Role In Lens Development Research Articles

Impact of α-crystallin protein loss on zebrafish lens development

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with mutations in each of the three α-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larvae at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated αBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.

Defect of LSS Disrupts Lens Development in Cataractogenesis.

Congenital cataract is one of the leading causes of blindness in children worldwide. About one-third of congenital cataracts are caused by genetic defects. LSS, which encodes lanosterol synthase, is a causal gene for congenital cataracts. LSS is critical in preventing abnormal protein aggregation of various cataract-causing mutant crystallins; however, its roles in lens development remain largely unknown. In our study, we generated a mouse model harboring Lss G589S mutation, which is homologous to cataract-causing G588S mutation in human LSS. LssG589S/G589S mice exhibited neonatal lethality at postal day 0 (P0), whereas these mice showed severe opacity in eye lens. Also, we found that cataract was formed at E17.5 after we examined the opacity of embryonic lens from E13.5 to E18.5. Moreover, disrupted lens differentiation occurred at E14.5 prior to formation of the opacity of eye lens, shown as delayed differentiation of lens secondary fiber and disordered lens fiber organization. In addition, RNA-seq analysis indicated that cholesterol synthesis signaling pathways were significantly downregulated. Overall, our findings provide clear evidence that a mouse model harboring a homozygous Lss G589S mutation can recapitulate human congenital cataract. Our study points out that LSS functions as a critical determinant of lens development, which will contribute to better understanding LSS defects in cataractogenesis and developing therapies for cataracts.

Protective Effect and Mechanism of Bone Morphogenetic Protein-4 on Apoptosis of Human Lens Epithelium Cells under Oxidative Stress.

Bone morphogenetic proteins (BMPs), a member of the transforming growth factor β (TGF-β) superfamily, are abundant in human ocular tissues and play an important role in lens development. Targeted deletion of BMP-4 in mice results in failure of lens placode formation. Following lens maturation, the formation of senile cataracts is demonstrably associated with free radical-related oxidative stress. Previous studies reported that BMPs play an antiapoptotic role in cells under oxidative stress, and the BMP-4 signal is important in inflammation regulation and homeostasis. BMP-4 evidently suppressed the apoptosis of human lens epithelial cells (HLECS) under oxidative stress induced by H2O2. This protective antiapoptotic effect is partly due to a decrease in caspase-3 activity and reactive oxygen species (ROS) level. Furthermore, the expression of activating transcription factor- (ATF-) 6 and Krüppel-like factor- (KLF-) 6 increased under oxidative stress and decreased after BMP-4 treatment.

Lens-specific conditional knockout of Msx2 in mice leads to ocular anterior segment dysgenesis via activation of a calcium signaling pathway

Ocular anterior segment dysgenesis (ASD) is a failure of normal development of anterior structures of the eye, leading to lens opacification. The underlying mechanisms relating to ASD are still unclear. Previous studies have implicated transcriptional factor muscle segment homeobox 2 (Msx2) in ASD. In this study, we used Msx2 conditional knockout (CKO) mice as a model and found that Msx2 deficiency in surface ectoderm induced ASD. Loss of Msx2 function specifically affected lens development, while other eye structures were not significantly affected. Multiple lines of evidence show that calcium signaling pathways are involved in this pathogenesis. Our study demonstrates that Msx2 plays an essential role in lens development by activating a yet undetermined calcium signaling pathway.

