Mitochondrial oxygen metabolism in primary human lens epithelial cells: Association with age, diabetes and glaucoma.

PURPOSE: The hypoxic environment around the lens is important for maintaining lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption to assess the role of human LECs in maintaining hypoxia around the lens, as well as the impact of systemic and ocular diagnosis on these cells. METHODS: Baseline cellular respiration was measured in rabbit LECs (NN1003A), canine kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and bovine corneal endothelial cells (CCEE) using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro. Following informed written consent, lens capsule epithelial cells were obtained from patients during cataract surgery and were divided into small explants in 96-well plates. Capsules were removed when LECs became confluent. OCR was normalized to the number of cells per well using rabbit LECs as a standard. The effect of patient age, sex, race, and presence of diabetes or glaucoma on oxygen consumption was assessed by using the Mann-Whitney U test and multivariate regression analysis. RESULTS: Primary LECs were obtained from 69 patients. The OCR from donors aged 70 and over was lower than that of those under 70 years (2.21±1.037 vs. 2.86±1.383 fmol/min/cell; p<0.05). Diabetic patients had lower OCR than non-diabetic patients (2.02±0.911 vs. 2.79±1.332fmol/min/cell; p<0.05), and glaucoma patients had lower OCR than non-glaucoma patients (2.27±1.19 vs. 2.83±1.286 fmol/min/cell; p<0.05). Multivariate regression analysis confirmed that donors aged 70 and over (p<0.05), diabetic patients (p<0.01), and glaucoma patients (p<0.05) had significantly lower OCR, independent of other variables. Gender and race had no significant effect on OCR. CONCLUSIONS: The lower oxygen consumption rate of human LECs in older donors and patients with diabetes or glaucoma could contribute to cataract development. Diabetes and glaucoma are particularly important factors associated with decreased OCR, independent of age. Ongoing studies are examining pO2 at the anterior surface of the lens in vivo and oxygen consumption in the patient's LECs.

Changes in adhesion complexes define stages in the differentiation of lens fiber cells.

PURPOSE: During their differentiation, lens fiber cells elongate, detach from the lens capsule, associate at the sutures, and degrade all cytoplasmic membrane-bound organelles. Changes in the expression or organization of cell adhesion and cytoskeleton-associated proteins were correlated with these events during fiber cell differentiation in chicken embryos. METHODS: Fixed or living lenses were sliced with a tissue slicer, permeabilized or extracted with detergents, stained with antibodies or fluorescent-labeled phalloidin, and viewed with a confocal microscope. The distribution of N-cadherin in elongating and mature fiber cells was determined by Western blot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the distribution of vinculin and paxillin transcripts. RESULTS: Staining for N-cadherin and band 4.1 protein decreased soon after fiber cells detached from the capsule. Detergent extraction of lens sections and Western blots of dissected lens regions showed that much of this decrease in staining was due to epitope masking. Vinculin immunoreactivity was barely detectable on the lateral membranes of elongating fiber cells but increased markedly once these cells reached their maximum length and formed the sutures. Staining for paxillin was also low in elongating fiber cells but increased late in fiber cell differentiation, just before the cells destroyed their membrane-bound organelles. Spectrin and ankyrin immunoreactivity did not change when fiber cells reached the sutures. Staining for F-actin increased transiently in cells that had just reached the sutures. Messenger RNAs for vinculin and paxillin were more abundant in maturing than in elongating fiber cells. CONCLUSIONS: The adhesion complexes of lens fiber cells change in organization and composition soon after these cells finish elongating and detach from the capsule. Increased staining for vinculin and paxillin defines distinct stages of fiber cell differentiation that are intermediate between the completion of cell elongation and the time when lens fiber cells degrade their membrane-bound organelles. Remodeling adhesion complexes during fiber cell maturation may assure the stability of fiber-fiber associations, once these cells are no longer transcriptionally active.

Alpha-smooth muscle actin expression in cultured lens epithelial cells.

