Chicken lens delta-crystallin gene expression and methylation in several non-lens tissues.

RNA sequences coding for the most abundant chicken lens proteins, delta-crystallin, were detected at very low levels in day old post hatch chick lung, heart, kidney and liver, and in 6 day embryo headless bodies. The pattern of cytosine methylation within the CCGG sequences of the delta-crystallin genes was also examined and shown to vary in several non-lens tissues, from several stages of development. Embryonic neural retina, which expresses a higher level of delta-crystallin RNA than the above tissues, is no less methylated in the sites studied than the tissues which have no association with the eye, and is actually more heavily methylated than the kidney. Thus no obvious correlation was found between undermethylation and gene expression.     

An attempt to assay the state of determination by using transfected genes as probes in transdifferentiation of neural retina into lens

Hybrid genes coding for chloramphenicol acetyltransferase (CAT) with a non-specific retroviral, lens-specific delta-crystallin or lens-specific alpha-crystallin promoters were constructed to transfect the transdifferentiating (lentoidogenic) and non-transdifferentiating (non-lentoidogenic) cultures of chicken embryonic neural retina for assaying the state of determination towards lens differentiation. The expression occurred only when CAT genes with lens-specific promoters were transfected to the cultures maintained in the conditions permissive to lentoidogenesis. The expression of these exogenous, lens-specific CAT genes began at stages of culturing that were earlier than the expression of endogenous crystallin. Presumably, there are two steps in the transdifferentiation of neural retina into lens; acquisition of capacity to express crystallin genes and derepression of the endogenous crystallin genes.     

Relative levels of methylation in human growth hormone and chorionic somatomammotropin genes in expressing and non-expressing tissues.

It has been shown that the extent of methylation of cytosine in vertebrate DNA is inversely correlated with gene expression. We studied cytosine methylation in and around the homologous human growth hormone (GH) and chorionic somatomammotropin (CS) genes to determine if these genes are undermethylated in DNA from tissues in which they are expressed (pituitary and placenta, respectively) compared to other tissues. Hpa II and Hha I (which cleave only unmethylated 5' CCGG 3' and 5' GCGC 3' respectively) and Msp I (which cleaves CCGG and CmeCGG) were used to digest DNA samples followed by gel electrophoresis, Southern transfer and hybridization with a GH cDNA probe. The extent of methylation of Hpa II and Hha I sites in the GH and CS genes was leukocyte much greater than pituitary greater than placenta = hydatidiform mole. Taken as a whole, our data support the hypothesis that undermethylation is a necessary but not sufficient condition for gene expression since placental and pituitary DNAs are less methylated than leukocyte DNA in this region. However, the correlation between gene expression and undermethylation is imperfect since (1) hydatiform mole DNA has a very similar methylation pattern compared to placental DNA even though moles make little or no CS and (2) the level of methylation of the GH gene compared to the CS gene does not vary in a tissue-specific manner.     

Variable methylation of the ribosomal RNA genes of the rat.

Both the pattern and level of rRNA gene methylation vary in the rat. This variation reflects stages in the maturation process and perhaps the level of gene expression in different tissues. We studied methylation at a common site, the inner cytosine of the sequence CCGG, by hybridizing 32P-rRNA to DNA digests obtained with endonuclease Msp I (which cleaves CCGG and CMCGG) and its isochizomer, HpaII (which cleaves only CCGG). In the liver, the changing pattern of rRNA gene methylation reflected the late stages of development: the rRNA genes were mostly unmethylated at 14 days gestation; by 18 days gestation, about 30% of them were methylated, and this level persisted into adulthood. In 18-day DNA, the methylation was uniform, but in adult DNA, the methylation pattern was discontinuous, because otherwise methylated genes contained a demethylated region. Similar developmental changes were observed in brain DNA. In a tissue culture cell line, the change from the continuous to the discontinuous pattern of the methylation could be induced by transformation with Kirsten sarcoma virus. And, in adult tissues, the lowest level of rRNA gene methylation was found in rapidly growing jejunal epithelium, and the highest level, in non-growing spermatozoa.     

