|Molecular Vision 2006;
Received 8 September 2006 | Accepted 21 December 2006 | Published 26 December 2006
Gene expression profiling in embryonic mouse lenses
Lisa D. White,2
Donald A. Fox,1
Paul A. Overbeek,2
1College of Optometry, University of Houston, Houston, TX; 2Department of Molecular and Cellular Biology, Department of Molecular & Human Genetics and Microarray Core Facility, Baylor College of Medicine; 3Department of Pediatrics-Hematology & Oncology, Texas Children Hospital, Baylor College of Medicine, Houston, TX
Correspondence to: Qin Chen, M.D., College of Optometry, University of Houston, 4901 Calhoun, Houston, TX, 77204-2020; phone: (713) 743-1998; FAX: (713) 743-2053; email: firstname.lastname@example.org
Purpose: In this study, we used laser capture microdissection (LCM) and microarray hybridization technology to compare the gene expression profiles of mouse embryonic days 10 and 12 lenses (E10 and E12).
Methods: Lens cells of C57/BL6 mouse embryos at E10 and E12 were harvested using the PixCell II LCM System. Total RNA was extracted, amplified, labeled, and hybridized to the 430 2.0 mouse chip (Affymetrix) according to the manufacturer's instructions. Data extracted from the images were analyzed using different software programs. Regulated expression of selected genes was confirmed by real-time PCR (RT-PCR).
Results: Analysis of the microarray data from E10 and E12 lenses identified 1,573 genes that showed a two fold or greater change in expression level. Among these 1,573 genes, 956 genes were downregulated and 617 were upregulated in E12 lenses. In addition to the upregulated expression of beta- and gamma-crystallin genes, genes that regulate the cell cycle showed significant changes of gene expression during the E10 (lens pit) to E12 (primary fiber cell induction) time period. Genes involved in insulin-like growth factor (IGF) signaling and Wnt (a family of secreted glycoproteins related to the Drosophila segment polarity gene, wingless, and to the proto-oncogene, int-1) signaling were also differentially regulated. In particular, positive regulators of Wnt signaling were downregulated and negative regulators were upregulated, indicating that modulation of Wnt signaling is important for normal lens morphogenesis.
Conclusions: Our results provide new information about differential regulation of gene expression during early lens development. Analysis of global gene expression profiles in embryonic mouse lenses has allowed us to identify several molecular pathways that are differentially regulated during early lens development.
The ocular lens provides a classical model system for studying molecular mechanisms that regulate cell proliferation and differentiation during development. The lens arises from the surface ectoderm. The morphogenesis of the mouse lens begins with the formation of the lens placode at embryonic day 9 (E9). At E10, the lens placode begins to invaginate, forming first the lens pit, and subsequently a hollow sphere of epithelial cells termed the lens vesicle. By E12, the cells located at the posterior surface of the lens vesicle are induced to differentiate into primary lens fiber cells. The fiber cells elongate to fill the lens vesicle, while cells in the anterior portion of the vesicle remain as a single-layered epithelium overlaying the fiber cells, thus producing an intact lens with a distinctive polarity [1,2].
Initiation of lens fiber cell differentiation is accompanied by cell cycle exit, upregulation of expression of the cyclin-dependent kinase inhibitor Kip2 (p57), and activation of expression of fiber cell-specific proteins including the β- and γ-crystallins. Many transcription factors, including Pax-6, Six-3, Sox-2, c-Maf, Prox-1, and Sox-1, and the transcriptional coactivators CBP/p300 have been identified to be required for lens fiber cell differentiation and for regulation of crystallin gene expression [1-3]. Fiber cell differentiation is thought to be controlled by a variety of extracellular signals, including exposure to secreted proteins of the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) families . In addition, Wnts and their related signaling molecules may regulate lens development and/or lens morphogenesis. One study showed that a null mutation in a Wnt coreceptor (Lrp6) resulted in the formation of an abnormal lens epithelium , suggesting a positive role for Wnt signaling in the regulation of lens cell differentiation. In contrast, a recent study found that loss of function of β-catenin, a key component of the classical Wnt signaling pathway, did not alter lens fate in central ocular ectoderm . Moreover, overexpression of activated β-catenin in presumptive lens tissue inhibited lens formation , implying that normal lens development requires the critical regulation of canonical Wnt signaling.
