Regenerative medicine for retinal diseases: activating endogenous repair mechanisms.

The retina is subject to degenerative diseases that often lead to significant visual impairment. Non-mammalian vertebrates have the remarkable ability to replace neurons lost through damage. Fish, and to a limited extent birds, replace lost neurons by the dedifferentiation of Müller glia to a progenitor state followed by the replication of these neuronal progenitor cells. Over the past five years, studies have investigated whether regeneration can be stimulated in the mouse and rat retina. Several groups have reported that at least some types of neurons can be regenerated in the mammalian retina in vivo or in vitro, and that the regeneration of neurons can be stimulated using growth factors, transcription factors or subtoxic levels of excitatory amino acids. These recent results suggest that some part of the regenerative program that occurs in non-mammalian vertebrates remains in the mammalian retina, and could provide a basis to develop new strategies for retinal repair in patients with retinal degenerations.

AIPL1, a protein implicated in Leber's congenital amaurosis, interacts with and aids in processing of farnesylated proteins.

The most common form of blindness at birth, Leber's congenital amaurosis (LCA), is inherited in an autosomal recessive fashion. Mutations in six different retina-specific genes, including a recently discovered gene, AIPL1, have been linked to LCA in humans. To understand the molecular basis of LCA caused by aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) mutations, and to elucidate the normal function of AIPL1, we performed a yeast two-hybrid screen using AIPL1 as bait. The screen demonstrated that AIPL1 interacts specifically with farnesylated proteins. Mutations in AIPL1 linked to LCA compromise this activity. These findings suggest that the essential function of AIPL1 within photoreceptors requires interactions with farnesylated proteins. Analysis of isoprenylation in cultured human cells shows that AIPL1 enhances the processing of farnesylated proteins. Based on these findings, we propose that AIPL1 interacts with farnesylated proteins and plays an essential role in processing of farnesylated proteins in retina.

A comparative study of neurogenesis in the retinal ciliary marginal zone of homeothermic vertebrates.

The retina of many fish and amphibians grows throughout life, roughly matching the overall growth of the animal. The new retinal cells are continually added at the anterior margin of the retina, in a circumferential zone of cells, known as the ciliary marginal zone, or CMZ. Recently, Fischer and Reh [Dev. Biol. 220 (2000) 197] have found that new neurons are added to the retina of the chicken via proliferation and subsequent differentiation of neurons and glia at the retinal margin in a zone highly reminiscent of the CMZ of lower vertebrates. In addition, other groups have reported that putative retinal stem cells could be isolated from the ciliary margin of the adult mouse. In light of these findings, we have re-investigated the eyes of three additional species to determine whether other homeothermic vertebrates also possess CMZ cells and whether we could detect evidence for addition of neurons at the retinal margin in mature animals. We examined one additional avian species, the quail, one marsupial, the opposum, and one mammal, the mouse. We find that the CMZ cells have been gradually diminished during vertebrate evolution. The quail has a reduced CMZ as compared to the chicken, while the opposum has only a few cells likely related to the CMZ and we failed to find evidence of CMZ cells at the margin of the mouse retina.

Immunocytochemical characterization of cysts in the peripheral retina and pars plana of the adult primate.

PURPOSE: To better characterize the cellular constituents of cysts in the peripheral retina and pars plana of the adult monkey. METHODS: Frozen sections of the peripheral retinal margin and pars plana from monkeys (Macaca nemestrina) between 1 and 15 years of age were stained with toluidine blue or immunolabeled with a variety of glia- and neuron-specific antibodies. RESULTS: In animals 1 to 2 years of age, the nonpigmented inner layer of the pars plana is a pseudostratified columnar epithelium. In these young animals, the peripheral retina had distinct layers and did not contain cysts. In animals 6 years of age or older, there were numerous cysts in the pars plana and in the peripheral retina. In the peripheral retina, neurons were randomly distributed and did not have a laminar organization. Cells surrounding cysts were immunoreactive for different types of markers for retinal neurons. Some of the cells surrounding cysts in the pars plana were also unexpectedly immunoreactive for antigens normally expressed only in retinal neurons and glia. CONCLUSIONS: Cysts form in the peripheral retina and pars plana in adult monkeys. The peripheral retinal cysts disrupt the normal lamination of the cells, but all types of retinal neurons are still present in the cysts. In an unexpected finding, cysts in the pars plana also contained cells immunoreactive for a few of the markers of retinal cells, suggesting that neurogenesis may occur in the pars plana of the adult primate.

