Coordinating cell proliferation and migration in the lens and cornea.

Migration is a complex process for epithelial tissues, because the epithelium must move as an intact sheet to preserve its barrier function. The requirement for structural integrity is met by coupling cell-to-matrix and cell-to-cell adhesion at the cellular level, and by coordinating cell proliferation and cell migration in the tissue as a whole. Proliferation is suppressed at the migrating cell front, allowing cells in this region to remain tightly packed while advancing rapidly. At the same time, proliferation is enhanced in a region behind the advancing cell front to expand the epithelial cell sheet. This review considers the extracellular signals and intracellular signaling pathways that regulate these processes in the lens and corneal epithelium, with emphasis on the commonalities that link these tissues.

Regulation of c-fos induction in lens epithelial cells by 12(S)HETE-dependent activation of PKC.

PURPOSE: 12(S)-Hydroxyeicosatetraenoic acid (12(S)HETE), a 12-lipoxygenase metabolite of arachidonic acid, is required for epidermal growth factor (EGF)-dependent DNA synthesis and c-fos induction in lens epithelial cells. The present study was undertaken to identify signal transduction events upstream of c-fos induction that may be regulated by 12(S)HETE. METHODS: The rabbit lens epithelial cell line, N/N1003A, was cultured in serum-free medium, with or without EGF. Activation of PKC and other selected enzymes was examined in the presence of the lipoxygenase inhibitor baicalein and/or exogenous 12(S)HETE. Relative abundance of PKC isoforms in subcellular fractions was determined by immunoblot analysis with isoform-specific antibodies. PKC activity in subcellular fractions was measured by peptide substrate phosphorylation, with and without pseudosubstrate peptide inhibitor. Phosphorylated enzymes were detected by immunoblot analysis. Relative levels of c-fos mRNA were determined by RT/PCR with internal standard. RESULTS: Baicalein blocked EGF-dependent translocation and activation of PKC, without affecting phosphorylation of Erk1/2. Of several PKC isoforms investigated (alpha, betaI, betaII, and gamma), only PKCalpha and betaII were significantly activated by EGF and inhibited by baicalein. 12(S)HETE, in combination with EGF, countered the effect of lipoxygenase inhibitors on PKC activation, and 12(S)HETE in the absence of EGF stimulated PKC translocation. Also of note, 12(S)HETE alone activated PKCgamma, an isoform that was not significantly activated by EGF. Inhibiting PKC activation with GF109203X blocked induction of c-fos by EGF but did not affect EGF-stimulated phosphorylation of Erk1/2, indicating that the effect of PKC on c-fos induction is independent of the Erk1/2 pathway. CONCLUSIONS: In lens epithelial cells, 12(S)HETE-dependent activation of PKCalpha and betaII acts in concert with other EGF-dependent signals to induce c-fos mRNA.

The rabbit lens epithelial cell line N/N1003A requires 12-lipoxygenase activity for DNA synthesis in response to EGF.

PURPOSE: Cultured rat lenses and primary human lens epithelial cells (HLECs) express12-lipoxygenase (12-LOX) and require a 12-LOX metabolite of arachidonic acid for growth in response to EGF and insulin. This study seeks to identify an established cell line with these characteristics. METHODS: Immunoblotting was used to screen eight lens epithelial cell lines for 12-LOX expression: the human line, HLE-B3; mouse lines alphaTN4, 17EM15, 21EM15, and MLE6, and rabbit lines N/N1003A, LEP2 and B3. DNA synthesis was measured as incorporation of 3H-thymidine into DNA. Expression of c-fos mRNA was detected by RT-PCR. The involvement of 12-lipoxygenase metabolites was determined using the lipoxygenase inhibitors baicalein, cinnamyl 3,4-dihydroxy-alpha-cyanocinnamate (CDC), or nordihydroguiairetic acid (NDGA). RESULTS: 12-LOX was detected only in the rabbit lines N/N1003A, LEP2 and B3. N/N1003A cells were chosen for further study. 12-LOX inhibitors blocked DNA synthesis in response to EGF with or without insulin. Inhibition of EGF-stimulated DNA synthesis was reversed by 0.3 microM to 3 microM 12(S)hydroxyeicosatetraenoic acid (HETE), but not by equivalent concentrations of 5(S)HETE, 8(S)HETE, 15(S)HETE, or 12(R)HETE. Baicalein prevented EGF induction of c-fos mRNA. The transformed HLEC line, HLE-B3, showed little stimulation of DNA synthesis in response to EGF and was unaffected by the presence of 12-LOX inhibitors. CONCLUSIONS: N/N1003A cells, like primary cultured human lens epithelial cells or neonatal rat lenses, require 12-LOX activity for EGF dependent growth. This line will be useful for studies of the mechanism of action of 12(S)HETE.

