Benzo(a)pyrene and X-rays induce reversions of the pink-eyed unstable mutation in the retinal pigment epithelium of mice

The pink-eyed unstable (p(un)) mutation is the result of a 70kb tandem duplication within the murine p gene. Homologous deletion/recombination of the locus to wild-type occurs spontaneously in embryos and results in pigmented spots in the fur and eye that persist for life. Such deletion events are also inducible by a variety of DNA damaging agents, as we have observed previously with the fur spot assay. Here, we describe the use of the retinal pigment epithelium (RPE) of the eye to detect reversion events induced with two differently acting agents. Benzo(a)pyrene (B(a)P) induces a high frequency, and X-ray exposure a more modest increase, of p(un) reversion in both the fur and the eye. The eye-spot assay requires fewer mice for significant results than the fur spot assay. Previous work had elucidated the cell proliferation pattern in the RPE and a position effect variegation phenotype in the pattern of p(un) reversions, which we have confirmed. Acute exposure to B(a)P or X-rays resulted in an increased frequency of reversion events. The majority of the spontaneous reversions lie toward the periphery of the RPE whereas induced events are found more centrally, closer to the optic nerve head. The induced distribution corresponds to the major sites of cell proliferation in the RPE at the time of exposure, and further advocates the proposal that dividing cells are at highest risk to develop deletions.     

p21 controls patterning but not homologous recombination in RPE development

p21/WAF1/CIP1/MDA6 is a key cell cycle regulator. Cell cycle regulation is an important part of development, differentiation, DNA repair and apoptosis. Following DNA damage, p53 dependent expression of p21 results in a rapid cell cycle arrest. p21 also appears to be important for the development of melanocytes, promoting their differentiation and melanogenesis. Here, we examine the effect of p21 deficiency on the development of another pigmented tissue, the retinal pigment epithelium. The murine mutation pink-eyed unstable (p(un)) spontaneously reverts to a wild-type allele by homologous recombination. In a retinal pigment epithelium cell this results in pigmentation, which can be observed in the adult eye. The clonal expansion of such cells during development has provided insight into the pattern of retinal pigment epithelium development. In contrast to previous results with Atm, p53 and Gadd45, p(un) reversion events in p21 deficient mice did not show any significant change. These results suggest that p21 does not play any role in maintaining overall genomic stability by regulating homologous recombination frequencies during development. However, the absence of p21 caused a distinct change in the positions of the reversion events within the retinal pigment epithelium. Those events that would normally arrest to produce single cell events continued to proliferate uncovering a cell cycle dysregulation phenotype. It is likely that p21 is involved in controlling the developmental pattern of the retinal pigment. We also found a C57BL/6J specific p21 dependent ocular defect in retinal folding, similar to those reported in the absence of p53.     

Ku86 deficiency leads to reduced intrachromosomal homologous recombination in vivo in mice

Ku70 and Ku86 together with DNA-PKcs form the DNA-dependent protein kinase (DNA-PK) complex that is involved in DNA double-strand break repair by nonhomologous end joining. We investigated the effect of Ku86 mutation on intrachromosomal homologous recombination (HR) resulting in deletions in vivo in mice. We quantified such deletion events using a phenotypic pigmentation assay. Deletion of one copy of a 70 kb DNA duplication in the pink-eyed unstable (pun) allele results in reversion to the wildtype pink-eyed dilution (p) gene, allowing black pigment accumulation in cells of the retinal pigment epithelium (RPE). We found that the frequency of homologous recombination was significantly reduced in Ku86 deficient mice. Furthermore, the proliferation of cells in which recombination events occurred was reduced and developmentally delayed in the Ku86 deficient mice. These data indicate a role for Ku86 directly or indirectly in homologous recombination in vivo.     