To date, target genes (other than transcription factors) that are regulated by these different signaling pathways in lens cells have not been well defined. In particular, little is known about the overall changes in gene expression that occur during the earliest stages of lens cell differentiation. Previously laser capture microdissection (LCM) [6,7] was used to perform transcriptional profiling on mature lens fibers [8,9]. In the current study, we used LCM, RNA amplification, and microarray hybridizations to compare gene expression profiles between E10 and E12 lenses.
C57/BL6 mice were housed according to NIH guidelines (NIH Publication No. 86-23, 1985) at the University of Houston. All experiments were performed in compliance with the ARVO statement for use of animals in ophthalmic and vision research. The male and female mice were put into a mating cage around 5:00 PM. The females were inspected for the presence of a vaginal plug the next morning. The date of the vaginal plug was defined as gestation day zero. Embryos were harvested by dissection at two different embryonic ages, E10 and E12.
The heads of E10 and E12 mouse embryos were frozen in OCT at -80 °C, and were cryo-sliced into 10 μm thick sections. To minimize contamination of lens cells by the adjacent vascular capsule (especially at E12), the tissue slides containing mid-frontal lens sections were selected and fixed in absolute ethanol, washed in DEPC water, and dehydrated serially in ethanol and Xylene. The tissue slides were then dried in a vacuum chamber and used for LCM.
Lens microdissection and RNA extraction
Lens cells were dissected out using laser capture microdissection system (PixCell II LCM System, Arcturus, Sunnyvale, CA). To avoid contamination of lens cells by the adjacent non-lens tissue, the following steps were used during laser capture process: (1) Paper Prepar strips (Arcturus) were used before placing LCM Cap to remove tissues that were not well fixed on the sections; (2) Cells around the edge of lens sections were removed first using control LCM Cap. Total RNA from the dissected lens cells was extracted and purified using the PicoPureTM RNA Isolation Kit (Arcturus) according to the manufacturer's protocol. Independent samples at E10 or E12 were collected by pooling several age-matched lens slides together. Each sample contains 80 to 150 ng of total RNA extracted from 2,000 to 5,000 dissected lens cells. RNA quality was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). For each age group, three independent RNA samples with high quality were used for amplification and labeling for microarray chip hybridization. Another three were used for real-time PCR assays.
Complementary RNA (cRNA) labeling and hybridization for microarray
The quality and size distribution of the RNAs were assessed with the Agilent 2100 Bioanalyzer system using the RNA 6000 Nano LabChip kit (Agilent Technologies). Expression Profiling experiments were performed by the Microarray Core Facility at Baylor College of Medicine. Typically, 15-50 ng of the total RNAs were used to generate biotin-labeled cRNA using Affymetrix two-cycle target labeling kit following the manufacturer's protocol. In brief, total RNAs were first reverse-transcribed using a T7-oligo (dT) promoter primer for the first-strand cDNA synthesis, followed by the second-strand cDNA synthesis to make double stranded cDNA. The double stranded cDNA was used as a template for an in vitro transcription (IVT) reaction to generate unlabeled complementary RNA (cRNA), which was then reverse-transcribed into cDNA using random primers. The T7-oligo (dT) promoter primer was used to generate double stranded cDNAs that contained T7 promoter sequences. The double stranded cDNAs served as a template to generate biotin-labeled cRNAs in the second IVT reaction. The biotin-labeled cRNAs were fragmented and hybridized to the Mouse Genome 430 2.0 array chips (Affymetrix Inc., Santa Clara, CA) for 16 h at 45 °C. After hybridization, arrays were washed and processed using an Affymetrix Fluidic Station F450 following the manufacturer's instructions.
