Embryonic Lens Research Articles

New perspectives on embryonic lens induction

Abstract Embryologists at the turn of the century discovered the process of embryonic induction while investigating the mechanisms of lens determination. The simple view of lens induction from that period has been substantially altered in recent years as new insights into the tissue interactions and genes involved in this process have been revealed.

HIV type 1 Nef perturbs eye lens development in transgenic mice.

HIV transgenic mice often display lens cataracts as a consequence of viral-specific expression of HIV gene products in the developing lens. Cataractous mouse lines encoding either HIV-1 proviral DNA, HIV delta Gag/Pol] proviral DNA, or the HIV-1 nef gene alone were examined to ascertain the effect of Nef on murine lens development. Ocular disease was characterized by a progressive architectural disorganization within the lens fiber cell compartment developing in 100% of HIV-positive mice in five reported transgenic lines. Late embryonic stage transgenic lenses featured a mild microphthalmia, pyknotic nuclei within the lens fiber department, ballooning lens fiber cells, and elongated lens epithelial cells. Increased DNA fragmentation was evident in transgenic embryonic lenses, suggesting that cell death occurred by apoptosis. As studied in HIV delta Gag/Pol] transgenic mice, HIV transcription was developmentally linked to alpha A- and alpha B-crystallin gene expression, preceded disease development (in E14.5-E16.5 embryos), and persisted for weeks after birth. HIV-1 Nef was the predominant HIV gene product detected in the lens fiber cells of this line and was expressed almost to the exclusion of other HIV gene products. Nef was implicated as a major determinant of disease because (1) cataracts developed in mice transgenic for Nef alone and (2) the expression of other HIV gene products in wild-type HIV provirus transgenic mice occurred without a concomitant change in lens pathology.

Reduced levels of γ-crystallin transcripts during embryonic development of murine Cat2 nop mutant lenses

From previous experiments it is known that the murine dominant cataract mutants carrying the gene Cat2 have a decreased content of gamma-crystallin-specific transcripts in the juvenile lens, when the cataract is completely expressed. Moreover, the mutant locus has been mapped recently to chromosome 1, closely linked to the gamma E-crystallin gene (map distance 0.3 +/- 0.3 cM). In the present paper we describe the phenotypic changes and the gamma-crystallin expression in embryonic lenses of the Cat2nop mutants as an example for the Cat2 allelic series. The technique of in situ hybridization was applied using a probe from the murine gamma D-crystallin gene, and, for control, from the murine alpha A-crystallin gene. Simultaneously, a series of lens sections was examined histologically. The presence of gamma-crystallin mRNA was demonstrated from embryonic day 13.5 (E13.5) onward, but in the mutants to a lower extent than in the wild-type lenses. However, the first morphological abnormality in the mutant lenses was observed as swelling of lens fibers at day E15.5. Progressive degeneration of the lens core followed, leading to a cataracta immatura. The reduced level of gamma-crystallin transcripts is the first alteration observable during the embryonic development of the Cat2 mutant lenses: it precedes the morphological changes. This result represents an additional line of argument that the gamma-crystallin genes may be the target of the mutation in the Cat2 mice.

Temporally distinct patterns of p53-dependent and p53-independent apoptosis during mouse lens development.

Programmed cell death, or apoptosis, is a critical event in the development of multicellular organisms, and its perturbation is implicated in many diseases including cancer. The tumor suppressor protein p53 is known to mediate apoptosis induced by the DNA tumor virus oncoproteins, adenovirus E1A (AdE1A) and SV40 T antigen (SV40 Tag). We have recently demonstrated that the E6 and E7 oncoproteins of human papillomavirus type 16 (HPV-16) modulate apoptosis when expressed in the lens of transgenic mice. In this study we have identified the pathways that mediate E7 induction and E6 inhibition of apoptosis during different stages in the development of the lens. E7 transgenic mice made p53-null were only partially rescued in their apoptotic phenotype, indicating that both p53-dependent and -independent pathways mediate E7-induced apoptosis in the lens. The E6 transgene and p53-null genotype acted additively to reduce levels of apoptosis induced by E7 in neonatal lenses, indicating that E6 modulates apoptosis at least in part through p53-independent mechanisms. The partial reduction in E7-induced apoptosis by the p53-null genotype correlated with an increased incidence of lens tumors in adult E7 transgenic mice. Analyses of embryonic lenses at E13.5, E15.5, and E17.5 revealed a temporally distinct activation of p53-dependent and -independent apoptosis in the E7 lens. During the early stages of lens development, apoptosis was highly p53-dependent, whereas at later stages, apoptosis occurred through both p53-independent and -dependent pathways. This later time correlates temporally with the time of normal fiber cell denucleation, which can be inhibited by E6 through a p53-independent mechanism. These data suggest a similarity between the mechanism regulating E7-induced, p53-independent apoptosis and the apoptotic-like developmental process of fiber cell denucleation, and the mechanisms through which E6 suppresses both processes.

