Proliferation Of Retinal Progenitor Cells Research Articles

Redefining Low Lead Levels

Vol. 119, No. 5 PerspectivesOpen AccessRedefining Low Lead Levels Ivo Iavicoli Edward J. Calabrese Ivo Iavicoli Search for more papers by this author and Edward J. Calabrese Search for more papers by this author Published:1 May 2011https://doi.org/10.1289/ehp.1103489Cited by:1AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit In the January 2011 issue of EHP, Giddabasappa et al. (2011) reported that gestational lead exposure (GLE) of C57BL/6 mice produced selective nonmonotonic increases in the numbers of rods and cone bipolar cells (BCs) in the adult retina. Interestingly, this increase was characterized by an inverted U-shaped dose–response curve. Moreover, findings of this study showed that GLE increases and prolongs proliferation of retinal progenitor cells (RPCs) without decreasing apoptosis. Consequently, this phenomenon produced an adult retina with normal lamination and a selectively increased number of rods and BCs. These results should be considered to define a more adequate risk assessment at low levels of lead exposure. In fact, other published articles have indicated that lead induced a biphasic dose–response relationship (Calabrese and Baldwin 2003).In experiments in Swiss mice using low-level lead exposures similar to and lower than those used by Giddabasappa et al. (2011), we observed an increase in the number of red blood cells, in female gestational parameters, and in Th1 cytokine levels (Iavicoli et al. 2003, 2004, 2006a, 2006b). For this reason, it would be interesting if Giddabasappa et al. could verify this increase in the number of neurons in the rod-signaling pathway at even lower blood lead levels (< 10 μg/dL). The findings of our studies were also implicated over several generations.In any case, we agree with Giddabasappa et al. (2011) that their findings, as ours, raise complex issue for toxicologists, pediatricians, public health regulators, and risk assessors who need to incorporate the occurrence of such U-shaped dose responses in the hazard and risk assessment process. In this context, these findings could be explained by the hormesis phenomenon, which is a dose–response relationship characterized by low-dose stimulation and high-dose inhibition (Calabrese 2008, 2009).ReferencesCalabrese EJ. 2008. Hormesis: why it is important to toxicology and toxicologists. Environ Toxicol Chem 27:1451-147418275256. Crossref, Medline, Google ScholarCalabrese EJ. 2009. Getting the dose-response wrong: why hormesis became marginalized and the threshold model accepted. Arch Toxicol 83:227-24719234688. Crossref, Medline, Google ScholarCalabrese EJ, Baldwin LA. 2003. Inorganics and hormesis. Crit Rev Toxicol 33:215-30412809427. Crossref, Medline, Google ScholarGiddabasappa A, Hamilton WR, Chaney S, Xiao W, Johnson JE, Mukherjee Set al.. 2011. Low-level gestational lead exposure increases retinal progenitor cell proliferation and rod photoreceptor and bipolar cell neurogenesis in mice. Environ Health Perspect 119:71-7720840909. Link, Google ScholarIavicoli I, Carelli G, Stanek EJ, Castellino N, Calabrese EJ. 2003. Effects of low doses of dietary lead on red blood cell production in male and female mice. Toxicol Lett 137:193-19912523962. Crossref, Medline, Google ScholarIavicoli I, Carelli G, Stanek EJ, Castellino N, Calabrese EJ. 2006a. Below background levels of blood lead impact cytokine levels in male and female mice. Toxicol Appl Pharmacol 210:94-9916289177. Crossref, Medline, Google ScholarIavicoli I, Carelli G, Stanek EJ, Castellino N, Calabrese EJ. 2004. Effects of low doses of dietary lead on puberty onset in female mice. Reprod Toxicol 19:35-4115336710. Crossref, Medline, Google ScholarIavicoli I, Carelli G, Stanek EJ, Castellino N, Li Z, Calabrese EJ. 2006b. Low doses of dietary lead are associated with a profound reduction in the time to the onset of puberty in female mice. Reprod Toxicol 22:586-59016713174. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetailsCited By Corsetti G, Romano C, Stacchiotti A, Pasini E and Dioguardi F (2016) Endoplasmic Reticulum Stress and Apoptosis Triggered by Sub-Chronic Lead Exposure in Mice Spleen: a Histopathological Study, Biological Trace Element Research, 10.1007/s12011-016-0912-z, 178:1, (86-97), Online publication date: 1-Jul-2017. Vol. 119, No. 5 May 2011Metrics About Article Metrics Publication History Originally published1 May 2011Published in print1 May 2011 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. 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Lead Doesn’t Spare the Rod: Low-Level Exposure Supercharges Retinal Cell Production in Mice