PURPOSE: Lens epithelial cells transdifferentiate to myofibroblasts during the formation of anterior subcapsular cataracts and secondary cataracts. One of the defining characteristics of myofibroblasts is the expression of alpha-smooth muscle actin (alpha-SMA). This study investigated some of the factors that influence alpha-SMA expression in lens epithelial cells. METHODS: Bovine, rabbit, and human lens epithelial explants or cells were cultured with or without serum. Immunohistochemistry and immunoblotting were used to detect and quantitate alpha-SMA expression. RESULTS: Cells from all species studied expressed alpha-SMA in primary explant culture with or without serum. Immunostaining for alpha-SMA first appeared in a diffuse granular pattern, then accumulated at the cell cortex, and eventually was detected along stress fibers. When lens epithelial cells migrated onto cell-free regions of the capsule or were transferred to a plastic culture dish, alpha-SMA expression increased significantly. Expression of alpha-SMA positively correlated with cell size and cell migration. CONCLUSIONS: Expression of alpha-SMA is a common feature of cultured mammalian lens epithelial cells. Because alpha-SMA expression occurred without the addition of exogenous factors, the fibrosis seen in anterior subcapsular cataracts or secondary cataracts may reflect the intrinsic properties of lens epithelial cells. Interaction between lens epithelial cells and their substratum appears to be an important regulator of myofibroblast formation. Understanding the factors that regulate alpha-SMA expression in lens epithelial cells could lead to the development of methods for preventing secondary cataracts and anterior subcapsular cataracts.

The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye.

During the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium.

Human age-related cataract and lens epithelial cell death.

PURPOSE: To evaluate the importance of lens epithelial cell death in age-related cataract. To determine whether the large percentage of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive lens epithelial cells previously reported in human capsulotomy specimens results from apoptosis or necrosis. METHODS: Capsulotomy specimens from patients who had undergone cataract surgery and epithelia from cataractous lenses of eye bank eyes were compared with epithelia from noncataractous lenses of eye bank eyes. DNA fragmentation was assayed using the TUNEL method. Cell membrane integrity was tested using a fluorescent stain for DNA, BOBO-3, that is excluded from living cells. Cell proliferation was assayed by labeling with 5-bromo-2'-deoxyuridine (BrdU). The number of cells in different regions of the lens epithelium was measured by digital imaging and computerized counting of nuclei after staining with methyl green. RESULTS: TUNEL-positive cells were sometimes detected adjacent to denuded regions of capsulotomy specimens, especially when epithelia were not fixed immediately after surgery. TUNEL-stained cells usually stained with BOBO-3, indicating loss of plasma membrane integrity. No BrdU-labeled cells were detected in capsulotomy specimens. Cell density in cataractous lens epithelia was similar to that in normal lens epithelia. In cataractous lenses from eye bank eyes, cell density in the region of the epithelium overlying the cataract was higher than cell density in the region of the epithelium overlying the transparent part of the lens. No correlation was found between cell density and cataract severity or between cell density and age. CONCLUSIONS: TUNEL staining of lens epithelial cells in capsulotomy specimens most likely results from necrotic cell death caused by damage during or soon after cataract surgery. Loss of cells from the lens epithelium, by apoptosis or other mechanisms of cell death, does not seem to play a major role in age-related cataract formation.

Activation of the Jak-STAT-signaling pathway in embryonic lens cells.