Changes in cyclin dependent kinase expression and activity accompanying lens fiber cell differentiation.

Previous studies from this laboratory have shown that differentiating lens fiber cells contain two active cyclin dependent kinases (Cdks), Cdk1 and Cdk5. The present study was undertaken to explore the expression and regulation of six additional members of the Cdk family (Cdk2, Cdk3, Cdk4, Cdk6, Cdk7 and Cdk8) during lens differentiation. Differentiating lens fiber cells were separated from lens epithelial cells by microdissection of developing rat lenses [embryonic day 16 (E16) to postnatal day 8 (P8)] and Cdk expression was assessed by RT-PCR and immunoblotting. Two Cdks (Cdk3 and Cdk6) were not expressed in lens fiber cells or epithelial cells during this developmental period. In the lens epithelium, we detected proteins and mRNAs corresponding to all other Cdks examined (Cdk2, Cdk4, Cdk7, Cdk8) throughout this developmental period. Epithelial cells showed significant Cdk2 activity, which decreased with developmental age, but no significant activity was detected for Cdk4, Cdk7, or Cdk8. Fiber cells contained all four Cdk proteins and the corresponding Cdk mRNAs except for Cdk2 mRNA. None of the Cdks examined showed significant kinase activity in fiber cells. Immunoprecipitates of Cdk2 and Cdk4 from fiber cells contained p57(kip2), supporting the view that this Cdk inhibitor blocks the activity of these Cdks in lens fibers. In contrast, p57(kip2)did not co-immunoprecipitate with Cdk5 from lens fibers. These findings suggest that the differential affinity of p57(kip2)for members of the Cdk family may provide a mechanism for specific regulation of individual Cdks during fiber cell differentiation.

pRb and p107 regulate E2F activity during lens fiber cell differentiation.

During growth arrest and differentiation, activity of the E2F family of transcription factors is inhibited by interactions with pRb and the related proteins, p107 and p130. To determine which members of the E2F and pRb families may contribute to growth arrest as lens epithelial cells differentiate into fiber cells, we examined the expression of individual E2F species and characterized the E2F protein complexes formed in rat lens epithelia and fibers. RT/PCR detected all five known members of the E2F family in lens epithelial cells, but only E2F-1, E2F-3, and E2F-5 in fiber cells. Proteins extracted from lens epithelia of newborn rats formed at least two specific complexes with an E2F consensus oligonucleotide. Proteins from lens fiber cells formed three specific complexes, one of which comigrated with an epithelial cell complex. Incubation of epithelial and fiber cell extracts with an antibody specific for p107 demonstrated that two fiber cell complexes and one epithelial cell complex contained p107. Although the remaining fiber cell complex did not react with antibodies to pRb or p130 in this assay, a strong reaction with pRb antibody was observed when the electromobility shifted complexes were subsequently immunoblotted (shift/Western assay). Immunocytochemistry confirmed that pRb protein is present in the nuclei of both epithelial cells and fiber cells. Immunoblotting of whole cell extracts with pRb antibody showed multiple, phosphorylated forms of pRb in the epithelial cells, but predominantly hypophosphorylated pRb in the fiber cells. None of the complexes formed with E2F were recognized exclusively by the p130 antibody, although the previously identified p107 complexes reacted weakly. The absence of p130/E2F complexes was correlated with the presence of multiple ubiquitinated forms of p130, especially in the fiber cells. Thus, although p130/E2F complexes are implicated in the terminal differentiation of many cell types, in differentiating lens fiber cells pRb and p107 seem to be the primary regulators of E2F activity.

Cyclins, cyclin-dependent kinases and differentiation.

Cyclin-dependent kinases and their regulatory subunits, the cyclins, are known to regulate progression through the cell cycle. Yet these same proteins are often expressed in non-cycling, differentiated cells. This review surveys the available information about cyclins and cyclin-dependent kinases in differentiated cells and explores the possibility that these proteins may have important functions that are independent of cell cycle regulation.

Expression of Cdk5, p35, and Cdk5-associated kinase activity in the developing rat lens.