Microarray data collection and analysis
The arrays were scanned with an Affymetrix GeneChip Scanner 3000 7G and the images quantified using Affymetrix GeneChip Operating Software (GCOS). The raw data from two E10 and two E12 samples with good signals were first loaded onto dChip using model-based expression index method (MBEI) [10-12]. The "PM-only model" [10,11] was used for normalization and analysis. Probe sets were selected based on the following criteria: (1) At least one of the mean expression values equal or greater than 50; and (2) fold changes in the mean expression values between E10 and E12 equal or greater than 2. The second line analysis was performed using R with the relevant packages. Normalization and hypothesis test were performed with GCRMA  and LIMMA . The normalized data were filtered based on criteria that the expression values (in log2 scale) were equal or greater than the 10th percentile on at least two chips. A probe set with a q-value, which was equal or less than 0.01, was defined to be significantly different in gene expression. The probe sets selected from dChip were further compared and verified with the selected probe sets from R analysis. Genes with mean expression changes greater than 2.0 were selected, and were further submitted to the NIH gene annotation software DAVID  for gene ontology analysis. The selected genes were classified according to Gene Ontology category "biological process". Some of the individual genes were selected and included in Table 1, Table 2, and Table 3. The preliminary data of selected genes can be found in Appendix 1.
cRNA amplification for real-time PCR
In order to confirm the microarray results, expression levels of selected genes were quantitated by real-time PCR. Three independent RNA samples from each age group were used for linear mRNA amplification to generate the amplified RNA (aRNA) as a template for real-time PCR assay. Two rounds of linear mRNA amplification were performed using the RiboAmpTM RNA Amplification Kit (Arcturus). In brief, each round of amplification was a five-step process: (1) First strand cDNA synthesis was done using 10 ng total RNA and an oligo (dT) primer containing a T7-RNA polymerase binding site. (2) Second strand cDNA synthesis was done using random oligonucleotides and DNA polymerase to generate double stranded cDNAs (ds cDNA). (3) The ds cDNAs were purified using specially designed purification columns. (4) Using T7-RNA polymerase, in vitro RNA transcription was performed to generate cRNA, also termed amplified RNA (aRNA). (5) The residual ds cDNAs were destroyed using DNase, and the aRNAs were isolated using purification columns.
Real-time RT-PCR and data analysis
One ug of aRNA templates from each sample were used to synthesize cDNAs using SuperScriptTM III Reverse Transcriptase kit (Strategene, La Jolla, CA). Quantitative Real-time PCR (qRT-PCR) was performed using an iCycler (BioRad), selected gene primers, cDNA template and iQ SYBR Green Supermix (BioRad, Hercules, CA) under the following conditions: 95 °C for 3 min, followed by 40 cycles of 95 °C for 30 s and 60 °C for 30 s. All reactions were performed in triplicate. β-Actin was included in each assay as a loading control. Primer pairs were designed using Oligo Analysis software (Integrated DNA Technologies, Inc., Coraville, IA). The primer sequences and the length of the corresponding amplified products are shown in Table 4. For each gene, the qRT-PCR experiments were performed with three independent batches of cDNAs. Changes (x-fold) in gene expression level were calculated by the 2ΔΔct method [16,17]. ANOVA statistical analysis was performed using Excel software (Microsoft, Cupertino, WA).
