Multipotent and Stem Cells in the Developing, Definitive, and Regenerating Vertebrate Eye

This is a review of the experimental studies on the vertebrate retina neurogenesis. Data are provided on the distribution and localization of multipotent and stem cells in the developing, definitive, and regenerating eye. At the early stages of retina development, the neuroepithelial cells divide synchronously, thus leading to the accumulation of a certain number of the retinal rudiment cells. Synchronous divisions precede the asynchronous ones, when the differentiation of the retinal cells is initiated. The neuroepithelial cells are multipotent: the neuroblast is a source of the cells of different types, for example, neurons and glial cells. The proliferating multipotent cells are preserved in the ciliary-terminal zone of the retina of amphibians, fish, and chickens during their entire life. The differentiated pigment epithelium cells also proliferate in this area of the eye. The multipotent cells of the retinal ciliary-terminal zone and cells of the pigment epithelium in the eye periphery provide for the growth of amphibian and fish eyes during the entire life of these animals. In adult mammals, clonable and self-renewable cells were found among the pigmented differentiated cells in the ciliary folds. In a culture, the stem cells form spheroids consisting of depigmented and proliferating cells. Upon transdifferentiation, the cells of spheroids form rods, bipolar cells, and ganglion and glial cells, thus suggesting the possible regenerative potencies of the stem cells in the ciliary body of the mammalian eye. The main event of retinal regeneration in newts is the transdifferentiation of the pigment epithelium cells. The results of comparative analysis suggest that the stem cells of the ciliary body in the mammalian eye and pigment epithelium cells in lower vertebrates exhibit similar potencies and use similar mechanisms during the formation of the cells of the neural series.     

Neural stem cell properties of Müller glia in the mammalian retina: regulation by Notch and Wnt signaling

The retina in adult mammals, unlike those in lower vertebrates such as fish and amphibians, is not known to support neurogenesis. However, when injured, the adult mammalian retina displays neurogenic changes, raising the possibility that neurogenic potential may be evolutionarily conserved and could be exploited for regenerative therapy. Here, we show that Müller cells, when retrospectively enriched from the normal retina, like their radial glial counterparts in the central nervous system (CNS), display cardinal features of neural stem cells (NSCs), i.e., they self-renew and generate all three basic cell types of the CNS. In addition, they possess the potential to generate retinal neurons, both in vitro and in vivo. We also provide direct evidence, by transplanting prospectively enriched injury-activated Müller cells into normal eye, that Müller cells have neurogenic potential and can generate retinal neurons, confirming a hypothesis, first proposed in lower vertebrates. This potential is likely due to the NSC nature of Müller cells that remains dormant under the constraint of non-neurogenic environment of the adult normal retina. Additionally, we demonstrate that the mechanism of activating the dormant stem cell properties in Müller cells involves Wnt and Notch pathways. Together, these results identify Müller cells as latent NSCs in the mammalian retina and hence, may serve as a potential target for cellular manipulation for treating retinal degeneration.     

Activation of peroxisome proliferator-activated receptor-α enhances fatty acid oxidation in human adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPARα in adipocytes have been unclarified. We examined the functions of PPARα using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPARα by GW7647, a potent PPARα agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPARγ, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPARα activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPARγ is activated. On the other hand, PPARα activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPARα-dependent manner. Moreover, PPARα activation increased the production of CO₂ and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPARα stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPARα agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPARα activation are very valuable for managing diabetic conditions accompanied by obesity, because PPARγ agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.     

Trying to understand axonal regeneration in the CNS of fish

In contrast to the situation in mammals and birds, neurons in the central nervous system (CNS) of fish—such as the retinal ganglion cells—are capable of regenerating their axons and restoring vision. Special properties of the glial cells and the neurons of the fish visual pathway appear to contribute to the success of axonal regeneration. The fish oligodendrocytes lack the axon growth inhibiting molecules that interfere with axonal extension in mammals. Instead, fish optic nerve oligodendrocytes support—at least in vitro—axonal elongation of fish as well as that of rat retinal axons. Moreover, the fish retinal ganglion cells re-express upon injury a set of growth associated cell surface molecules and equip the regenerating axons throughout their path and up into their target, the tectum opticum with these molecules. This may indicate that the injured fish ganglion cells reactivate the cellular machinery necessary for axonal regrowth and pathfinding. Furthermore, the target itself provides positional marker molecules even in adult fish. These marker molecules are required to guide the regenerating axons back to their retinotopic home territory within the tectum. © 1992 John Wiley & Sons, Inc.     