Müller glia are a potential source of neural regeneration in the postnatal chicken retina.

The retina of warm-blooded vertebrates is believed to be incapable of neural regeneration. Here we provide evidence that the retina of postnatal chickens has the potential to generate new neurons. In response to acute damage, numerous Müller glia re-entered the cell cycle, and shortly thereafter, expressed CASH-1, Pax6 and Chx10, transcription factors expressed by embryonic retinal progenitors. These progenitor-like cells transiently expressed neurofilament. Newly formed cells became distributed throughout the inner and outer nuclear layers of the retina, and remained for at least three weeks after damage. Some of these newly formed cells differentiated into retinal neurons, a few formed Müller glia, and most remained undifferentiated, with continued expression of Pax6 and Chx10. These cells continued to proliferate when grown in culture, with some differentiating into retinal neurons or Müller glia. We propose that, in response to damage, Müller glia in the retina are a potential source of neural regeneration.

A thyroid hormone receptor that is required for the development of green cone photoreceptors.

Color vision is facilitated by distinct populations of cone photoreceptors in the retina. In rodents, cones expressing different opsin photopigments are sensitive to middle (M, 'green') and short (S, 'blue') wavelengths, and are differentially distributed across the retina. The mechanisms that control which opsin is expressed in a particular cone are poorly understood, but previous in vitro studies implicated thyroid hormone in cone differentiation. Thyroid hormone receptor beta 2 (TR beta 2) is a ligand-activated transcription factor that is expressed in the outer nuclear layer of the embryonic retina. Here we delete Thrb (encoding Tr beta 2) in mice, causing the selective loss of M-cones and a concomitant increase in S-opsin immunoreactive cones. Moreover, the gradient of cone distribution is disturbed, with S-cones becoming widespread across the retina. The results indicate that cone photoreceptors throughout the retina have the potential to follow a default S-cone pathway and reveal an essential role for Tr beta 2 in the commitment to an M-cone identity. Our findings raise the possibility that Thrb mutations may be associated with human cone disorders.

FGFR3 expression during development and regeneration of the chick inner ear sensory epithelia.

Several studies suggest fibroblast growth factor receptor 3 (FGFR3) plays a role in the development of the auditory epithelium in mammals. We undertook a study of FGFR3 in the developing and mature chicken inner ear and during regeneration of this epithelium to determine whether FGFR3 shows a similar pattern of expression in birds. FGFR3 mRNA is highly expressed in most support cells in the mature chick basilar papilla but not in vestibular organs of the chick. The gene is expressed early in the development of the basilar papilla. Gentamicin treatment sufficient to destroy hair cells in the basilar papilla causes a rapid, transient downregulation of FGFR3 mRNA in the region of damage. In the initial stages of hair cell regeneration, the support cells that reenter the mitotic cycle in the basilar papilla do not express detectable levels of FGFR3 mRNA. However, once the hair cells have regenerated in this region, the levels of FGFR3 mRNA and protein expression rapidly return to approximate those in the undamaged epithelium. These results indicate that FGFR3 expression changes after drug-induced hair cell damage to the basilar papilla in an opposite way to that found in the mammalian cochlea and may be involved in regulating the proliferation of support cells.

Transdifferentiation of pigmented epithelial cells: a source of retinal stem cells?

In urodeles, larval anurans, embryonic chicks and rodents, the retinal pigmented epithelium (RPE) is capable of transdifferentiation and generating new neurons. Recent evidence suggests that pigmented cells in the ciliary body of the adult rodent eye are capable of producing new neurons in vitro. Here we provide data to suggest that the pigmented epithelium at the retinal margin of postnatal chickens is similar to that found in the embryonic retina. Pigmented cells at the retinal margin expressed mitf and pax6, transcription factors that are transiently expressed by the developing RPE. Furthermore, these pigment cells at the retinal margin express high levels of proliferating cell nuclear antigen and accumulate bromodeoxyuridine, indicating that they continue to proliferate long after embryonic stages of development. Exogenous fibroblast growth factor-2 (FGF2) or insulin alone did not affect the proliferation of these cells, while FGF2 plus insulin induced their proliferation and loss of pigmentation. We propose that the pigmented cells at the retinal margin of the postnatal chicken are similar to those found in the embryonic eye, and these cells could be a source of neural regeneration under appropriate conditions.