We have investigated the expression of Cdk5 and its regulatory subunit, p35, in the developing rat lens from embryonic day 16 (E16) to postnatal day 8 (P8). Reverse transcription and polymerase chain reaction (RT/PCR) detected Cdk5 and p35 mRNA expression in lens epithelial cells and in differentiating lens fibers throughout this developmental period. Subsequent sequencing of the RT/PCR products confirmed their identifies. In sity hybridization with Cdk5 and p35 riboprobes showed especially high expression of both mRNAs in the newly formed lens fiber cells in the bow region of the lens. Immunocytochemistry at E18 showed that Cdk5 was present in the cytoplasm of lens epithelial cells and fiber cells, with especially strong immunostaining at the anterior ends of the fibers. Fiber cells in the final stages of maturation, immediately prior to nuclear degeneration, showed positive staining for Cdk5 in the nucleus. Immunoprecipitation of proteins with Cdk5 antibody followed by immunoblotting with either N-terminal specific or C-terminal specific p35 antibodies demonstrated that p35 is complexed with Cdk5 in lens epithelial cells and lens fibers. Immunoprecipitates of Cdk5 from epithelia and fibers showed kinase activity in vitro using histone H1 as a substrate. These findings demonstrate that p35/Cdk5 activity is not restricted to neurons and raise the possibility that this kinase may play a role in lens fiber cell differentiation.

Expression of alternatively spliced PCTAIRE-1 mRNA in PC12 cells and neonatal rat brain.

A rat PCTAIRE-1 cDNA clone was isolated by immunoscreening of a PC12 cDNA library, followed by 5' RACE (rapid amplification of cDNA ends) to determine the 5' end. The rat PCTAIRE-1 cDNA sequence is 96% identical to mouse PCTAIRE-1 and contains an alternatively spliced exon of 131 bp near the 5' end. Although a mouse cDNA containing this exon has been reported, examination of several mouse cell lines provided no evidence for expression of the corresponding mRNA (Okuda et al., 1992). In contrast, reverse transcription and polymerase chain reaction (RT/PCR) across this region using RNA from proliferating, differentiated, and apoptotic PC12 cells demonstrated that alternatively spliced forms of PCTAIRE-1 mRNA with and without this exon are expressed. Both forms of PCTAIRE-1 mRNA are also expressed in vivo in neonatal rat brain, although other tissues examined contained only the form lacking the alternatively spliced exon. In the absence of the alternatively spliced exon PCTAIRE-1 mRNA contains an open reading frame of 1488 bp, corresponding to a 55-kDa protein that is 97% identical to mouse PCTAIRE-1 protein. When the alternatively spliced exon is present, this open reading frame is terminated by a stop codon and a second open reading frame is initiated, predicting a second PCTAIRE-1 protein of 52 kDa. The two predicted PCTAIRE-1 proteins are identical downstream of the splice site, but share no homology at their N-terminal ends.

A role for 12(S)-HETE in the response of human lens epithelial cells to epidermal growth factor and insulin.

PURPOSE: To determine whether the 12-lipoxygenase pathway of arachidonic acid metabolism is present in the human lens and whether 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) plays a role in regulating proto-oncogene expression and DNA synthesis in human lens epithelial cells (HLECs). METHODS: Second- and third-passage primary cultures of HLECs were used for analysis. Human cataract epithelia were obtained from surgery. 12-lipoxygenase mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the PCR product was sequenced. The 12-lipoxygenase protein was detected by immunoblotting. 12(S)-HETE was detected in HLEC-conditioned medium by radioimmunoassay. For studies of growth factor-induced mitogenesis, HLECs were serum starved, then stimulated with 15 ng/ml epidermal growth factor (EGF) and 1 microgram/ml insulin or with 0.3 microM 12(S)-HETE. The 12-lipoxygenase inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC, 10 microM) was used to block endogenous 12-lipoxygenase activity. Expression of c-fos mRNA was determined by RT-PCR, and DNA synthesis was measured by 3H-thymidine incorporation. RESULTS: 12-lipoxygenase mRNA and protein were detected in HLECs and in human cataract tissues. 12(S)-HETE was released into the medium by HLECs in the presence of EGF-insulin. Stimulation of c-fos mRNA expression and DNA synthesis by EGF-insulin was inhibited when the 12-lipoxygenase pathway was blocked by CDC. This inhibition was reversed completely by exogenously added 12(S)-HETE. However, exogenous 12(S)-HETE was unable to stimulate HLEC DNA synthesis in the absence of growth factors. CONCLUSIONS: The 12-lipoxygenase pathway of arachidonic acid metabolism is present in human lens epithelial cells. 12(S)-HETE does not stimulate HLEC DNA synthesis in the absence of growth factors but enables the cellular response to EGF and insulin.