Adult neurogenesis and neuronal regeneration in the brain of teleost fish

Whereas adult neurogenesis appears to be a universal phenomenon in the vertebrate brain, enormous differences exist in neurogenic potential between “lower” and “higher” vertebrates. Studies in the gymnotiform fish Apteronotus leptorhynchus and in zebrafish have indicated that the relative number of new cells, as well as the number of neurogenic sites, are at least one, if not two, orders of magnitude larger in teleosts than in mammals. In teleosts, these neurogenic sites include brain regions homologous to the mammalian hippocampus and olfactory bulb, both of which have consistently exhibited neurogenesis in all species examined thus far. The source of the new cells in the teleostean brain are intrinsic stem cells that give rise to both glial cells and neurons. In several brain regions, the young cells migrate, guided by radial glial fibers, to specific target areas where they integrate into existing neural networks. Approximately half of the new cells survive for the rest of the fish’s life, whereas the other half are eliminated through apoptotic cell death. A potential mechanism regulating development of the new cells is provided by somatic genomic alterations. The generation of new cells, together with elimination of damaged cells through apoptosis, also enables teleost fish rapid and efficient neuronal regeneration after brain injuries. Proteome analysis has identified a number of proteins potentially involved in the individual regenerative processes. Comparative analysis has suggested that differences between teleosts and mammals in the growth of muscles and sensory organs are key to explain the differences in adult neurogenesis that evolved during phylogenetic development of the two taxa.     

Transdifferentiation Potential of Ciliary and Pigment Epithelial Cells in Lower Vertebrates and Mammals

Cellular composition of the peripheral region of the eye in amphibians and mammals as well as embryonic fissure in amphibians was studied. Different distributions of proliferating cells in retinal pigment epithelium have been revealed in adult amphibians (newt, axolotl, and Xenopus). Single cells incorporated [³H]thymidine in the newt and Xenopus; 0.4% cells, in the axolotl. An embryonic fissure was observed in the eye of the axolotl. Pigment epithelial cells in the embryonic palpebral region actively proliferated: about 20% cells incorporated [³H]thymidine. Proliferating cells were also localized in the ciliary marginal zone of the retina in all studied amphibians, particularly, in the axolotl. In newborn hamsters, [³H]thymidine-labeled cells have been revealed in the pigment epithelium as well as in the outer pigmented and inner unpigmented layers of the ciliary body. Proliferative activity of the peripheral regions of the eye is due to eye growth in adult amphibians and newborn hamsters. After retinectomy, the retina is regenerated from the cells of the growth ciliary marginal zone in all amphibians, pigment epithelial cells in the newt, and pigment epithelial cells of the embryonic fissure in the axolotl. Heterogeneous composition of the pigment epithelium in the newt and axolotl reflects high transdifferentiation potential of these regions. Structural comparison of the peripheral region of the eye in amphibians and mammals demonstrate that the ciliary body of mammals containing stem cells is homologous to the ciliary marginal zone of amphibians containing multipotent cells.     

Neurogenesis in the Adult Mammalian Brain

The concept of the CNS cell composition stability has recently undergone significant changes. It was earlier believed that neurogenesis in the mammalian CNS took place only during embryonic and early postnatal development. New approaches make it possible to prove that neurogenesis takes part even in the adult brain. The present review summarizes the data about the neural stem cell. It has been demonstrated that new neurons are constantly formed in adult mammals, including man. In two brain zones, subventricular zone and dentate gyrus, neurogenesis appears to proceed throughout the entire life of mammals, including man. The newly arising neurons are essential for some important processes, such as memory and learning. Stem cells were found in the subependymal and/or ependymal layer. They express nestin and have a low mitotic activity. During embryogenesis, the stem cell divides asymmetrically: one daughter cell resides as the stem cell in the ependymal layer and another migrates to the subventricular zone. There it gives rise to a pool of dividing precursors, from which neural and glial cells differentiate and migrate to the sites of final localization. The epidermal and fibroblast growth factors act as mitogens for the neural stem cell. The neural stem cell gives rise to the cells of all germ layers in vitro and has a wide potential for differentiation in the adult organism. Hence, it can be used as a source of various cell types of the nervous tissue necessary for cellular transplantation therapy.     

