A novel antigen is shared by retinal pigment epithelium and pigmented neural crest

 Pigment cells in vertebrate embryos are formed in both the central and peripheral nervous system. The neural crest, a largely pluripotent population of precursor cells derived from the embryonic neural tube, gives rise to pigment cells which migrate widely in head and trunk.The retinal pigment epithelium is derived from the optic cup, which arises from ectoderm of the neural tube. We have generated an antibody, ips6, which stains an antigen common to pigment cells of retinal pigment epithelium and neural crest. Ips6 stains retinal pigment epithelium and choroid as well as a subset of crest cells that migrate in pathways typical of melanoblasts. Immunoreactivity is seen first in the eye and later in a subset of migrating crest cells. Crest cells in the amphibian embryo migrate along specific, stereotyped routes; ips6 immunoreactive cells are found in some but not all of these pathways. In older wild-type embryos, cells expressing ips6 appear coincident with pigment-containing cells in the flank, head, eye and embryonic gut. In older animals, staining in the eye extends to the intraretinal segment of optic nerve and interstices between photoreceptors and cells at the retinal periphery. We suggest that the ips6 antibody defines an antigen common to pigment cells of central and peripheral origin. Received: 22 January 1996/Accepted: 15 July 1996     

Fibroblast growth factor receptor deficiency in dystrophic retinal pigmented epithelium

The retinal pigmented epithelium (RPE) is known to be site of the primary lesion in inherited retinal dystrophy in the Royal College of Surgeons (RCS) rat, a model for retinitis pigmentosa. Although the only functional defect so far detected in these cells is their failure to efficiently phagocytose shed photoreceptor outer segment debris, the actual cause of photoreceptor cell death is still unknown. Recently the possibility of “trophic factors” important in photoreceptor survival produced by normal RPE but not by dystrophic RPE has been suggested. Hence we decided to investigate the presence and abundance of two candidate diffusible factors, the acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), as well as their high affinity cell surface receptors (FGF-R). mRNA was isolated from primary cultures of purified normal and dystrophic RPE and analyzed by PCR amplification using specific oligonucleotide primers for aFGF and bFGF: the size and abundance of amplified fragments was similar for both cell types. Also, aFGF protein, detected by immunocytochemistry using specific antisera, appeared to be present in approximately equal amounts and distributed in a similar pattern. However, scatchard analysis of radio-labelled bFGF binding to primary cultures of normal and dystrophic rat RPE revealed that dystrophic RPE possess only 29% the number of surface receptors compared to congenic normal cells. Furthermore, the level of expression of FGF-R2 mRNA, but not that of FGF-R1, was significantly different. Other parameters measured (receptor affinity, profile of ligand internalization and degradation, receptor molecular weight and mitogenic activity) did not show any significant differences between normal and dystrophic RPE. The precise role of FGF-R deficiency in the etiology of the disease hence remains to be determined, but it indicates the importance of trophic factors in the normal functioning of the retina. © 1993 Wiley-Liss, Inc.     

In vitro stimulation of cartilage in embryonic chick neural crest cells by products of rentinal pigmented epithelium.

The retinal pigmented epithelium of the chick embryo influences head neural crest mesenchymal cells to form the scleral cartilage of the eye. The possible role of extracellular matrix in this interaction was studied. Extracellular matrix was deposited on Millipore filters in vitro by pigmented epithelial cells which were then killed by distilled water lysis. When grown on the Millipore filters which had carried pigmented epithelium, clonal neural crest and periocular mesenchyme “target” cells formed cartilage in 61 of 155 experiments. Cartilage was not formed when the cells were grown on naked filters nor did gels of purified Type I and Type II collagen promote chondrogenesis. It is concluded that extracellular matrix deposited by the pigmented epithelium in vitro is a potent stimulus for the induction of chondrogenesis in competent mesenchyme, and that living pigmented epithelial cells need not be present for such induction.     

Basic fibroblast growth factor induces retinal pigment epithelium to generate neural retina in vitro.