Low-level gestational lead exposure has been shown to increase the electrical response of the rod signaling pathway in the retinas of children, monkeys, and rats, which could in turn contribute to retinal disease. Now researchers demonstrate the phenomenon underlying this effect: increased proliferation of retinal progenitor cells, which give rise to functionally differentiated retinal cells that sense and transmit visual information [EHP 119(1):71–77; Giddabasappa et al.]. Using a previously described mouse model of low-level gestational lead exposure, the researchers set out to test the hypothesis that such exposure selectively increases rod photoreceptors and bipolar cells in the rod signaling pathway. (The rod signaling pathway detects gradations of light, as opposed to the cone signaling pathways, which detect colors.) Female mice were given water containing varying concentrations of lead: 0 ppm (control), 27 ppm (“low” dose), 55 ppm (“moderate” dose), or 109 ppm (“high” dose). The exposures were administered for 2 weeks before mating, during pregnancy, and through postnatal day 10—a model for the human gestation period. On postnatal day 10, unspiked water replaced the water–lead mixtures for all groups. The adult mammalian retina consists of six types of neurons and a Muller glial cell. These cell types develop in one of two distinct phases: primarily in utero (“early-born”) or primarily after birth (“late-born”). In examining controls and exposed mice at postnatal day 60, the researchers found that late-born rod photoreceptors and rod and cone bipolar cells increased by 16–30% in exposed offspring, whereas Muller glial cells (also classified as late-born retinal cells) did not increase. Low and moderate lead doses showed the greatest effects. Gestational lead exposure also increased and prolonged retinal progenitor cell proliferation but did not alter developmental apoptosis (programmed cell death), indicating that the higher numbers of rods and bipolar cells were due to increased production, not decreased apoptosis. These results demonstrate that gestational lead exposure resulting in blood lead levels of 10 μg/dL alters retinal development by selectively promoting the development of rod photoreceptor cells and bipolar cells. The authors speculate that the increased number of rods and bipolar cells in the lead-exposed animals could accelerate age-related retinal degeneration. These nonmonotonic dose–response results raise complex issues for neurotoxicology, risk assessment, public health, and children’s health.

The expression and roles of inhibitor of DNA binding helix-loop-helix proteins in the developing and adult mouse retina

Inhibitor of DNA binding (Id) proteins bind to and inhibit the function of basic helix-loop-helix (bHLH) transcription factors including those that regulate retinal development. However, little is known about the role of Id proteins in the growth and differentiation of the retina during development. The purpose of this study is to observe the expression of Id proteins in the developing and adult mouse retinas as the first step in investigating the functions of Id family members in the eye. The expression of Id1–4 was examined by real-time PCR, Western blot, and immunohistochemistry in wild-type and Id1/Id3 double-knockout mice. Id1–4 genes and proteins showed high expression levels in the retina at embryonic and early postnatal stages, whereas declined in the adult. Expression of Id proteins was observed in the inner neuroblastic layer (NBL) at embryonic (E) day 13.5 through 16.5. Id4 expression began at E18.5. By E18.5 and postnatal day 1, the expression of Id1–4 exhibited distinct yet overlapping patterns in the ganglion cell layer and inner part of NBL. In the adult, Ids were expressed in retinal ganglion cells, amacrine cells, bipolar cells, and horizontal cells. No Id expression was found in Müller cells. Id1 and Id3 double-knockout mice (Id1−/−/Id3−/−) showed smaller retinal size compared to wild-type or heterozygous littermates. However, histological analyses in Id1 and Id3 single-knockout retinas revealed no obvious defects in developmental phenotype. Our results indicate that the expression of the Id family may play an important role in regulating retinal progenitor cell proliferation and differentiation.