Sall1 plays pivotal roles for lens fiber cell differentiation in mouse

Mammals possess four Sall transcription factors that play various roles in organogenesis. Previously, we found that Sall1 is expressed in microglia in the central nervous system, and it plays pivotal roles in microglia maturation. In the eye, Sall1 was also expressed in the developing lens, and we examined its role in lens development. A knock-in mouse harboring the EGFP gene in the Sall1 locus (Sall1-gfp) was used to analyze the Sall1 expression pattern. In Sall1-gfp/wild, EGFP was expressed throughout the presumptive lens at E11.5, and subsequently the expression in the lens epithelium became weaker. After birth, signals were observed in the equator region. The effects of Sall1 knockout on lens development were examined in Sall1-gfp/gfp. Lens sections revealed small vacuole-like holes and gaps in the center of the lens fibers at E14.5. Subsequently, the vacuoles appeared in most regions of the fiber cells. Electron microscopic analysis indicated that the vacuoles were between the fiber cells, leading to huge gaps. In addition, contact between the lens epithelium and apical end of the fiber cell was disrupted, and there were gaps between the adjoining lens epithelial cells. However, gap junction structure was observed by electron microscopic analysis, and immunostaining of Zo1 showed rather appropriate expression pattern. Immunohistochemistry indicated that the major lens transcription factors Prox1 and Pax6 were expressed in relatively normal patterns. However, although the expression of Prox1 and Pax6 decreased in nuclei in the control lens, it remained in Sall1-gfp/gfp. In addition, lower expression level of c-Maf protein was observed. Therefore, Sall1 is strongly expressed in the lens from the early developmental stage and plays an essential role in the maintenance of fiber cell and lens epithelium adhesion.

A novel MAF missense mutation leads to congenital nuclear cataract by impacting the transactivation of crystallin and noncrystallin genes

The transcription factor v-maf avain musculoaponeurotic fibrosarcoma oncogene homolog (MAF) plays an important role in lens development. It contains a unique extended homology region (EHR) in the DNA binding domain. MAF mutations are associated with phenotypically distinct forms of congenital cataract and show different effects on the transactivation of target genes. Mutations in the MAF EHR region were rarely reported and their corresponding phenotype and impact on target genes' transactivation were not evaluated. A three- generation Chinese family with congenital cataract was recruited. The patients in the family present non-syndromic congenital nuclear and lamellar opacities. A novel MAF mutation (c.812 T > A, p.Val271Glu) was identified by targeted next-generation sequencing. The mutation is in highly conserved EHR region of MAF and co-segregates with the cataract in the family. It is predicted to be pathogenic by multiple algorithms and is absent in a control population. Dual luciferase activity assay shows the mutation significantly impair the transcriptional activity of four crystallin genes (CRYAA, CRYBA4, CRYBA1, and CRYGA) and two non-crystallin genes (HMOX1 and KDELR2). Herein, we report a novel missense mutation in the MAF EHR region of the DNA binding domain in a family with congenital cataract. The mutation is associated with non-syndromic bilateral nuclear cataract and impacts the transactivation of cataract associated genes involved in lens structure and stress response.

Eaf2 protects human lens epithelial cells against oxidative stress‑induced apoptosis by Wnt signaling

The tumor suppressor protein ELL-associated factor 2 (Eaf2) serves an important role in lens development and maturation; however, its role in oxidative stress-induced cataract formation remains unclear. In the present study, an in vitro apoptosis model was constructed by treating HLE-B3 cells with 50 µM hydrogen peroxide (H2O2), and was confirmed by flow cytometry. Subsequently, overexpression of Eaf2 was induced in H2O2-induced HLE-B3 cells by ligating Eaf2 cDNA to a pcDNA3.0 plasmid and the role of Wnt3a in the function of Eaf2 was also assessed by inhibiting the expression of the gene in Eaf2-overexpression cells. The expression levels of glycogen synthase kinase 3β, β-catenin, Eaf2, caspase 3, Wnt3a, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein were examined using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Immunocytochemistry was used to locate Eaf2 and Wnt3 protein expression in the H2O2-induced HLE-B3 cells. The results indicated that Eaf2 was able to effectively suppress H2O2-induced apoptosis of HLE cells via inhibition of caspase 3 production and activation of Wnt3a signaling. In addition, knockdown of Wnt3a in Eaf2-overexpression cells evidently counteracted the effect of Eaf2 in antagonizing H2O2-induced apoptosis. Taken together, these findings suggested that Eaf2 may suppress oxidative stress-induced apoptosis of HLE-B3 cells exerted through the activation of Wnt3a signaling.