Induction of cyclin B and H1 kinase activity in apoptotic PC12 cells.

The present study examines whether cyclin B may be involved in apoptosis of neuronally differentiated PC12 cells following withdrawal of NGF. Cyclin B mRNA increased approximately 10-fold 4 days after NGF withdrawal, as indicated by competitive RT/PCR. Sequencing of the PCR product confirmed that it was derived from cyclin B mRNA. Cyclin B protein increased in parallel with cyclin B mRNA, as shown by immunoblotting. Immunoprecipitation with anti-cyclin B antibody demonstrated that cyclin B was associated with H1K activity, which reached a maximum 5 days after NGF withdrawal. When proteins immunoprecipitated with anti-cyclin B antibody were immunoblotted with anti-PSTAIR antibody, a protein with apparent molecular weight of 34 kDa was detected. This protein was identified as p34cdc2 on the basis of immunoreactivity with antibody against the C-terminal portion of mouse p34cdc2. Since cyclin B/p34cdc2 complexes are known to catalyze chromosomal condensation and nuclear envelope breakdown during mitosis, these results suggest that cyclin B/p34cdc2 may play some role in the nuclear changes accompanying apoptosis of PC12 cells.

Insulin regulates expression of c-fos and c-jun and suppresses apoptosis of lens epithelial cells.

This study investigates whether insulin (a differentiation factor for lens epithelial cells) acts as a survival factor. In the absence of insulin, 6-day embryonic chicken lens epithelial explants undergo apoptosis as shown by changes in cell morphology, DNA fragmentation, and loss of trypan blue exclusion. Insulin inhibits these changes and promotes survival of the cells. Aurintricarboxylic acid suppresses the apoptosis of lens explants. In contrast to 6-day embryonic explants, 19-day embryonic explants survive in the absence of insulin, presumably due to an endogenous survival factor. To explore the mechanism of the action of insulin as a survival factor for 6-day embryonic lens explants, we compared the pattern of cell cycle markers (c-fos, c-jun, c-myc, p53, histone H3, thymidine kinase, and cyclin B) in both apoptotic and differentiating lens explants. In the presence of insulin, the expression of c-fos and c-jun was down-regulated after an initial induction. Expression of these genes was also induced in the absence of insulin, but mRNA levels remained elevated as the cells underwent apoptosis. In contrast, expression of c-myc, p53, histone H3, thymidine kinase, and cyclin B showed only minor differences in differentiating and apoptotic cells. Since c-fos and c-jun have been shown to play a role in apoptosis in other cell types, the ability of insulin to regulate expression of these genes may be central to its ability to act as a survival factor for lens epithelial cells.

Effects of c-Jun and a negative dominant mutation of c-Jun on differentiation and gene expression in lens epithelial cells.

We have used a retroviral vector (RCAS) to overexpress wild-type chicken c-Jun or a deletion mutant of chicken c-Jun (Jun delta 7) lacking the DNA binding region to investigate the possible role of c-Jun in lens epithelial cell proliferation and differentiation. Both constructs were efficiently expressed in primary cultures of embryonic chicken lens epithelial cells. Overexpression of c-Jun increased the rate of cell proliferation and greatly delayed the appearance of "lentoid bodies," structures which contain differentiated cells expressing fiber cell markers. Excess c-Jun expression also significantly decreased the level of beta A3/A1-crystallin mRNA, without affecting alpha A-crystallin mRNA. In contrast, the mutated protein, Jun delta 7, had no effect on proliferation or differentiation but markedly increased the level of alpha A-crystallin mRNA in proliferating cell cultures. These results suggest that c-Jun or Jun-related proteins may be negative regulators of alpha A- and beta A3/A1-crystallin genes in proliferating lens cells.

Cyclin B, p34cdc2, and H1-kinase activity in terminally differentiating lens fiber cells.