An in vivo study of basic fibroblast growth factor on activation and proliferation of retinal progenitor cell in RCS rats

To investigate the effect of intravitreal basic fibroblast growth factor(bFGF) on activation and proliferation of endogenous retinal progenitor cells in the Royal College of Surgeons(RCS) rats. Twenty-four rats were studied after the 30th postnatal day(≥30). Eighteen affected rats were randomly divided into 3 groups: bFGF-treated, vehicle-treated and untreated group, and 6 unaffected rats were used as normal controls. Six μl of bFGF (5μg/10 μl) or vehicle was injected into the vitreous on days 31, 33 and 35 after birth (P31, P33, P35) in the bFGF group and vehicle group, and no injection was administered in the untreated and control groups. All the rats were euthanized, and their eyes were enucleated, hemisected and fixed at 50 d after birth for immunohistochemistry and measurement of outer nuclear layer thickness. Nestin and Chx10 were positively expressed in all retinal layers, intravitreous injection of bFGF in retina-dystrophic RCS(RCS-p+/Lav) rats induced intense labeling for the retinal progenitor cell markers Chx10 and Nestin, which were highly colocalized. Fluorescence intensity for both labels was slightly less in the control rats, and much less in the vehicle-injected rats as well as in the untreated RCS rats. The outer nuclear layer (ONL) was significantly thicker in bFGF group than that of vehicle-treated or untreated group(p<0.01), but thinner than that of the control group(p<0.01). No significant difference was observed in the ONL thicknesses between the vehicle group and untreated group(P>0.05). bFGF may contribute to the activation of retinal progenitor cells in RCS rats, thus counteract degeneration by promoting the proliferation of the progenitor cells.

Low-Level Gestational Lead Exposure Increases Retinal Progenitor Cell Proliferation and Rod Photoreceptor and Bipolar Cell Neurogenesis in Mice

BackgroundGestational lead exposure (GLE) produces novel and persistent rod-mediated electroretinographic (ERG) supernormality in children and adult animals.ObjectivesWe used our murine GLE model to test the hypothesis that GLE increases the number of neurons in the rod signaling pathway and to determine the cellular mechanisms underlying the phenotype.ResultsBlood lead concentrations ([BPb]) in controls and after low-, moderate-, and high-dose GLE were ≤ 1, ≤ 10, approximately 25, and approximately 40 μg/dL, respectively, at the end of exposure [postnatal day 10 (PND10)]; by PND30 all [BPb] measures were ≤ 1 μg/dL. Epifluorescent, light, and confocal microscopy studies and Western blots demonstrated that late-born rod photoreceptors and rod and cone bipolar cells (BCs), but not Müller glial cells, increased in a nonmonotonic manner by 16–30% in PND60 GLE offspring. Retinal lamination and the rod:cone BC ratio were not altered. In vivo BrdU (5-bromo-2-deoxyuridine) pulse-labeling and Ki67 labeling of isolated cells from developing mice showed that GLE increased and prolonged retinal progenitor cell proliferation. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and confocal studies revealed that GLE did not alter developmental apoptosis or produce retinal injury. BrdU birth-dating and confocal studies confirmed the selective rod and BC increases and showed that the patterns of neurogenesis and gliogenesis were unaltered by GLE.ConclusionsOur findings suggest two spatiotemporal components mediated by dysregulation of different extrinsic/intrinsic factors: increased and prolonged cell proliferation and increased neuronal (but not glial) cell fate. These findings have relevance for neurotoxicology, pediatrics, public health, risk assessment, and retinal cell biology because they occurred at clinically relevant [BPb] and correspond with the ERG phenotype.

Pituitary adenylyl cyclase‐activating polypeptide controls the proliferation of retinal progenitor cells through downregulation of cyclin D1

During retinal development, cell proliferation and exit from the cell cycle must be precisely regulated to ensure the generation of the appropriate numbers and proportions of the various retinal cell types. Previously, we showed that pituitary adenylyl cyclase-activating polypeptide (PACAP) exerts a neuroprotective effect in the developing retina of rats, through the cAMP-cAMP-dependent protein kinase (protein kinase A) (PKA) pathway. Here, we show that PACAP also regulates the proliferation of retinal progenitor cells. PACAP, PACAP-specific receptor (PAC1), and the receptors activated by both PACAP and vasoactive intestinal peptide (VIP), VPAC1 and VPAC2, are expressed during embryonic and postnatal development of the rat retina. Treatment of retinal explants with PACAP38 reduced the incorporation of [(3)H]thymidine as well as the number of 5-bromo-2'-deoxyuridine-positive and cyclin D1-positive cells. Pharmacological experiments indicated that PACAP triggers this antiproliferative effect through the activation of both PAC1 and VPACs, and the cAMP-PKA pathway. In addition, PACAP receptor activation decreased both cyclin D1 mRNA and protein content. Altogether, the data support the hypothesis that PACAP is a cell-extrinsic regulator with multiple roles during retinal development, including the regulation of proliferation in a subpopulation of retinal progenitor cells.

