Tissue-specific regulation of a chicken delta-crystallin gene in mouse cells: involvement of the 5' end region.

A cloned delta-crystallin gene of the chicken is preferentially expressed in lens cells after introduction into various mouse tissues. The level of expression in the lens epithelium is 20 times higher than in fibroblasts. Taking advantage of this system, we attempted to define regulatory regions of the delta-crystallin gene using a variety of deletion and substitution mutants. The results indicate that tissue-specific regulation of the delta-crystallin gene is mediated by the 5' end region of the gene; sequences upstream from -93 are not required for expression and sequences downstream from +58 are not involved in tissue specificity. The high expression in lens cells requires 5' flanking sequences of 80-bp long from the cap site, whereas the low expression in fibroblasts requires an additional 12 bp upstream sequence. Expression of both types is lost in a mutant with only 51 bp of the 5' flanking sequence. Thus, fine deletion analysis demonstrated that expression in lens cells and expression in fibroblasts are distinct not only in level but in regulation.     

Specific expression of the chicken delta-crystallin gene in the lens and the pyramidal neurons of the piriform cortex in transgenic mice

Two transgenic mice, 5-8 and 7-5, carrying the chicken delta-crystallin gene were produced by microinjecting cloned genes into male pronuclei. The mice were analyzed at 8 weeks of age with respect to gene integration and expression by means of blotting techniques and immunohistochemistry. Southern blot analysis indicated that both mice carried, on average, 50 copies of intact delta-crystallin gene per cell. Histological analysis of the mice using DNA-DNA in situ hybridization indicated that mouse 5-8 carried the delta-crystallin gene in every cell while mouse 7-5 was mosaic, with 20-40% of the cells of various tissues carrying the gene. Western blot analysis indicated that in both mice delta-crystallin is expressed in the lens and the cerebrum, but not in any other tissue examined. Immunohistological analysis revealed that, in the cerebrum of the mice, delta-crystallin was expressed specifically in pyramidal neurons located in layer IIb of the anterior piriform cortex. Thus, our results with transgenic mice not only demonstrate the primary specificity of delta-crystallin gene expression in authentic lens tissue, but reveal the unexpected specificity of this chicken gene in the central nervous system of the mouse.     

Conservation of δ-crystallin gene structure between ducks and chickens

A cloned chicken delta-crystallin cDNA was used to identify two putative delta-crystallin genes in the duck by Southern blot hybridization. A DNA fragment containing most of one of these genes was isolated from a library made in bacteriophage lambda Charon 28A containing genomic DNA from 14-day-old embryonic ducks. Electron microscopy, partial gene sequencing, primer extension analysis using duck mRNA, and comparison with the well-characterized chicken delta-crystallin genes suggest that our cloned duck delta-crystallin gene, like the chicken delta-crystallin genes, is 8-10 kb long and contains 17 exons. Hybridization and sequencing data show great similarity between the homologous 5' untranslated and coding exons of the duck and chicken delta-crystallin genes. Overall, the homologous introns also appear to have approximately 30% sequence similarity, and have been subject to deletion/insertion events. Our partial characterization of duck delta-crystallin gene sequences suggests that this avian and reptilian crystallin family has been conserved during evolution, as have the other crystallin gene families that are expressed in the eye lens.     

Structural and functional similarities of delta-crystallin messenger ribonucleic acids from duck and chicken lenses

delta-Crystallin of the embryonic duck lens was compared with that of the embryonic chicken lens with respect to polypeptide composition, synthesis, and messenger ribonucleic acid (mRNA) sequences. Labeling experiments with [35S]methionine revealed that the duck delta-crystallin is composed of minor amounts of polypeptides with molecular weights near 50000 (50K) and 49000 (49K) and much greater amounts of polypeptides with molecular weights near 48000 (48K) and 47000 (47K), as judged by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. All four sizes of polypeptides were synthesized in similar relative proportions as found in vivo in a rabbit reticulocytes lysate supplemented with delta-crystallin mRNA isolated from the embryonic duck lens. Synthesis of the 48K and 47K delta-crystallin polypeptides was differentially reduced in duck lenses cultured in the presence of ouabain. This is similar to the differential reduction of synthesis of the lower molecular weight delta-crystallin peptides in embryonic chicken lenses demonstrated previously. R loops formed between duck or chicken delta-crystallin mRNA and a cloned chicken delta-crystallin cDNA and heteroduplexes formed between duck or chicken delta-crystallin mRNA and cloned chicken genomic DNAs containing delta-crystallin sequences showed that, except for the putative 5' leader sequence, the duck and chicken delta-crystallin mRNAs have extremely similar nucleotide sequences. These data indicate considerable conservation of delta-crystallin throughout the approximately 100 million years of divergence between ducks and chickens. The findings also suggest a possible relationship between the structure of delta-crystallin mRNA and the differential reduction in synthesis of the lower molecular weight delta-crystallin polypeptides in ouabain-treated lenses of ducks and chickens.     