DNA methylation at a CCGG sequence in the large intron of the rabbit β-globin gene: tissue-specific variations

We have analysed DNA modification in a HapII site (CCGG) present in the major intron of the discontinuous rabbit β-globin gene. In most somatic tissues, including erythroid and non-erythroid tissues, about 50% of the DNA is resistant to cleavage at this site by HapII, though 100% cleavage is found with the isoschizomer MspI. Since the former enzyme is unable to cleave CCGG sites if the internal C residue is 5-methyl C (and since methylation is the only form of CpG modification documented in animal DNA), while the latter enzyme cleaves DNA irrespective of methylation at this residue, we infer that 50% of the CCGG sites in the β-globin gene intron are methylated in these tissues.     

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina

Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)⁺ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.     

Expression of Toxin Receptors on Cell Surfaces in Transdifferentiating Cultures of Neural Retina

It has often been asked which of the cell types found during the early stages of culturing embryonic chick neural retina can undergo transdifferentiation into lens in vitro. Since neuronal cell-surface toxin receptors are maintained in NR cultures for much longer than internal neuronal enzymes (e.g. choline acetyltransferase), and since the transdifferentiation process can be greatly accelerated by preparing reaggregates of neural retina cells after about 10 days of preculture as "monolayers", a direct test of this question became feasible. 7 or 9 day embryonic chick neural retina cells, precultured for 10–12 days as monolayers, were dissociated and reaggregated under continuous gyration. Reaggregates were maintained for 8 days in the presence of either tetanus toxin or FITC-conjugated α-bungarotoxin, to permit surface-bound toxins to become internalised via receptor turnover. The reaggregates were then dissociated, stained with rabbit antitoxin and FITC-conjugated anti-antibody in the case of tetanus toxin-labelled material, and restained with a rat or mouse antibody against chick δ crystallin followed by the appropriate rhodamine-conjugated anti-antibody. Although both FITC/toxin-labelled cells (putative neurones) and rhodamine/δ crystallin-labelled cells (transdifferentiated lens cells) were abundant, no examples of double-labelled cells were observed with 9 day starting material, and only a very few with 7 day starting material. We conclude that the vast majority of differentiated neuronal cells expressing surface receptors for these toxins do not transdifferentiate directly into lens cells.     

Immunohistochemical characterization of delta crystallin-containing retina/optic nerve "boundary" cells in the chick embryo

The term "transdifferentiation" has been used to describe the apparent phenotypic conversion of chick embryo neural retina Müller glial cells into lens-like cells in vitro. This phenotypic conversion is characterized by expression of such lens-specific proteins as delta crystallin and has been viewed as an example of cells transforming from the phenotype of a given tissue to that of another. We have identified a population of neuroglia-like cells in the embryonic chick retina which express high levels of delta crystallin as a function of normal development. The position and morphology of these cells is quite distinctive in that they form a loose meshwork which defines the boundary between the neural retina and the optic nerve head. These "boundary" cells are detectable as early as Day 5 of development through hatching. However, the meshwork structure formed by the cells is only readily observed between Days 8 and 9 of development. Double-immunolabeling procedures comparing delta crystallin staining to that of glial and neuronal markers suggest that these cells are a form of retinal Müller glial cell. The results show that under appropriate microenvironmental conditions, expression of delta crystallin falls into the normal repertoire of retinoblast cells. The results also demonstrate the presence of a cellular boundary defining the junction between the neural retina and the optic nerve, tissues that are ontogenetically and structurally continuous but functionally distinct.     