Bmi1 Distinguishes Immature Retinal Progenitor/Stem Cells from the Main Progenitor Cell Population and Is Required for Normal Retinal Development

The developing mammalian retina is generated by the proliferation and differentiation of multipotent retinal progenitor cells (RPCs) giving rise to neuronal and glial lineages. Whether an immature progenitor/stem cell subpopulation is present in the developing mammalian retina remains undefined. Deficiency in the polycomb group gene Bmi1 results in reduced proliferation and postnatal depletion of neural and hematopoietic stem cells. Here, we show that Bmi1 is required for the self-renewal of most immature RPCs and for postnatal retinal development. In the embryo, Bmi1 is highly enriched in a rare stage-specific embryonic antigen-1-positive RPC subpopulation expressing the stem cell markers Sox2, Lhx2, and Musashi. Gain-of-function experiments revealed that Bmi1 overexpression could convert RPCs having limited proliferation capacity into RPCs showing extensive proliferation and multiple differentiation capacities over time. At all developmental stages analyzed using the neurosphere assay, Bmi1 deficiency resulted in reduced proliferation and self-renewal of most immature RPCs. Reduced RPCs proliferation was also observed in the peripheral retina of Bmi1(-/-) fetus and newborn mice. The biological impact of these developmental anomalies was revealed by the reduced retinal diameter of Bmi1-deficient pups. P19(Arf) and p16(Ink4a) were upregulated in vivo and in vitro and coinactivation of p53, which lies downstream of p19(Arf), partially restored Bmi1-deficient RPCs self-renewal phenotype. Bmi1 thus distinguishes immature RPCs from the main RPC population and is required for normal retinal development.

Roles of paroxetine and corticosterone on adult mammalian ciliary body cell proliferation.

The neurogenesis in retina of adult mammals is generally abolished, and this renders the retina lack of regenerative capacity. Despite this, there is a small population of nestin-positive cells in the ciliary epithelium which retains neurogenic potential. The present study aimed at investigating the effect of two drugs, corticosterone and paroxetine, on the cell proliferation of the ciliary body. Adult Sprague-Dawley rats were given vehicle, corticosterone, paroxetine, or both corticosterone and paroxetine treatment for 14 days. Cell proliferation in the ciliary body was quantified using 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. Co-labelling of BrdU and stem cell marker was used to phenotype the BrdU immunoreactive cells. Corticosterone treatment suppressed while paroxetine treatment increased the cell proliferation of the ciliary body. Co-labelling with cell markers revealed that the BrdU positive cells also showed nestin expression but not glial fibrillary acidic protein (GFAP). The results illustrate that proliferation of retinal progenitor cells situated in ciliary body are subjected to regulation by selective serotonin reuptake inhibitors (SSRI) and corticosteroid, which is similar to our previous findings in neurogenic regions in central nervous system (CNS). Paroxetine treatment could reverse the suppressive effect of corticosterone on ciliary body cell proliferation. This provides information for future investigation of retinal stem cell biology and potential treatment of retinal degenerative diseases.

Temporal Requirement of the Protein TyrosinePhosphatase Shp2 in Establishing the Neuronal Fatein Early Retinal Development

FGF signaling is critical in the development of the vertebrate retina, which differentiates in a wave-like pattern similar to that found in the Drosophila eye. In this study, we investigated the mechanism of FGF signaling in vertebrate eye development by identifying Shp2, a protein tyrosine phosphatase, as a novel factor in orchestrating retinal morphogenesis. Using a series of Shp2 conditional mutants, we have shown that Shp2 is specifically required for the initiation of retinal neurogenesis but not for the maintenance of the retinal differentiation program. By mosaic deletion of Shp2, we further demonstrated that Shp2 ablation did not prevent the spreading of the retinal differentiation wave. Shp2 instead controls the patterning of the optic vesicle by regulating the retinal progenitor factors and cell proliferation. In ex vivo culture models and genetic rescue experiments, we showed that Shp2 acts downstream to FGF signaling in retinal development and that it can be functionally substituted by activated Ras signaling. Together, these results demonstrate that Shp2 mediates FGF-Ras signaling to control retinal progenitor cell fate.