Previous studies showed that lens epithelial cells proliferate rapidly in the embryo and that a lens mitogen, most likely derived from the blood, is present in the anterior chamber of the embryonic eye (Hyatt, G. A., and Beebe, D. C., Development 117, 701-709, 1993). Messenger RNAs for several growth factor receptors have been identified in embryonic lens epithelial cells. We tested several growth factors that are ligands for these receptors for their ability to maintain lens cell proliferation. Embryo serum, PDGF, GM-CSF, and G-CSF maintained lens cell proliferation, but NGF, VEGF, and HGF did not. This and a previous study (Potts, J. D., Harocopos, G. J., and Beebe, D. C., Curr. Eye Res. 12, 759-763, 1993) detected members of the Janus kinase family (Jaks) in the developing lens. Because Jaks are central players in the Jak-STAT-signaling pathway, we identified STAT proteins in the lens and tested whether they were phosphorylated in response to mitogens. STAT1 and STAT3, but not STAT 5 were detected in chicken embryo lens epithelial cells. Only STAT3 was found in terminally differentiated lens fiber cells. STAT1 and STAT3 were phosphorylated in lens cells analyzed immediately after removal from the embryo and when lens epithelial explants were treated with embryo serum, PDGF, or GM-CSF, but not with NGF. Chicken embryo vitreous humor or IGF-1, factors that stimulate lens cell differentiation, but not proliferation, did not cause STAT phosphorylation. When lens epithelial cells were cultured for 4 h in unsupplemented medium, STAT1 and STAT3 declined to nearly undetectable levels. Treatment with PDGF or embryo serum for an additional 15 min restored STAT1 and -3 levels. This recovery was blocked by cycloheximide, but not actinomycin D, suggesting that STAT levels are regulated at the level of translation. STAT levels were maintained in epithelial explants by lens mitogens, but not by factors that stimulated lens fiber differentiation. Both factors that stimulated lens cell proliferation and those that caused fiber differentiation protected cultured lens epithelial cells from apoptosis. These data suggest that the factor(s) responsible for lens cell proliferation in vivo activates the Jak-STAT-signaling pathway. They also indicate that growth factors maintain STAT protein levels in lens epithelial cells by promoting the translation of STAT mRNA, an aspect of STAT regulation that has not been described previously. Signaling by most of the growth factors and cytokines known to activate the Jak-STAT pathway has been disrupted in mice by mutation or targeted deletion. Consideration of the phenotypes of these mice suggests that the factor responsible for lens cell proliferation in vivo may be a growth factor or cytokine that has not yet been described.

Collagen type IX and developmentally regulated swelling of the avian primary corneal stroma.

A critical event in avian corneal development occurs when the acellular primary stroma swells and becomes populated by mesenchymal cells that migrate from the periphery. These cells then deposit the mature stromal matrix that exhibits the unique features necessary for corneal function. Our previous work correlated the disappearance of collagen type IX immunoreactivity at stage 27 (5 1/2-6 days) with matrix swelling and invasion. To investigate further the mechanism of this disappearance, we employed immunohistochemistry after tissue fixation with Histochoice, a non-crosslinking fixative, immunoblot analysis of protein extracts, and gel substrate chromatography (zymography) to detect endogenous proteolytic activity. We found that corneas fixed in Histochoice retain immunoreactivity for type IX collagen for 1-2 days after corneal swelling. This immunoreactivity, however, becomes extractable from tissue sections of unfixed corneas at the time of initiation of stromal swelling and mesenchymal cell invasion. Immunoblot analysis confirmed that, following swelling, immunoreactivity for collagen IX decreased substantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extracts indicated that, at the time of swelling, much of the immunoreactivity for type IX collagen in cornea shifted from the AS precipitate (containing high molecular weight molecules) to the AS supernatant (containing smaller fragments). In contrast, collagen IX immunoreactivity from the vitreous was precipitated by ammonium sulfate throughout the period of study. Collagen type II, a major fibrillar collagen in both the corneal stroma and vitreous, remained in the high molecular weight fraction at all times examined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP-2) before corneal swelling and invasion (4 days), and both the latent and active forms of the enzyme after corneal swelling. This suggests tissue-specific, developmentally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling.

Isolation and expression of homeobox genes from the embryonic chicken eye.

PURPOSE: To identify homeobox-containing genes that may play a role in the differentiation of ocular tissues. METHODS: Total RNA was isolated from microdissected chicken embryo eye tissues at 3.5 days of development (embryonic day 3.5; E3.5). An "anchor-oligo-dT primer" was used for the synthesis of cDNA. Degenerate oligonucleotides designed from highly-conserved sequences in the third helix of the homeobox and the "anchor-primer" were used to amplify cDNAs by polymerase chain reaction (PCR). PCR products were cloned and sequenced. The spatial and temporal expression of selected transcripts was mapped by whole-mount in situ hybridization and northern blot analysis. RESULTS: After sequencing eighteen clones we identified a member of the distal-less family (dlx-3) in cDNA from presumptive neural retina and three chicken homologs of the Xenopus "anterior neural fold" (Xanf-1) in cDNA from anterior eye tissue. Dlx transcripts were mapped by in situ hybridization. Expression began at Hamburger and Hamilton stage 14 (E2.5) and was widely distributed in embryonic mesenchyme on E3 and E4. Expression increased in the retina during early development and persisted until after hatching. The one anf clone selected for further study was not detected by in situ or northern blot analysis. CONCLUSIONS: It is feasible to isolate homeobox cDNAs directly from microdissected embryonic tissues. Chicken dlx-3 mRNA has a wider distribution in the embryo than expected, based on the expression of the mouse homolog. Dlx-3 may play a role in establishing or maintaining the differentiation of the retina.