Analysis of DNA methylation in different maize tissues

DNA methylation plays an important role in gene expression regulation during biological development and tissue differentiation in plants. This study adopted methylation-sensitive Amplified fragment length polymorphism （AFLP） to compare the levels of DNA cytosine methylation at CCGG sites in tassel, bracteal leaf, and ear leaf from maize inbred lines, 18 White and 18 Red, respectively, and also examined specific methylation patterns of the three tissues. Significant differences in cytosine methylation level among the three tissues and the same changing tendency in two inbred lines were detected. Both MSAP （methylation sensitive amplification polymorphism） ratio and full methylation level were the highest in bracteal leaf, and the lowest in tassel. Meanwhile, different methylation levels were observed in the same tissue from the inbred lines, 18 White and 18 Red. Full methylation of internal cytosine was the dominant type in the maize genome. The differential methylation patterns in the three tissues were observed. In addition, sequencing of nine differentially methylated fragments and the subsequent blast search revealed that the cytosine methylated 5 ＇ -CCGG-3 ＇ sequences were distributed in repeating sequences, in the coding and noncoding regions. Southern hybridization was used to verify the methylation polymorphism. These results clearly demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the maize genome, and the complexity of DNA methylation change during plant growth and development. The different methylation levels may be related to specific gene expression in various tissues.     

pp60c-src expression in transdifferentiating cultures of embryonic chick neural retina cells

Chick embryo neural retinal cells transdifferentiate extensively into lens cells when cultured in Eagle's MEM containing horse and fetal calf sera (FHMEM). Such cultures express elevated levels of pp60c-src-associated tyrosine kinase activity relative to parallel cultures prevented from transdifferentiating by the addition of supplementary glucose (FHGMEM) or replacement of MEM by medium 199 (F199). Northern blotting and in vitro translation studies suggest that c-src mRNA levels are only slightly higher in late transdifferentiating (FHMEM) cultures as compared to parallel blocked (FHGMEM or F199) cultures. By immunocytochemical staining, we show that pp60c-src protein is largely localized in cell groups undergoing conversion into lens (i.e. expressing delta crystallin) in late FHMEM cultures. Initial studies of pp60c-src in chick lens tissues during development indicate that higher kinase activity is found in the epithelial cells relative to mature lens fibres. Thus pp60c-src may be expressed both during the differentiation of lens cells in vivo and during the transdifferentiation of neural retina cells into lens in vitro.     

Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.     

The ability of the epithelium of diencephalic origin to differentiate into cells of the ocular lens.

After the discovery that in adult salamanders following lentectomy a new, functional lens develops by transdifferentiation (cell-type conversion) of previously depigmented epithelial cells of the iris (Wolffian lens regeneration), this phenomenon has been intensively studied by various experimental approaches. During the last two decades it was shown that pleiomorphic aggregates of atypical lens cells (lentoids) differentiated in reaggregates of dissociated cells of the chick neural retina and in spread cell cultures of the pigmented epithelium of the iris and retina, of the neural retina and the pineal gland of the chick embryo. The neural retina of human fetuses and adults also displayed this capacity. We showed that lentoids developed at a low incidence in renal isografts of rat embryonic shields or isolated embryonic ectoderm and of lentectomized eyes of rat fetuses, as well as in organ cultures of rat embryonic shields in chemically defined media. The addition of transferrin significantly increased the incidence of differentiation of lentoids in explants. In both renal isografts and explants in vitro a continuous transformation of retinal epithelial cells into atypical lens cells was observed. In renal isografts lentoids were also observed to originate from the ependyma of the brain ventricle. All tissues having the capacity to convert into lens cells belong to the diencephalon in a broad sense. Evolutionary aspects of this feature are discussed.     

A DNA signal from the Thy-1 gene defines de novo methylation patterns in embryonic stem cells.

Although DNA can be extensively methylated de novo when introduced into pluripotent cells, the CpG island in the Thy-1 gene does not become methylated either in the mouse embryo or in embryonic stem cells. A 214-base-pair region near the promoter of the Thy-1 gene protects itself as well as heterologous DNA sequences from de novo methylation. We propose that this nucleotide sequence is representative of a class of important signals that limits de novo methylation in the embryo and establishes the pattern of hypomethylated CpG dinucleotides found in somatic tissues.