Developmentally Regulated Expression of KLF6 in the Mouse Cornea and Lens

Abstract

purpose. To examine the expression of transcription factor Krüppel-like factor-6 (KLF6) in the cornea and lens of mouse eyes throughout developmental stages.

methods. 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.

results. 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.

conclusions. 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.

Krüppel-like factor-6 (KLF6, also known as Zf9 or CPBP) is a member of the family of Krüppel-like factors, which is composed of 16 nuclear transcription factors sharing a highly conserved C-terminal DNA-binding domain containing three zinc fingers. ^{1 2} The three carboxyl-terminal C₂H₂ zinc fingers interact directly with the promoter of target genes through a GC box element. Cloned originally from cDNA libraries of placenta, human KLF6 mRNA is ubiquitously expressed, with a high level of expression in the liver, lung, intestine, prostate, and placenta. ^{3 4} KLF6 has been shown to play a crucial role in regulation of genes involved in tissue development, differentiation, angiogenesis, hematopoiesis, cell cycle control, and proliferation. ⁵ Target genes of KLF6 reported so far include collagen α1(I), ⁶ keratin 4, ⁷ placental glycoprotein PSG5, ³ urokinase, ⁸ the human immunodeficiency virus long-terminal repeat, ⁹ transforming growth factor (TGF)-β type I and II receptors, ¹⁰ a collagen-specific molecular chaperone heat shock protein 47 (HSP47), ¹¹ nitric-oxide synthase, ¹² leukotriene C4 synthase, ⁷ and endoglin. ¹³  

Recently, KLF6 was found to be expressed in human corneal epithelial cells. ¹⁴ It was also demonstrated that KLF6 can upregulate the transcription of the keratin 12 (K12) gene by binding directly to a CTCCACCCA sequence at the promoter region of the gene in human corneal epithelial cells. ¹⁴ KLF6 thus joins two other transcription factors, Pax6 and ESE-1, as a factor capable of modulating the expression of K12. ^{15 16} Both Pax6 and ESE-1 are expressed in the cornea and the lens, and both have been shown to be involved in lens and corneal differentiation and development. ^{16 17} By contrast, whether KLF6 has a role in corneal and/or lens differentiation or development is still unknown. To gain further insight into the developmental implications of KLF6, we undertook the present study to examine the spatial and temporal expression of KLF6 in the cornea and lens of mouse eyes through various developmental stages. 

Materials and Methods

Mouse Preparation

C57BL/6 mice were raised within the Biological Resources Laboratory of the University of Illinois at Chicago with a 14-hour light cycle and a standard chow diet (Nakamura H, et al. IOVS 2002;43:ARVO E-Abstract 3204). Mice were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For timed pregnancies, the day on which plugs were observed was considered the first day of gestation. The normal gestation period for the mice was 20 or 21 days. Because diurnal variations are known to occur in the expression of transcription factors, including CLOCK and BMAL1, ¹⁸ all animals were killed between 9 and 11 AM. Embryos or eyes were collected on embryonic day (E)10.5, E12.5, E15.5, and E18.5, and postnatal day (P)0, P7, P 11, P15, P30, and P60. 

Immunofluorescence Analysis

Embryos or eyes were fixed at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 6 hours and were processed for paraffin-embedded sectioning. ¹⁹ Four series of paraffin-embedded sections were prepared at each time point. 

For immunohistochemistry, 5-μm-thick paraffin-embedded sections were deparaffinized, rehydrated, and unmasked with a heat-induced epitope retrieval technique with 10 mM sodium citrate buffer (pH 6.0; NeoMarkers, Fremont, CA). Sections were incubated at room temperature sequentially with polyclonal rabbit anti-KLF6 (R-173) antibody (diluted 1:100; Santa Cruz Biotechnology, Santa Cruz, CA) for 90 minutes, biotinylated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) for 45 minutes, and dichlorotriazinyl aminofluorescein (DTAF)–conjugated extravidin (Jackson ImmunoResearch Laboratories) for 45 minutes. For the negative control, sections were incubated with normal rabbit IgG in place of anti-KLF6. After mounting in an aqueous mounting fluid (Vectashield; Vector, Burlingame, CA), the fluorescence staining was scored by three masked observers on a scale of 0 to 4, with 0 indicating no staining and 4 the most intense staining. The observers independently scored the same set of slides using the same microscope (Axioskop; Carl Zeiss Meditec, Jena, Germany), with identical settings and without any quenching of the fluorescence. Two sections were examined for each embryonic and postnatal time point. Experiments were repeated three times. For the purpose of comparison, fluorescence micrographs in each set of experiments were captured (AxioCam; Carl Zeiss Meditec) with the same exposure time. 

