Retinal stem cells in vertebrates

In fish and amphibia, retinal stem cells located in the periphery of the retina, the ciliary marginal zone (CMZ), produce new neurons in the retina throughout life. In these species, the retina grows to keep pace with the enlarging body. When birds or mammals reach adult proportions, however, their retinas stop growing so there appears to be no need for such a proliferative area with stem cells. It is a surprise, therefore, that recent data suggest that a region similar to the CMZ of fish and amphibia exists in the postnatal chick and the adult mouse.     

Genetic dissection of the zebrafish retinal stem-cell compartment

In a large-scale forward-genetic screen, we discovered that a limited number of genes are required for the regulation of retinal stem cells after embryogenesis in zebrafish. In 18 mutants out of almost 2000 F2 families screened, the eye undergoes normal embryonic development, but fails to continue growth from the ciliary marginal zone (CMZ), the post-embryonic stem-cell niche. Class I-A mutants (5 loci) display lower amounts of proliferation in the CMZ, while nearly all cells in the retina appear differentiated. Class I-B mutants (2 loci) have a reduced CMZ with a concomitant expansion in the retinal pigmented epithelium (RPE), suggesting a common post-embryonic stem cell is the source for these neighboring cell types. Class II encompasses three distinct types of mutants (11 loci) with expanded CMZ, in which the progenitor population is arrested in the cell cycle. We also show that in at least one combination, the reduced CMZ phenotype is genetically epistatic to the expanded CMZ phenotype, suggesting that Class I genes are more likely to affect the stem cells and Class II the progenitor cells. Finally, a comparative mapping analysis demonstrates that the new genes isolated do not correspond to genes previously implicated in stem-cell regulation. Our study suggests that embryonic and post-embryonic stem cells utilize separable genetic programs in the zebrafish retina.     

A large scale screen for neural stem cell markers in Xenopus retina

Neural stem cell research suffers from a lack of molecular markers to specifically assess stem or progenitor cell properties. The organization of the Xenopus ciliary marginal zone (CMZ) in the retina allows the spatial distinction of these two cell types: stem cells are confined to the most peripheral region, while progenitors are more central. Despite this clear advantage, very few genes specifically expressed in retinal stem cells have been discovered so far in this model. To gain insight into the molecular signature of these cells, we performed a large-scale expression screen in the Xenopus CMZ, establishing it as a model system for stem cell gene profiling. Eighteen genes expressed specifically in the CMZ stem cell compartment were retrieved and are discussed here. These encode various types of proteins, including factors associated with proliferation, mitotic spindle organization, DNA/RNA processing, and cell adhesion. In addition, the publication of this work in a special issue on Xenopus prompted us to give a more general illustration of the value of large-scale screens in this model species. Thus, beyond neural stem cell specific genes, we give a broader highlight of our screen outcome, describing in particular other retinal cell markers that we found. Finally, we present how these can all be easily retrieved through a novel module we developed in the web-based annotation tool XenMARK, and illustrate the potential of this powerful searchable database in the context of the retina.     

Asymmetric growth and development of the Xenopus laevis retina during metamorphosis is controlled by type III deiodinase

During the metamorphosis of the Xenopus laevis retina, thyroid hormone (TH) preferentially induces ventral ciliary marginal zone (CMZ) cells to both increase their proliferation and give rise to ipsilaterally projecting ganglion cells. Here we show that dorsal CMZ cells express type III deiodinase (D3), an enzyme that inactivates TH. The dorsal CMZ cells can be induced to proliferate if deiodinase activity is inhibited. D3 or dominant-negative thyroid hormone receptor transgenes inhibit both TH-induced proliferation of the ventral CMZ cells and the formation of the ipsilateral projection. Thus, the localized expression of D3 in the dorsal CMZ cells accounts for the asymmetric growth of the frog retina.     