Anterior segment dysgenesis correlation with epithelial-mesenchymal transition in Smad4 knockout mice.

To explore the molecular mechanisms in lens development and the pathogenesis of Peters anomaly in Smad4 defective mice. Le-Cre transgenic mouse line was employed to inactivate Smad4 in the surface ectoderm selectively. Pathological techniques were used to reveal the morphological changes of the anterior segment in Smad4 defective eye. Immunohistochemical staining was employed to observe the expression of E-cadherin, N-cadherin and α-SMA in anterior segment of Smad4 defective mice and control mice at embryonic (E) day 16.5. Real-time quantitative polymerase chain reaction (qPCR) was performed to detect the expression of Snail, Zeb1, Zeb2 and Twist2 in lens of Smad4 defective mice and control mice at E16.5. Statistical evaluations were performed using the unpaired Student's t-test (two-tailed) by SPSS 11.0 software. Conditional deletion of Smad4 on eye surface ectoderm resulted in corneal dysplasia, iridocorneal angle closure, corneolenticular adhesions and cataract resembling Peters anomaly. Loss of Smad4 function inhibited E-cadherin expression in the lens epithelium cells and corneal epithelium cells in Smad4 defective eye. Expression of N-cadherin was up-regulated in corneal epithelium and corneal stroma. Both E-cadherin and N-cadherin were down-regulated at the future trabecular meshwork region in mutant eye. The qPCR results showed that the expression of Twist2 was increased significantly in the mutant lens (P<0.01). Smad4 is essential to eye development and likely a candidate pathogenic gene to Peters anomaly by regulating epithelial-mesenchymal transition. Twist2 can be regulated by Smad4 and plays an essential role in lens development.

BCAS2 interacts with HSF4 and negatively regulates its protein stability via ubiquitination

Heat shock factor 4 (HSF4) is an important transcriptional factor that plays a vital role in lens development and differentiation, but the mechanism underlying the regulation of HSF4 is ambiguous. BCAS2 was reported to be an essential subunit of pre-mRNA splicing complex. Here, we identified BCAS2 as a novel HSF4 interacting partner. High expression of BCAS2 in the lens epithelium cells and the bow region of mouse lens was detected by immunohistochemistry. In human lens epithelial cells, BCAS2 negatively regulates HSF4 protein level and transcriptional activity, whereas in BCAS2 knockdown cells, HSF4 protein stability was increased significantly. We further demonstrated that the prolonged protein half-time of HSF4 in BCAS2 knockdown cells was due to reduced ubiquitination. Moreover, we have identified the lysine 206 of HSF4 as the key residue for ubiquitination. The HSF4-K206R mutant blocked the impact of BCAS2 on HSF4 protein stability. Taken together, we identified a pathway for HSF4 degradation through the ubiquitin–proteasome system, and a novel function for BCAS2 that may act as a negative regulatory factor for modulating HSF4 protein homeostasis.

Sfrp1 and Sfrp2 are not involved in Wnt/β-catenin signal silencing during lens induction but are required for maintenance of Wnt/β-catenin signaling in lens epithelial cells

During eye lens development, regulation of Wnt/β-catenin signaling is critical for two major processes: initially it must be silent in the lens placode for lens development to proceed, but subsequently it is required for maintenance of the lens epithelium. It is not known how these different phases of Wnt/β-catenin activity/inactivity are regulated. Secreted frizzled related protein-2 (Sfrp2), a putative Wnt-Fz antagonist, is expressed in lens placode and in lens epithelial cells and has been put forward as a candidate for regional Wnt/β-catenin pathway regulation. Here we show its closely-related isoform, Sfrp1, has a complimentary pattern of expression in the lens, being absent from the placode and epithelium but expressed in the fibers. As mice with single knockouts of Sfrp1 or Sfrp2 had no defects in lens formation, we examined lenses of Sfrp1 and Sfrp2 double knockout (DKO) mice and showed that they formed lens placode and subsequent lens structures. Consistent with this we did not observe ectopic TCF/Lef activity in lens placode of DKOs. This indicates that Sfrp1 and Sfrp2 individually, or together, do not constitute the putative negative regulator that blocks Wnt/β-catenin signaling during lens induction. In contrast, Sfrp1 and Sfrp2 appear to have a positive regulatory function because Wnt/β-catenin signaling in lens epithelial cells was reduced in Sfrp1 and Sfrp2 DKO mice. Lenses that formed in DKO mice were smaller than controls and exhibited a deficient epithelium. Thus Sfrps play a role in lens development, at least in part, by regulating aspects of Wnt/β-catenin signaling in lens epithelial cells.