Stem cells in the vertebrate retina.

The capacity for retinal regeneration in cold-blooded vertebrates has long been recognized. Regeneration occurs, in part, through a population of retinal stem cells residing at the peripheral margin of the retina. It has generally been thought that homeothermic vertebrates, such as birds and mammals, lack this so-called ciliary marginal zone. Recent studies have, however, provided evidence that birds too possess a zone of cells at the retinal margin analogous to the ciliary marginal zone of fish and amphibians. In addition, there is an indication that, under certain conditions, Müller glia of the chicken retina can transdifferentiate into retinal progenitor/stem cells. These progenitor/stem cells then generate certain types of retinal neurons. Taken together, these studies have revealed an unexpected capacity for retinal regeneration in birds.

Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick.

The vertebrate eye develops from the neuroepithelium of the ventral forebrain by the evagination and formation of the optic vesicle. Classical embryological studies have shown that the surrounding extraocular tissues - the surface ectoderm and extraocular mesenchyme - are necessary for normal eye growth and differentiation. We have used explant cultures of chick optic vesicles to study the regulation of retinal pigmented epithelium (RPE) patterning and differentiation during early eye development. Our results show that extraocular mesenchyme is required for the induction and maintenance of expression of the RPE-specific genes Mitf and Wnt13 and the melanosomal matrix protein MMP115. In the absence of extraocular tissues, RPE development did not occur. Replacement of the extraocular mesenchyme with cranial mesenchyme, but not lateral plate mesoderm, could rescue expression of the RPE-marker Mitf. In addition to activating expression of RPE-specific genes, the extraocular mesenchyme inhibits the expression of the neural retina-specific transcription factor Chx10 and downregulates the eye-specific transcription factors Pax6 and Optx2. The TGF(&bgr;) family member activin can substitute for the extraocular mesenchyme by promoting expression of the RPE-specific genes and downregulating expression of the neural retina-specific markers. These data indicate that extraocular mesenchyme, and possibly an activin-like signal, pattern the domains of the optic vesicle into RPE and neural retina.

Soluble factors and the development of rod photoreceptors.

Photoreceptors are the most abundant cell type in the vertebrate neural retina. Like the other retinal neurons and the Müller glia, they arise from a population of precursor cells that are multipotent and intrinsic to the retina. Approximately 10 years ago, several studies demonstrated that retinal precursor cells (RPCs) are competent to respond to environmental factors that promote cell type determination and differentiation. Since those studies, significant effort has been directed at identifying the molecular nature of these environmental signals and understanding the precise mechanisms they employ to drive RPCs towards the different retinal fates. In this review, we describe the recent progress toward understanding how environmental factors influence the development of vertebrate rod photoreceptors.

p27(Kip1) regulates cell cycle withdrawal of late multipotent progenitor cells in the mammalian retina.

The cyclin-dependent kinase inhibitor protein, p27(Kip1), is necessary for the timing of cell cycle withdrawal that precedes terminal differentiation in oligodendrocytes of the optic nerve. Although p27(Kip1) is widely expressed in the developing central nervous system, it is not known whether this protein has a similar role in neuronal differentiation. To address this issue, we have examined the expression and function of p27(Kip1) in the developing retina, a well-characterized part of the central nervous system. p27(Kip1) is expressed in a pattern coincident with the onset of differentiation of most retinal cell types. In vitro analyses show that p27(Kip1) accumulation in retinal cells correlates with cell cycle withdrawal and differentiation, and when overexpressed, p27(Kip1) inhibits proliferation of the progenitor cells. Furthermore, the histogenesis of photoreceptors and Müller glia is extended in the retina of p27(Kip1)-deficient mice. Finally, we examined the adult retinal dysplasia in p27(Kip1)-deficient mice with cell-type-specific markers. Contrary to previous suggestions that the dysplasia is caused by excess production of photoreceptors, we suggest that the dysplasia is due to the displacement of reactive Müller glia into the layer of photoreceptor outer segments. These results demonstrate that p27(Kip1) is part of the molecular mechanism that controls the decision of multipotent central nervous system progenitors to withdraw from the cell cycle. Second, postmitotic Müller glia have a novel and intrinsic requirement for p27(Kip1) in maintaining their differentiated state.