Functional roles of Otx2 transcription factor in postnatal mouse retinal development

We previously reported that Otx2 is essential for photoreceptor cell fate determination; however, the functional role of Otx2 in postnatal retinal development is still unclear although it has been reported to be expressed in retinal bipolar cells and photoreceptors at postnatal stages. In this study, we first examined the roles of Otx2 in the terminal differentiation of photoreceptors by analyzing Otx2; Crx double-knockout mice. In Otx2+/-; Crx-/- retinas, photoreceptor degeneration and downregulation of photoreceptor-specific genes were much more prominent than in Crx-/- retinas, suggesting that Otx2 has a role in the terminal differentiation of the photoreceptors. Moreover, bipolar cells decreased in the Otx2+/-; Crx-/- retina, suggesting that Otx2 is also involved in retinal bipolar-cell development. To further investigate the role of Otx2 in bipolar-cell development, we generated a postnatal bipolar-cell-specific Otx2 conditional-knockout mouse line. Immunohistochemical analysis of this line showed that the expression of protein kinase C, a marker of mature bipolar cells, was significantly downregulated in the retina. Electroretinograms revealed that the electrophysiological function of retinal bipolar cells was impaired as a result of Otx2 ablation. These data suggest that Otx2 plays a functional role in the maturation of retinal photoreceptor and bipolar cells.     

Creatine transporter localization in developing and adult retina: importance of creatine to retinal function

Creatine and phosphocreatine are required to maintain ATP needed for normal retinal function and development. The aim of the present study was to determine the distribution of the creatine transporter (CRT) to gain insight to how creatine is transported into the retina. An affinity-purified antibody raised against the CRT was applied to adult vertebrate retinas and to mouse retina during development. Confocal microscopy was used to identify the localization pattern as well as co-localization patterns with a range of retinal neurochemical markers. Strong labeling of the CRT was seen in the photoreceptor inner segments in all species studied and labeling of a variety of inner neuronal cells (amacrine, bipolar, and ganglion cells), the retinal nerve fibers and sites of creatine transport into the retina (retinal pigment epithelium, inner retinal blood vessels, and perivascular astrocytes). The CRT was not expressed in Müller cells of any of the species studied. The lack of labeling of Müller cells suggests that neurons are independent of this glial cell in accumulating creatine. During mouse retinal development, expression of the CRT progressively increased throughout the retina until approximately postnatal day 10, with a subsequent decrease. Comparison of the distribution patterns of the CRT in vascular and avascular vertebrate retinas and studies of the mouse retina during development indicate that creatine and phosphocreatine are important for ATP homeostasis. photoreceptor; development; glutamine synthetase; neurochemistry     

Retinal homeobox genes and the role of cell proliferation in cavefish eye degeneration

The teleost Astyanax mexicanus exhibits eyed surface dwelling (surface fish) and blind cave dwelling (cavefish) forms. Despite lacking functional eyes as adults, cavefish embryos form eye primordia, which later arrest in development, degenerate and sink into the orbit. We are comparing the expression patterns of various eye regulatory genes during surfacefish and cavefish development to determine the cause of eye degeneration. Here we examine Rx and Chx/Vsx family homeobox genes, which have a major role in cell proliferation in the vertebrate retina. We isolated and sequenced a full-length RxcDNA clone (As-Rx1) and part of a Chx/Vsx(As-Vsx2) gene, which appear to be most closely related to the zebrafish Rx1 and Alx/Vsx2 genes respectively. In situ hybridization shows that these genes have similar but non-identical expression patterns during Astyanax eye development. Expression is first detected in the optic vesicle, then throughout the presumptive retina of the optic cup, and finally in the ciliary marginal zone (CMZ), the region of the growing retina where most new retinoblasts are formed. In addition, As-Rx1 is expressed in the outer nuclear layer (ONL) of the retina, which contains the photoreceptor cells, and As-Vsx2 is expressed in the inner nuclear layer, probably in the bipolar cells. With the exception of reduced As-Rx-1 expression in the ONL, the As-Rx1 and As-Vsx2 expression patterns were unchanged in the developing retina of two different cavefish populations, suggesting that cell proliferation is not inhibited. These results were confirmed by using PCNA and BrdU markers for retinal cell division. We conclude that the CMZ is active in cell proliferation long after eye growth is diminished and is therefore not the major cause of eye degeneration.     