In Situ Hybridization

Paraffin-embedded sections were deparaffinized and rehydrated. The sections were digested at 37°C with proteinase K (10 μg/μL; Promega, Madison, WI) for 11 minutes, and postfixed at 4°C with 1% paraformaldehyde in water treated with diethyl pyrocarbonate for 5 minutes. The slides were hybridized at 72°C overnight with digoxigenin-labeled KLF6 sense and antisense riboprobes, synthesized as previously described. ⁴ The tissue sections were further incubated with alkaline-phosphatase–conjugated anti-digoxigenin (Roche Diagnostics, Indianapolis, IN), and the color was developed by reaction with nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl phosphate (NBT/BCIP; Roche). The sections were mounted, and the staining was scored as described for immunohistochemical analysis. Two sections for each embryonic and postnatal time point were included for each experiment, and experiments were repeated three times. The staining intensity was scored by three masked observers. 

Relative Quantitative RT-PCR

Eyes were enucleated at each embryonic stage, and the corneas and the lens were dissected from the eyes at postnatal stages. Total RNA was isolated from embryonic eyes, postnatal corneas, and lenses (Absolutely RNA RT-PCR Miniprep Kit; Stratagene, La Jolla, CA). RT was performed with 0.5 μg total RNA from each sample by using random hexamers and a DNA synthesis kit (SuperScript First-Strand Synthesis System; Invitrogen, Carlsbad, CA). For quantitative analysis of the KLF6 transcript, relative quantitative RT-PCR was performed according to the manufacturer’s instructions (QuantumRNA Universal kit; Ambion, Austin, TX), with KLF6-specific primers (5′-GGACCAAATTCATTCTAGCTCGGG-3′ and 5′-AGGCGTCGCCATTACCCTTG-3′) and primer sets for 18S ribosomal RNA (Ambion) used as an internal standard. PCR reactions were run at 94°C, 64°C, and 72°C for 30 seconds each, followed by a 7-minute extension at 72°C. The number of reaction cycles was 27, 29, and 29 cycles for embryonic eyes, postnatal corneas, and lenses, respectively. The PCR products were visualized on 1.75% agarose gels after ethidium bromide staining. Band intensities were analyzed by densitometry (Digital Science Image Station, model 440CF; Eastman Kodak, Rochester, NY). The KLF6 band intensity was normalized against that of the 18S ribosomal RNA. The expected sizes of amplified DNA fragments for KLF6 and 18S RNA were 384 and 315 bp, respectively. The sequence of the KLF6 PCR product was then verified (CEQ 2000XL DNA Analysis System; Beckman Coulter, Fullerton, CA). 

Results

Developmental Expression of KLF6

By immunofluorescence (Fig. 1) , the KLF6 staining that was distributed throughout the cytoplasm was observed in the lens pit (future lens epithelium) ²⁰ at E10.5 and in the ectoderm (future corneal epithelium), the mesenchyme (future corneal stroma and endothelium), and the lens epithelium at E12.5. At these stages, the nuclei lacked staining. At E15.5, the KLF6 staining was predominantly non-nuclear, although some appeared in the nuclei of the corneal epithelium and stromal keratocytes. The non-nuclear KLF6 staining was also first observed in a layer of mesothelium (corneal endothelium) at this stage. The presence of KLF6 in the nuclei became evident in the corneal endothelium and the lens epithelium at E18.5 (Fig. 1) , whereas the non-nuclear staining remained. In the basal corneal epithelial cells and keratocytes, the nuclear staining of KLF6 was quite abundant throughout stages between E18.5 and P60 (Figs. 1 2) with the non-nuclear staining diminishing after P7. In the lens epithelium, nuclear staining of KLF6 was found at P0 (data not shown), P7, and P11 (Fig. 1) . The intensity of the nuclear staining declined subsequently. The non-nuclear staining also subsided after P0 (Figs. 1 2) . 