Results & Discussion
Changes in gene expression during primary fiber cell formation
The lens pit at E10 is composed of proliferating lens progenitor cells. By E12, these progenitor cells have developed into an intact lens that contains two types of cells: anterior proliferating epithelial cells and posterior elongating fiber cells. To determine the global patterns of gene expression during this transition, we took advantage of the LCM and Affymetrix microarray techniques. LCM enables the capture and harvesting of defined sets of cells from tissue sections on slides. Using this technique, we dissected lens pits at E10 (progenitor cells before differentiating into epithelial and primary fiber cells) and lens cells at E12 (both epithelial and primary fiber cells after progenitor cell differentiation). Well fixed 10 μm thick tissue sections (single cell layer) and the experimental principle of "adjacent cell removal first" enabled us to minimize the possible contamination with non-lens cells. Total RNAs were extracted from the captured sample cells for in vitro amplification and labeling. Linear RNA amplification was done using T7-RNA polymerase-based technique [6,7,9], followed by labeling and hybridization to the Affymetrix chip. After normalization and analysis of microarray data using the PM-only model in dChip and R packages [10-12], the selected genes were further analyzed using the NIH gene annotation software DAVID  to identify their functional classification. We found that 1,573 genes showed a two fold or greater change in expression in the E12 lens cells compared with E10 lens cells. Among these 1,573 genes, 956 genes showed decreased expression, and 617 showed increased expression in E12 lenses, indicating that downregulation of gene expression is a prominent aspect of the fiber cell differentiation program. Gene expression was verified by qRT-PCR. Eighty out of 1,573 genes were selected for qRT-PCR assay, and half of these were confirmed. Some of the selected qRT-PCR data are shown in Table 5 and Figure 1. According to the Gene Ontology analysis, genes that promote the cell cycle were most commonly downregulated between E10 and E12 (Table 1), consistent with the notion that progenitor cells exit the cell cycle when they initiate differentiation. The selected cell cycle genes and crystallins (markers of lens fiber cell differentiation) will be discussed in detail in the following sections. Genes that are involved in Wnt and insulin-like growth factor (IGF) signaling pathway showed significant changes in expression. Some of these are described in more detail in the next section.
Change of cell cycle-related gene expression
Differentiation of epithelial cells into fibers is accompanied by cell cycle exit, downregulation of cell cycle promoting genes, and upregulation of negative cell cycle regulators. In this study, 16 cell cycle-related genes with fold change in expression values greater than 3 in E12 lenses have been identified, and are listed in Table 1. Among these genes, 15 were downregulated, and one was upregulated at E12.
It has been known that the retinoblastoma (Rb) protein plays an important role in the regulation of lens cell cycle exit and fiber cell differentiation . In one study, immunocytochemistry showed that Rb protein is present in both lens epithelial and fiber cells, with the phosphorylated forms in the epithelial cells and the predominantly hypophosphorylated forms in the fiber cells . In our microarray study, we found that transcription of Rb gene was significantly increased in the E12 lenses (Table 1), indicating that initiation of lens fiber cell differentiation is accompanied by upregulation of Rb expression. Members of the E2F family are key players in the regulation of cell cycle progression. The E2F proteins pair with a heterodimeric partner (Dp1 or Dp2) to form an active complex, which promotes expression of genes involved both in progression through the G1 phase of the cell cycle and in DNA replication. Among the eight E2F genes identified in mammals, E2F3 has been suggested to make a major contribution toward the in vivo phenotypic consequences of Rb protein deficiency . Consistent with this study, we found that E2F3, as well as Dp1, were significantly downregulated at E12 after initiation of lens fiber cell differentiation (Table 1). Cyclins are proteins that positively regulate the cell cycle. For example, cyclin B1/Cdc2 can promote the G2/M transition of the cell cycle. We previously showed that expression of cyclin B1 in mouse lens is restricted to the proliferating epithelial cells at E12 (unpublished data) and E15 . In this study, we found that cyclin B1 expression was significantly downregulated during E10 to E12 transition (Table 1). Since cyclin B1 does not express (or expresses at undetectable level) in the differentiating lens fiber cells , downregulation of cyclin B1 at E12 suggests that genes driving cell proliferation in the lens epithelium are less active than those in E10 lens progenitor cells. Cyclin T2/Cdk9 complexes have been shown to be involved in the regulation of terminal differentiation in muscle cells . We found that in mouse lens cells expression of cyclin T2, like cyclin B1, was also downregulated at E12 (Table 1). Decreased expression of cyclins B1 and T2 was verified using qRT-PCR (Figure 1).