PPARα is essential for retinal lipid metabolism and neuronal survival

Background

Peroxisome proliferator activated receptor-alpha (PPARα) is a ubiquitously expressed nuclear receptor. The role of endogenous PPARα in retinal neuronal homeostasis is unknown. Retinal photoreceptors are the highest energy-consuming cells in the body, requiring abundant energy substrates. PPARα is a known regulator of lipid metabolism, and we hypothesized that it may regulate lipid use for oxidative phosphorylation in energetically demanding retinal neurons.

Results

We found that endogenous PPARα is essential for the maintenance and survival of retinal neurons, with Pparα ^-/- mice developing retinal degeneration first detected at 8 weeks of age. Using extracellular flux analysis, we identified that PPARα mediates retinal utilization of lipids as an energy substrate, and that ablation of PPARα ultimately results in retinal bioenergetic deficiency and neurodegeneration. This may be due to PPARα regulation of lipid transporters, which facilitate the internalization of fatty acids into cell membranes and mitochondria for oxidation and ATP production.

Conclusion

We identify an endogenous role for PPARα in retinal neuronal survival and lipid metabolism, and furthermore underscore the importance of fatty acid oxidation in photoreceptor survival. We also suggest PPARα as a putative therapeutic target for age-related macular degeneration, which may be due in part to decreased mitochondrial efficiency and subsequent energetic deficits.

     

Antibodies against pax6 immunostain amacrine and ganglion cells and neuronal progenitors,but not rod precursors,in the normal and regenerating retina of the goldfish

Pax6 is a developmental regulatory gene that plays a key role in the development of the embryonic brain, eye, and retina. This gene is also expressed in discrete groups of neurons within the adult brain. In this study, antibodies raised against a fusion protein from a zebra fish pax6 cDNA were used to investigate the expression of the pax6 gene in the mature, growing, and regenerating retina of the goldfish. On western blots of retinal proteins, the pax6 antibodies recognize a single band at the approximate size of the zebra fish pax6 protein. In retinal sections, the antibodies label the nuclei of mature amacrine and some ganglion cells. At the retinal margin, where neurogenesis and cellular differentiation continually occur in goldfish, the antibodies label neuronal progenitors and the newly postmitotic neurons. Following injury and during neuronal regeneration, the antibodies label mitotically active progenitors of regenerating neurons. Rod precursors, proliferating cells that normally give rise solely to rod photoreceptors and are the presumed antecedents of the injury-stimulated neuronal progenitors, are not immunostained by antibodies to the pax6 protein. The results of this study document the identity of pax6-expressing cells in the mature retina and demonstrate that in the goldfish pax6 is expressed in neuronal progenitors during both retinal growth and regeneration. © 1996 John Wiley & Sons, Inc.     

Smn deficiency causes neuritogenesis and neurogenesis defects in the retinal neurons of a mouse model of spinal muscular atrophy

The eye is an excellent model for the study of neuronal development and pathogenesis of central nervous system disorders because of its relative ease of accessibility and the well‐characterized cellular makeup. We have used this model to study spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease caused by deletions or mutations in the survival of motor neuron 1 gene (SMN1). We have investigated the expression pattern of mouse Smn mRNA and protein in the neural retina and the optic nerve of wild type mice. Smn protein is present in retinal ganglion cells and amacrine cells within the neural retina as well as in glial cells in the optic nerve. Histopathological analysis in phenotype stage SMA mice revealed that Smn deficiency is associated with a reduction in ganglion cell axon and glial cell number in the optic nerve, as well as compromised cellular processes and altered organization of neurofilaments in the neural retina. Whole mount preparation and retinal neuron primary culture provided further evidence of abnormal synaptogenesis and neurofilament accumulation in the neurites of Smn‐deficient retinal neurons. A subset of amacrine cells is absent, in a cell‐autonomous fashion, in the retina of SMA mice. Finally, the retinas of SMA mice have altered electroretinograms. Altogether, our study has demonstrated defects in axodendritic outgrowth and cellular composition in Smn‐depleted retinal neurons, indicating a role for Smn in neuritogenesis and neurogenesis, and providing us with an insight into pathogenesis of SMA. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 153‐169, 2011     