In situ hybridization experiments detected positive hybridization products for KLF6 transcript (Fig. 3) in the lens pit at E10.5. The KLF6 transcript was also found in the ectoderm and mesenchyme at E12.5 (Fig. 3) and in the mesothelium at E15.5 (data not shown). The KLF6 mRNA in the lens epithelium was more abundant than that found in the corneal layers during embryonic stages. Compared with the corneal endothelium, the basal corneal epithelium and the stroma contained a higher level of KLF6 transcript. The level in each corneal layer was rather constant between E15.5 and P60, whereas that in the lens epithelium was diminished after P11 (Fig. 4) . The developmental expression pattern was similar to that observed by immunostaining for KLF6 protein levels (Figs. 1 2) . 

Relative Quantitative RT-PCR

The 384-bp KLF6 PCR product was found in all embryonic eyes and in postnatal corneas and lenses. In the embryonic eyes, the intensity of the KLF6 band relative to the 18S band increased between E10.5 and E15.5 and then leveled off. After birth, the KLF6-to-18S ratio in the cornea varied somewhat but stayed within a similar range between P7 and P60. The ratio in the lenses peaked at P0 and gradually declined to a low level at P60 (Fig. 5) , confirming in situ hybridization results. 

Discussion

KLF6/Zf9/CPBP is a zinc finger transcription factor that contributes to mammalian development and physiology. It has been shown to participate in important biological events, including extracellular matrix homeostasis, angiogenesis, cellular differentiation, and proliferation equilibrium and apoptosis. ² By regulating the expression of K12 ¹⁴ and the collagen-specific chaperone HSP47 ¹¹ genes and being involved in the TGFβ pathway, ¹³ KLF6 appears to be a key factor in eye development and maintenance. In the present investigation, we studied the spatiotemporal expression pattern of KLF6 during mouse eye development. Both KLF6 transcript and protein were detected as early as E10.5 in the lens pit and at E12.5 in the ectoderm and mesenchyme. The immunostaining data are consistent with the in situ hybridization and relative quantitative RT-PCR results, suggesting that the changes observed at the protein level are related to the mRNA level under transcriptional regulation. 

By immunofluorescence, distinct non-nuclear staining of KLF6 was observed during embryonic stages in the lens epithelium and until P7 in the corneal epithelium and stroma. Although transcription factors are known to be functional in the nucleus, their subcellular localization out of the nucleus and their shift between the nucleus and cytoplasm have been well documented—for instance, for Yin Yang-1 (YY1), one of the zinc finger transcription factors. ^{21 22 23} This multiple subcellular localization and shift of YY1 have been shown to be related to viral infection, ²¹ cell cycle, ²² and development. ²³ In the eye, the paired homeodomain transcription factor Otx2 was detected in the nuclei of retinal pigment epithelial and bipolar cells. In rod photoreceptors, however, Otx2 was present in the cytoplasm. This pattern of subcellular localization was suggested to participate in the determination of cell fate during retinal development. ²⁴ As for KLF6, there is one report in which non-nucleus localization and translocation between the nucleus and cytoplasm is noted. ²⁵ In this report, KLF6 was found to be distributed throughout the cytoplasm of human umbilical vein endothelial cells and was demonstrated to be shifted from the cytoplasm to nuclei after injury. Our demonstration of the non-nuclear presence of KLF6, especially in early developmental stages, suggests that subcellular localization may participate in the initial differentiation and development of the cornea and lens. 