Growth and development of the mouse retinal pigment epithelium. II. Cell patterning in experimental chimaeras and mosaics

The retinal pigment epithelium (PE), with pigmentation as a cell-autonomous marker, was analyzed in three types of mice: congenic pigmented----albino chimaeras, X-inactivation mosaics (Cattanach's translocation), and mosaics homozygous for the pink-eyed unstable mutation, which contain rare fully pigmented cells. In 10 chimaeric and 34 X-inactivation eyes, the proportionate mix in the right and left eyes of an individual animal was similar, the mix was approximately constant in all parts of a given eye, average patch size was larger toward the periphery of the PE, and peripheral patches tended to be elongated in the radial dimension. In all 44 whole mounts from pink-eyed unstable mutants, patches of 1-12 pigmented cells, each representing a single clone, were scattered throughout the PE; they tended to be larger with increasing distance from the optic nerve head. The collective data are consistent with significant cell mixing prior to specification of the two eye fields, during early organ-forming stages, and during later development of the PE. The tendency of peripheral patches to orient radially reflects the edge-biased pattern of cell proliferation in the PE. Cell mixing appears to be more prominent posteriorly in the PE sheet; growth proceeds anteriorly for more generations.     

Transgenerational genomic instability as revealed by a somatic mutation assay using the medaka fish

We previously established a somatic mutation assay of the medaka wl (white leucophores) locus based on visual inspection, and showed that somatic mutations at paternally derived alleles frequently arise during the development of F1 embryos fertilized by sperm/late spermatids that had been exposed to gamma-rays. To further study such delayed mutations, we determined the frequency of mutant embryos obtained from three different crosses between irradiated males and non-irradiated females. When sperm and late spermatids were irradiated, the mutant frequency within non-irradiated maternally derived alleles was approximately 3 times higher than in the control group. In the F2 generation, however, no increase in mutant frequency was observed. Similarly, there was no significant increase in the F1 mutant frequency when stem spermatogonia were irradiated. These data suggest that irradiation of sperm and late spermatids can induce indirect mutations in F1 somatic cells, supporting the idea that genomic instability arises during F1 embryonic development. Moreover, such instability apparently arises most frequently when eggs are fertilized just after the sperm are irradiated.     

Induction of homologous recombination following in utero exposure to DNA-damaging agents

Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures.     

The accumulation of lipid peroxidation products in the eye structures of mice under whole-body x-ray irradiation

In studying the effect of whole-body X-irradiation on the accumulation of lipid peroxidation products (conjugated dienes, TBA-active products, and Schiff bases) in retina and retinal pigmented epithelium of pigmented and nonpigmented mice it was shown that irradiation of dark-pigmented mice does not cause even a slight accumulation of lipid peroxidation products as compared to that in the controls. Albino mice exhibited a marked increase in the level of lipid peroxidation products which was manifested soon after irradiation and persisted for at least 3 months after irradiation. Melanine is suggested to participate in protecting eye structures against pro-oxidizing action of ionizing radiation.     

A comparative study of the effect of pigment on drug toxicity in human choroidal melanocytes and retinal pigment epithelial cells

The aim of this study was to investigate whether the presence of pigment affects the sensitivity of pigmented cells of the eye, retinal pigment epithelium (RPE) and choroidal melanocytes (CMs) to the cytotoxic effects of xenobiotic drugs. Two approaches were used to compare pigmented versus unpigmented cells: RPE cells were repigmented by phagocytosis of synthetic melanin; UVB irradiation was used to induce an increase in pigment in both RPE and CMs. Three drugs known to induce toxicity in the eye, tamoxifen, chloroquine and thioridazine, were used to assess the sensitivity of cells to xenobiotic drugs. RPE cells were more resistant than CMs to the cytotoxic effects of all three drugs by a factor of 5-fold for tamoxifen, 7-fold for thioridazine and 30-fold for chloroquine. When RPE cells were repigmented using synthetic melanin, their sensitivity to tamoxifen was unchanged, they showed a slightly improved response to thioridazine (after 3 days of incubation with this drug), but they showed greatly increased toxicity to chloroquine (after 1 and 3 days of exposure to the drug), suggesting accumulation of this latter drug on the synthetic melanin. UVB irradiation was used to achieve an increase in the pigment content of both RPE and CMs. CMs were much more sensitive to UVB than RPE cells. CMs appeared to synthesise pigment via DOPA oxidase activity; RPE cells showed an increase in fluorescent material independent of any detectable DOPA oxidase activity. Irrespective of the nature of the pigment that UVB induced in melanocytes and RPE cells, their subsequent response to thioridazine and chloroquine was unchanged by the presence of this pigment.     