Transcriptional role of cyclin D1 in development revealed by a genetic–proteomic screen

Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas-an organ that critically requires cyclin D1 function-cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1(-/-)) retinas. Transduction of an activated allele of Notch1 into Ccnd1(-/-) retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. Our approach, which we term 'genetic-proteomic', can be used to study the in vivo function of essentially any protein.

Developmental sources of conservation and variation in the evolution of the primate eye

Conserved developmental programs, such as the order of neurogenesis in the mammalian eye, suggest the presence of useful features for evolutionary stability and variability. The owl monkey, Aotus azarae, has developed a fully nocturnal retina in recent evolution. Description and quantification of cell cycle kinetics show that embryonic cytogenesis is extended in Aotus compared with the diurnal New World monkey Cebus apella. Combined with the conserved mammalian pattern of retinal cell specification, this single change in retinal progenitor cell proliferation can produce the multiple alterations of the nocturnal retina, including coordinated reduction in cone and ganglion cell numbers, increase in rod and rod bipolar numbers, and potentially loss of the fovea.

Cyclin D1 fine-tunes the neurogenic output of embryonic retinal progenitor cells

BackgroundMaintaining the correct balance of proliferation versus differentiation in retinal progenitor cells (RPCs) is essential for proper development of the retina. The cell cycle regulator cyclin D1 is expressed in RPCs, and mice with a targeted null allele at the cyclin D1 locus (Ccnd1-/-) have microphthalmia and hypocellular retinas, the latter phenotype attributed to reduced RPC proliferation and increased photoreceptor cell death during the postnatal period. How cyclin D1 influences RPC behavior, especially during the embryonic period, is unclear.ResultsIn this study, we show that embryonic RPCs lacking cyclin D1 progress through the cell cycle at a slower rate and exit the cell cycle at a faster rate. Consistent with enhanced cell cycle exit, the relative proportions of cell types born in the embryonic period, such as retinal ganglion cells and photoreceptor cells, are increased. Unexpectedly, cyclin D1 deficiency decreases the proportions of other early born retinal neurons, namely horizontal cells and specific amacrine cell types. We also found that the laminar positioning of horizontal cells and other cell types is altered in the absence of cyclin D1. Genetically replacing cyclin D1 with cyclin D2 is not efficient at correcting the phenotypes due to the cyclin D1 deficiency, which suggests the D-cyclins are not fully redundant. Replacement with cyclin E or inactivation of cyclin-dependent kinase inhibitor p27Kip1 restores the balance of RPCs and retinal cell types to more normal distributions, which suggests that regulation of the retinoblastoma pathway is an important function for cyclin D1 during embryonic retinal development.ConclusionOur findings show that cyclin D1 has important roles in RPC cell cycle regulation and retinal histogenesis. The reduction in the RPC population due to a longer cell cycle time and to an enhanced rate of cell cycle exit are likely to be the primary factors driving retinal hypocellularity and altered output of precursor populations in the embryonic Ccnd1-/- retina.

The role of Xenopus Rx-L in photoreceptor cell determination

Members of the Rx ( retinal homeobo x) gene family play vital roles during eye development. In Xenopus, as in most other vertebrates, two Rx-type genes have been described. While Rx1 deficiency led to loss of optic vesicles and impaired the proper development of ventral forebrain structures, a recently isolated second Rx-gene, Rx-L, seems to function in late retinogenesis. Here, we report that the specific suppression of Xenopus Rx-L function impaired the formation of the photoreceptor layer and reduced the expression of photoreceptor specific genes. Overexpression of Xenopus Rx-L induced ectopic expression of photoreceptor specific genes, but did only marginally promote the proliferation of retinal progenitor cells. Targeted overexpression of Rx-L in developing retinoblasts in vivo led to an increased fraction of photoreceptor cells at the expense of amacrine and bipolar cells and revealed that Rx-L acts as a transcription activator. A phylogenic analysis of all reported Rx-type genes revealed that they could be grouped into three categories, including an “invertebrate Rx” group, a “classical vertebrate Rx” group, and a “vertebrate Qrx/Rx-L” group. Taken together, Rx-L, unlike Rx1, is required for the determination of retinal cell types, especially photoreceptors, rather than for proliferation of retinal progenitors.