During embryogenesis, the cells of the eye primordium are initially capable of giving rise to either neural retina or pigmented epithelium (PE), but become restricted to one of these potential cell fates. However, following surgical removal of the retina in embryonic chicks and larval amphibians, new neural retina is generated by the transdifferentiation, or phenotypic switching, of PE cells into neuronal progenitors. A recent study has shown that basic fibroblast growth factor (bFGF) stimulates this process in chicks in vivo. To characterize further the mechanisms by which this factor regulates the phenotype of retinal tissues, we added bFGF to enzymatically dissociated chick embryo PE. We found that bFGF stimulated proliferation and caused several morphological changes in the PE, including the loss of pigmentation; however, no transdifferentiation to neuronal phenotypes was observed. By contrast, when small sheets of PE were cultured as aggregates on a shaker device, preventing flattening and spreading on the substratum, we found that a large number of retinal progenitor cells were generated from the PE treated with bFGF. These results indicate that bFGF promotes retinal regeneration in vitro, as well as in ovo, and suggest that the ability of chick PE to undergo transdifferentiation to neuronal progenitors appears to be dependent on the physical configuration of the cells.     

Proteomic analysis of mature melanosomes from the retinal pigmented epithelium

The protein content of melanosomes in the retinal pigment epithelium (RPE) was analyzed by mass spectrometry. More than 100 proteins were found to be common to two out of three variations of sample preparation. Some proteins normally associated with other organelles were detected. Several lysosomal enzymes were detected, with the presence of cathepsin D confirmed by immunoelectron microscopy, thus supporting the previously suggested notion that melanosomes may contribute to the degradation of ingested photoreceptor outer segment disks.     

Gene transfer and expression studies in cultured avian neural crest cells differentiating into melanocytes

Neural crest cells obtained from explanted neural tubes take up, express, and retain exogenous DNA applied by the CaPO4 co-precipitation method during their differentiation into melanocytes. High efficiencies of gene transfer were obtained with both supercoiled DNA and intact phage particles; linear DNA or DNA from the phage yielded very low efficiencies. There is some evidence that transferred gene expression is differentiation dependent. The system should be useful for studies concerned with the analysis of cell developmental genes and their regulatory elements.     

A mart-1::Cre transgenic line induces recombination in melanocytes and retinal pigment epithelium

The number of transgenic mouse lines expressing Cre in either type of pigment cells (melanocytes and retinal pigment epithelium, RPE) is limited, and the available lines do not always offer sufficient specificity. In this study, we addressed this issue and we report on the generation of a MART‐1::Cre BAC transgenic mouse line, in which the expression of Cre recombinase is controlled by regulatory elements of the pigment cell‐specific gene MART‐1 (mlana). When MART‐1::Cre BAC transgenic mice were bred with the ROSA26‐R reporter line, ß‐galactosidase expression was observed in RPE from E12.5 onwards, and in melanocyte precursors from E17.5, indicating that the MART‐1::Cre line provides Cre recombinase activity in pigment‐producing cells rather than in a particular lineage. In addition, breeding of this mouse line to mice carrying a conditional allele of RBP‐Jκ corroborated the reported phenotypes in both pigment cell lineages, inducing hair greying and microphthalmia. Our results thus suggest, that the MART‐1::Cre line may serve as a novel and useful tool for functional studies in melanocytes and the RPE.genesis 49:403–409, 2011. © 2011 Wiley‐Liss, Inc.     

Hypoxia-inducible factor and vascular endothelial growth factor in the neuroretina and retinal blood vessels after retinal ischemia

Retinal ischemia arises from circulatory failure. As the retinal blood vessels are key organs in circulatory failure, our aim was to study the retinal vasculature separately from the neuroretina to elucidate the role of hypoxia-inducible factor (HIF) 1α and 1β and vascular endothelial growth factor (VEGF) in retinal ischemia. Retinal ischemia was induced in porcine eyes by applying an intraocular pressure, followed by 12 h of reperfusion. HIF-1α mRNA expression was not affected by ischemia, while immunofluorescence staining was higher after ischemia in the neuroretina. HIF-1β immunoreactivity and mRNA expression were unaffected. VEGF protein levels in the vitreous humor and VEGF staining in the neuroretina were more pronounced in eyes subjected to ischemia than in the sham eyes. VEGF may be activated downstream of HIF-1 and is known to stimulate retinal neovascularization, which causes sight-threatening complications. These results emphasize the need for pharmacological treatment to block the HIF and VEGF signaling pathways in retinal ischemia.     

Activin signaling limits the competence for retinal regeneration from the pigmented epithelium

Regeneration of the retina in amphibians is initiated by the transdifferentiation of the retinal pigmented epithelium (RPE) into neural progenitors. A similar process occurs in the early embryonic chick, but the RPE soon loses this ability. The factors that limit the competence of RPE cells to regenerate neural retina are not understood; however, factors normally involved in the development of the eye (i.e. FGF and Pax6) have also been implicated in transdifferentiation. Therefore, we tested whether activin, a TGFbeta family signaling protein shown to be important in RPE development, contributes to the loss in competence of the RPE to regenerate retina. We have found that addition of activin blocks regeneration from the RPE, even during stages when the cells are competent. Conversely, a small molecule inhibitor of the activin/TGFbeta/nodal receptors can delay, and even reverse, the developmental restriction in FGF-stimulated neural retinal regeneration.     