Congenic strains of RCS rats with inherited retinal dystrophy.

Two congenic strains of RCS rats, RCS-p/+ and RCS-c, have been developed that differ from the parental strain at genetic loci affecting pigmentation. Inbred RCS rats are pink-eyed, while RCS-p/+ rats produce segregating litters of pink-eyed (p/p) and black-eyed (p/+) offspring, and RCS-c rats are albinos. All the strains are homozygous for the mutant form of the retinal dystrophy gene. The black eye pigment in RCS-p/+ rats slows the progression of the retinal degeneration by about 10 days in the posterior retina and by about 30-35 days in the peripheral retina in the superior half of the eye. No slowing of the disease occurs in the inferior half of the eye along the vertical meridian. All the strains are similar in body weight and litter size, and show a low incidence of cataract and microphthalmia.     

Effects of gamma-radiation on the differentiation of mouse melanocytes in the hair follicles

Pregnant mice were whole-body irradiated with a single acute dose of gamma-rays (60Co) to investigate the effect of gamma-radiation on embryonic melanoblasts. The effect was studied by scoring changes in the differentiation of melanocytes in the hair follicles of mice heterozygous for the recessive coat color mutation pink-eyed dilution (p). Abnormal round melanocytes were found in the hair matrix and the dermal papilla of F1 offspring 3.5 days after birth. However, these round melanocytes possessed a melanin deposition of similar intensity to normal hair follicular melanocytes. The frequency of the abnormal hair follicles increased in a dose-dependent manner. Moreover, higher frequencies were found in the animals irradiated at earlier stages of embryonic development. These results indicate that gamma-radiation affects dendritogenesis and the location of mouse melanocytes in the hair follicles, with greater effects seen at the earlier stages of development.     

Extrinsic modulation of retinal ganglion cell projections: analysis of the albino mutation in pigmentation mosaic mice

Tyrosinase is a key enzyme involved in the synthesis of melanin in the retinal pigment epithelium (RPE). Mice that are homozygous for the albino allele at the tyrosinase locus have fewer retinal ganglion cells with uncrossed projections at the optic chiasm. To determine the site of the albino gene action we studied the projections of retinal ganglion cells in two types of pigmentation mosaic mice. First, we generated mosaic mice that contain a translocated allele of the wild-type tyrosinase on one X chromosome but that also have the lacZ reporter transgene on the opposite X chromosome. In these lacZ/tyrosinase mice, which are homozygous for the albino allele on chromosome 7, X-inactivation ensures that tyrosinase cannot be functional within 50% of the retinal ganglion cells and that these individual cells can be identified by their expression of the lacZ reporter gene product, beta-galactosidase. The proportion of uncrossed retinal ganglion cells expressing beta-galactosidase was found to be identical to the proportion that did not express it, indicating that the albino mutation associated with axonal behavior at the optic chiasm must affect ganglion cells in a cell-extrinsic manner. Second, to determine whether the RPE is the source of the extrinsic signal, we generated aggregation chimeras between pigmented and albino mice. In these mosaic mice, the extent of the uncrossed projection corresponded with the amount of pigmented cells within the RPE, but did not correspond with the genotypes of neural retinal cells. These studies demonstrate that the albino mutation acts indirectly upon retinal ganglion cells, which in turn respond by making axonal guidance errors at the optic chiasm.     

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.     

