The initiation of melanogenesis in the chick retinal pigment epithelium.

Observations have been made on chick pigment retinal epithelium between 2 and 5 days of development. 2-Thiouracil has been demonstrated to be an effective agent for measuring the rate of melanin synthesis.Using [³H]thymidine and colcimid, we have found that the cells undergo a marked withdrawal from the cell cycle between 3 and 3.5 days of incubation in ovo, indicating that a majority of the population is synchronized. This withdrawal is followed, approximately 24 hr later, by a rapid rise in melanin synthesis from the basal level which first appears at approximately 3 days.5-Bromodeoxyuridine (BUdR) has been used to determine the time at which melanin synthesis is initiated. When BUdR is administered as early as 2 days in ovo, it is incapable of blocking the appearance of basal levels of melanin even though the cells divide at least three times in the presence of this thymidine analog. However, BUdR is capable of delaying the rapid rise in the rate of melanin synthesis first observed at 4.5 days. This delay has been found to correlate, using [³H]BUdR, with a delay in the withdrawal of the cells from the division cycle.In pursuing the idea of a correlation between withdrawal and the rapid increase of melanin formation, 5-fluorodeoxyuridine (FUdR) was used. Histological and biochemical evidence suggests that those cells which have been prevented from dividing by FUdR increase their rate of melanin synthesis to the high level of the postmitotic control cells described above.Therefore, it seems that (1), in light of work done by others, the initial decision to make melanin is made prior to 2 days in ovo, and (2) the mechanism by which cells shift their synthetic capabilities to high levels of melanin production is withdrawal mediated.     

The relationship between cell division and melanocyte differentiation in epidermal cultures from mouse embryos

Cultures of 14-day embryonic mouse epidermis that include melanoblasts initiate melanin synthesis 30 hr after plating, a schedule that is 2.5 days earlier than in vivo. In order to determine if the accelerated differentiation of melanoblasts is related to a cessation of cell proliferation in the cultures, a study of [³H]thymidine incorporation by melanoblasts and melanocytes was made. Autoradiograms of 14-day epidermal cultures grown for 48 hr in medium containing [³H]thymidine revealed that melanoblasts continue to proliferate during this time period. A second population of melanoblasts that did not incorporate [³H]thymidine was also present in these cultures. The relative numbers of dividing and nondividing melanoblasts change with the age of the epidermis cultured. Ninety-one percent of the melanoblasts in 13-day epidermis take up [³H]thymidine, 63% incorporate [³H]thymidine in 14-day cultures, and only 29% take up label in cultures of 15-day epidermis. It appears from these results that melanoblasts during their migration from the neural crest are proliferative cells and that during the early invasion of the epidermis a nonproliferative population of melanoblasts is established. Both populations coexist in the epidermis and subsequently undergo differentiation on the same time schedule.     

INHIBITION OF MYOBLAST FUSION AFTER ONE ROUND OF DNA SYNTHESIS IN 5-BROMODEOXYURIDINE

The thymidine analogue 5-bromodeoxyuridine (BUdR) has a differential effect on the synthesis of tissue-specific products and molecules required for growth and division. Proliferating myogenic cells cultured in BUdR fail to fuse and fail to initiate the synthesis of contractile protein filaments. Conversely, BUdR has but a minor effect on cell viability and reproductive integrity. Low concentrations of BUdR result in an enhancement of cell number relative to the controls; higher concentrations are cytotoxic. Suppression of myogenesis is reversible after at least 10 cell generations of growth in the analogue. Cells that do not synthesize DNA, such as postmitotic myoblasts and myotubes, are not affected by BUdR. Incorporation of BUdR for one round of DNA synthesis was accomplished by first incubating myogenic cells, prior to fusion, in 5-fluorodeoxyuridine (FUdR) to block DNA synthesis and collect cells in the presynthetic phase. The cells were then allowed to synthesize either normal DNA or BU-DNA for one S period by circumventing the FUdR block with BUdR or BUdR plus thymidine (TdR). The cultures were continued in FUdR to prevent dilution of the incorporated analogue by further division. After 3 days, the cultures from the FUdR-BUdR series showed the typical BUdR effect; the cells were excessively flattened and few multinucleated myotubes formed. Cells in the control cultures were of normal morphology, and multinucleated myotubes were present. These results were confirmed in another experiment in which BUdR-³H was added to 2-day cultures in which myotubes were forming. Fusion of thymidine-³H-labeled cells begins at 8 hr after the preceding S phase. In contrast, cells which incorporate BUdR-³H for one S period do not fuse with normal myotubes.     