The developmental processes of the eye are known to be controlled by a complex network of regulatory genes highly conserved throughout evolution. ²⁶ In a previous study, an ancestral homologous gene encoding for KLF6 and KLF7 called Luna has been shown to be expressed during ocular development in Drosophila melanogaster. ²⁷ The conservation of KLF6 expression between fruit fly and vertebrate eyes may argue for a central role for KLF6 in eye development and physiology. 

In nonocular tissues, KLF6 has been shown to be expressed in human placenta ⁴ and in the embryonic gut, heart, lung, and kidney. ²⁸ In the kidney, ²⁹ KLF6 expression could be detected not only during developmental but also at adult stages. Such a stable expression throughout developmental and adult life has also been described for UKLF/KLF7, another member of the KLF family. ²⁸  

Our data indicate that the levels of KLF6 transcripts and protein are relatively stable throughout corneal development. By contrast, KLF6 is more abundant during the embryonic stage than the postnatal stages in the lens. This suggests that KLF6 may be more involved in the developmental regulation of the lens than the cornea. KLF6 may join several other previously described transcription factors to have a role in lens induction or differentiation. There are two principal components of the embryonic eye: the neural ectoderm of the optic vesicle, which forms the retina, and the overlying surface ectoderm, which forms the lens and cornea. Transcription factors such as Otx2, ³⁰ Pax6, ³¹ Foxf1, ³² Lens1, ³⁰ and FoxE3 ³³ have been demonstrated to be essential for the formation of lens placodes from the surface ectoderm, whereas maf, ³⁰ and the Sox ³¹ family are responsible for the initiation of lens differentiation programs, including crystalline expression and cell elongation. KLF6 has also been shown to reduce cellular proliferation significantly ³⁴ in a p53-independent manner. This transcription factor could also be involved in lens cell cycle control, a cellular event that occurs during lens development from the presumptive lens ectoderm. 

To regulate the cornea-specific K12 gene, KLF6 may have to be expressed at a relatively constant level in both the embryonic and postnatal corneal epithelia. Transcription factor activating protein (AP)-2 has also been shown to be expressed in embryonic and postnatal corneal epithelium, regulating genes involved in epithelial differentiation, such as the keratins and cadherins. ^{35 36} In addition, a KLF family member, KLF4, has been described to be a target gene of the AP-2 pathway in induction of terminal differentiation and growth regulation. ^{36 37} The cross-talk between AP-2 and KLF family members including KLF6 could thus represent another level of molecular signaling during corneal differentiation. Furthermore, Pax6, ³⁸ a factor crucial for corneal morphogenesis, ³⁹ regulates epithelial target genes including K12. ¹⁵ The interaction between KLF6 and Pax6 for regulation of K12 and more globally for corneal morphogenesis and homeostasis is thus also of high interest and merits additional investigation. 

Another transcription factor from the zinc finger family, Sp1, is also expressed in the lens and the cornea (Nakamura H, et al. IOVS 2002;43:ARVO E-Abstract 3204) during mouse development. The developmental expression pattern of Sp1, however, is different from that of KLF6. In the lens, the Sp1 expression level is low throughout the embryonic and postnatal stages. In the cornea, Sp1 is abundant in the epithelium and keratocytes from early development to P11. It is markedly downregulated at P15, right after eye lid opening at P13 or P14. The disparity observed in expression patterns suggests that KLF6 and Sp1 have different roles in the development of the lens and cornea. These two factors have been shown to act in concert in regulating the expression of genes such as endoglin. ¹³ It is possible that the changing levels of KLF6 and Sp1 coordinate expression of various genes, thereby influencing the developmental programs. From the expression patterns observed, it appears that KLF6 may be a key factor in the development of lens, whereas Sp1 may be more active in the cornea. 

In conclusion, KLF6 is dynamically expressed during embryonic and postnatal stages in the cornea and the lens. This information is a prerequisite for the understanding of the roles of KLF6 in eye development and physiology. Deficiencies of KLF6 activation due to gene mutations or deletions have been described in prostate cancer. ³⁴ Because expression abnormalities of a few other transcription factors, including Pax6, ⁴⁰ PITX2, ⁴¹ and FKHL7, ⁴² have already been implicated in anterior chamber disorders such as aniridia ⁴¹ and Axenfeld-Rieger syndrome, ^{41 42} the current findings may provide baseline insight for further study of KLF6 in relation to anomalies in the anterior chamber of the eye. 