Activin A promotes progenitor differentiation into photoreceptors in rodent retina.

Activins are TGF beta-like proteins that were first discovered for their actions on the reproductive system, but have subsequently been shown to play a role in a variety of developmental processes. Previous studies have demonstrated that activins and their receptors are present in the developing retina, as well as other regions of the embryonic nervous system. We used both in vitro and in vivo approaches to test for functions of activin during retinal development. We found that activin A treatment of embryonic day 18 rat retinal cultures causes the progenitor cells in the cultures to exit the cell cycle and differentiate into rod photoreceptors. This effect is dose-dependent and the promotion of rod photoreceptor differentiation is specific, since the other primary retinal neurons generated in these cultures, the C1+ amacrine cells, are not affected by activin A treatment. Mice with homozygous deletion of the activin betaA gene show a specific decrease in the number of rod photoreceptors compared to wild-type or heterozygous littermates. These data demonstrate that activin A is an important regulator of photoreceptor differentiation in the developing retina.

Identification of a proliferating marginal zone of retinal progenitors in postnatal chickens.

In warm-blooded vertebrates it is generally accepted that after early stages of development new neurons are not added to the retina. Contrary to this belief, we show here that hatched chickens have a zone of proliferating cells at the peripheral margin of the retina, similar to that of fish and amphibians. We found that cells at the peripheral edge of the retina incorporated the thymidine analog BrdU and expressed the cell cycle regulator proliferating cell nuclear antigen (PCNA). Furthermore, cells in the ciliary epithelium and retinal margin coexpressed the homeodomain transcription factors Pax6 and Chx-10, similar to multipotent progenitors of embryonic retina. Expression of PCNA, Pax6, and Chx-10 in cells at the retinal margin was maintained in adult birds. Double-labeling studies showed that BrdU-labeled cells that were integrated into the retina expressed proteins found only in differentiated neurons. Increased rates of ocular growth, induced by visual deprivation, resulted in increased numbers of BrdU-labeled cells at the retinal margin. Unlike the progenitors in the retinal marginal zone of fish and amphibians, the progenitors of the chick retina do not increase their rate of proliferation in response to acute damage. Furthermore, insulin, insulin-like growth factor-I, and epidermal growth factor increased proliferation of progenitors at the retinal margin, while basic fibroblast growth factor had no effect. These results indicate that the avian retina has a marginal growth zone containing proliferating cells that share similarities with multipotent embryonic retinal progenitors and the retinal stem cells of cold-blooded vertebrates.

The development of the pattern of retinal ganglion cells in the chick retina: mechanisms that control differentiation.

Neurons in both vertebrate and invertebrate eyes are organized in regular arrays. Although much is known about the mechanisms involved in the formation of the regular arrays of neurons found in invertebrate eyes, much less is known about the mechanisms of formation of neuronal mosaics in the vertebrate eye. The purpose of these studies was to determine the cellular mechanisms that pattern the first neurons in vertebrate retina, the retinal ganglion cells. We have found that the ganglion cells in the chick retina develop as a patterned array that spreads from the central to peripheral retina as a wave front of differentiation. The onset of ganglion cell differentiation keeps pace with overall retinal growth; however, there is no clear cell cycle synchronization at the front of differentiation of the first ganglion cells. The differentiation of ganglion cells is not dependent on signals from previously formed ganglion cells, since isolation of the peripheral retina by as much as 400 microm from the front of ganglion cell differentiation does not prevent new ganglion cells from developing. Consistent with previous studies, blocking FGF receptor activation with a specific inhibitor to the FGFRs retards the movement of the front of ganglion cell differentiation, while application of exogenous FGF1 causes the precocious development of ganglion cells in peripheral retina. Our observations, taken together with those of previous studies, support a role for FGFs and FGF receptor activation in the initial development of retinal ganglion cells from the undifferentiated neuroepithelium peripheral to the expanding wave front of differentiation.