The Matured Eye of Xenopus laevis Tadpoles Produces Factors That Elicit a Lens-Forming Response in Embryonic Ectoderm

Previous studies have indicated that the outer cornea can undergo transdifferentiation to form a lens in the tadpole larva of Xenopus laevis following removal of the original lens. This transformation appears to require an interaction with the neural retina. In the present study, we carried out a series of experiments to determine if the matured tadpole eye can also elicit lens formation in embryonic ectoderm. Labeled embryonic ectoderm was removed from the presumptive lens-forming region, or from the belly region (ventral ectoderm), at various stages of development (stages 11-19, gastrula to neural tube stages) and implanted into the eye cavity (posterior chamber) of advanced stage 52-55 tadpoles. After 3 days, we examined the tadpoles and their implanted tissues for lens cell formation using lens-specific antibodies. Implanted presumptive lens ectoderm differentiated lens cells in a large number of cases. The percentage of cases forming lens cells and the extent of morphological differentiation increased with increasing age of the implanted tissue. Implanted ventral ectoderm also formed lens cells, although at a reduced frequency and with limited morphological differentiation. These results indicate that the environment of the matured tadpole eye cavity stimulates lens cell formation in both presumptive lens and nonlens ectoderm. The development of the implanted tissues was compared to that found in previous studies where these tissues were cultured as explants or transplanted to lens-forming regions during early development and subjected to various periods of embryonic lens induction. Together, these findings suggest that the process of embryonic lens formation is related to that involved in transdifferentiation of the tadpole cornea during "lens regeneration." However, the inductive effect of the matured tadpole eye is qualitatively different from that of the early period of embryonic lens induction and, while more intense, may be more closely related to that which takes place via the optic vesicle during the later phase of embryonic lens induction.

Transient expression of glutamate decarboxylase and gamma-amino butyric acid in embryonic lens fibers of the rat.

We have determined the localization and developmental expression of glutamate decarboxylase (GAD67) in the rat lens. Immunofluorescence experiments showed that GAD67 was transiently expressed in the nuclear fiber cells of the lens between embryonic days (E) 15 and 20, with maximal immunostaining occurring on E17 and E18. gamma-amino butyric acid (GABA) co-localized with GAD67 in the embryonic nuclear fiber cells. Reverse transcription-polymerase chain reaction (RT-PCR) tests showed that at least three alternatively spliced forms of GAD67 mRNA, including mRNAs with and without the I80 and the I86 insert, were transiently co-expressed with GAD67 in the embryonic lens. The major GAD67 protein in the lens was 67 kDa. We conclude that enzymatically active GAD67 is transiently expressed in the lens nuclear fiber cells of the embryonic rat. The transient expression is regulated by transcriptional and/or posttranscriptional processes. We speculate on the basis of possible common gene regulatory elements for glutamate and ornithine decarboxylases and the involvement of these enzymes with polyamine synthesis, that the transient expression of GAD67 may be connected to nuclear and/or DNA breakdown during lens fiber cell differentiation.

Ocular lens gap junctions: protein expression, assembly, and structure-function analysis.