Terminal differentiation of lens fiber cells is marked by chromatin condensation, abrupt dissolution of the nuclear lamina, vesicularization of the nuclear membrane, and complete degradation of the nucleus and other organelles. Since these events resemble the chromosomal condensation and nuclear envelope breakdown associated with mitosis, we investigated whether a similar biochemical mechanism might be involved by testing for the presence of cyclin B/p34cdc2 complexes and p34cdc2-associated histone kinase activity in differentiating lens fiber cells. A coupled reverse transcription/polymerase chain reaction using RNA from E7, E15, or E20 embryonic chicken lens fibers amplified a cyclin B product of the expected size, whose identity was confirmed by sequencing. In situ hybridization showed that cyclin B mRNA was present in nucleated lens fiber cells at E19. Immunoblotting of proteins isolated from E6 or E15 lens fibers by p13-agarose affinity chromatography with anti-cyclin B antibody detected the 45-kDa cyclin B protein, while immunoblotting with anti-PSTAIRE antibody detected a single, 34-kDa band, identified as p34cdc2. The p13-affinity purified fraction from E6 or E15 lens fibers showed histone H1 kinase activity in vitro. Immunocytochemistry of E6 lenses with anti-chicken cyclin B antiserum showed positive staining in the nuclei of postmitotic annular pad and fiber cells. These results demonstrate that cyclinB/p34cdc2 complexes and p34cdc2-associated histone kinase activity are present in postmitotic, differentiating lens fiber cells and support the possibility that phosphorylation of specific nuclear substrates by p34cdc2 may play a role in the denucleation of lens fiber cells.

12(S)-hydroxyeicosatetraenoic acid regulates DNA synthesis and protooncogene expression induced by epidermal growth factor and insulin in rat lens epithelium.

Neonatal rat lens epithelium has a high 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] synthetic capacity, which decreases as epithelial cell proliferation decreases with age. To determine whether products of the 12-lipoxygenase pathway are involved in lens cell proliferation, we measured the effect of 12-lipoxygenase inhibitors on endogenous 12-HETE production, epidermal growth factor/insulin-stimulated DNA synthesis and protooncogene expression in cultured neonatal rat lens epithelial cells. Incubation of neonatal rat lenses in epidermal growth factor plus insulin, which stimulated endogenous 12-HETE production 8- to 10-fold, also produced a transient induction of c-fos and c-myc mRNAs after 2 to 3 h, followed by a round of DNA synthesis approximately 20 h later. The lipoxygenase inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, strongly inhibited both the endogenous 12-HETE synthesis and growth factor-stimulated DNA synthesis with a half-maximal inhibition between 10 and 20 microM. Cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (10 microM) also inhibited the expression of c-fos and c-myc mRNA and, to a lesser extent, c-jun mRNA. The inhibitory effects of cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate on protooncogene expression and DNA synthesis were prevented by 0.3 microM 12(S)-HETE but not by equivalent concentrations of either 5(S)-HETE or 15(S)-HETE. These findings suggest that endogenously synthesized 12(S)-HETE may mediate epidermal growth factor/insulin-stimulated DNA synthesis in neonatal rat lens epithelial cells by regulating protooncogene expression.

Expression of HSP70 mRNA in the embryonic chicken lens: association with differentiation.

The heat shock protein, HSP70 is constitutively expressed in the embryonic chicken lens. The present study investigates the localization of HSP70 mRNA in the lens as a function of developmental age to determine whether the state of proliferation or differentiation of the cells is correlated with expression of this gene. Using reverse transcription and quantitative PCR for HSP70 mRNA in RNA samples derived from microdissected regions of the lens, we have found that the concentration of HSP70 mRNA is highest in the annular pad cells at 6 days of development, and in the annular pad and fiber cells at 14 and 19 days of development, suggesting that expression is associated with fiber cell formation. This association was confirmed by studies of HSP70 transcription and HSP70 mRNA levels in cultured explants of 6 day embryonic chicken lens epithelia differentiating in culture. Nuclear run-on transcription experiments indicated that transcription of HSP70 is elevated after 5 and 24 hr of differentiation in vitro. Other genes whose transcription was monitored, c-myc and delta-crystallin, showed distinct patterns of transcriptional regulation, indicating that the changes in HSP70 transcription are specific. RNA extracted from cultured explants at these same times showed increased levels of a 3.4 kb mRNA which hybridized at high stringency to a chicken HSP70 cDNA probe. These results demonstrate that HSP70 mRNA in the embryonic chicken lens is associated primarily with cells in the early stages of fiber formation, and that increased transcription of this gene is part of the differentiation process.

Differential expression of the two delta-crystallin genes in lens and non-lens tissues: shift favoring delta 2 expression from embryonic to adult chickens.