Retinoic acid promotes rod photoreceptor differentiation in rat retina in vivo.

Previous studies have indicated that retinoic acid (RA) promotes rod photoreceptor differentiation in dissociated cultures of rat retina and in zebrafish embryos. To determine whether RA will have the same affect in the mammalian retina in vivo, pregnant rats were given single i.p. injections of RA on the 18th and 20th days of gestation, and the retinas of the pups were analyzed for rods. HPLC showed that i.p. injections of RA substantially increased levels of retinal RA in the embryos. Embryonic exposure to RA caused an increase in the number of cells that differentiated as rod photoreceptors. There was a comparable decrease in the number of cells that differentiated as amacrine cells. These results demonstrate that RA promotes the differentiation of rods in vivo and further support the hypothesis that differentiation of rods is normally controlled partly by the RA concentration in the developing retina or RPE.

A diffusible factor from normal retinal cells promotes rod photoreceptor survival in an in vitro model of retinitis pigmentosa.

Transgenic mice expressing a dominant mutation in the gene for the phototransduction molecule rhodopsin undergo retinal degeneration similar to that experienced by patients with the retinal degenerative disease, retinitis pigmentosa (RP). Although the mutation is thought to cause photoreceptor degeneration in a cell-autonomous manner, the fact that rod photoreceptor degeneration is slowed in chimeric wild-type/mutant mice suggests that cellular interactions are also important for maintaining photoreceptor survival. To more fully characterize the nature of the cellular interactions important for rod degeneration in the RP mutant mice, we have used an in vitro approach. We found that when the retinas of the transgenic mice were isolated from the pigmented epithelium and cultured as explants, the rod photoreceptors underwent selective degeneration with a similar time course to that observed in vivo. This selective rod degeneration also occurred when the cells were dissociated and cultured as monolayers. These data indicate that the mutant rod photoreceptors degenerate when removed from their normal cellular relationships and without contact with the pigmented epithelium, thus confirming the relative cell autonomy of the mutant phenotype. We next tested whether normal retinal cells could rescue the mutant photoreceptors in a coculture paradigm. Coculture of transgenic mouse with wild-type mouse or rat retinal cells significantly enhanced transgenic rod photoreceptor survival; this survival-promoting activity was diffusible through a filter, was heat labile, and not present in transgenic retinal cells. Several peptide growth factors known to be present in the retina were tested as the potential survival-promoting molecule responsible for the effects of the conditioned medium; however, none of them promoted survival of the photoreceptors expressing the Pro23His mutant rhodopsin. Nevertheless, we were able to demonstrate that the mutant photoreceptors could be rescued by an antagonist to a retinoic acid receptor, suggesting that the endogeneous survival-promoting activity may function through this pathway. These data thus confirm and extend the findings of previous work that local trophic interactions are important in regulating rod photoreceptor degeneration in retinitis pigmentosa. A diffusible factor found in normal but not transgenic retinal cells has a protective effect on the survival of rod photoreceptors from Pro23His mutant rhodopsin mice.

The nuclear receptor transcription factor, retinoid-related orphan receptor beta, regulates retinal progenitor proliferation.

We examined the role of retinoid-related orphan receptor (ROR)beta, a member of the nuclear receptor family of transcription factors, in retinal neurogenesis. In situ hybridization studies showed that RORbeta is expressed in retinal progenitor cells in the embryonic rat retina. Further studies demonstrated that RORbeta colocalizes with Chx10, a transcription factor thought to influence retinal progenitor proliferation (Burmeister, M., Novak, J., Liang, M-Y., Basu, S., Ploder, L., Hawes, N.L. Vidgen, D., Hoover, F., Goldman, D. , Kalnins, V.I., Roderick, T.H., Taylor, B.A., Hankin, M.H. and McInnes, R.R., 1996. Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation. Nature Genetics 12, 376-383). Northern analysis reveals that RORbeta expression is dramatically decreased in the ocular retardationJ mutant, which possesses a defect in the Chx10 gene. Overexpression of RORbeta in retinal progenitors by biolistic transfection results in an increase in the number of large cell clones. These data support a role for RORbeta in regulating retinal progenitor proliferation, possibly via the Chx10 gene.