Recent advances in understanding lens fiber gap junction formation are reviewed. These include studies of junctional protein expression in the embryonic lens, and of age related changes affecting gap junction structure and composition in the adult lens. An in vitro assembly system based on detergent solubilized pore complexes and endogenous lipids has been developed to provide information on the molecular interactions involved in gap junction formation and to provide material for structure analysis. Important information on the electrical properties of lens gap junction channels is obtained using electrophysiological techniques including planar lipid bilayer analysis and patch clamping.

α-crystallin polypeptides in developing chicken lens cells

We provide evidence that the different cells that form the chicken lens have isoelectric variants of alpha-crystallins at early and late developmental stages. We separated the alpha A and alpha B-crystallin subclasses by sodium dodecylsulphate polyacrylamide gel electrophoresis and then further resolved each by isoelectric focusing and assays with specific anti alpha-crystallin antibodies. We found that the annular pad, cortical and nuclear fibers, as well as the epithelial cells, contain alpha A and alpha B native chains and their respective isoelectric variants. These results on adult and embryonic lenses obtained a short time after the onset of alpha-crystallin expression suggest that lens cells, having different phenotypes, are able to produce post-translational modifications of the alpha A and alpha B chains as a part of their developmental program.

Expression of transforming growth factor β in the embryonic avian lens coincides with the presence of mitochondria

During their maturation, lens cells lose all membrane bound organelles, including mitochondria. In chicken embryos this process begins in the central lens fibers beginning around embryonic day 12 (E12). Transforming growth factor beta (TGF beta) is a multipotent growth modulator thought to play a role in numerous developmental processes. TGF beta 1 has been localized to mitochondria in rat liver cells and muscle cells. In the present study, we examined the expression of TGF beta isoform mRNAs and proteins during chicken embryonic lens development. PCR analysis demonstrated TGF beta 2 and TGF beta 3 transcripts in the lens epithelium and fibers throughout pre- and post-hatching development. TGF beta isoforms were detected throughout the lens epithelium and fibers early in development (E6). However by E19, the distribution of TGF beta 2 and TGF beta 3 transcripts and proteins coincided with regions of the lens that contained mitochondria. In addition, intense TGF beta staining was observed in the basal portions of the equatorial epithelial cells, a region with abundant mitochondria. Transcripts for TGF beta 1 and TGF beta 4 were not detected in any tissue or time frame examined. Similarly, no immunostaining for TGF beta 1 was observed.

β1 Integrins in Epithelial Tissues: A Unique Distribution in the Lens

Integrins have been shown to play a role in directing and maintaining cell differentiation and polarization. The embryonic lens provides a good system in which to examine their role in epithelial cell differentiation, because all stages of lens development are represented in an individual embryonic lens. Therefore, we examined the expression and distribution of β1 integrin heterodimers in both the lens epithelium and the differentiated lens fiber cells. In lens epithelial cells β1 integrin was found to be localized to all membrane surfaces. Lens fiber cells contained β1 integrin all along their lateral borders as well as at the site of their attachment to the lens capsule and at their interface with lens epithelial cells. The distribution of β1 integrin in the lens was distinct from that observed in simple epithelia, the retinal pigment epithelium (RPE), kidney, and intestine, where it was limited to a basal lateral localization. We examined the specific β1 integrin heterodimers expressed in the lens by Western blot analysis for the integrin α subunits following β1 immunoprecipitation and compared them with those expressed in RPE cells. In the lens we detected α 3 and α 6 subunits but not α 1, α 5, or α V. When the lens was separated into epithelial and fiber cells, we found that α 3 was expressed at a higher level in the epithelial cells, while α 6 was primarily associated with the fiber cells. In the RPE the primary β1 integrin detected was α 3. Unlike in lens and kidney, α 6β 1 integrin in RPE cells was expressed only at a low level, α V was also expressed in RPE cells but not as a β 1 heterodimer. As in the lens, neither α 5β 1 nor α 1β 1 integrin was detected in RPE. Both lens and RPE cells express a specific subset of β 1 integrin heterodimers which are likely to be important to the initiation and maintenance of their differentiated phenotype.