Putative stem cells and the lineage of rod photoreceptors in the mature retina of the goldfish

The retinas of teleost fish grow continuously, in part, by neuronal hyperplasia and when lesioned will regenerate. Within the differentiated retina, the growth-associated hyperplasia results in the generation of new rod photoreceptors only, whereas injury-induced neurogenesis results in the regeneration of all retinal cell types. It is believed, however, that both new rod photoreceptors and regenerated neurons originate from the same populations of intrinsic progenitors. Experiments are described here that attempt to identify in the normal retina of goldfish neuronal progenitors intrinsic to the retina, particularly those which have remained cryptic because they divide infrequently. Long-term, systemic exposure to bromodeoxyuridine (BrdU) was used to label these cells. Five populations of proliferative cells were labeled: microglia, which are briefly described but not studied further; retinal progenitors in the circumferential germinal zone (CGZ); and rod precursors in the outer nuclear layer (ONL), both of which have been well characterized previously; and two populations of slowly-dividing cells in the inner nuclear layer (INL). The majority of these cells have a fusiform morphology, whereas the remaining ones are spherical. Longitudinal BrdU labeling suggests that the fusiform cells migrate to the ONL to replenish the pool of rod precursors. A subset of the spherical cells express pax6, although none are stained with markers of differentiated amacrine or bipolar cells. It is hypothesized that these rare, pax6-expressing cells are retinal stem cells, which give rise to the pax6-negative fusiform cells. Based on these data, two models are proposed: the first describes the lineage of rod photoreceptors in goldfish; the second is a consensus model of neurogenesis in the retinas of all teleosts.     

Serine racemase expression and D-serine content are developmentally regulated in neuronal ganglion cells of the retina

d -Serine, the endogenous ligand for the glycine modulatory binding site of the NMDA receptor, and serine racemase, the enzyme that converts l -serine to d -serine, have been reported in vertebrate retina; initial reports suggested that localization was restricted to Müller glial cells. Recent reports, in which d -serine and serine racemase were detected in neurons of the brain, prompted the present investigation of neuronal expression of d -serine and serine racemase in retina and whether expression patterns were developmentally regulated. RT-PCR, in situ hybridization, western blotting, immunohistochemistry, and immunocytochemical methods were used to localize d -serine and serine racemase in intact retina obtained from 1 to 3 day, 3 week, and 18 week mouse retinas and in primary ganglion cells harvested by immunopanning from neonatal mouse retina. Results of these analyses revealed robust expression of d -serine and serine racemase in ganglion cells, both in intact retina and in cultured cells. The levels appear to be developmentally regulated with d -serine levels being quite high in ganglion cells of neonatal retinas and decreasing rapidly postnatally. Serine racemase levels are also developmentally regulated, with high levels detected during the early postnatal period, but diminishing considerably in the mature retina. This represents the first report of neuronal expression of d -serine and serine racemase in the vertebrate retina and suggests an important contribution of neuronal d -serine during retinal development.     

The hedgehog pathway is a modulator of retina regeneration

The embryonic chick has the ability to regenerate its retina after it has been completely removed. Here, we provide a detailed characterization of retina regeneration in the embryonic chick at the cellular level. Retina regeneration can occur in two distinct manners. The first is via transdifferentiation, which is induced by members of the Fibroblast growth factor (Fgf) family. The second type of retinal regeneration occurs from the anterior margin of the eye, near the ciliary body (CB) and ciliary marginal zone (CMZ). We show that regeneration from the CB/CMZ is the result of proliferating stem/progenitor cells. This type of regeneration is also stimulated by Fgf2, but we show that it can be activated by Sonic hedgehog (Shh) overexpression when no ectopic Fgf2 is present. Shh-stimulated activation of CB/CMZ regeneration is inhibited by the Fgf receptor (Fgfr) antagonist, PD173074. This indicates that Shh-induced regeneration acts through the Fgf signaling pathway. In addition, we show that the hedgehog (Hh) pathway plays a role in maintenance of the retina pigmented epithelium (RPE), as ectopic Shh expression inhibits transdifferentiation and Hh inhibition increases the transdifferentiation domain. Ectopic Shh expression in the regenerating retina also results in a decrease in the number of ganglion cells present and an increase in apoptosis mostly in the presumptive ganglion cell layer (GCL). However, Hh inhibition increases the number of ganglion cells but does not have an effect on cell death. Taken together, our results suggest that the hedgehog pathway is an important modulator of retina regeneration.     

Antisense peptide nucleic acids conjugated to somatostatin analogs and targeted at the n-myc oncogene display enhanced cytotoxity to human neuroblastoma IMR32 cells expressing somatostatin receptors

Peptide nucleic acid (PNA) sequences are synthetic versions of naturally occurring oligonucleotides which display improved binding properties to DNA and RNA, but are still poorly internalized across cell membranes. In an effort to employ the rapid binding/internalization properties of somatostatin agonist analogs and the over-expression of somatostatin receptors on many types of tumor cells, PNAs complementary to target sites throughout 5'-UTR, translation start site and coding region of the n-myc oncogene were conjugated to a somatostatin analog (SSA) with retention of high somatostatin biological potency. IMR32 cells, which over-express somatostatin receptor type 2 (SSTR2) and contain the n-myc oncogene, were treated with these PNA-SSA conjugates. The results show that PNA conjugates targeted to the 5'-UTR terminus and to regions at or close to the translation start site could effectively inhibit n-myc gene expression and cell growth, whereas the non-conjugate PNAs were without effect at similar doses. The most potent inhibition of cell growth was achieved with PNAs binding to the translation start site, but those complementary to the middle coding region or middle upstream site between 5'-UTR and translation start site displayed no inhibition of gene expression. These observations were extended to four other cell lines: GH3 cells which express SSTRs with the n-myc gene, SKNSH cells containing a silent n-myc gene without SSTR2, HT-29 cells carrying the c-myc but no n-myc gene, and CHO-K1 cells lacking SSTR2 with n-myc gene. The results show that there was almost no effect on these four cell lines. Our study indicates that PNAs conjugated to SSA exhibited improved inhibition of gene expression possibly due to facilitated cellular uptake of the PNAs. These conjugates were mRNA sequence- and SSTR2-specific suggesting that many other genes associated with tumor growth could be targeted using this approach and that SSA could be a novel and effective transportation vector for the PNA antisense strategy.     

Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation

Continuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/β-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina.     

Adhesive recognition between developing retinal cells and the optic tecta of the chick embryo.

An assay is described that can measure the rates of adhesion of isotopically labeled cell bodies from either the dorsal or ventral regions of the chick embryo retina to dorsal and ventral tectal halves. Immediately after dissociation of the retina, both ventral and dorsal retinal cells adhere preferentially to ventral tectal halves. With increasing time after dissociation, however, the preference of the ventral retinal cells shifts to dorsal tectal halves. The dorsal retinal cells continue to show a specificity for ventral tectal halves regardless of the length of time after their dissociation.When the developmental age of the tectal halves is varied from 8–14 days, there is no change in the specificities shown by retinal cells toward these tecta.When retinal age is varied, ventral retinal cells do not adhere preferentially to dorsal tecta until day 6 and older. Dorsal retinas show their specificity toward ventral tecta as early as day three.Control experiments include the use of nonretinal tissues, noninnervated tectal halves and pigmented retinal cells.     