Transient expression of a 'lens-specific' gene, delta-crystallin, in the embryonic chicken adenohypophysis

A characteristic protein of the lens, delta-crystallin, has been reported previously to be present in the embryonic chicken adenohypophysis. We confirmed this earlier finding by biochemical detection of delta-crystallin protein using a monoclonal antibody and delta-crystallin mRNA using a specific cDNA probe. We estimate the concentration of delta-crystallin and its mRNA in the 3.5-day embryonic chicken adenohypophysis to be approximately 1/3,000 and 1/5,000 of the respective value found in lens. Tissue culture revealed that cells positive for delta-crystallin comprise about 30% of embryonic adenohypophysis and are randomly scattered in this organ. No lentoid formation was observed during the culture period.     

The pattern of DNA methylation in the delta-crystallin genes in transdifferentiating neural retina cultures

Terminally differentiated lens fibre cells are formed in the vertebrate lens throughout life. Lens fibre cells may also be obtained by an in vitro process termed transdifferentiation, from certain tissues of different developmental origin from lens, such as embryo neural retina. delta-Crystallin is the major protein in the chick embryo lens fibre cells, and also in transdifferentiated lens cells obtained from cultured embryonic neural retina. Lens crystallin proteins and mRNA are present at low levels in the intact embryonic neural retina but are no longer detectable in the early stages of neural retina cell culture. However, levels rise steeply in the later stages and crystallins become the major products in terminally transdifferentiating neural retina cultures. We have used this system to test the hypothesis that the patterns of DNA methylation in particular genes are correlated with gene expression. A number of developmentally regulated genes have been found to be undermethylated in tissues where they are expressed, and methylated in tissues where they are not. However this correspondence does not always hold true. Eight-day-old embryonic neural retina was cultured for the period of time during which crystallin gene expression increases 100-fold. DNA methylation in the delta-crystallin gene region was analysed at several stages of cell culture by using the restriction endonucleases HpaII and MspI which cleave at the sequence CCGG. The former enzyme cannot cleave internally methylated cytosine (CmCGG) while the latter cannot cleave externally methylated cytosine (mCCGG). We detect no change in the methylation of CCGG sites within the delta-crystallin gene regions during transdifferentiation. Since dramatic changes in delta-crystallin gene expression occur during this process we conclude that large scale alterations in the pattern of DNA methylation are not a necessary accompaniment to changes in gene activity.     

Two delta-crystallin polypeptides are derived from a cloned delta 1-crystallin cDNA

Previous studies have shown that there are 2 similar delta-crystallin genes (delta 1 and delta 2) and at least 2 delta-crystallin polypeptides in the chicken eye lens. We show here that both delta-crystallin polypeptides can be synthesized from mRNA transcribed in vitro from a cloned delta 1-crystallin cDNA. Both polypeptides co-migrate in SDS-urea-polyacrylamide electrophoresis with their authentic counterparts isolated from 15-day-old embryonic chicken lenses, and both react with sheep anti-chicken delta-crystallin serum. Screening nearly 900 delta-crystallin cDNA clones from a 15-day-old embryonic lens library with an oligonucleotide probe specific for exon 2 of the delta 2-crystallin gene failed to detect any delta 2 cDNA clones, indicating that the delta 2 gene produces little or no mRNA in the lens at this stage of development. Our results suggest that both of the observed delta-crystallin polypeptides are derived from mRNA transcribed from the delta 1 gene, with heterogeneity arising at the translational or co-translational level.     

Reliability of delta-crystallin as a marker for studies of chick lens induction

Induction of a lens by the optic vesicle of the brain was the first demonstration of how tissue interactions could influence cell fate during development. However, recent work with amphibians has shown that the optic vesicle is not the primary inducer of lens formation. Rather, an earlier interaction between anterior neural plate and presumptive lens ectoderm appears to direct lens formation. One problem with many early experiments was the absence of an unambiguous assay for lens formation. Before being able to test whether the revised model of lens induction applies to chicken embryos, we examined the suitability of using delta-crystallin as a marker of lens formation. Although delta-crystallin is the major protein synthesized in the chick lens, one or both of the two delta-crystallin genes found in chickens is transcribed in many non-lens tissues as well. In studies of lens formation where appearance of the delta-crystallin protein is used as a positive assay, synthesis of delta-crystallin outside of the lens could make experiments difficult to interpret. Therefore, polyacrylamide gel electrophoresis, immunoblotting, and immunofluorescence were used to determine whether the delta-crystallin messenger RNA detected in non-lens tissues is translated into protein, as it is in the lens. On Coomassie-blue-stained gels of several tissues from stage-22 embryos, a prominent protein was observed that co-migrated with delta-crystallin. However, on immunoblots, none of the non-lens tissues tested contained detectable levels of delta-crystallin at this stage. By imunofluorescence, delta-crystallin was observed in Rathke's pouch and in a large area of oral ectoderm near Rathke's pouch, yet none of the cells in these non-lens tissues showed the typical elongated morphology of lens fiber cells. When presumptive lens ectoderm or other regions of ectoderm from stage-10 embryos were cultured and tested for lens differentiation, both cell elongation and delta-crystallin synthesis were observed, or neither were observed. The results suggest that delta-crystallin synthesis and cell elongation together serve as useful criteria for assessing a positive lens response.