Symposium 10: Differentiation Plasticity of Stem Cells. Direct differentiation of radial glial cells from embryonic stem cells

The major role of radial glial cells in neuronal development is to provide support and guidance for neuronal migration. In vitro, neurons, astrocytes and oligodendrocytes have also been generated from neural stem cells and embryonic stem cells, but the generation of radial glial cells in vitro has not yet been reported. Since radial glial cells can lead to neurons and astrocytes during brain development, neurogenesis and gliogenesis of stem cells in vitro may at least in part also utilize the same mechanisms. To test this hypothesis, we utilized five different clones of embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glial cells can be generated from ES/EC cell lines. These ES/EC cell‐derived radial glial cells are similar in morphology to radial glial cells in vivo. They also express several cytoskeletal markers that are characteristics of radial glial cells in vivo. The processes of these in vitro‐generated radial glial cells are organized into scaffolds that appear to support the migration of newly generated neurons in culture. Like radial glial cells in vivo, they appear to differentiate subsequently into astrocytes. Differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system may facilitate the investigation of regulation of radial glial cell differentiation and its biological function. Acknowledgements: Supported by USPHS Grant NS11853 and a grant from the Children's Medical Research Foundation.     

Age‐related changes in stem cell dynamics,neurogenesis, apoptosis,and gliosis in the adult brain: A novel teleost fish model of negligible senescence

Adult neurogenesis, the generation of new neurons in the adult central nervous system, is a reported feature of all examined vertebrate species. However, a dramatic decline in the rates of cell proliferation and neuronal differentiation occurs in mammals, typically starting near the onset of sexual maturation. In the present study, we examined possible age‐related changes associated with adult neurogenesis in the brain of brown ghost knifefish (Apteronotus leptorhynchus), a teleost fish distinguished by its enormous neurogenic potential. Contrary to the well‐established alterations in the mammalian brain during aging, in the brain of this teleostean species we could not find evidence for any significant age‐related decline in the absolute levels of stem/progenitor cell proliferation, neuronal and glial differentiation, or long‐term survival of newly generated cells. Moreover, there was no indication that the amount of glial fibrillary acidic protein or the number of apoptotic cells in the brain was altered significantly over the course of adult life. We hypothesize that this first demonstration of negligible cellular senescence in the vertebrate brain is related to the continued growth of this species and to the lack of reproductive senescence during adulthood. The establishment of the adult brain of this species as a novel model of negligible senescence provides new opportunities for the advancement of our understanding of the biology of aging and the fundamental mechanisms that underlie senescence in the brain. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 514–530, 2014     

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye^1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment⁴; however the cells are still capable of forming the different cell types found within the neural retina^1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury⁵, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent^6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation^9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.     

Growth and differentiation of the retina and the optic tectum in the medaka fish requires olSfrp5

Secreted Frizzled‐Related Proteins (SFRPs) are extracellular modulators of Wnt and Bmp signaling. Previous studies in birds and fishes have shown that Sfrp1, a member of this family, is strongly expressed throughout the development of the eye contributing to the specification of the eye field, retina neurogenesis and providing guidance information to retina ganglion cell axons. Here, we report that in medaka fish (Oryzias latipes) the expression of olSfrp5, which is closely related to olSfrp1, largely overlaps with that of olSfrp1 in the eye, but is additionally expressed in the developing midbrain and gut primordium. Morpholino‐based interference with olSfrp5 expression causes microphthalmia and reduction of the tectum size associated with an increase in apoptotic cell death in these structures. Furthermore, interference with the levels of olSfrp5 expression impairs the patterning of the ventral portion of the optic cup, leading in some cases to a fissure coloboma. These early defects are followed by an abnormal retinal and tectal neurogenesis. In particular, only reduced numbers of photoreceptor and RGC were generated in olSfrp5 morphants retinas. The results point to an important role of olSfrp5 in visual system formation and indicate that olSfrp1 and olSfrp5, despite their overlapping expression, have only partially redundant function during eye development. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization

Galectin‐1/LGALS1, a newly recognized angiogenic factor, contributes to the pathogenesis of diabetic retinopathy (DR). Recently, we demonstrated that glucocorticoids suppressed an interleukin‐1β‐driven inflammatory pathway for galectin‐1 expression in vitro and in vivo. Here, we show glucocorticoid‐mediated inhibitory mechanism against hypoxia‐inducible factor (HIF)‐1α‐involved galectin‐1 expression in human Müller glial cells and the retina of diabetic mice. Hypoxia‐induced increases in galectin‐1/LGALS1 expression and promoter activity were attenuated by dexamethasone and triamcinolone acetonide in vitro. Glucocorticoid application to hypoxia‐stimulated cells decreased HIF‐1α protein, but not mRNA, together with its DNA‐binding activity, while transactivating TSC22 domain family member (TSC22D)3 mRNA and protein expression. Co‐immunoprecipitation revealed that glucocorticoid‐transactivated TSC22D3 interacted with HIF‐1α, leading to degradation of hypoxia‐stabilized HIF‐1α via the ubiquitin‐proteasome pathway. Silencing TSC22D3 reversed glucocorticoid‐mediated ubiquitination of HIF‐1α and subsequent down‐regulation of HIF‐1α and galectin‐1/LGALS1 levels. Glucocorticoid treatment to mice significantly alleviated diabetes‐induced retinal HIF‐1α and galectin‐1/Lgals1 levels, while increasing TSC22D3 expression. Fibrovascular tissues from patients with proliferative DR demonstrated co‐localization of galectin‐1 and HIF‐1α in glial cells partially positive for TSC22D3. These results indicate that glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced retinal glial galectin‐1/LGALS1 expression via HIF‐1α destabilization, highlighting therapeutic implications for DR in the era of anti‐vascular endothelial growth factor treatment.     

DRF 2655: A Unique Molecule that Reduces Body Weight and Ameliorates Metabolic Abnormalities

Objective: Preclinical evaluation of DRF 2655, a peroxisome proliferator‐activated receptor alpha (PPARα) and PPARγ agonist, as a body‐weight lowering, hypolipidemic and euglycemic agent. Research Methods and Procedures: DRF 2655 was studied in different genetic, normal, and hyperlipidemic animal models. HEK 293 cells were used to conduct the reporter‐based transactivation of PPARα and PPARγ. To understand the biochemical mechanism of lipid‐, body‐weight‐, and glucose‐lowering effects, activities of key β‐oxidation and lipid catabolism enzymes and gluconeogenic enzymes were studied in db/db mice treated with DRF 2655. 3T3L1 cells were used for adipogenesis study, and HepG2 cells were used to study the effect of DRF 2655 on total cholesterol and triglyceride synthesis using [¹⁴C]acetate and [³H]glycerol. Results: DRF 2655 showed concentration‐dependent transactivation of PPARα and PPARγ. In the 3T3L1 cell‐differentiation study, DRF 2655 and rosiglitazone showed 369% and 471% increases, respectively, in triglyceride accumulation. DRF 2655 showed body‐weight lowering and euglycemic and hypolipidemic effects in various animal models. db/db mice treated with DRF 2655 showed 5‐ and 3.6‐fold inhibition in phosphoenolpyruvate carboxykinase and glucose 6‐phosphatase activity and 651% and 77% increases in the β‐oxidation enzymes carnitine palmitoyltransferase and carnitine acetyltransferase, respectively. HepG2 cells treated with DRF 2655 showed significant reduction in lipid synthesis. Discussion: DRF 2655 showed excellent euglycemic and hypolipidemic activities in different animal models. An exciting finding is its body‐weight lowering effect in these models, which might be mediated by the induction of target enzymes involved in hepatic lipid catabolism through PPARα activation.     

Germline stem cells are critical for sexual fate decision of germ cells

Egg or sperm? The mechanism of sexual fate decision in germ cells has been a long‐standing issue in biology. A recent analysis identified foxl3 as a gene that determines the sexual fate decision of germ cells in the teleost fish, medaka. foxl3/Foxl3 acts in female germline stem cells to repress commitment into male fate (spermatogenesis), indicating that the presence of mitotic germ cells in the female is critical for continuous sexual fate decision of germ cells in medaka gonads. Interestingly, foxl3 is found in most vertebrate genomes except for mammals. This provides the interesting possibility that the sexual fate of germ cells in mammals is determined in a different way compared to foxl3‐possessing vertebrates. Considering the fact that germline stem cells are the cells where foxl3 begins to express and sexual fate decision initiates and mammalian ovary does not have typical germline stem cells, the mechanism in mammals may have been co‐evolved with germline stem cell loss in mammalian ovary.