Chicken argininosuccinate lyase (ASL)/delta-crystallin, a lens enzyme-crystallin, is encoded in two linked genes (delta 1 and delta 2); only the delta 2 polypeptide contains ASL activity. Here we have quantified delta 1- and delta 2-crystallin mRNA in the lens, cornea, neural retina, heart, and brain at different stages of embryonic development and in 1-wk-old and 1-yr-old chickens by the polymerase chain reaction using internal delta 1 and delta 2 RNA standards. The delta 1/delta 2 mRNA ratio differed for every tissue and was regulated during development. In the embryo there was more delta 1 than delta 2 mRNA in the lens (50-100 times), cornea (3-4 times), and neural retina (2-20 times), about equal amounts of delta 1 and delta 2 mRNA in the heart, and more delta 2 mRNA in the brain (15 times). delta 1-Crystallin mRNA differentially decreased in every tissue after hatching; by contrast, the delta 2 mRNA remained about the same except for the lens, where it decreased 50-fold between 1 wk and 1 yr after hatching. In the 1-yr-old chicken, the delta 2/delta 1 mRNA ratios were 7 in the lens, 175 in the cornea, 22 in the neural retina, 107 in the heart, and 136 in the brain, indicating that delta 2-crystallin is strongly favored in all adult tissues of the chicken. The excess of delta 1 to delta 2 mRNA in the embryonic lens, cornea, and neural retina is intriguing, and suggests some connection with developing transparent eye tissues. Finally, we raise the possibility that expression of both delta-crystallin genes may create tetrameric ASL isoenzymes (perhaps with different specific activities). The unexpected predominance of delta 2 mRNA in the 1-yr-old lens suggests that both the enzymatic and refractive functions of ASL/delta-crystallin are operative and spatially separated, with the enzymatic role present in the cortical fibers and the refractive role in the center of the lens.

A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens.

A discrete 10-kDa polypeptide (10K) is expressed from early stages in the embryonic chicken lens. Since this has potential as a marker for lens cell development, chicken 10K and its homologues from mouse and human lenses were identified by protein sequencing and cloning. Surprisingly, lens 10K proteins appear to be identical to a lymphokine, macrophage migration inhibitory factor (MIF), originally identified in activated human T cells. Using microdissection and PCR techniques, we find that expression of 10K/MIF is strongly correlated with cell differentiation in the developing chicken lens. Northern blot analysis shows that 10K/MIF is widely expressed in mouse tissues. These results suggest that proteins with MIF activity may have roles beyond the immune system, perhaps as intercellular messengers or part of the machinery of differentiation itself. Indeed, partial sequence of other small lens proteins identifies another MIF-related protein (MRP8) in calf lens. The relatively abundant expression of MIF in lens may have clinical significance, with the possibility of involvement in ocular inflammations that may follow damage to the lens.

Cell cycle synchrony in the developing chicken lens epithelium.

Synchronous oscillations of DNA synthesis and histone 2B mRNA expression occur during normal development of 13- to 16-day-old embryonic chicken lens epithelium. At least four cycles were observed with peak values of DNA synthesis and histone 2B mRNA 5 to 10 times greater than baseline values. Fourier analysis of DNA synthesis identified a statistically significant oscillatory period of 18 hr, the approximate length of the cell cycle at this age. Minor components of 7-9 and 12 hr were also identified in the data sets. Lenses labeled with 3H-thymidine and analyzed by autoradiography at 13.8 days of embryogenesis revealed more than twice the number of labeled nuclei at this time than in lenses labeled 9 hr later; histone 2B mRNA followed this same pattern. These findings demonstrate that a significant population of cells is synchronized with respect to the cell cycle in the developing lens epithelium in ovo. The temporal pattern of mitosis may be the basis of the fiber cell architecture and consequent lens transparency.

Contrasting patterns of c-myc and N-myc expression in proliferating, quiescent, and differentiating cells of the embryonic chicken lens.

The present study uses the polymerase chain reaction and in situ hybridization to examine c-myc and N-myc mRNA in the embryonic chicken lens at 6, 10, 14 and 19 days of development and compares the pattern of expression obtained with the developmental pattern of cell proliferation and differentiation. In the central epithelium, c-myc mRNA levels were proportional to the percentage of proliferating cells throughout development. N-myc mRNA expression in this region was relatively low and showed no correlation with cell proliferation. The ratio of N-myc to c-myc mRNA increased markedly with the onset of epithelial cell elongation and terminal fiber cell differentiation, although both c-myc and N-myc mRNAs continued to be expressed in postmitotic, elongating cells of the equatorial epithelium and in terminally differentiating lens fiber cells. Thus, increased expression of N-myc, a gene whose protein product may compete with c-myc protein for dimerization partners, accompanies the dissociation of c-myc expression and cell proliferation during terminal differentiation of lens fiber cells.