Cell lineage and the differentiation of corneal epithelial cells.

PURPOSE: Studies were designed to determine whether cell division and cell differentiation are linked directly in the corneal epithelium. To obtain these data, corneal basal epithelial cells were labeled during DNA synthesis, and the resultant daughter cells were followed for as long as 2 weeks. METHODS: Adult rats were injected with 5-bromo-deoxyuridine (BrdU), a thymidine analog, and were killed 6 hours to 14 days later. Corneas were fixed and permeabilized, and BrdU-labeled nuclei were detected with a monoclonal antibody to BrdU and a fluorescent-labeled secondary antibody. Fluorescent nuclei were visualized in three dimensions in corneal whole-mounts using a laser scanning confocal microscope. Optical sections were collected and displayed as serial images, three-dimensional anaglyphs, color-encoded projections, or three-dimensional reconstructions. Data were confirmed using 3H-thymidine autoradiography of epithelia sectioned parallel to the corneal surface. RESULTS: Cells synthesizing DNA at the time of injection incorporated BrdU into their DNA. Pairs of labeled nuclei were produced by the division of cells that had been labeled with BrdU. These daughter cells remained in the basal layer of the epithelium for a variable period of time. Some daughter cells continued to divide, producing clusters of labeled basal cells. When labeled daughter cells left the basal layer and began the process of terminal differentiation, they nearly always did so together. The synchronous differentiation of daughter cells was evident from the pairs of labeled nuclei seen throughout the depth of the epithelium from 2 to 14 days after labeling. CONCLUSIONS: Cell division and differentiation are not linked directly in the corneal epithelium. After cell division, daughter cells either remain in the basal layer, where they may undergo additional rounds of cell division, or both cells differentiate synchronously. When the daughter cells of a mitosis differentiate, the time between the previous cell division and differentiation is highly variable. This suggests that the coordination of cell division and differentiation in the corneal epithelium involves a complex regulatory network.

Differential temporal and spatial expression of insulin-like growth factor binding protein-2 in developing chick ocular tissues.

PURPOSE: To determine the developmental expression and localization of mRNA for insulin-like growth factor binding protein-2 (IGFBP-2), a major binding protein of IGF-I and IGF-II, in ocular tissues of the embryonic and early posthatched chick. METHODS: In situ hybridization and northern blot analysis were used to analyze the cellular origin and relative expression of IGFBP-2 mRNA in ocular tissues. RESULTS: Wholemount in situ hybridization reveals that, as early as 3.5 days of embryonic development (E3.5), IGFBP-2 mRNA is already expressed in many areas of the embryo, including surface ectoderm, certain regions of the brain, pharyngeal clefts, somites, and limb buds. In the eye, IGFBP-2 mRNA is expressed only in the presumptive corneal epithelium at this time. By E6, IGFBP-2 mRNA expression is present in both the corneal epithelium and endothelium. By E12, IGFBP-2 mRNA is detected clearly in the corneal stroma as well as in several other ocular structures, such as the sclera, eyelid, and ciliary body. In the neural retina, a low, diffuse expression of IGFBP-2 mRNA is found at E6, which becomes more localized to the nuclear layers by E12. Northern blot analysis confirms that a high level of IGFBP-2 expression is present in the cornea and sclera by E8 to E12. A high level of IGFBP-2 mRNA expression, however, is not observed in the retina until E18. At posthatch day 2 (P2), northern blot analyses of ocular tissues reveal that the cornea contains the highest ocular level of IGFBP-2 mRNA expression, a value equal to that of brain and liver. CONCLUSIONS: The early appearance, along with differential temporal and spatial expression of IGFBP-2 mRNA in developing ocular tissues, suggests a role for IGFBP-2 in the regulation of growth and differentiation of several ocular tissues, including the cornea, sclera, and retina.