Mosaic: a position-effect variegation eye-color mutant in the mosquito Anopheles gambiae

The Mosaic (Mos) mutation, isolated in the F1 of 60Co-irradiated mosquitoes, confers variegated eye color to third and fourth instar larvae, pupae, and adults of the mosquito Anopheles gambiae. Mos is recessive in wild pink eye (p+) individuals, but is dominant and confers areas of wild-type pigment in mutant pink eye backgrounds. Mos is located 14.4 cM from pink eye on the X chromosome and is associated with a duplication of division 2B euchromatin that has been inserted into division 6 heterochromatin. Various combinations of Mos, pink eye alleles, and the autosomal mutation red eye were produced. In all cases, the darker pigmented regions of the eye in Mos individuals show the phenotypic interactions expected if the phenotype of those regions is due to expression of a p+ allele. Expression of Mos is suppressed by rearing larvae at 32 degrees C relative to 22 degrees C. All of these characteristics are consistent with Mos being a duplicated wild copy of the pink eye gene undergoing position-effect variegation.     

Clonal analysis of the development of the pigment epithelium of the eye in chimeric or/or----AKR mice

The development of cell clones was studied in the retinal pigment epithelium of chimaeric mice or/or----AKR. The clonal analysis suggests that on the 13th day of gestation the cells in the retinal pigment epithelium are distributed almost randomly while in the adults they are grouped in small coherent clones. In the retinal pigment epithelium the AKR cell predominated over the or/or cells. The interaction of mutant and normal clones during the eye development leads, in most cases, to the normalization of eyes in chimaeras. Cases of microphthalmia and asymmetry in distribution of clones suggest irregular and random distribution of these clones in the embryo.     

A novel function for Hedgehog signalling in retinal pigment epithelium differentiation

Sonic hedgehog is involved in eye field separation along the proximodistal axis. We show that Hh signalling continues to be important in defining aspects of the proximodistal axis as the optic vesicle and optic cup mature. We show that two other Hedgehog proteins, Banded hedgehog and Cephalic hedgehog, related to the mouse Indian hedgehog and Desert hedgehog, respectively, are strongly expressed in the central retinal pigment epithelium but excluded from the peripheral pigment epithelium surrounding the ciliary marginal zone. By contrast, downstream components of the Hedgehog signalling pathway, Gli2, Gli3 and X-Smoothened, are expressed in this narrow peripheral epithelium. We show that this zone contains cells that are in the proliferative state. This equivalent region in the adult mammalian eye, the pigmented ciliary epithelium, has been identified as a zone in which retinal stem cells reside. These data, combined with double labelling and the use of other retinal pigment epithelium markers, show that the retinal pigment epithelium of tadpole embryos has a molecularly distinct peripheral to central axis. In addition, Gli2, Gli3 and X-Smoothened are also expressed in the neural retina, in the most peripheral region of the ciliary marginal zone, where retinal stem cells are found in Xenopus, suggesting that they are good markers for retinal stem cells. To test the role of the Hedgehog pathway at different stages of retinogenesis, we activated the pathway by injecting a dominant-negative form of PKA or blocking it by treating embryos with cyclopamine. Embryos injected or treated at early stages display clear proximodistal defects in the retina. Interestingly, the main phenotype of embryos treated with cyclopamine at late stages is a severe defect in RPE differentiation. This study thus provides new insights into the role of Hedgehog signalling in the formation of the proximodistal axis of the eye and the differentiation of retinal pigment epithelium.     

Developmental interactions in the pigmentary system of the tip of the mouse tail: effects of coat-color genes on the expression of a tail-spotting gene

The tails of agouti C3H/HeJmsHir mice are completely pigmented, whereas the tails of black C57BL/10JHir animals possess unpigmented tips. Genetic analysis indicates that white tail-tipping is due to an autosomal recessive gene, with incomplete penetrance, that segregates independently from the gene for agouti with a maternal influence in the F1 generation. To analyze the influence of specific coat-color genes on the expression of tail-spotting in mice, five congenic lines of C57BL/10JHir with different coat colors were prepared. No influence was observed on the occurrence of tail-spotting in agouti (A/A) or dilute (d/d) mice or in F1 mice from crosses between black and albino (c/c), or in F1 mice from crosses between black and pink-eyed dilution (p/p). However, the frequency of tail-spotting was dramatically decreased in brown (b/b) mice. These results suggest that the mutant allele (b) at the brown locus is involved in determining the extent of pigmented areas in the tail tips of mice through an interaction with the tail-spotting gene.     