Discrepant Expression of MicroRNAs in Transparent and Cataractous Human Lenses

Age-related cataracts are considered to be a pathological condition that arise as senescence progresses. However, little is known about the function of microRNAs (miRNAs) in the formation of age-related cataracts. The purpose of this study was to identify possible differences in miRNA expression in the central epithelium of transparent and age-related cataractous human lenses. Microarrays were used to determine the miRNA expression profiles of both transparent and cataractous lenses. The results were analyzed by significance analyses performed by the microarray software, and the results were confirmed by stem-loop RT-PCR. Algorithms were used to predict the target genes of the differentially expressed miRNAs. Two hundred and six miRNAs were identified in all human lenses. The top eight miRNAs according to expression levels were miR-184, let-7b, miR-923, miR-1826, miR-125b, miR-1308, miR-26a, and miR-638 in transparent lenses. In contrast, the top eight miRNAs in cataractous lenses were miR-184, miR-1826, let-7b/c, miR-24, miR-23b, miR-923, and miR-23a. The expression levels of 20 miRNAs were increased and the levels of 12 miRNAs were decreased by more than 2-fold in transparent lenses relative to the levels in cataractous lenses. These findings were confirmed by stem-loop RT-PCR. In addition, several genes that were predicted to be targets of the identified miRNAs have been reported to be involved in lens development or cataract formation. The authors report, for the first time, the distinct expression profiles of miRNAs in the central epithelium of transparent and age-related cataractous human lenses. Significant differences in miRNA expression were identified, and the genes targeted by the relevant miRNAs were predicted. The differential expression of miRNAs suggests that these miRNAs have potential roles in lens development and/or cataract formation.

MIP/Aquaporin 0 Represents a Direct Transcriptional Target of PITX3 in the Developing Lens

The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations.

Polymorphisms of the gamma crystallin A and B genes among Indian patients with pediatric cataract

Previous familial studies have reported co-segregation of mutation in gamma crystallin A and B CRYGA and CRYGB genes with childhood cataract. We investigated association of nucleotide variations in these genes in subjects with and without pediatric cataract from India. The study included 195 pediatric subjects including healthy children with no ocular defects and pediatric cataract cases. Subjects were genotyped by PCR-RFLP method for exonic and intronic genetic variations in CRYGA and CRYGB. The association of these polymorphisms with cataract was estimated by two way contingency tables and the risk allele was also analyzed for their functional impact using in silico tools. Results : No significant difference was observed between cases and control subjects for the frequencies of SNPs G198A (Intron A), T196C (Exon 3) of CRYGA and G449T (Exon 2) of CRYGB gene. -47C allele of rs2289917 in CRYGB showed the strongest association with cataract (Odd Ratio-OR=3.34, 95% Confidence Interval-CI 95% =1.82-6.12, P=0.00007). In silico analyses revealed that this polymorphism lies in a phylogenetically conserved region and impacts binding of a transcription factor, viz. progesterone receptor (PR) to CRYGB promoter. rs2289917 risk allele showed a strong association with increased vulnerability for pediatric cataract. The findings suggest that this association may be a secondary phenomenon related to genetic variation playing critical role in lens development during perinatal and/or pediatric growth. Present exploratory study provides a basis for further defining the role of PR as a regulator of CRYG locus in lens formation/transparency.