We identified additional cell cycle-related genes that were downregulated including Incenp, Ube2j2, Cks2, Cdc7, Ppp1cc, Ube2c, Clspn, Rad51, Mycbp, Rbbp4, and Rbbp6 (Table 1). Incenp (inner centromere protein) has been found in a complex with the Aurora-B kinase and is required for alignment of metaphase chromosomes during cell division. It has been shown that overexpression of either full-length or an N-terminally truncated mutant of Incenp in Drosophila eye imaginal discs reduces the size of the adult eye . Among the protein serine-threonine kinases, Protein phosphotase 1, catalytic subunit (Ppp1cc) mRNAs were found in both epithelial and fiber cells of adult rat lenses, but the Ppp1cc protein was mainly present in the epithelial cells . Rad51 is an important enzyme in DNA repair and recombination. It has been shown that overexpression of Rad51 in Drosophila eye imaginal discs leads to disruption of cell cycle progression and apoptosis . These studies indicate that Incenp, Ppp1cc, and Rad51 play important roles during eye development. Downregulation of these genes in E12 lenses identified in this study implies that they may be required for lens progenitor cell cycle regulation. Ube2j2, Cks2, Cdc7, Ube2c, Clspn, Mycbp, Rbbp4, and Rbbp6 genes have not been shown to express in the eye and play important roles in lens development. Downregulation of these genes in E12 lenses suggests that some of them may be important in regulation of lens formation.
Taken together, our microarray findings indicate that proliferation of lens progenitor cells is accompanied by higher expression of genes which control the G1/S and G2/M transitions, and that expression of most of these genes decreases when fiber cell differentiation begins.
Expression of fiber cell differentiation markers
Within the developing and the mature lens there is a distinctive pattern of crystallin gene expression . In the mouse, transcripts of αA- and αB-crystallins are observed in the lens pit at E10. At later developmental stages, αA-crystallin transcripts are mainly detected in the fiber cells, while αB-crystallin mRNAs are preferentially present in the epithelial cells . Our microarray data showed that expression of αA-crystallin was highly upregulated at E12 (more than 30 fold increase; Table 2). The β-crystallins can be divided into βA- (A1, A2, A3, and A4) and βB- (B1, B2, and B3) crystallins, and are early markers of lens fiber cell differentiation . We found that expression of βA1-, βA4-, βB1-, and βB3-crystallin genes was highly upregulated in E12 lenses (435-, 17-, 25-, and 35 fold increase, respectively; Table 2). Among the β-crystallin subfamily, βA1-crystallin was more highly expressed than the rest of the β-crystallin family members at E12 (Table 2). βA1-crystallin expression was confirmed by qRT-PCR (Figure 1 and Table 5). The γ-crystallins are encoded by a cluster of six genes (γA-γF-crystallin) . Previously, it had been thought that γ-crystallins were only expressed in lens fiber cells. A recent study shows that these proteins are also present in the epithelial cells . In our studies, expression of γA-, γB-, γD-, and γF-crystallins were detected in E10 lens progenitor cells (Table 2), and was significantly upregulated at E12 (88-, 2-, 19-, and 86 fold increase, respectively; Table 2). γA- and γF-crystallin are the γ-crystallin family members expressed at the highest levels during early primary fiber cell differentiation (Table 2). Taken together, these results indicate that our microarray approach has generated a reliable gene expression profile.
Cell signaling during early embryonic lens development
Lens induction and morphogenesis are complex processes which involve multiple signaling pathways [2,29]. In the case of IGF signaling, it is not clear whether or how this signaling influences lens fiber cell differentiation in vivo. Previous in vitro studies showed that IGF-1 can promote lens cell differentiation and crystallin gene expression [29,30]. In addition, previous studies have detected expression of IGF-1, IGF-2 and their high-affinity binding proteins (IGFbp2-6) in most differentiating rat eye tissues, but not in the lens [31,32]. However, overexpression of IGF-1 in vivo in the mouse lens was found to cause a delay in the differentiation of lens epithelial cells . This observation suggests that endogenous IGF signaling may be involved in the maintenance of lens epithelial cell proliferation, but not differentiation. We identified two other IGF signaling genes with significant changes in expression at E12: IGF2bp1 was downregulated, while IGFbp7 was upregulated. The changes in expression of these genes were verified by qRT-PCR (Figure 1 and Table 5). Although IGFbps have been known to be extracellular modulators of IGF signaling that bind IGFs and regulate their biological activities, it is not yet known how IGFbp7 and IGF2bp1 proteins modulate the IGF signaling. A recent study shows that IGFbp7 highly expresses in colonic adenocarcinoma tissue, and its protein can inhibit cell growth rate and induce apoptosis, suggesting that IGFbp7 may function as a potential tumor suppressor . Upregulation of IGFbp7 expression in mouse E12 lenses implies that IGFbp7 may be required for lens cell cycle exit and initiation of fiber cell differentiation during early lens development. Currently, there is not much information about IGF2bp1 expression pattern and function. Downregulation of this gene in the E12 lenses suggests that it may play a role in the regulation of lens progenitor cell proliferation.