Toll-like receptor 4 restricts retinal progenitor cell proliferation

Toll-like receptor 4 restricts retinal progenitor cell proliferation

Toll-like receptor 4 restricts retinal progenitor cell proliferation

Retinal neurogenesis ceases by the early postnatal period, although retinal progenitor cells (RPCs) persist throughout life. In this study, we show that in the mammalian eye, the function of Toll-like receptor 4 (TLR4) extends beyond regulation of the innate immune response; it restricts RPC proliferation. In TLR4-deficient mice, enhanced proliferation of cells reminiscent of RPCs is evident during the early postnatal period. In vitro experiments demonstrate that TLR4 acts as an intrinsic regulator of RPC fate decision. Increased TLR4 expression in the eye correlates with the postnatal cessation of cell proliferation. However, deficient TLR4 expression is not sufficient to extend the proliferative period but rather contributes to resumption of proliferation in combination with growth factors. Proliferation in vivo is inhibited by both MyD88-dependent and -independent pathways, similar to the mechanisms activated by TLR4 in immune cells. Thus, our study attributes a novel role to TLR4 as a negative regulator of RPC proliferation.

Sox2 Plays a Role in the Induction of Amacrine and Müller Glial Cells in Mouse Retinal Progenitor Cells

The transcription factor Sox2 plays important roles in both human and mouse retinal development. Although loss-of-function mutations in Sox2 have been studied in mice, gain-of-function experiments in the neural retina have been lacking. The detailed expression pattern of Sox2 in the developing mouse retina was examined by immunohistochemistry. Then, Sox2 was expressed in a retinal explant culture prepared from E17 mouse embryos by retrovirus-mediated gene transfer to examine its role in retinal development. In addition, shRNA was used to suppress Sox2 in a retinal explant using a retrovirus-mediated system. Sox2 was expressed throughout the neuroblastic layer in the embryonic retina, but only in the inner nuclear layer in the mature retina. Double immunostaining revealed that Sox2 was expressed in Müller glial cells and in a subset of amacrine cells. Forced expression of Sox2 in a mouse retinal explant culture resulted in the dramatic accumulation of amacrine cells in the inner nuclear layer; in addition, cells expressing amacrine cell markers were also found on the innermost side of the outer nuclear layer. The expression of Pax6, which plays an important role in amacrine cell differentiation, was observed in the Sox2-expressing cells, and Sox2 activated the Pax6 promoter to drive luciferase expression in Y79 cells. A decrease in retinal progenitor cell proliferation accompanied these effects. The suppression of Sox2 expression by shRNA resulted in a decreased number of cells in the inner nuclear layer. Therefore, ectopic Sox2 expression can induce amacrine cells in the mouse retina from stage E17 onward, possibly by facilitating cell cycle exit.

Interleukin-4 blocks proliferation of retinal progenitor cells and increases rod photoreceptor differentiation through distinct signaling pathways

Interleukin-4 (IL-4), an anti-inflammatory cytokine, has been related to the differentiation of the rodent retina in vitro, but constitutive presence of either IL-4 or of IL-4 receptor in the retina has not been reported. In this work we examined the expression of IL-4 and its specific receptor α subunit (IL-4Rα). IL-4Rα is expressed both in neural retina and non-neural ocular tissue, while IL-4 was found mainly in non-neural tissue. We characterized a novel trophic effect of IL-4 upon the retina. We showed that IL-4 can inhibit the proliferation of retinal cells (~ 40%) through the cAMP-PKA pathway and associated with a reduction of cyclin D1 and increase of p27 kip1. IL-4 also promotes the differentiation of rod photoreceptors. Activation of tyrosine kinases, protein kinase C, and mitogen-activated kinases of the Erk family were required for IL-4-induced rod photoreceptor differentiation, independent of the release of other trophic factors in culture. Taken together, our results show, for the first time, that IL-4 directly modulates proliferation of retinal cells and rod photoreceptor differentiation, through distinct signaling pathways.

