retinal regeneration from the pigmented epithelium in amphibians and chick embryos results in an oppositely oriented retina. The optic vesicle is shown for orientation, with a basal surface and an apical surface. During retinal regeneration in the amphibian, one of the first stages in the process is when the RPE cells detach from their basement membrane (Bruch’s membrane), migrate into the vitreous and become repolarized when they make contact with remnants of the vitreal basement membrane. The progenitor cells that are produced by the RPE thus have the normal retinal polarity. By contrast, in embryonic chicks and mammals, there is direct conversion of the RPE cell layer into neural retina, without the cells ever detaching from Bruch’s membrane. As a result, the regenerated retina retains the polarity of the original RPE and thus the retina is inverted from its ‘normal’ orientation.

no RPE remains. Thus, while the RPE’s ability to convert to neural retina in embryonic chicks and mammals indicates that some parts of the regeneration process of amphibians are retained in higher vertebrates, it is clear that there are important differences that need to be understood if the process is to be stimulated in the mammalian retina.

Although our understanding of the molecular events critical for regeneration are far from complete, several key events in the process appear to mirror aspects of normal development (Moshiri et al., 2004). Signalling molecules, including fibroblast growth factors (FGFs), bone morphogenetic proteins and hedgehogs, have been shown to be critical signalling molecules in both retinal development and regeneration. Coulombre first noted that the transdifferentiation of the RPE in chick embryos required that a small amount of neural retinal tissue remain in the eye (Coulombre and Coulombre, 1965). In Xenopus tadpoles