Aquaporin 0 enhances gap junction coupling via its cell adhesion function and interaction with connexin 50

Both connexin 50 (Cx50) and aquaporin 0 (AQP0) have important roles in lens development and homeostasis, and their mutations are associated with human congenital cataracts. We have previously shown that Cx50 directly interacts with AQP0. Here, we demonstrate the importance of the Cx50 intracellular loop (IL) domain in mediating the interaction with AQP0 in the lens in vivo. AQP0 significantly increased (~20-30%) the intercellular coupling and conductance of Cx50 gap junctions. However, this increase was not observed when the IL domain was replaced with those from other lens connexins. The Cx50-AQP0 interaction had no effect on Cx50 hemichannel function. A fusion protein containing three extracellular loop domains of AQP0 efficiently blocked the cell-to-cell adhesion of AQP0 and attenuated the stimulatory effect of AQP0 on Cx50 gap junction conductance. These data suggest that the specific interaction between Cx50 and AQP0 enhances the coupling of Cx50 gap junctions, but not hemichannels, through the cell adhesion function of AQP0. This result establishes a physiological role of AQP0 in the functional regulation of gap junction channels.

The transcription factor D‐Pax2 regulates crystallin production during eye development in Drosophila melanogaster

The generation of a functioning Drosophila eye requires the coordinated differentiation of multiple cell types and the morphogenesis of eye-specific structures. Here we show that D-Pax2 plays a significant role in lens development through regulation of the Crystallin gene and because Crystallin is also expressed in D-Pax2(+) cells in the external sensory organs. Loss of D-Pax2 function leads to loss of Crystallin expression in both eyes and bristles. A 2.3 kilobase (kb) upstream region of the Crystallin gene can drive GFP expression in the eye and is dependent on D-Pax2. In addition, D-Pax2 binds to an evolutionarily conserved site in this region that, by itself, is sufficient to drive GFP expression in the eye. However, mutation of this site does not greatly affect the regulatory region's function. The data indicate that D-Pax2 acts to promote lens development by controlling the production of the major protein component of the lens. Whether this control is direct or indirect remains unresolved.

09-P038 Pax6 roles in lens development

09-P038 Pax6 roles in lens development

Apoptosis gene profiling reveals spatio-temporal regulated expression of the p53/Mdm2 pathway during lens development

Evidence is emerging for apoptosis gene expression in the lens during development. Therefore, here we used a filter array to assess expression of 243 apoptosis-related genes in the developing postnatal mouse lens using 33P labelled cDNA synthesized from p7 and p14 mouse lenses. We demonstrated that 161 apoptosis-related genes were expressed at levels significantly above background and 20 genes were potentially significantly differentially expressed (P<0.05) by at least 2-fold between p7 and p14. We used RT-PCR to confirm expression of these genes in newborn, p7, p14 and 4 wk mouse lens cDNA samples. Expression of 19/20 of the genes examined was confirmed, while 5 genes (Huntingtin, Mdm2, Dffa, galectin-3 and Mcl-1) were confirmed as differentially regulated between p7 and p14. RT-PCR was also used to examine the expression of the chick homologues of the most-highly expressed and/or potentially differentially regulated genes in chick embryo lenses at E6–E16. The majority of genes expressed in the postnatal mouse lens were also expressed in the chick embryo lens. Western blotting confirmed developmentally regulated expression of Axl and Mcl-1 during mouse lens development and of Mdm2, Mdm4/X and p53 during mouse and chick lens development. Western blotting also revealed the presence of p53 and Mdm4/X splice variants and/or proteolytic cleavage products in the developing lens. Since Mdm2 is a regulator of the tumour suppressor gene p53, we chose to thoroughly investigate the spatio-temporal expression patterns of p53, Mdm2 and the functionally related Mdm4/X in mouse lens development at E12.5–E16.5 using immunocytochemistry. We also examined Mdm2 expression patterns during chick lens development at E6-E16 and Mdm4/X and p53 at E14. Expression of Mdm2, Mdm4/X and p53 was spatio-temporally regulated in various compartments of the developing lens in both mouse and chick, including lens epithelial and lens fibre cells, indicating potential roles for these factors in regulation of lens epithelial cell proliferation and/or lens fibre cell differentiation This study provides a thorough initial analysis of apoptosis gene expression in the postnatal mouse lens and provides a resource for further investigation of the roles in lens development of the apoptosis genes identified. Furthermore, building on the array studies, we present the first spatio-temporal analysis of expression of p53 pathway molecules (p53, Mdm2 and Mdm4/X) in both developing mouse and chick lenses, suggesting a potential role for the p53/Mdm2 pathway in lens development, which merits further functional analysis.