Identification of negative-acting and protein-binding elements in the mouse αA-crystallin −1556/−1165 region

The mouse αA-crystallin-encoding gene (α A-cry) is expressed in a highly lens-preferred manner. To date, it has been shown that this lens-preferred expression is controlled by four proximal positive-acting transcriptional regulatory elements: DEI (−111/−97), αA-CRYBPl (−66/−57), PE1/TATA (−35/−19) and PE2 (+24/+43). The present study extends our knowledge of mouse α A-cry transcriptional regulatory elements to the far upstream region of that gene by demonstrating that the − 1556 to − 1165 region contains negative-acting sequence elements which function in transfected lens cells derived from mouse, rabbit and chicken. This is the first negative-acting regulatory region identified in α A-cry. The − 1556 to − 1165 region contains sequences similar to repressor/silencer elements identified in other genes, including those highly expressed in the lens, such as the δl-crystallin (δ1- cry) and vimentin ( vim) genes. The − 1480 to − 1401 region specifically interacts with nuclear proteins isolated from the αTN4-1 mouse lens cell line. Co within this protein-binding region and positioned at − 1453 to − 1444 is a sequence (RS1) similar to the chicker intron 3 repressor, and which competes for the formation of − 1480 to − 1401 DNA-protein complexes. Our findings suggest that lens nuclear proteins bind to the mouse α A-cry RS1 region. We demonstrate that the chicken δ1- cry intron represser binds similar nuclear proteins in chicken embryonic lens cells and mouse αTN4-1 lens cells. However, the mouse α A-cry RS1 region does not appear to bind the same protein(s) that bind to the chicken δ1- cry intron repressor, suggesting the existence of a family of silencer proteins that recognize a similar site in the diverse cry genes.

Lens-specific activity of the chicken delta 1-crystallin enhancer in the mouse.

A lens-specific enhancer was identified in the third intron of the chicken delta 1-crystallin gene by analysis based on transient transfection of primary-cultured cells. To assess the significance of this enhancer's activity in embryonic lens cells during development, tkCAT gene carrying the enhancer was introduced into mouse embryos utilizing ES (embryonic stem) cell-mediated gene transfer. In the undifferentiated culture condition, ES lines with enhancer-carrying tkCAT did not express any significant level of CAT (chloramphenicol acetyltransferase). However, when the ES cells were injected into a blastocyst and allowed to differentiate into various somatic cells of an embryo, CAT expression was observed exclusively in lens, and the expression was dependent upon the delta 1-crystallin enhancer. We concluded that the delta 1-crystallin enhancer alone is sufficient for eliciting lens-specific gene expression in developing mouse embryos and that the mechanism of lens-specific regulation effected by the delta 1-crystallin enhancer is conserved between the chicken and the mouse.

Neurofilament expression in lens cells of the house shrew, Suncus murinus.

Immunohistochemical analysis demonstrated that lens cells of the laboratory house shrew, Suncus murinus, expressed many intracellular filaments that immunoreacted with pooled monoclonal antibodies against 70-kDa, 160-kDa, and 210-kDa neurofilament triplet proteins. Immunopositive filaments in lens cells first appeared in day 13 embryos, while the invaginating lens placode was thickening, and this immunoreactivity was still present in immature lens fiber cells of the adult animal. Western blot analysis showed that the immunopositive molecule was a low-molecular-weight neurofilament protein that appeared in the neural tissues of this animal. The immunoreactive pattern of lens cells was quite similar to that of neurons, although there were some peculiar aspects. When the cells of the lens vesicle differentiated into the lens epithelium and fibers, immunoreactivity occurred in both, suggesting that the neurofilaments in the lens cells do not directly relate to lens fiber elongation nor to a determinant of the fiber caliber. The strong immunoreactivity in the embryonic lens and weak expression of this protein in the immature lens fiber cells of the adult animal suggest that low-molecular-weight neurofilament protein is transiently expressed in the differentiating lens cells. This may be a common feature of placode-derived cells.