Modification of pigmentation patterns in allophenic mice by the W gene

Mouse embryos heterozygous at the W locus were combined with embryos which were wild type at this locus but homozygous for albino. The resulting allophenics displayed an unusual pigmentation phenotype consisting of entirely white fur and ruby-coloured eyes. Microscopic examination showed the eye pigment to be located exclusively in the retinal epithelium, which was a mosaic of black and white sectors. This ruby-eyed white pattern corresponds to what would have been expected for WWCC in equilibrium wwcc mosaics but not for WwCC in equlibrium wwcc mice. WW mice are black-eyed whites, but Ww mice have black eyes and black fur, except for a small ventral white spot. These results suggest that melanocytes of the Ww genotype, although capable of producting normally pigmented fur in Ww animals, fail to populate hair follicles when the competition with wwcc (albino) melanocytes that are wild type at the W locus. The genotype of these WwCC in equilibrium wwcc alophenes was proved by progeny testing. This is apparently the first report of a single gene change affecting the competitive ability of cells in allophenic mice, and suggests that such changes may play a significant role in the clonal selection of embryonic cells during development.     

Interaction of the Murine Dilute Suppressor Gene (Dsu) with Fourteen Coat Color Mutations

The murine dilute suppressor gene, dsu, was previously shown to suppress the dilute coat color phenotypes of mice homozygous for the dilute (d), leaden (ln), and ashen (ash) mutations. Each of these mutations produce adendritic melanocytes, which results in an abnormal transportation of pigment granules into the hair shaft and a diluted coat color. The suppression of each mutation is associated with the restoration of near normal melanocyte morphology, indicating that dsu can compensate for the absence of normal d, ln and ash gene products. In experiments described here, we have determined whether dsu can suppress the coat color phenotype of 14 additional mutations, at 11 loci, that affect coat color by mechanisms other than alterations in melanocyte morphology. In no case was dsu able to suppress the coat color phenotype of these 14 mutations. This suggests that dsu acts specifically on coat color mutations that result from an abnormal melanocyte morphology. Unexpectedly, dsu suppressed the ruby eye color of ruby-eye (ru) and ruby-eye-2 (ru-2) mice, to black. The exact nature of the defect producing these two mutant phenotypes is unknown. Histological examination of the pigmented tissues of the eyes of these mice indicated that dsu suppresses the eye color by increasing the overall level of pigmentation in the choroid but not the retinal pigmented epithelium. Choroid melanocytes, like those in the skin, are derived from the neural crest while melanocytes in the retinal pigmented epithelium are derived from the optic cup. This suggests that dsu may act specifically on neural crest-derived melanocytes. These studies have thus identified a second group of genes whose phenotypes are suppressed by dsu and have provided new insights into the mechanism of action of dsu.     

An analysis of the distribution of clone cells in the retinal pigment epithelium from chimeric mice

The quantity of pigmented and unpigmented cells was estimated in the retinal pigment epithelium (RPE) in ten newborn and five 20-days old aggregated chimaeric mice C/C----c/c. A strong correlation was shown in the proportion of cells of the paternal genotypes either in the whole RPE of right and left eyes or in its separate regions, i.e. dorsal, central, ventral. Random distribution was revealed in these RPE cells clones. A high correlation was shown between the number of RPE pigmented cells and percentage of coat pigmentation.     

Culture of rat retinal pigment epithelium.

A method of preparing monolayer cultures of retinal pigment epithelium from normal pigmented neonatal rats is described. Critical features include incubating the eyes in balanced salt solution and treating with trypsin before dissecting the eyes. The tissue also has been culured from RCS rats with inherited retinal degeneration. Since the pigment epithelium has been shown to be the primary site of action of the gene for retinal dystrophy in the RCS rat, the method should be usefull in studying the defect(s) associated with this mutation.