Cloning and characterization of a chick embryo cDNA and gene for IGF-binding protein-2.

We have isolated and characterized a cDNA for IGF-binding protein-2 (IGFBP-2) and its gene from the chick embryo. Using primers from a conserved region of the mammalian IGFBP-2 sequence, a cDNA clone (1.6 kb) was isolated from an embryonic day-18 chick retina cDNA library. Although the clone was truncated at the 5' end, the complete coding sequence was obtained from 5' rapid amplification of cDNA ends and genomic sequencing. The open reading frame encoded a 311 amino acid precursor protein which contains a putative 36 residue signal peptide. The mature 275 amino acid protein had a predicted M(r) of 33,500 and exhibited 71, 68, 68 and 66% identity to rat, bovine, ovine and human IGFBP-2 cDNA respectively, with conservation of all 18 cysteines. The cDNA contained an RGD peptide but lacked a putative ATP-binding motif. A single transcript of approximately 2.3 kb was present in embryonic day-15 eye, brain, skeletal muscle, heart and intestine, but was virtually absent from embryonic day-15 liver. The chicken IGFBP-2 gene spanned approximately 38 kb, consisted of four exons, and was similarly organized to that of the rat and human. Southern blot analysis of chicken genomic DNA suggested that it is encoded by a single gene. The sequence information from the avian IGFBP-2 should be of value in examining the role of IGFBP-2 in vertebrate development.

Expression of transforming growth factor beta 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.

Expression of platelet-derived growth factor receptors in the developing chicken lens.

PURPOSE: Platelet-derived growth factor (PDGF) has previously been shown to influence lens growth and transparency. The purpose of the present study was to investigate the expression of the PDGF receptor and its mRNA during lens development in chicken embryos. METHODS: To examine the expression of PDGF receptor mRNAs, the authors used a combination of polymerase chain reaction, Northern blot analyses, and RNase protection assays. Platelet-derived growth factor receptors were studied using Western blot analyses and immunofluorescence-confocal microscopy. The mitogenic effects of PDGF were assayed using immunochemical detection of 5-bromo-2'-deoxyuridine incorporation into explanted lens epithelia. RESULTS: The authors' studies strongly suggest that the PDGF alpha receptor is the only PDGF receptor present in the embryonic chicken lens during development because they were unable to detect the PDGF beta receptor using two different approaches. Northern blots showed that the chicken lens alpha receptor mRNA is similar in size to the mRNA encoding the human and frog alpha receptor, and Western blots detected a single band with a molecular weight of 170 kDa, close to the reported molecular weight for the human alpha receptor. After staining with specific antibodies, confocal microscopy localized the immunoreactivity to the membranes of the epithelial and annular pad cells. At early stages of development (E6), the PDGF alpha receptor was present throughout the epithelium. At later stages, immunoreactivity was restricted to the peripheral epithelium and the annular pad. The addition of human recombinant PDGF (AA or BB) to tissue cultured E6 chicken lens epithelia sustained cell proliferation significantly better than basal medium alone. CONCLUSIONS: The authors' studies provide the first evidence for PDGF alpha receptors in the chicken lens. Localization of these receptors to the peripheral lens epithelium during development and the mitogenic effect of exogenously added PDGF suggest a role for this growth factor in the control of lens growth.

Intercellular communication between epithelial and fiber cells of the eye lens.