An essential role for FGF receptor signaling in lens development

Since the days of Hans Spemann, the ocular lens has served as one of the most important developmental systems for elucidating fundamental processes of induction and differentiation. More recently, studies in the lens have contributed significantly to our understanding of cell cycle regulation and apoptosis. Over 20 years of accumulated evidence using several different vertebrate species has suggested that fibroblast growth factors (FGFs) and/or fibroblast growth factor receptors (FGFRs) play a key role in lens development. FGFR signaling has been implicated in lens induction, lens cell proliferation and survival, lens fiber differentiation and lens regeneration. Here we will review and discuss historical and recent evidence suggesting that (FGFR) signaling plays a vital and universal role in multiple aspects of lens development.

Macrophage localization in the developing lens primordium of the mouse embryo – An immunohistochemical study

In mammals, macrophages are known to play an important role in lens development. Macrophages in the embryonic lens are positive for F4/80 monoclonal antibody, and, from 10.5 days to 12 days of gestation, numerous macrophages were observed in the ectoderm, lens vesicle, lens cavity and surrounding mesenchymal tissue, phagocytosing and removing degenerating epithelial cells. During primary lens fiber differentiation, the narrowing lens cavity contained numerous macrophages. Most of the macrophages in the cavity attached to the anterior epithelial wall of the lens vesicle, but a few macrophages were found within the lens epithelial cell layer. Conversely, the thickening posterior wall of the vesicle did not contain any positive cells. After the lens cavity was filled, intralental positive cells disappeared. These characteristic localizations of macrophages in the developing lens to remove apoptotic dead cells may indicate that cell death took place mainly in the anterior wall of the lens vesicle, that is, in the lens epithelium.

Developmentally Regulated Expression of KLF6 in the Mouse Cornea and Lens

To examine the expression of transcription factor Kruppel-like factor-6 (KLF6) in the cornea and lens of mouse eyes throughout developmental stages. C57BL/6 mice were set up for timed mating. Embryos on embryonic day (E)10.5, E12.5, E15.5, and E18.5 and eyes from mice on postnatal day (P)0, P7, P11, P15, P30, and P60 were collected for immunofluorescence staining. Transcript of KLF6 was detected by in situ hybridization with digoxigenin-labeled RNA probes. Relative quantitative reverse transcription (RT)-PCR was performed to measure the KLF6 transcript level in eyes enucleated at embryonic stages, and the corneas and lenses isolated at postnatal stages. Staining for the KLF6 protein was observed at E10.5 in the lens pit and at E12.5 in the ectoderm, the mesenchyme, and the lens epithelium. Nuclear staining for KLF6 protein was evident at E15.5 in the corneal epithelium and the stroma. The staining was abundant between E18.5 and P60. In the lens epithelium, nuclear staining was detected at P0, P7, and P11. The staining intensity declined subsequently. The KLF6 transcript was found in the lens pit at E10.5 and in the ectoderm and mesenchyme at E12.5. The KLF6 mRNA level in corneal layers remained relatively constant until P60. In the lens epithelium, it was high during embryonic stages but decreased with development. KLF6 is expressed in both the cornea and the lens in mouse eyes. Its expression in the lens is temporally regulated, suggesting that it has a central role in lens development.