Wnt signaling is quite complex. In the mouse, there are 19 known Wnt ligands and 10 frizzled receptors. There are also at least three signaling cascades: the Wnt/β-catenin pathway, the Wnt/Ca2+ pathway, and the Wnt/planar cell polarity (PCP) pathway. There is evidence that Wnt signaling plays an important role in lens morphogenesis. For example, mice lacking the co-receptor LRP6 have abnormal lens development . In addition, several Wnt ligands (including Wnt2, Wnt2b, Wnt5a, Wnt 5b, Wnt7a, and Wnt7b) and Frizzled receptors (Fzds 1-8) are expressed in developing mouse lenses [4,35]. However, little is known about changes in expression of these genes and their downstream targets. In our microarray study, we detected 17 Wnt ligands and 10 frizzled receptors in dChip with expression values equal or greater than 50 in at least one developmental stage. After analysis using R packages, we identified eight Wnt signaling-related genes that were downregulated in E12 lenses (Table 3). As of now, these genes have not been shown to play important roles in lens development. Particularly, it is not clear whether Ldlr, Mitf, Slc9a3r1, and Catnal1 are truly involved in Wnt signaling. Casein kinase II and Bcl9 have been shown to be positive regulators of Wnt signaling [36,37]. Downregulation of expression of these genes, together with decreased expression of Fzd4 and Fzd7 (Table 3), suggests that Wnt signaling may need to be suppressed during lens fiber cell differentiation. Altered expression of Fzd4 and Fzd7 was confirmed by qRT-PCR (Figure 1 and Table 5). In addition, inhibitors of Wnt signaling (Wif1 and DKK3) were found upregulated at E12 (less than two fold, data not shown). Wnt inhibitory factor-1 (Wif1) can directly bind to Wnt ligands and is a secreted antagonist of Wnt signaling . Dickkopf (DKK) proteins are important negative regulators of β-catenin signaling . In the absence of Wnt, a multiprotein complex containing glycogen synthase kinase-3β (Gsk3β), axin, the adenomatous poliposis coli (APC) protein, and phosphoprotein phosphatase 2A (PP2A) promotes β-catenin degradation [40-42]. Upregulation of Wif1 and Dkk3 further indicates that the Wnt/β-catenin pathway is downregulated when lens progenitor cells are induced to differentiate into fiber cells. This finding is consistent with a previous study showing that overexpression of activated β-catenin can abolish lens formation . Since Lrp6-/- mice show defects in their lens epithelium , our results suggest that either the Wnt/Ca2+ pathway and/or the planar cell polarity (PCP) pathway are critical for Wnt signaling in the lens.
Taken together, the changes in gene expression profiles identified in this microarray study provide new information about differential regulation of gene expression during early differentiation of lens progenitor cells. Our next study will focus on the differences of gene expression profiles between lens epithelial and primary fiber cells. Analysis of the global gene expression profiles in embryonic mouse lenses has allowed us to identify several molecular pathways that are differentially regulated during early lens development. Based on the gene expression information obtained from this study, further investigation will also focus on characterization of the roles for these candidates in regulation of lens development.
This study was supported by NIH grants R03 EY015764 (Q.C.) and R01 ES012482 (D.A.F.) and UHCO's Core Grant (P30 EY007551). We thank Dr. Wei Yu (CodonBioscience, Houston, Texas) for technical assistance.
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