Bullwhip neurons in the retina regulate the size and shape of the eye

Bullwhip and mini-bullwhip cells are unconventional types of retinal neurons that utilize the neuropeptides glucagon, glucagon-like peptide 1 (GLP1) and substance P. These cells have been implicated in regulating the proliferation of neural progenitors in the circumferential marginal zone (CMZ) of the chicken retina. The purpose of this study was to investigate the roles of the bullwhip cells in regulating ocular size and shape. We found that intravitreal delivery of colchicine at postnatal day 7 destroys the vast majority (approximately 98%) of the bullwhip and mini-bullwhip cells and their peptidergic terminals that are concentrated in the CMZ near the equator of the eye. Interestingly, colchicine-treatment resulted in excessive ocular growth that involved the expansion of equatorial diameter, but not axial length. Intraocular injections of glucagon completely prevented the equatorial expansion that occurs with colchicine-treatment. In eyes with undamaged retinas, exogenous glucagon suppressed equatorial eye growth, whereas glucagon receptor antagonists caused excessive equatorial growth. Furthermore, visual stimuli that increase or decrease rates of ocular growth caused a down- or up-regulation, respectively, of the immediate early gene Egr1 in the bullwhip cells; indicating that the activity of the bullwhip cells is regulated by growth-guiding visual cues. We found that the glucagon receptor was expressed by cells in the fibrous and cartilaginous sclera in equatorial regions of the eye. Taken together, these findings suggest that glucagon peptide released from the terminals of the bullwhip and mini-bullwhip cells regulates the growth of the equatorial sclera in a vision-dependent manner. Although the bullwhip and mini-bullwhip cells are not abundant, less than 1000 cells per retina, their influence on the development of the eye is substantial and includes vision-guided ocular growth.     

Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: relevance to optic nerve coloboma.

The presence of astrocyte precursor cells (APCs) and time course and topography of astrocyte differentiation during development were investigated by triple-label immunohistochemistry with intact fetal and adult human retinas. Throughout retinal development and adulthood, expression of Pax2 was restricted to cells of the astrocytic lineage. Three distinct stages of astrocytic differentiation were identified during development: i) Pax2+/vimentin+/GFAP- APCs; ii) Pax2+/vimentin+/GFAP+ immature perinatal astrocytes; and iii) Pax2+/vimentin-/GFAP+ mature perinatal astrocytes. In adult, cells with the antigenic phenotype of mature perinatal astrocytes were restricted to a region surrounding the optic nerve head (ONH), whereas cells at a fourth stage of differentiation, adult astrocytes (Pax2-/vimentin-/GFAP+), were apparent throughout the vascularized retina. APC appearance was centered around the ONH and preceded the appearance of perinatal astrocytes. A cluster of Pax2+ somas was also present in a small region surrounding the ONH at the ventricular surface of the developing retina, which suggests the existence of two distinct sites of astrocytic differentiation. The coincidence in the location of APCs and perinatal astrocytes at the ventricular zone with that of optic nerve colobomas, together with the association of Pax2 gene mutations with this condition, suggests that coloboma formation may result from impaired astrocyte differentiation during development.     

Retinal ganglion cell differentiation in cultured mouse retinal explants

The availability of genetically engineered mice harboring specific mutations in genes affecting one or more retinal cell types affords new opportunities for investigating the genetic regulatory mechanisms of vertebrate retina formation. When identifying critical regulatory genes involved in retina development it is often advantageous to complement in vivo analysis with in vitro characterization. In particular, by combining classical techniques of retinal explant culturing with gene transfer procedures relying on herpes simple virus (HSV) amplicon vectors, gain-of-function analysis with genes of interest can be performed quickly and efficiently. Here, details are provided for isolating and culturing explants containing retinal progenitor cells and for infecting the explants with HSV expression vectors that perturb or rescue retinal ganglion cells, the first cell type to differentiate in the retina. In addition, the availability of sensitive techniques to monitor gene expression, including detection of reporter gene expression using antibodies and detection of endogenous marker gene expression using quantitative RT-PCR, provides an effective means for comparing wild-type and mutant retinas from genetically engineered mice.