Differentiation and angiogenic growth factor message in two mammalian lens epithelial cell lines

Abstract Lens epithelial cells in culture can sometimes be induced to form spheroid aggregates termed lentoid bodies, composed of cells exhibiting various characteristics of the more highly differentiated lens fiber cells. However, lentoid bodies are often slow to form, and the ability to produce them declines with serial subculture. It was therefore of interest to establish and/or characterize lens epithelial cell lines capable of forming lentoid bodies. The differentiation state was assessed in lentoid bodies formed by each of two lens epithelial cell lines, the transformed αTN4 cell line from mouse and the nontransformed N/N1135A cell line from rabbit. Lentoid and monolayer cultures of each cell line were examined for transcripts of the lens-specific αA-crystallin (“αA”), γD-crystallin (“γD”; formerly γ 1-crystallin) and MP26 genes. αTN4 lentoid bodies contained 2.5 times the αA RNA found in monolayer cells, but lacked detectable γD and MP26 RNA. None of the three markers were detected in either lentoid or monolayer N/N1135A cultures grown under the conditions described. Lentoid body formation alone, therefore, does not indicate the extent of differentiation occurring. At least some of the changes in cell adhesion occurring during lentoid body formation involve laminin-like and fibronectin-like interactions, and are reminiscent of those observed during embryonic lens formation. Finally, vascular endothelial growth factor mRNA was absent from the lens but present in αTN4 cells, suggestin a mechanism whereby the lens tumors of the founder mouse became vascularized.

Xenopus gamma-crystallin gene expression: evidence that the gamma-crystallin gene family is transcribed in lens and nonlens tissues.

Crystallins, the major gene products of the lens, accumulate to high levels during the differentiation of the vertebrate lens. Although crystallins were traditionally thought to be lens specific, it has recently been shown that some are also expressed at very low levels in nonlens tissues. We have examined the embryonic expression pattern of gamma-crystallins, the most abundant crystallins of the embryonic lens in Xenopus laevis. The expression profile of five Xenopus gamma-crystallin genes mirrors the pattern of lens differentiation in X. laevis, exhibiting on average a 100-fold increase between tailbud and tadpole stages. Four of these genes are also ubiquitously expressed outside the lens at a very low level, the first demonstration of nonlens expression of any gamma-crystallin gene; expression of the remaining gene was not detected outside the head region, thus suggesting that there may be two classes of gamma-crystallin genes in X. laevis. Predictions regarding control mechanisms responsible for this dual mode of expression are discussed. This study raises the question of whether any crystallin, on stringent examination, will be found exclusively in the lens.

DNA strand breakage during physiological apoptosis of the embryonic chick lens: free 3' OH end single strand breaks do not accumulate even in the presence of a cation-independent deoxyribonuclease.

Epithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosomes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single-strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP-ribose)n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP-ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double-strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation.

Development of the lens in human embryos: a histochemical and ultrastructural study.

The early development of the lens was examined, using 36 externally normal human embryos at Carnegie stages 13-23 (4 to 8 weeks of gestation). Twenty-two embryos were sectioned serially and stained with periodic acid-Schiff and a modified method of PAS. In 14 embryos, not only the differential distribution of glycogen but also the ultrastructural change in the developing lens, with special reference to junctional complexes, were examined electron microscopically. At stage 15, when the lens vesicle was formed, glycogen was observed in the cytoplasm of the lens epithelium, especially in the posterior lens epithelium. From stages 16 to 18, when the posterior lens epithelium was differentiated into the primary lens fibers and elongated toward the anterior lens epithelium, the amount of glycogen increased in the basal cytoplasm of the primary lens fiber, where the intracellular organelles, such as the tubular vesicles, mitochondria and multivesicular bodies, began to aggregate. At stage 20, when the lens cavity was obliterated, glycogen was also present in the anterior lens epithelium. At stage 21, as the formation of the secondary lens fibers proceeded, glycogen was noted in the secondary lens fibers in the equator region. These findings suggest that the distribution of glycogen is associated with the formation of the primary and secondary lens fibers. In addition, we provide additional information that a lot of glycogen is distributed in the region where many intracellular organelles aggregate in the embryonic lens vesicles.

Characterization of Xenopus laevis γ-crystallin-encoding genes

In order to gain insight into crystallin (Cry)-encoding gene ( cry) evolution and developmental function, we have determined the gene structure and sequence of several Xenopus laevis γ- cry. These encode the most abundant Cry in the embryonic lens. Four of the X. laevis γ- cry, which are part of a multigene family, were isolated from a X. laevis genomic library and demonstrated to have the same gene structure as γ- cry from other vertebrates, thereby providing further evidence that the split between β and γ members of the βγ cry family occurred relatively early in evolution. Sequence comparisons indicate that these X. laevis genes share 88–90% nucleotide sequence identity in the protein coding regions, which is slightly higher than the identity observed between γ- cry of other species. The 5' upstream regions of X, laevis γ- cry contain a few short stretches of homology and one putative promoter element conserved among all cry genes but lack other regions common to γ- cry promoters from other organisms. The deduced amino acid sequences of all four genes and one cDNA suggest that the structure of X. laevis γ-Cry is highly conserved with that of other vertebrate γ-Cry, as deduced from the known three-dimensional structure of bovine γB Cry.

Transcriptional Control of δ-Crystallin Gene Expression in the Chicken Embryo Lens: Demonstration by a New Method for Measuring mRNA Metabolism

Crystallins are proteins that accumulate to very high concentrations in the fiber cells of the lens of the eye. Crystallins are responsible for the transparency and high refractive index that are essential for lens function. In the chicken embryo, delta-crystallin accounts for more than 70% of the newly synthesized lens proteins. We used density labeling and gene-specific polymerase chain reaction (PCR) to determine the mechanism regulating the expression of the two very similar delta-crystallin genes. Newly synthesized RNA was separated from preexisting RNA by incubating the lenses with 15N- and 13C-labeled ribonucleosides and then separating newly synthesized, density-labeled RNA from the bulk of light RNA by equilibrium density centrifugation in NaI-KI gradients. The relative abundances of the two crystallin mRNAs in the separated fractions were then determined by PCR. This method permitted the quantitation of newly synthesized processed and unprocessed delta-crystallin mRNAs. Additional studies used intron- and gene-specific PCR primers to determine the relative expression of the two delta-crystallin genes in processed RNA and unprocessed RNA extracted from different regions of the embryonic lens. Results of these tests indicated that the differential expression of the delta-crystallin genes was regulated primarily at the level of transcription. This outcome was not expected on the basis of the results of previous studies, which used in vitro transcription and transfection methods to evaluate the relative strengths of delta-crystallin promoter and enhancer sequences. Our data suggest that the cultured cells used in these earlier studies may not have provided an accurate view of delta-crystallin regulation in the intact lens.

Transcriptional control of delta-crystallin gene expression in the chicken embryo lens: demonstration by a new method for measuring mRNA metabolism.

Crystallins are proteins that accumulate to very high concentrations in the fiber cells of the lens of the eye. Crystallins are responsible for the transparency and high refractive index that are essential for lens function. In the chicken embryo, delta-crystallin accounts for more than 70% of the newly synthesized lens proteins. We used density labeling and gene-specific polymerase chain reaction (PCR) to determine the mechanism regulating the expression of the two very similar delta-crystallin genes. Newly synthesized RNA was separated from preexisting RNA by incubating the lenses with 15N- and 13C-labeled ribonucleosides and then separating newly synthesized, density-labeled RNA from the bulk of light RNA by equilibrium density centrifugation in NaI-KI gradients. The relative abundances of the two crystallin mRNAs in the separated fractions were then determined by PCR. This method permitted the quantitation of newly synthesized processed and unprocessed delta-crystallin mRNAs. Additional studies used intron- and gene-specific PCR primers to determine the relative expression of the two delta-crystallin genes in processed RNA and unprocessed RNA extracted from different regions of the embryonic lens. Results of these tests indicated that the differential expression of the delta-crystallin genes was regulated primarily at the level of transcription. This outcome was not expected on the basis of the results of previous studies, which used in vitro transcription and transfection methods to evaluate the relative strengths of delta-crystallin promoter and enhancer sequences. Our data suggest that the cultured cells used in these earlier studies may not have provided an accurate view of delta-crystallin regulation in the intact lens.