Results of electrical, dye-coupling and morphological studies have previously suggested that gap junctions mediate communication between the anterior epithelium of the lens and the underlying lens fiber cells. This connection is believed to permit 'metabolic cooperation' between these dissimilar cell types and may be of particular importance to the fiber cells, which are thought incapable of autonomous ionic homeostasis. We reinvestigated the nature of the connection between epithelial and fiber cells of the embryonic chicken lens using fluorescence confocal microscopy and freeze-fracture analysis. In contrast to earlier studies, our data provided no support for gap-junction-mediated transport from the lens epithelium to the fibers. Fluorescent dyes loaded biochemically into the lens epithelium were retained there for more than one hour. There was a decrease in epithelial fluorescence over this period, but this was not accompanied by an increase in fiber cell fluorescence. Diffusional modeling suggested that these data were inconsistent with the presence of extensive epithelium-fiber cell coupling, even if the observed decrease in epithelial fluorescence was attributed exclusively to the diffusion of dye into the fiber mass via gap junctions. Furthermore, the rate of loss of fluorescence from isolated epithelia was indistinguishable from that measured in whole lenses, suggesting that decreased epithelial fluorescence resulted from photobleaching and leakage of dye rather than diffusion, via gap junctions, into the fibers. Analysis of freeze-fracture replicas of plasma membranes at the epithelial-fiber cell interface failed to reveal evidence of gap-junction plaques, although evidence of endocytosis was abundant. These studies were done under conditions where the location of the fracture plane was unambiguous and where gap junctions could be observed in the lateral membranes of neighboring epithelial and fiber cells. Paradoxically, tracer molecules injected into the fiber mass were able to pass into the epithelium via a pathway that was not blocked by incubation at 4 degrees C or by treatment with octanol and which excluded large (approximately 10 kDa) molecular mass tracers. Together with previous measurements of electrical coupling between fiber cells and epithelial cells, these data indicate the presence of a low-resistance pathway connecting these cell types that is not mediated by classical gap junctions.

Differential localization of collagen type IX isoform messenger RNAs during early ocular development.

PURPOSE: To determine the temporal and spatial localization of the messenger RNAs (mRNAs) for the two collagen alpha 1(IX) isoforms during early development of the embryonic chicken eye. METHODS: The reverse transcription-polymerase chain reaction method was used to amplify mRNAs for the two collagen alpha 1(IX) isoforms. mRNA was extracted from optic vesicles of chicken embryos at stages 14 to 19 and from microdissected ocular tissues of older embryonic eyes. After synthesis of complementary DNA, the polymerase chain reaction was performed for 20, 25, or 30 cycles. This ensured a reliable estimate of the relative abundance of the two mRNAs at different stages and in different ocular tissues. Data from the polymerase chain reaction were confirmed by Northern blot analysis. RESULTS: mRNA for the shorter alpha 1(IX) chain was present in the optic vesicle as early as stage 14, whereas mRNA for the longer alpha 1(IX) chain was not detectable until after the optic vesicle and lens started to invaginate (stage 15; day 2.5). In later stages of development, mRNAs for both alpha 1(IX) chains were present predominantly in the presumptive ciliary epithelium. They were just detectable in the neural retina at stages 20 and 23 (days 3 to 3.5). By E6 no mRNA for the shorter alpha 1(IX) isoform was detected in the retina, although a trace of the longer alpha 1(IX) isoform was still present. In the lens, mRNA for neither isoform was detectable at any stage. CONCLUSIONS: The two isoforms of alpha 1(IX) collagen mRNA are expressed differentially in space and time during early development of the embryonic chicken eye. These molecules may serve as markers for the early specialization of ciliary epithelium as a distinct region of the optic cup.

Identification of receptor tyrosine kinases in the embryonic chicken lens.

Protein phosphorylation plays a critical role in the control of growth and regulation of many eukaryotic cells. Members of the protein tyrosine kinase (PTK) family of peptides function as growth factor receptors and oncoproteins. A common feature of members of the PTK family is a highly conserved intracellular catalytic domain. We analyzed the chicken lens epithelium, which responds to several known growth factors, for the presence of receptor PTK's. Using reverse transcription polymerase chain reaction (rtPCR) and degenerate primers made to conserved regions within kinase domains, we amplified RNA from embryonic day 6 (E6) lens epithelium and sequenced 135 cDNA clones. Sixteen distinct kinase sequences were obtained. Eight of these sequences represented kinase domains of known mammalian growth factor receptors, and six represented intercellular kinases. Two sequences appeared to code for new kinases. The amino acid identity of the chicken homologs ranged from 80-100% when compared to their mammalian counterparts.

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.