Neural retinal regeneration in the anuran amphibian Xenopus laevis post-metamorphosis: transdifferentiation of retinal pigmented epithelium regenerates the neural retina

In urodele amphibians like the newt, complete retina and lens regeneration occurs throughout their lives. In contrast, anuran amphibians retain this capacity only in the larval stage and quickly lose it during metamorphosis. It is believed that they are unable to regenerate these tissues after metamorphosis. However, contrary to this generally accepted notion, here we report that both the neural retina (NR) and lens regenerate following the surgical removal of these tissues in the anuran amphibian, Xenopus laevis, even in the mature animal. The NR regenerated both from the retinal pigment epithelial (RPE) cells by transdifferentiation and from the stem cells in the ciliary marginal zone (CMZ) by differentiation. In the early stage of NR regeneration (5-10 days post operation), RPE cells appeared to delaminate from the RPE layer and adhere to the remaining retinal vascular membrane. Thereafter, they underwent transdifferentiation to regenerate the NR layer. An in vitro culture study also revealed that RPE cells differentiated into neurons and that this was accelerated by the presence of FGF-2 and IGF-1. The source of the regenerating lens appeared to be remaining lens epithelium, suggesting that this is a kind of repair process rather than regeneration. Thus, we show for the first time that anuran amphibians retain the capacity for retinal regeneration after metamorphosis, similarly to urodeles, but that the mode of regeneration differs between the two orders. Our study provides a new tool for the molecular analysis of regulatory mechanisms involved in retinal and lens regeneration by providing an alternative animal model to the newt, the only other experimental model.     

Transgenic mouse models for tumors of melanocytes and retinal pigment epithelium

Cutaneous and ocular melanomas are due to malignant transformation of neural crest-derived melanocytes. The rising incidence of this tumor in humans has stimulated experiments to devise suitable mouse models. In the past years, transgenic mouse lines have been generated using different oncogenes - Ha-ras, SV40 T antigen (Tag), ret - which develop benign lesions of melanocytes, melanoma, and/or eye tumors. Pigment cell tumors in humans, although rather rare, can also develop from the retinal pigment epithelium (RPE), a cell layer of neuroectodermal origin. We, therefore, established transgenic models for this ocular tumor. Regulated by the promoter of tyrosinase-related protein-1 (TRP-1), two oncogenes, ret and SV40 Tag, were targeted to the developing RPE in transgenic mice. The TRP-1/ret transgenic mice displayed microphthalmia and benign tumors of the RPE. Expression of SV40 T antigen (TRP-1/Tag) led to malignant tumors, which were invasive and metastasized to inguinal lymph node and spleen.     

A novel in vitro retinal differentiation model by co-culturing adult human bone marrow stem cells with retinal pigmented epithelium cells

Human retinal pigment epithelium (HRPE) cells are important in maintaining the normal physiology within the neurosensory retina and photoreceptors. Recently, transplantation of HRPE has become a possible therapeutic approach for retinal degeneration. By negative immunoselection (CD45 and glycophorin A), in this study, we have isolated and cultivated adult human bone marrow stem cells (BMSCs) with multilineage differentiation potential. After a 2- to 4-week culture under chondrogenic, osteogenic, adipogenic, and hepatogenic induction medium, these BMSCs were found to differentiate into cartilage, bone, adipocyte, and hepatocyte-like cells, respectively. We also showed that these BMSCs could differentiate into neural precursor cells (nestin-positive) and mature neurons (MAP-2 and Tuj1-positive) following treatment of neural selection and induction medium for 1 month. Furthermore, the plasticity of BMSCs was confirmed by initiating their differentiation into retinal cells and photoreceptor lineages by co-culturing with HRPE cells. The latter system provides an ex vivo expansion model of culturing photoreceptors for the treatment of retinal degeneration diseases.     

Isolation and culture of neural crest cells from embryonic murine neural tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types. NC also has the unique ability to influence the differentiation and maturation of target organs. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. NC multipotency was first described from explants of the avian neural tube. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter. The method presented here is adapted from protocols optimized for the culture of rat NC. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC.