February 2004
Volume 45, Issue 2
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Cornea  |   February 2004
Postnatal Gene Expression in the Normal Mouse Cornea by SAGE
Author Affiliations
  • Barbara Norman
    From the Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
  • Janine Davis
    From the Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
  • Joram Piatigorsky
    From the Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
Investigative Ophthalmology & Visual Science February 2004, Vol.45, 429-440. doi:10.1167/iovs.03-0449
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      Barbara Norman, Janine Davis, Joram Piatigorsky; Postnatal Gene Expression in the Normal Mouse Cornea by SAGE. Invest. Ophthalmol. Vis. Sci. 2004;45(2):429-440. doi: 10.1167/iovs.03-0449.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To provide a detailed gene expression profile of the normal postnatal mouse cornea.

methods. Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization.

results. A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) ω-1, and GST α-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription.

conclusions. In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea.

The transparent cornea is on the outer surface of the eye and serves a role in vision and barrier function. Multiple mechanisms contribute to the transparent and refractive properties of the mammalian cornea that are essential for proper image formation to the retina, an essential step in vision. 1 While allowing light in, the cornea simultaneously functions as a protective barrier for the eye, much like the skin does for the rest of the body. Adverse environmental influences such as light, pollutants (including oxygen), abrasion, and invasion by pathogens require defense systems that reduce chemical and oxidative stress, impart mechanical strength, and provide an immunologic surveillance system. 
Formation of the mature cornea depends on an intrinsic developmental program as well as later extrinsic cues from the environment that initiate after eye opening 2 weeks after birth. The anterior surface of an adult cornea is covered with stratified, squamous epithelium that resides on top of a collagen-rich stroma containing a small number of specialized fibroblastic cells known as keratocytes. A one-cell-thick endothelial layer resides at the posterior surface of the cornea, adjacent to the anterior chamber of the eye. 2 Like other sites in the body covered with stratified squamous epithelium, such as the epidermis, gut, and esophagus, 3 the corneal epithelium undergoes continuous self-renewal, turning over, on average, every 7 to 10 days under normal homeostasis, 4 5 and increasing after injury. 6 7 8 9 10 A delicate balance exists between proliferating basal cells, early differentiated suprabasal cells, and terminally differentiated superficial squamous cells, ultimately proceeding through a complex differentiation program while migrating centripetally from a peripheral reservoir of stem cells and upward to the anterior surface to be sloughed. 11 12 13 14 15 The mature corneal epithelium, therefore, is composed at all times of cells in various stages of differentiation. Approximately 2 weeks after birth, stratification of mouse corneal epithelium commences, with the expansion from 1–2 to 8–10 cell layers by 6 weeks of age. 
The onset of epithelial stratification coincides with two other major postnatal developments in the mouse cornea. First, the eyelid opens exposing the eye to new and changing levels of oxygen, light, moisture, and pathogens. Coincident with eye opening and stratification is the upregulation of several abundant soluble corneal proteins including aldehyde dehydrogenase III (ALDH3) and transketolase (TKT), which constitute 44% and 10% of the total soluble protein of the adult mouse cornea, respectively. 16 17 ALDH3 mRNA is not detected at birth, but increases robustly by postnatal day (PN)14 (Kays and Piatigorsky, unpublished results, 1996). A time course of ALDH3 protein showed very small amounts of ALDH3 protein, beginning at PN9, with a sharp increase at PN13, coincident with the time of eye opening (Davis and Piatigorsky, unpublished results, 2002). A sizable increase in the levels of mRNA and protein for TKT has also been observed in the mouse cornea at PN14. 16 Although environmental and developmental stimuli are thought to contribute to the increase in levels of these proteins, 18 19 the identity of the molecules that are important in their regulation is unknown. 
Indeed, the gene expression profile of the cornea has not been examined in detail yet. Neither the molecular determinants that subserve the various protective and optical functions of the transparent cornea nor the molecular components that distinguish the cornea before and after eye opening are known. Of particular interest is that both the developmental regulation and the function of the transparent cornea involve a tight interaction between intrinsically programmed and environmentally controlled events. This interaction is especially clear by the profound structural (i.e., epithelial stratification) and biochemical (i.e., major increases in ALDH3 and TKT gene expression) changes that take place precisely at eye opening, the time at which the corneal surface comes in direct contact with the oxidative and other stresses of the environment. Consequently, in the present study, we used the powerful method of serial analysis of gene expression (SAGE) 20 to identify the repertoire of genes expressed in the mouse cornea before eye opening at PN9 and in the mature cornea at 6 weeks after birth. SAGE provides several advantages over the more commonly used microarray analysis. It detects without bias all the genes that are expressed, not just those that are represented on the microarray chip, allows quantitative analysis of mRNA contents for each species, and provides probes for further analysis, such as gene isolation, in situ hybridization, and comparison with expressed sequence tags (ESTs) from other sources. 
Our results provide many new findings of fundamental importance that contribute to the molecular description of the mouse cornea. These data give the first gene expression signature for the mature mouse cornea and classify the expressed genes into broad provisional functional classes. In addition, we found that the large repertoire of genes expressed before eye opening was surprisingly different quantitatively from that expressed in the mature cornea. Indeed, two thirds of the expressed genes differed at the two stages, with only one third overlapping the two stages. Of the numerous (n > 200) transcripts in the cornea that change expression levels after eye opening, a small cluster (n = 25) of transcription factors were identified that may mediate corneal development, providing specific directions for future experiments. Finally, in situ hybridization provided new molecular markers for distinct layers of the stratified corneal epithelium. One of these, glutathione S-transferase ω-1, is the first molecular marker to be reported for middle wing cells of the corneal epithelium. In summary, in addition to giving a comprehensive profile of expressed genes in the mature mouse cornea, the present SAGE libraries demonstrate global and unexpectedly dynamic changes in corneal gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development and function, and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. 
Materials and Methods
Corneal Tissue
Corneas were isolated from C57Bl/6 mice killed at PN9 and 6 weeks (adult) after birth. The 6-week-old mice were awake with their eyes open before death. All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The 6-week-old mice were male whereas the sex of the PN9 mice was not determined. Whole eyes were enucleated and rinsed in ice-cold 1× phosphate-buffered saline (PBS), and the cornea was immediately removed (within 10 minutes) under a dissection microscope by introducing a small opening at the limbus with fine forceps and separating the cornea from the conjunctiva and sclera by pulling on either side of the opening. The corneas were trimmed of any remaining noncorneal tissues with a scalpel blade, frozen on dry ice, and stored at −80°C until further use. 
SAGE Library Construction, Sequencing, and Analysis
Total RNA was isolated from 24 adult and 98 PN9 whole corneas, using a total RNA isolation kit (SNAP; Invitrogen, Carlsbad, CA) according to the manufacturer’s directions, except that the corneas were first homogenized in lysis buffer in small glass homogenizers. Each library was constructed from 20 μg of total RNA using a SAGE kit (I-SAGE; Invitrogen) according to the manufacturer’s instructions, except that the amplification of the ditags was performed in two stages. 21 The first round of PCR included 27 cycles of amplification, and the 100-bp product was purified and used as template for the second round of PCR, which included 9 cycles of amplification. 
Bacterial colonies were transferred with sterile toothpicks into 100 μL low-salt antibiotic medium (Zeocin; Invitrogen) and incubated at 37°C for 2 hours. The bacterial culture (5 μL) was amplified with the −47 primer (CEQ2000 Quick Start; Beckman-Coulter, Fullerton, CA) and reverse primer (5′CAGGAAACAGCTATGACC3′) for 30 cycles. Amplified PCR products were purified with a PCR cleanup kit (Qiagen, Valencia, CA) and eluted with 10 mM Tris (pH 8.0). Templates were sequenced with the −47 primer using a sequencing kit (CEQ2000 Quick Start; Beckman-Coulter) on a DNA analysis system (CEQ2000; Beckman-Coulter). 
Sequencing results were analyzed using sequence-analysis software (SAGE2000 ver. 4.0; Invitrogen). Using this software, tag numbers in the two libraries were compared pair-wise with a test based on a Monte Carlo simulation of tag counts. 22 Tag counts were normalized to 62,206 unless otherwise indicated in the table footnotes. The sequences of novel tags (tags that have no database match) are indicated in the “Description” columns. 
Tag-to-gene assignments were made using the reliable mouse tag database of July 2, 2002 (Mus musculus UniGene Build 108; http://www.ncbi.nlm.nih.gov/UniGene; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). In the event of multiple gene assignments, several strategies were used to determine the most likely assignment for a tag. The “probable extra base,” determined by the sequence-analysis software (SAGE2000; Invitrogen), was used to eliminate some of these. Also, if a tag matched only one deposited sequence in a UniGene cluster, that gene assignment was eliminated. If a single most likely gene assignment could not be determined, the tag was eliminated from further consideration. If a gene was represented by more than one tag, the counts of all tags were added to obtain a count for the gene. 
The number of duplicate ditags were 3276 (10.2%) and 1659 (5.3%) in the PN9 and adult libraries, respectively. The GC content of a randomly selected group of tag sequences was 46.7%. 
In Situ Hybridization
Fresh, frozen, 10 μm eye sections from 6-week-old mice were fixed, treated with proteinase K (0.2 μg/mL PBS) for 8 to 10 minutes, and processed for in situ hybridization. Riboprobes were synthesized using a digoxygenin (DIG) RNA labeling kit (Sp6/T7; Roche Molecular Biochemicals, Indianapolis, IN) with linearized, proteinase-K–treated, plasmid cDNA templates. Random sequencing of a total corneal C57Bl/6 cDNA library provided us with cDNAs encoding epithelial membrane protein 1 (EMP; X98403; bases 782-1215), aquaporin 5 (AF087654; bases 960-1533), SLURP1 (NM_020519; bases 24-525), calcyclin (X66449; bases 106-526), calizzarin (NM_009112; bases 16-422), glutathione S-transferase (GST) α-4 (NM_010357; bases 22-437), GST ω-1 (U80819; bases 62-548), annexin A2 (BC003327; bases 31-517), and PSCA (AF319173; bases 14-504). Hybridizations were performed as described. 23 The reaction was allowed to proceed until purple color was visible, approximately 30 to 60 minutes, at which time reactions for both the sense and antisense riboprobes generated from a given cDNA were terminated. 
Results
SAGE Libraries of PN9 and Adult Corneas
SAGE was performed to obtain qualitative and quantitative gene expression profiles from PN9 and 6-week-old adult C57Bl/6 wild-type mouse corneas. These developmental time points were chosen to encompass the period during which the cornea undergoes significant postnatal changes. Several measurements support the fact that these corneal libraries are of high quality (see the Methods section).2425 The two libraries were submitted to the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus as accession numbers GSM13196 and GSM13195. Mouse corneal SAGE data are provided in Supplemental Tables 1A and 1B. The UniGene numbers can also be used to cross-reference specific accession numbers of interest, such as gene microarrays (e.g., Chipset MOE430; Affymetrix, Santa Clara, CA). 
Table 1 shows a summary of the sequenced SAGE libraries. The 64,272 PN9 and 62,206 adult total tags corresponded to 19,544 PN9 and 18,509 adult unique tags. Unique (different) SAGE tags continued to accumulate at the end of our analysis at the rate of 22% in both libraries. This measurement indicated that we had sampled more than 78% of the unique transcripts present in the cornea at each of the two developmental stages. When tags observed only once were eliminated from the analysis to compensate for possible sequencing errors, 6535 PN9 and 5877 adult unique tags with a frequency of more than 1 (n > 1) remained. 
Numerous studies have shown that the frequency of encountering a tag reflects the abundance of the transcript in vivo. 26 27 The most abundant tags occurred 592 (PN9) and 691 (adult) times. However, only a small fraction of unique tags (n >1; 1.0% PN9 and 1.2% adult) were present with more than 100 copies. Of the unique tags, 11.6% (PN9) and 11.8% (adult) occurred between 10 and 100 times, with the remaining 87.4% (PN9) and 87.1% (adult) appearing less than 10 times. Of the unique tags encountered more than once, 92.8% (PN9) and 92.7% (adult) matched known genes or ESTs, whereas the remainder did not match any GenBank sequences. Therefore, 473 and 431 novel transcripts were identified in the PN9 and adult corneas, respectively. The number of unmatched tags in the corneal SAGE libraries are within the range (5%–30%) of unmatched tags reported in other SAGE libraries. 28  
Abundant Tags Reflect Corneal In Vivo Functions
The most abundant tags encountered in the corneal SAGE libraries from the two developmental stages, minus those corresponding to ribosomal genes, are shown in Tables 2A (PN9) and 2B (adult). A ranking, the absolute number of times a tag was encountered (count), a corresponding UniGene number, and a descriptive name, where possible, was assigned to the top 100 tags in each library. In addition, each transcript was placed in a functional category that best represented its primary known or presumed function in the cornea. The categories were selected to reflect the normal physiological function of the cornea. Genes with protein products that play a role in redox, barrier, or transcriptional activities were separated into three categories. A fourth category comprised genes with housekeeping, metabolic, and signal transduction functions (HMT). The last category, termed unknown, represents tags that correspond to named genes with unknown function, ESTs, and novel tags. 
In this very high abundance class, 80% of the tags were expressed in both libraries, most of which were classified in the HMT category (Tables 2A 2B , green). Approximately 9% of the shared transcripts function in redox activities (Tables 2A 2B , yellow), including ALDH3, TKT, cytochrome oxidase 3, and two GSTs. Of the shared genes’ products, 23% contributed to the mechanical strength and barrier properties of the cornea (Tables 2A 2B , red) including thymosin beta 4, several keratins (5, 6a and 6b, 12, and 13), actin, tubulin, procollagen (α-1 and α-2), calizzarin, calcyclin, calpactin, SPARC, destrin, annexins (A1, A2, and A8), epithelial membrane protein1 (EMP1), and aquaporin 5. 
Notably, several shared tags corresponded to genes with unknown function in the cornea (Tables 2A 2B , gray). Six of these tags matched ESTs, one tag represented a novel gene, as no match was found in the database, and three tags corresponded to known members of the Ly-6/urokinase plasminogen activator receptor (uPAR) family (Ly-6/uPAR). These transcripts, corresponding to secreted Ly-6/uPAR related protein 1 (SLURP1), prostate stem cell antigen (PSCA), and hypothetical CD59 antigen containing protein, constitute 0.51% and 1.3% of the total mRNA in the PN9 and adult cornea, respectively. 
Cornea Enriched Genes: Defining the Cornea at the Molecular Level
To reveal the genes that distinguish the cornea from other tissues, each of the two corneal libraries was compared with eight publicly available mouse SAGE libraries from limb, brain, heart, and embryonic stem cells. A tag was considered enriched in the cornea if it was encountered five times more frequently in the corneal library under examination than in each of the other eight libraries. The corneal libraries contained 334 PN9 and 339 adult tags fitting this description; 172 of these tags were expressed in both stages of development. A complete tabulation of the cornea enriched genes is available in Supplemental Tables 2A and 2B
The proportion of enriched genes from the adult library comprising each of the major functional divisions is charted in Figure 1 . More than half of the enriched genes were placed in the unknown function category, whereas the remainder represented genes with known functions, many of which have been shown previously to play important roles in the cornea. For example, the cornea-specific roles of keratin 12, BIGH3, mucin 4, 29 keratocan, and lumican 30 31 are well documented. Of the enriched genes, 17% mediated housekeeping functions, 18% contributed to barrier integrity, and 4% were involved in redox activities. A small, but significant fraction of enriched genes appear to play a role in regulating gene transcription (described later). A similar functional distribution of corneal-enriched tags was observed at PN9. 
Novel Expression Patterns of Select, Abundant Cornea Enriched Genes
The localization of several of the genes that were both abundant and enriched in the adult cornea was examined by in situ hybridization, using cDNAs that were derived independently from the SAGE data. The abundance of these genes’ transcripts suggested by SAGE was corroborated by the short amount of time (30 minutes) required for the hybridization signal to develop. 
A diverse collection of expression patterns was observed for the nine genes examined, using antisense riboprobes (Fig. 2) . The control sense PSCA riboprobe did not hybridize to the cornea (Fig. 2I) , nor did any of the other sense riboprobes tested (data not shown). SLURP1 and PSCA (Figs. 2A 2B , respectively), two Ly6/uPAR family members, were expressed throughout the cornea. A hybridization signal of uniform intensity was observed in the cells of epithelium, keratocytes, and endothelium. In contrast, three calcium-binding proteins—calcyclin (Fig. 2C) , calpactin (Fig. 2D) , and annexin 2 (data not shown)—were restricted to the epithelial portion of the cornea where expression was homogeneous in all layers of the epithelium. Aquaporin 5, EMP1, GST ω-1, and GST α-4 were also localized exclusively to the epithelial portion of the cornea. Moreover, their expression patterns were restricted to different layers of the epithelium. Aquaporin 5 mRNA was observed in all anterior cell layers including the suprabasal cells; however, it was not detected in the basal cell layer of the epithelium (Fig. 2E) . EMP1, in contrast, was expressed at detectable levels throughout the epithelium, but highly enriched in the most superficial cells (Fig. 2F) . The most distinctive patterns of expression were for the two GSTs: GST ω-1 (Fig. 2G) was expressed only in the most superficial, epithelial cell layer, while GST α-4 (Fig. 2H) mRNA was found in the center, but not the basal or the superficial layers of the corneal epithelium. 
Developmentally Regulated Genes
The genes that are shared or expressed differentially at PN9 and adult stages are shown in a Venn diagram (Fig. 3) . Collectively, the two libraries identified 8929 unique (n > 1) sequence tags. Whereas 3483 (39%) of the 8929 unique tags were observed at both developmental stages, 3052 (34%) and 2394 (27%) were identified exclusively in PN9 or in adult corneas, respectively. 
Statistically significant differences between the gene profiles of the two developmental stages were observed. Two hundred and five transcripts were identified that showed differential expression (at least a fourfold difference with a P ≤ 0.01). A total of 109 transcripts were downregulated and 96 transcripts were upregulated in the adult relative to the PN9 cornea. These transcripts are shown in Supplemental Tables 3A and 3B. Differences in expression levels ranged from 4- to 143-fold (change multiples for each tag are listed in the supplemental tables) with the most dramatic changes occurring in the downregulated transcripts. ESTs accounted for 34% and 35% of the up- and downregulated transcripts, respectively. No sequence match was found for 6 (upregulated) and 15 (downregulated) tags, indicating that a significant number of putative novel genes show temporal changes in their expression levels. Any biological significance of these differences (or an apparent lack of a difference) in gene expression must be qualified by considering that the cornea is made up of different cell types whose proportions change over the period examined in this report. More information is needed to determine whether these differences reflect changes in expression levels per cell type. 
The differentially expressed transcripts were assigned to one of the functional categories described earlier (Fig. 1) . Although the functions of the majority of the differentially expressed transcripts were unknown, the assignment of the remaining transcripts revealed some general trends in cellular processes over this period. Most of the genes that were expressed at greater levels at PN9, relative to adult stages, were in the barrier integrity category. It appears that the extracellular stroma is synthesized early, in that six different procollagens subunits along with lysyl oxidase, glypican3, Ednra, and lumican are downregulated in the adult cornea. In contrast, a greater number of genes devoted to housekeeping/signal transduction/general metabolism were expressed at greater levels in the adult versus the PN9 cornea. The second most abundant class of differentially expressed genes was that involved in regulating gene transcription. 
Identification of Transcripts that Regulate Gene Transcription
Two hundred and thirty-four different genes (n > 1) involved in transcriptional regulation were detected in the combined libraries. A complete list, including the UniGene number, gene name, and abundance of each of these transcripts can be found in Supplemental Table 4. Genes that mediate both site-specific and global transcriptional regulation were identified, as were proteins that modulate transcription independent of DNA-binding such as coactivators and repressors and binding proteins. Also included in this category were general transcription components, such as RNA polymerases and elongation factors. 
A large number of the transcripts contained an Ets-, helix-loop-helix (HLH)-, zinc finger-, homeobox-, or basic leucine zipper (bZIP)-domain (Table 3) . Also, members of the interferon regulatory factor (IRF), SOX, and nuclear receptor families were well represented (Table 3) . The six most abundant transcriptional regulators at PN9 included myocyte enhancer factor 2 (MEF2; n = 38), kruppel-like factor 4 (KLF4; n = 33), Pax 6 (n = 28), retinoblastoma binding protein 4 (n = 25), cold shock domain protein A (n = 25), and HMG17 (n = 25). The six most abundant transcriptional regulators at the adult stage included IRF1 (n = 51), KLF4 (n = 48), serum response element binding factor-2 (SREBF-2; n = 25), Ets-like factor 3 (ELF3; n = 22), MEF 2 (n = 22), and suppressor of variegation 3-9 homolog 1 (n = 22). 
The prevalence of cornea enriched transcriptional regulators was examined by comparing the PN9 and adult corneal libraries to eight, non-eye mouse SAGE libraries as described earlier. Fifty-seven transcripts were enriched at least threefold in the cornea: 17 exclusively at PN9 and 15 exclusively at the adult stage (Table 4)
To identify transcription factors that may regulate postnatal corneal development, transcripts that showed differences in their levels of expression at the two developmental stages (at least twofold with P < 0.01) were investigated. The 12 transcripts with increased levels in the adult relative to PN9 cornea are listed in Table 5A , and the 13 transcripts with increased levels in the adult relative to the PN9 cornea are listed in Table 5B
Discussion
The expression profiles of the mouse cornea before and after eye opening revealed that the PN9 and adult corneal transcriptome is composed of at least 19,544 and 18,509 unique mRNAs, respectively. This number underestimates the true number of genes for several reasons. 32 First, we estimate that 78% of the unique tags were sequenced in this study. Second, the two libraries collectively identified 8929 different (n > 1) sequence tags. Although a sizable fraction of these tags appear to be expressed exclusively during one of the two developmental periods, it is possible that some are expressed at very low levels during the other developmental stage and simply were not encountered in this analysis. 25 This is particularly important, considering that 2755 PN9 and 2195 adult stage-specific tags occur themselves at low frequencies (two to four times). Notably, 758 tags from the two libraries had no match in any public database, and therefore represent novel genes. Similar to the SAGE of human normal 33 and Fuch’s dystrophy corneal endothelium, 34 the expression profiles from this study reveal a characteristic fingerprint of the mouse cornea. 
Representatives of the major functional classes of the cornea are among the 100 most abundant transcripts. More than half of these transcripts fall into the HMT category with more than 40 encoding distinct ribosomal components, reflecting a major requirement for protein synthesis. Abundant genes that may play prominent roles in cell signaling in the cornea include calmodulin-like protein, 35 Rack-1, 36 and stratifin. 37 38 Components of other signaling pathways including Notch, 39 40 Wnt, 41 TGF, 42 FGF, 43 Src kinase, 44 and EGF/ERB-B2 45 46 were present at lower amounts. The importance of some of these pathways in eye development has been described, 47 48 49 50 51 52 53 54 but their precise role in the postnatal cornea awaits further investigation. 
A significant number of the transcripts encode structural proteins that presumably impart mechanical strength and barrier properties to the cornea. Corneas lacking K12, one of the five different keratins to appear in the top 100 most abundant transcripts, are fragile, underscoring the importance of this corneal-specific keratin in conferring mechanical strength to the cornea. 55 A cytoskeletal role for microtubules is attested to by the abundance of transcripts encoding actin, myosin, tubulin, and the actin-binding proteins thymosin beta 4 56 and destrin. 57 Likewise, the abundant procollagens are pivotal to the formation of the tough, extracellular-rich stroma. 58 59 The importance of calcium in cellular signaling 60 and in epidermal stratification 61 62 63 is well documented, and the presence of several abundant calcium-binding proteins including calizzarin, calcyclin, and calpactin and the possible role of MEF2 in mediating calcium-dependent signaling pathways 64 portends a significant role for calcium in the cornea. Although EMP1 has been implicated in regulating cell growth and differentiation, 65 66 67 the intense localization of EMP1 to the most superficial layer of the corneal epithelium suggests that it may play a role in barrier formation as has been shown for another family member. 68 Proper water balance in the cornea maintains transparency and is mediated by water channels located principally in the endothelium. 69 Although aquaporins-1, -2, and -3 were identified in the SAGE library, the role of the abundant aquaporin 5 is unclear, as it was shown to localize exclusively in the upper layers of the corneal epithelium. 
More than one tenth of the 100 most abundant corneal transcripts are involved in defensive mechanisms that reduce the detrimental effects of environmental insults 70 known to be factors in corneal disease. 71 Most of these transcripts, including ALDH3, TKT, GST ω-1 and GST α-4, ferritin heavy chain, and hexokinase 1, were increased after eye opening suggesting that exposure to environmental stimuli induced this category of transcripts. ALDH3 and TKT mRNAs represent approximately 1% each of the total mRNA in the adult cornea by SAGE. Corroborating the numbers obtained by SAGE, 2.9% and 0.5% of 1103 randomly selected clones from a traditional cDNA library, constructed from adult mouse total cornea, corresponded to ALDH3 and TKT, respectively (Norman B, Davis J, Piatigorsky J, unpublished data, 2001). K12 was the most abundant sequence in the cDNA screen, representing 3% of the total clones. However, ALDH3 and TKT constitute 20% to 40% of the total water-soluble protein, 16 17 suggesting that their expression is under posttranscriptional regulation and/or that protein stabilization is a major regulatory mechanism controlling the protein profile in the cornea. A yeast analysis showed no correlation between levels of mRNA and levels of the corresponding protein, 72 supporting the idea that quantitative mRNA data, as generated by SAGE, may not reflect corresponding protein levels. 
The nonoverlapping pattern of expression of the two abundant GSTs is, to the best of our knowledge, unprecedented in the cornea. Expression in two separate layers of the anterior corneal epithelium suggests an underlying biochemical difference between these layers of epithelial cells. The promoters of these two genes may be useful in revealing a differentiation program in the cornea similar to that observed in skin. 73 Although these two GSTs are members of the phase II detoxification enzyme family important in xenobiotic inactivation, 74 75 76 they may serve different functions in the cornea—both bind glutathione, but only GST α-4 is enzymatically active. 77  
We were unable to assign a corneal function to three abundant transcripts belonging to the Ly6/uPAR family. 78 79 80 81 PSCA, a membrane protein, is best known as an indicator of prostate cancer. 82 No function has been described for the abundant hypothetical CD59 antigen containing protein; however, its namesake CD59 83 is an important inhibitor of the complement cascade in the eye. 84 SLURP1 is a secreted Ly6/uPAR family member resembling snake venom neurotoxins. 85 Mutations in SLURP1 and a newly discovered relative, SLURP2, which is present in lower amounts in the corneal libraries, are associated with the skin diseases Mal de Meleda 86 and psoriasis vulgaris, 87 respectively. The overlap between genes expressed in the cornea and in the skin, such as SLURP1 and -2, calmodulin-like protein, and stratifin, is noteworthy. 88 89 90 Several transcription factors were identified in the corneal SAGE libraries that have known roles in epidermal development, including mOVO, 91 CUX, 92 TSC-22, 93 grainyhead, 94 95 and p63. 96 97  
More than 200 genes that regulate transcription were identified by SAGE and one third of them are enriched in the cornea. The abundant Ets, KLF, and IRF families of transcription factors stand out. Ets factors are important in the transcriptional regulation of genes involved in diverse biological functions. 98 99 Among the six ets family members found in this study, four are enriched in the cornea. ELK3 and Ets domain protein are expressed exclusively at PN9, whereas two others, ELF3 and ets homologous factor, are two of the most abundant transcription factors in the cornea at both developmental stages. ELF3, an epithelium-specific ets transcription factor, 100 regulates expression of genes associated with differentiation 101 and may play a similar role in corneal epithelial differentiation. 102 ELF3 is also essential for normal gut and uterine epithelial cell maturation. 103 Other transcription factors pivotal to intestinal epithelial cell development 104 are also expressed in the cornea, including Nkx2.3, Tcf-4, Pax6, and KLF4, 105 perhaps reflecting a common course of self-renewal throughout life. 
The abundant KLF4 is one of the six members of the kruppel-like factor (KLF) zinc-finger–containing family members expressed in the mouse cornea as well as in the epithelia of skin, lung, and the gastrointestinal tract. 106 107 Goblet cells in the colon fail to differentiate in KLF4 null mice, 108 and the mice die shortly after birth because of a perturbation in the cornified envelope, a late-stage structure in epidermal differentiation necessary to establish the barrier function of the skin. 109 Other studies suggest that KLF4 may also have an important function in regulating growth-specific genes in epithelia. 110 111 The role of KLF4 in the cornea has not been investigated. 
The importance of an immune surveillance system in the cornea is suggested by the wide variety and abundance of IRFs, 112 113 expressed in the SAGE libraries. IRF1, 114 the most abundant IRF in the SAGE libraries, is an important regulator of IFN-mediated antibacterial response. 115 IRFs may also regulate the cell cycle, cell transformation, and apoptosis. 116  
The ability of the cornea to respond to continuously changing environmental conditions is suggested by the expression of redox-, hypoxia-, and light-sensitive transcriptional regulators whose binding sites reside in several corneal gene promoters. 19 117 118 119 Numerous transcriptional components of two major universal response pathways mediating oxidative stress, 120 121 NF-κB (tags for p50, p65, nuclear factor of activated T-cells 5, I-κBα, Bcl, and IKKβ) and AP-1 (tags for JunB; Mafs K, B, F, and G; JDP2; Fra2; and Nrf1), were identified in the SAGE corneal libraries. AP-1 and NF-κB play other major roles in inflammation, 122 123 cell differentiation and proliferation, and cell death. 124 The importance of these factors in eye development is illustrated by the phenotypes that develop when their expression is altered. 125 126  
The PAS-domain–containing genes, hypoxia-inducible factor1a (HIF1a) and period 1 and 2 (Per1 and 2), considered to be environmental sensors of oxygen and light, 127 128 129 respectively, were detected in the corneal libraries. Of particular interest is the observation that ALDH3 expression is downregulated under hypoxic conditions. 19 118  
Finally, we have identified 25 transcriptional regulators exhibiting differential expression during the developmental period examined in this study. Further studies are needed to examine their role(s) in epithelial stratification and in the regulation of the abundant corneal proteins, ALDH3 and TKT. 
In conclusion, the present SAGE libraries provide a foundation for the complexity and a catalog of genes expressed in the mouse cornea, including many genes implicated in corneal disease. The analysis shows a surprising dynamism of gene expression patterns in the postnatal cornea, while delineating a genomic signature of the mouse cornea. Finally, and most important, it has opened a plethora of avenues to pursue for understanding the development, maintenance, and disease of the mouse cornea. 
 
Table 1.
 
Summary of SAGE Libraries
Table 1.
 
Summary of SAGE Libraries
Total Tags Unique Tags Unique Tags (n > 1)
PN9 64,272 19,544 6,535
ADULT 62,206 18,509 5,877
Table 2A.
 
Most Abundant Genes in PN9 Cornea
Table 2A.
 
Most Abundant Genes in PN9 Cornea
  Most Abundant Genes in PN9 Cornea
Table 2B.
 
Most Abundant Genes in Adult Cornea
Table 2B.
 
Most Abundant Genes in Adult Cornea
  Most Abundant Genes in Adult Cornea
Figure 1.
 
Functional categories of the cornea enriched genes. A total of 339 adult sequence tags were considered to be enriched in the cornea (defined as five times more frequent in the corneal library than in eight other mouse SAGE libraries). Of the enriched genes, 58% were placed in the unknown function category, which contains tags that correspond to named genes with unknown function, ESTs, and novel tags. Genes encoding proteins with functions relating to barrier, HMT, transcription, or redox activities represent 17%, 17%, 4%, and 4%, of the remaining enriched genes, respectively.
Figure 1.
 
Functional categories of the cornea enriched genes. A total of 339 adult sequence tags were considered to be enriched in the cornea (defined as five times more frequent in the corneal library than in eight other mouse SAGE libraries). Of the enriched genes, 58% were placed in the unknown function category, which contains tags that correspond to named genes with unknown function, ESTs, and novel tags. Genes encoding proteins with functions relating to barrier, HMT, transcription, or redox activities represent 17%, 17%, 4%, and 4%, of the remaining enriched genes, respectively.
Figure 2.
 
Localization of abundant transcripts in the cornea by in situ hybridization. Expression of several abundant tags was investigated in adult mouse corneas by in situ hybridization using an antisense and sense riboprobe for each transcript. SLURP1 (A) and PSCA (B) mRNAs were detected in all cells of the cornea. Calcyclin (C) and calpactin (D) mRNAs were limited to the corneal epithelial cells. mRNAs for aquaporin 5 (E), EMP1 (F), GST ω-1 (G), and GST α-4 (H) were preferentially expressed in distinct corneal epithelial layers. No signal was detected when a sense PSCA riboprobe (I) was hybridized with adult cornea. No hybridization signal was observed for any of the other sense riboprobes (data not shown). ce, corneal epithelium; s, stroma; cn, corneal endothelium.
Figure 2.
 
Localization of abundant transcripts in the cornea by in situ hybridization. Expression of several abundant tags was investigated in adult mouse corneas by in situ hybridization using an antisense and sense riboprobe for each transcript. SLURP1 (A) and PSCA (B) mRNAs were detected in all cells of the cornea. Calcyclin (C) and calpactin (D) mRNAs were limited to the corneal epithelial cells. mRNAs for aquaporin 5 (E), EMP1 (F), GST ω-1 (G), and GST α-4 (H) were preferentially expressed in distinct corneal epithelial layers. No signal was detected when a sense PSCA riboprobe (I) was hybridized with adult cornea. No hybridization signal was observed for any of the other sense riboprobes (data not shown). ce, corneal epithelium; s, stroma; cn, corneal endothelium.
Figure 3.
 
Overlapping and nonoverlapping transcripts. Collectively, the two libraries identified 8929 unique (n > 1) sequence tags. Whereas 3483 (39%) of the 8929 unique tags were observed at both developmental stages, 3052 (34%) and 2394 (27%) were identified exclusively in PN9 and adult corneas, respectively.
Figure 3.
 
Overlapping and nonoverlapping transcripts. Collectively, the two libraries identified 8929 unique (n > 1) sequence tags. Whereas 3483 (39%) of the 8929 unique tags were observed at both developmental stages, 3052 (34%) and 2394 (27%) were identified exclusively in PN9 and adult corneas, respectively.
Table 3.
 
Transcription Factor Families Expressed in Mouse Cornea
Table 3.
 
Transcription Factor Families Expressed in Mouse Cornea
ETS: ELF3
Ets homologous factor
Ets variant gene 6
ELK3
Ets domain protein
Est 2
HLH: SREBF 1 and 2
HEB
E47
Tcf4
ID 1 and 2
USF2
HES1
Max
Max binding protein
Mad2
Per 1 and 3
Hif 1a
Dec 1 and 2
Lisch7
TFEB
TFE3
Mitf
Zinc finger: KLF 3, 4, 5, 6, 13, 16
Ring finger protein 4
MAZ
mOVO
Odd-skipped related 2
YY1 associated factor 2
Schnurri2
ZNF36
ZFP 62, 100, 103, 106, 144, 162, 185, 207, 216, 238, 276, 319, 326
LIM only 4
CLIM
Four and a half LIM domains
Homeobox: PAX6
ES cell derived homeobox
TGIF
CUX
PHTF1
Hox1.6
IRX3
PRX1
NKX2.3
bZIP: Jun, JunB, JunD
JDP2
Fra2
MafK, G, B, F
NFR1
CREB 2 and 3
ATF 1 and 2
OASIS
Nuclear receptor: Rev-erbA-α
EAR2
RXR, γ
Estrogen related receptor, α
N-CoR 1 and 2
Thyroid hormone receptor α
Nuclear receptor coactivator 1 and 4
CAR
Nur77
SOX: Sox 7, 15
IRF: IRF 1, 3, 6, 7
Table 4.
 