Transformation of avian myogenic cultures with myelocytomatosis virus strain 29

Summary Primary cultures of proliferating chick presumptive myoblasts were exposed of either to two RNA tumor viruses and shortly thereafter treated with 5-bromodeoxyuridine (BUdR) to suppress differentiation. The effect of a Rous sarcoma virus which was temperature-sensitive for transformation (tsRSV) has been characterized previously and was used as a reference for evaluating the effect of a myelocytomatosis virus (MC29) and its helper. Two subcultures following exposure, both infected cultures were extensively transformed as indicated by cell morphology. Relaxation of the BUdR block at this time resulted in cultures which still appeared transformed and did not contain myoblast or myotube-like cells or two of their molecular markers. In contrast, uninfected controls and tsRSV-infected cultures which were shifted-up to the nonpermissive temperature produced numerous spontaneously contracting myotubes. The results confirm previous evidence that infection of presumptive myoblasts by tsRSV at the premissive temperature preserves the extant state of differentiation of presumptive myoblasts and suggest, by analogy, that MC29-infection renders a similar effect.     

FLOW CYTOMETRIC CELL CYCLE ANALYSIS USING THE QUENCHING OF 33258 HOECHST FLUORESCENCE BY BROMODEOXYURIDINE INCORPORATION

Cultures of L cells were grown in medium containing 2.0 mg/l bromodeoxyuridine (BUdR) and stained with the fluorescent dye 33258 Hoechst for flow cytometric analysis. During exposure to BUdR, the cells replace thymidine by BUdR in the newly synthesized DNA. The new DNA is not stainable with 33258 Hoechst, which is highly specific for thymidine. The temporal development of the fluorescence distributions after addition of BUdR to the growth medium has been investigated in the flow cytometer, and the data were used to calculate the mean durations of the phases G₁, S and G₂+ M in exponentially growing cultures as well as the cycle transit times in synchronized cultures. The percentage of non-cycling cells was determined in each experiment.     

Effect of L-phenylalanine on I-methyladenine production and spontaneous oocyte maturation in starfish

5-Bromodeoxyuridine (BUdR)-resistant cells were obtained from N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-treated soybean protoplasts and cultured in liquid nutrient medium containing BUdR (20 μg/ml) and uridine (100 μg/ml). Addition of uridine to the medium improved growth of the BUdR-resistant cells. The growth of BUdR-resistant cells was partly inhibited when hypoxanthine, aminopterine, glycine and thymidine were added to the medium. Both BUdR-resistant and BUdR-sensitive cells exhibited thymidine kinase activity. CsCl density gradient analyses showed that the DNA of BUdR-resistant cells, which were cultured in the presence of BUdR, had a buoyant density of 1.703 g/ml, while the DNA of the parental soybean cells grown without BUdR had a buoyant density of 1.692 g/ml. Uptake of ³H-thymidine or ¹⁴C-BUdR by the cells occurred in both BUdR-resistant and BUdR-sensitive cells. CsCl density gradient patterns of labelled DNA also demonstrated that ¹⁴C-BUdR and ³H-thymidine were incorporated into the DNA of BUdR-resistant cells, as well as into that of BUdR-sensitive cells.     

Bromodeoxyuridine and light treatment enhances responsiveness of pokeweed mitogen-stimulated human lymphocytes to autologous and allogeneic determinants

We have investigated a novel system whereby lymphocytes from normal human subjects can be induced to develop exaggerated reactivity to histocompatibility antigens in vitro. Peripheral blood mononuclear cells (PBMC) stimulated with pokeweed mitogen (PWM) showed increased and accelerated subsequent proliferation to both autologous and allogeneic stimulators. Addition of bromodeoxyuridine (BUdR) during the period of maximal PWM-induced DNA synthesis followed by light exposure caused unexpected, but marked enhancement of this secondary proliferation. While untreated cultures contained a preponderance of T8⁺ cells after PWM activation, BUdR plus light-treated cultures were largely T4⁺ cells. Because removal of suppressor cells in nonsuicided cultures with anti-T8 and complement just before restimulation failed to unmask enhanced autoreactivity, events critical in the induction of the enhanced response must have occurred during priming. Cultures of PBMC with medium alone or concanavalin A, as well as purified T cells cultured with PWM, gave no enhanced autoproliferation after BUdR and light; thus T and non-T cells must be acted on by a T- and B-cell mitogenic stimulus to prime T cells for enhanced responsiveness. The interactions between T cells and activated B cells in this in vitro system may be relevant to regulatory mechanisms important in the induction of pathological autoimmune responses.     

An analysis of parthogenetic cell clones in chimeric C57BL/6(PG)<-->BALB/c mice

We studied the distribution of parthenogenetic cell clones in the retinal pigment epithelium and choroid of eyes on serial sections and in the brain, kidneys, and liver by electrophoretic analysis of glucose phosphate isomerase isozymes in 12 mouse chimeras C57BL/6(PG)<-->BALB/c obtained earlier. Asymmetry was noted in the distribution of the parthenogenetic cell clones in the eye structure, just as the earlier established asymmetry in the distribution of the parthenogenetic clones of epidermal melanoblasts. A high correlation was shown between the ratio of parthenogenetic to normal cells in the retinal pigment epithelium of the right or left eyes and epidermal melanoblasts in the hair cover of the corresponding body half of the chimera. These data suggest that there is a certain relationship between the processes leading to the characteristic distribution of the ectodermal parthenogenetic clones in the retinal pigment epithelium of the right and left eyes and epidermal melanoblasts in parthenogenetic chimeras. Electrophoretic analysis did not show parthenogenetic components in the liver or kidneys of any chimera, and the parthenogenetic component was found in the brain of only two chimeras, in which a high percentage of parthenogenetic cells of ectodermal origin was noted. In these cases, asymmetry was noted in the right and left cerebral hemispheres, just as in the retinal pigment epithelium of the right and left eyes. The data obtained suggest that, during the development of the chimeras, parthenogenetic C57BL/6 cells were actively eliminated from the tissues of endodermal and mesodermal origin. In adult chimeras C57BL/6(PG)<-->BALB/c, parthenogenetic cell clones of ectodermal origin are mostly preserved.     

Effects of 5-bromodeoxyuridine on metamorphosis and on cell cycle kinetics of ganglion cells in Drosophila melanogaster

In order to determine suitable experimental conditions for estimating the accurate spontaneous frequency of sister chromatid exchanges (SCEs) in vivo in somatic cells of Drosophila melanogaster, the effects of bromodeoxyuridine (BUdR) on metamorphosis as well as on cell cycle kinetics were examined. The rate of growth of third-instar larvae, fed on BUdR-containing synthetic medium, markedly delayed with increasing concentrations of BUdR, but this toxic effect of BUdR was not observed below 150 μg/ml.Furthermore, the rate of eclosion drastically decreased by the incorporation of BUdR: it was reduced to about one-half of that in the control when the larvae were exposed to 100 (μg/ml. On the other hand, little difference in the rate of pupation was found within the range of 0–800 μg/ml BUdR. These results indicate that the developmental stage from pupa to adult is the most sensitive phase to BUdR.To test the effect of BUdR on cell cycle, metaphase cells were classified as having undergone each replication cycle in the presence of different BUdR concentrations according to the pattern of differential staining of sister chromatids, and the proportion of each replication cycle cells examined. No inhibition of cellular kinetics was observed at BUdR concentrations below 200 μg/ml.On the basis of these results, 100 μg/ml was chosen as suitable BUdR concentration for the analysis of cell cycle kinetics and according to the distribution of replication cycle metaphase cells as a function of time after the initiation of BUdR treatment, the cell cycle duration of the third-instar larval ganglion cells was roughly estimated to be about 7–8 h, at least under our experimental conditions.     

Differences between diploid and transformed human cells in the incorporation of 5-bromodeoxyuridine into DNA

In diploid human cells, the DNA precursor pool equilibration times for exogenous thymidine are about twice those for the thymidine analogue 5-bromodeoxyuridine (BUdR); in cells that were either transformed chemically or derived from malignant tumours, the pool equilibration times are the same for thymidine and 5-bromodeoxyuridine and are closer in value to the shorter (bromodeoxyuridine) times of the diploid cells. Thymidine, if present in the culture medium with BUdR, is incorporated into DNA preferentially in diploid cells (by 2 or 3 to 1). Discrimination against bromodeoxyuridine is evident within 2 h of incubation of the two precursors with diploid cells, but is not observed even after 24 h in any of the transformed cell lines tested. Experiments were performed to test the effect of inhibitors of the mammalian DNA polymerases alpha (N-ethylmaleimide) and beta (incubation of cells at 45 °C) upon the ability of cells to synthesise DNA and to incorporate thymidine preferentially when present with equimolar BUdR. In diploid cells, overall in vivo DNA synthesis is more sensitive to N-ethylmaleimide and more resistant to 45 °C treatment than is DNA synthesis in the transformed cell lines. N-Ethylmaleimide decreases the capacity of diploid cells to discriminate against BUdR, whereas heating increases it. Transformed cells treated with N-ethylmaleimide remain unable to discriminate against BUdR; some transformed lines, when heated at 45 °C, become less incapable of such discrimination.     

Biofilm-Specific Cross-Species Induction of Antimicrobial Compounds in Bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10^T and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10^T and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.     

Transformation of chicken embryo retinal melanoblasts by a temperature-sensitive mutant of Rous sarcoma virus.

Retinal melanoblasts were transformed by a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV). At the permissive temperature for transformation, the cells cease melanin synthesis, degrade their melanosomes and release much of their accumulated melanin into the medium. At the nonpermissive temperature, the cells assume an epithelioid morphology, actively synthesize melanin and become difficult to distinguish from normal uninfected control cultures. Both the transformed phenotype and the differentiated cell phenotype are temperature-dependent. Infected retinal melanoblasts which are incubated at the nonpermissive temperature and which accumulate a large amount of melanin are unable to transform in response to a temperature shift; instead, the cells degenerate and die. Retinal melanoblasts can be infected by subgroups A, B, C and D of RSV; however, their level of susceptibility to infection is about 1/40 compared to fibroblasts. Cultures infected by ts-RSV produce virus at both temperatures, suggesting that cell phenotype does not regulate virus synthesis.     

Mutations in C10orf11, a Melanocyte-Differentiation Gene,Cause Autosomal-Recessive Albinism

Autosomal-recessive albinism is a hypopigmentation disorder with a broad phenotypic range. A substantial fraction of individuals with albinism remain genetically unresolved, and it has been hypothesized that more genes are to be identified. By using homozygosity mapping of an inbred Faroese family, we identified a 3.5 Mb homozygous region (10q22.2–q22.3) on chromosome 10. The region contains five protein-coding genes, and sequencing of one of these, C10orf11, revealed a nonsense mutation that segregated with the disease and showed a recessive inheritance pattern. Investigation of additional albinism-affected individuals from the Faroe Islands revealed that five out of eight unrelated affected persons had the nonsense mutation in C10orf11. Screening of a cohort of autosomal-recessive-albinism-affected individuals residing in Denmark showed a homozygous 1 bp duplication in C10orf11 in an individual originating from Lithuania. Immunohistochemistry showed localization of C10orf11 in melanoblasts and melanocytes in human fetal tissue, but no localization was seen in retinal pigment epithelial cells. Knockdown of the zebrafish (Danio rerio) homolog with the use of morpholinos resulted in substantially decreased pigmentation and a reduction of the apparent number of pigmented melanocytes. The morphant phenotype was rescued by wild-type C10orf11, but not by mutant C10orf11. In conclusion, we have identified a melanocyte-differentiation gene, C10orf11, which when mutated causes autosomal-recessive albinism in humans.     

Model system studies of the use of 5-bromo-2′-deoxyuridine for selection of deficient mutants in plant cell suspension and protoplast cultures

Experiments using separate growing and arrested cell suspensions showed that treatment with the appropriate level of BUdR selectively killed the growing cultures. This effect was not light-dependent. A model system comprising leaf protoplasts of 2 different mutants of Nicotiana tabacum, only one of which was sensitive to valine, was developed to test the ability of this method to select non-dividing from dividing cells in mixed cultures. Following treatment of cultures with BUdR in the presence of valine, colonies recovered were tested on media which clearly differentiated the cell types on the basis of valine resistance and the ability to green in the presence of streptomycin (the marker for the valine-sensitive line). BUdR treatment of valine-inhibited cultures increased the percentage of valine-sensitive colonies recovered from mixtures containing up to 99.9% valine-resistant cells, although recovery of sensitive colonies was poor where these cells only represented a small proportion of the culture.     

Effect of Light on Glucose Utilization by Euglena gracilis

The effect of light on glucose consumption by wild-type Euglena gracilis Z. and mutant cells has been studied. When dark- or light-grown wild-type cells are transferred from a medium containing sodium butyrate as the only carbon source to a glucose-containing medium, glucose consumption is blocked for 6 to 7 days when cultures are incubated under a light intensity of at least 600 lux. During this time cells multiply at the same rate as controls kept on media devoid of any utilizable organic carbon source. This light-induced inhibition of glucose consumption and of growth on glucose-containing medium is not related to photosynthesis since: (a) glucose consumption is inhibited by light intensities much lower than those required for high phototrophic growth; (b) the inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea does not overcome the inhibition of glucose consumption; and (c) nonphototrophic-growing mutants also show light-induced inhibition of glucose consumption and of growth on glucose-containing medium. This inhibition of growth by light might be explained by modification in the permeability of the cellular membrane.     

The Patchwork Mouse Phenotype: Implication for Melanocyte Replacement in the Hair Follicle

Mice homozygous for the recessive patchwork (pwk) mutation are characterized by a variegated pigment pattern with a mixture of unpigmented and normally pigmented hairs. The pigmented hair bulbs contain functional melanocytes. By contrast, the unpigmented hair bulbs contain no melanocytes. This lack results from the death of melanoblasts in the hair follicle at the end of embryogenesis. Here, we report that melanoblasts and melanocytes are found in the epidermis of pwk/pwk mice. Furthermore, these epidermal pigment cells are able to colonize new hair follicles after skin wounding. Despite the presence of epidermal pigment cells with a colonization potential, a follicle that had produced an unpigmented hair produces a new unpigmented hair during the successive hair growth cycles. This hair color continuity is also true for the pigmented hair follicles. Thus, in normal conditions, the hair acts as an independent functional unit as regards its pigment cells population.     

The Visual Cycle in the Inner Retina of Chicken and the Involvement of Retinal G-Protein-Coupled Receptor (RGR)

The vertebrate retina contains typical photoreceptor (PR) cones and rods responsible for day/night vision, respectively, and intrinsically photosensitive retinal ganglion cells (ipRGCs) involved in the regulation of non-image-forming tasks. Rhodopsin/cone opsin photopigments in visual PRs or melanopsin (Opn4) in ipRGCs utilizes retinaldehyde as a chromophore. The retinoid regeneration process denominated as “visual cycle” involves the retinal pigment epithelium (RPE) or Müller glial cells. Opn4, on the contrary, has been characterized as a bi/tristable photopigment, in which a photon of one wavelength isomerizes 11-cis to all-trans retinal (Ral), with a second photon re-isomerizing it back. However, it is unknown how the chromophore is further metabolized in the inner retina. Nor is it yet clear whether an alternative secondary cycle occurs involving players such as the retinal G-protein-coupled receptor (RGR), a putative photoisomerase of unidentified inner retinal activity. Here, we investigated the role of RGR in retinoid photoisomerization in Opn4x (Xenopus ortholog) (+) RGC primary cultures free of RPE and other cells from chicken embryonic retinas. Opn4x (+) RGCs display significant photic responses by calcium fluorescent imaging and photoisomerize exogenous all-trans to 11-cis Ral and other retinoids. RGR was found to be expressed in developing retina and in primary cultures; when its expression was knocked down, the levels of 11-cis, all-trans Ral, and all-trans retinol in cultures exposed to light were significantly higher and those in all-trans retinyl esters lower than in dark controls. The results support a novel role for RGR in ipRGCs to modulate retinaldehyde levels in light, keeping the balance of inner retinal retinoid pools.     

AGE-DEPENDENT CHANGE IN THE TRANSDIFFERENTIATION ABILITY OF CHICK NEURAL RETINA IN CELL CULTURE*

We examined how the transdifferentiation ability of neural retinal cells into lens and/or pigment cells in call culture is changed with the development of the donor. Cells dissociated from neural retinas of chick embryos ranging from 3-day-old to the stage immediately before hatching and of 3-day-old chicks were cultured for about 60 days. The results clearly indicated that the transdifferentiation ability decreased with age. The latest developmental stage at which the differentiation of lens cells took place was in 18-day-old embryos. A gradual decrease in this ability was shown by the comparison of crystallin content in cultures prepared from embryos at different stages. The differentiation of pigment cells was recognized in cultures of neural retinas earlier than in 15-day-old embryos. Such loss of the ability of neural retinal cells to transdifferentiate into pigment cells earlier than into lens cells can be partially attributed to inhibitory factors accumulated in medium conditioned with many neuronal cells present in cultures.