Isolation and characterisation of neural progenitor cells from the adult Chx10orJ/orJ central neural retina

Retinal stem cells have been isolated from the ciliary epithelium (CE) of the mammalian retina. However, the central neural retina (CNR) lacks the capability to regenerate, a phenomenon retained by lower vertebrates. Mutations in the Chx10 homeobox gene cause reduced proliferation of retinal progenitor cells during development, leading to microphthalmia. Recently, we showed that in Chx10 orJ/orJ mice, dividing cells persist in the adult CNR, suggesting the existence of a dormant progenitor population. Here, we show that these cells are proliferative and give rise to neurospheres in vitro, a characteristic of neural stem cells. However, these adult-derived CNR progenitors differ from those of the wildtype CE, leading to de-pigmented, larger and more numerous neurospheres expressing Müller glial cell markers. Our results suggest that lack of Chx10 leads to maintenance of a dormant neural progenitor population in the adult CNR. Furthermore, Chx10 is not required for in vitro proliferation of these progenitors.

Vsx2/Chx10 ensures the correct timing and magnitude of Hedgehog signaling in the mouse retina

Vertebrate retinal progenitor cells (RPCs) undergo a robust proliferative expansion to produce enough cells for the retina to form appropriately. Vsx2 (formerly Chx10), a homeodomain protein expressed in RPCs, is required for sufficient proliferation to occur. Sonic Hedgehog protein (SHH), secreted by retinal ganglion cells (RGCs), activates Hedgehog (Hh) signaling in RPCs and is also required for sufficient proliferation to occur. Therefore, we sought to determine if reduced Hh signaling is a contributing factor to the proliferation changes that occur in the absence of Vsx2. To do this, we examined Shh expression and Hh signaling activity in the homozygous ocular retardation J (orJ) mouse, which harbors a recessive null allele in the Vsx2 gene. We found that Shh expression and Hh signaling activity are delayed during early retinal development in orJ mice and this correlates with a delay in the onset of RGC differentiation. At birth, reduced expression of genes regulated by Hh signaling was observed despite the production of SHH ligand. orJ RPCs respond to pre-processed recombinant SHH ligand (SHH-N) in explant culture as evidenced by increased proliferation and expression of Hh target genes. Interestingly, proliferation in the orJ retina is further inhibited by cyclopamine, an antagonist of Hh signaling. Our results suggest that reduced Hh signaling contributes to the reduced level of RPC proliferation in the orJ retina, thereby revealing a role for Vsx2 in mediating mitogen signaling.

Retinal tissue engineering using mouse retinal progenitor cells and a novel biodegradable, thin-film poly(e-caprolactone) nanowire scaffold

Retinal progenitor cells (RPCs) can be combined with nanostructured polymer scaffolds to generate composite grafts in culture. One strategy for repair of diseased retinal tissue involves implantation of composite grafts of this type in the subretinal space. In the present study, mouse retinal progenitor cells (RPCs) were cultured on laminin-coated novel nanowire poly(e-caprolactone)(PCL) scaffolds, and the survival, differentiation, and migration of these cells into the retina of C57bl/6 and rhodospsin −/− mouse retinal explants and transplant recipients were analyzed. RPCs were cultured on smooth PCL and both short (2.5 μm) and long (27 μm) nanowire PCL scaffolds. Scaffolds with adherent mRPCs were then either co-cultured with, or transplanted to, wild-type and rhodopsin −/− mouse retina. Robust RPC proliferation on each type of PCL scaffold was observed. Immunohistochemistry revealed that RPCs cultured on nanowire scaffolds increased expression of mature bipolar and photoreceptor markers. Reverse transcription polymerase chain reaction revealed down-regulation of several early progenitor markers. PCL-delivered RPCs migrated into the retina of both wild-type and rhodopsin knockout mice. The results provide evidence that RPCs proliferate and express mature retinal proteins in response to interactions with nanowire scaffolds. These composite grafts allow for the migration and differentiation of new cells into normal and degenerated retina.