 Supported by Grants EY03890 (BYJTY) and EY05628 (BYJTY) and Core Grant EY01792 from the National Institutes of Health and a Poste Accueil grant from INSERM (FC).

 Submitted for publication March 30, 2004; revised July 28, 2004; accepted September 8, 2004.

 Disclosure: H. Nakamura, None; F. Chiambaretta, None; J. Sugar, None; V. Sapin, None; and B.Y.J.T. Yue, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Immunofluorescence staining of KLF6 in the cornea and lens of mouse eyes during various developmental stages. The fluorescence staining for KLF6 was visualized with DTAF. The non-nuclear KLF6 staining, which was distributed throughout the cytoplasm, was observed in the lens pit at E10.5 and in the ectoderm, the mesenchyme, and the lens epithelium at E12.5. The presence of KLF6 in the nuclei became evident in the corneal epithelium and stroma at E15.5 and in the corneal endothelium and lens epithelium at E18.5. The nuclear staining in basal corneal epithelial cells and stromal keratocytes was abundant from E18.5 onward to P60, whereas the staining in the lens epithelium declined. Arrow: the lens pit at E10.5 and the lens epithelium at subsequent stages. Arrowheads: the ectoderm at E12.5 and the corneal epithelium at later time points. Double arrows: the mesothelium at E15.5 and the corneal endothelium at later time points. The corneal epithelium (blue box), keratocytes (yellow box), and lens pit or lens epithelium (white box) are shown in higher magnification in insets in appropriate stages. A serial section of the P60 specimen was incubated with normal rabbit IgG instead of anti-KLF6 as a negative control (NC). S, stroma. Bar, 50 μm.

Figure 2.

 

Summary of immunostaining for KLF6 in the mouse cornea and the lens during developmental stages. The intensity of the fluorescence staining was scored on a scale of 0 to 4, with 0 indicating no staining and 4 the most intense staining. (○) Ectoderm at E12.5 or basal corneal epithelium after E15.5, (□) mesenchyme at E12.5 or corneal stroma after E15.5, and (▴) lens pit at E10.5 or lens epithelium after E12.5. Data are expressed as the mean ± SE of scores of the staining intensity. *Non-nuclear staining.

Figure 3.

 

In situ hybridization of KLF6 in the cornea and lens of mouse eyes at various developmental stages. With the antisense probe, positive hybridization products (purplish blue) of KLF6 transcript were detected in the lens pit at E10.5 and in the ectoderm and mesenchyme at E12.5. The KLF6 mRNA in the lens epithelium was more abundant than that in the corneal layers during embryonic stages. The mRNA level in each corneal layer was rather constant between E15.5 and P60, whereas that in the lens epithelium was diminished after P11. Arrow: the lens pit at E10.5 and the lens epithelium at subsequent stages. Arrowheads: the ectoderm at E12.5 and the corneal epithelium at later time points. Double arrows: the corneal endothelium. A serial section of the P7 specimen was incubated with the sense probe as a negative control (NC). S, the stroma. Bar, 50 μm.

Figure 4.

 

Summary of in situ hybridization results. The intensity of the purplish blue staining was scored on a scale of 0 to 4, with 0 indicating no staining and 4 the most intense staining. Symbols are as described in Figure 2 . Data are expressed as the mean ± SE from scores of the staining intensity.

Figure 5.

 

Relative quantitative RT-PCR for KLF6 transcripts in the mouse cornea and the lens. The expected sizes of amplified DNA fragments for KLF6 and 18S ribosomal RNA were 384 and 315 bp, respectively. The KLF6 band intensity was normalized against that of the 18S RNA (Intensity ratio). In embryonic eyes, the intensity ratio increased between E10.5 and E15.5 before leveling off. After birth, the intensity ratio in the cornea varied somewhat but stayed within a similar range between P7 and P60. The ratio in the lenses peaked at P0 and gradually declined to a low level at P60.