Cornea Enriched Transcriptional Regulators
Table 4.
 
Cornea Enriched Transcriptional Regulators
Adult Count PN9 Count Total Unigene Description
48 33 81 4325 KLF4
51 21 72 1246 IRF1
22 17 39 3963 ELF3
19 10 29 10724 Ets homologous factor
12 11 23 36894 HEB
13 7 20 30262 KLF5
12 8 20 1167 JunB
11 9 20 193097 Rev-erbA-alpha
11 9 20 4179 IRF6
10 8 18 968 Oxysterols receptor LXR-beta
4 13 17 3608 PAX6
11 3 14 162 MafK
13 13 3233 IRF7
13 13 221168 Y-box binding protein (oxyR)
7 4 11 10723 Per3
11 11 3931 Max
6 4 10 1161 ZFP185
10 10 67919 MafB
9 9 31170 Cbp/p300-interacting transactivator 2
4 4 8 176369 SOX15
2 6 8 25552 Grainyheadlike 1
8 8 195896 ES cell derived homeobox
8 8 27887 LAF4
7 7 38016 SREBF2
7 7 34510 Ets-domain protein
7 7 28170 NF-kappa-B, p65
6 6 23095 Chromatin accessibility complex 1
6 6 231867 Retinoblastoma-binding protein 1
4 2 6 2229 Eya2
4 2 6 25762 TFE3
3 3 6 27962 ZFP238
3 3 6 26587 Thyroid hormone receptor alpha
6 6 116802 NFAT1
6 6 3420 NF-kappa-B, p50
5 5 3146 Transcription factor 20
5 5 193526 Cold shock domain protein A
3 2 5 38323 mOVO
3 2 5 27663 Transcription elongation factor B3
2 3 5 334 Sox6 binding protein
5 5 7373 Per1
5 5 4454 ELK3
4 4 103560 JDP2
4 4 89172 Grainyheadlike 2
4 4 34554 E2F4
2 2 4 39039 IRX3
2 2 4 23704 Fra2
4 4 29891 Forkhead box O1
4 4 2032 Interferon dependent 3 gamma
4 4 3879 Hif1a
3 3 20827 Homeodomain-IP kinase 1
3 3 12891 ZFP326
3 3 42162 SOX7
3 3 28269 I-kappa-B kinase beta
3 3 4067 Lisch7
3 3 33797 HDAC 7A
3 3 180561 RBP-J kappa
3 3 10353 Oasis
Table 5A.
 
Transcriptional Regulators Differentially Down-Regulated in Adult Cornea
Table 5A.
 
Transcriptional Regulators Differentially Down-Regulated in Adult Cornea
PN9 Count Adult Count Fold P Unigene Description
12 0 12 0.0003 153618 KOX17
11 0 11 0.0006 3931 Max
10 0 10 0.001 67919 MafB
8 0 8 0.005 21534 Interleukin enhancer binding factor 2
8 0 8 0.005 195896 ES cell derived homeobox
7 0 7 0.008 25703 FUSE binding protein
7 0 7 0.008 34510 Ets-domain protein
7 0 7 0.008 1068 Bcl3
15 3 5 0.005 16421 HMGB1
28 6 4.7 0.0002 3608 PAX6
18 4 4.5 0.003 42240 ZFP162
25 6 4.2 0.0006 911 HMGN2
18 5 3.6 0.007 641 CREB2
Table 5B.
 
Transcriptional Regulators Differentially Up-Regulated in Adult Cornea
Table 5B.
 
Transcriptional Regulators Differentially Up-Regulated in Adult Cornea
PN9 Count Adult Count Fold P Unigene Description
0 10 10 0.0007 20827 Homeodomain-interacting protein kinase 1
0 9 9 0.002 31170 Cbp/p300-interacting transactivator 2
0 8 8 0.003 2436 Dec1
0 8 8 0.003 8884 I-kappa-B, alpha
2 13 6.5 0.003 3233 IRF7
0 6 6 0.01 25877 PHTF1
0 6 6 0.01 20927 TSC22
0 6 6 0.01 28261 Nuclear receptor coactivator 4
0 6 6 0.01 34554 E2F4
3 13 4.3 0.009 22688 Tumor susceptibility gene 101
21 51 2.4 0.0001 1246 IRF1
11 25 2.3 0.01 38016 SREBF2
Supplementary Materials
Supplemental Table 1A - (1.20 MB) Tag Counts From PN9 Mouse Cornea SAGE Library 
Supplemental Table 1B - (1.14 MB) Tag Counts From Adult Mouse Cornea SAGE Library 
Supplemental Table 2A - (40.5 KB) Genes 5X Enriched in the PN9 Cornea 
Supplemental Table 2B - (40.5 KB) Genes 5X Enriched in the Adult Cornea 
Supplemental Table 3A - (21.5 KB) Genes Differentially Down-Regulated in Adult Cornea 
Tag counts were not normalized. The fold difference was at least 4.0 and p <_0.01 p-chance="p-chance" as="as" determined="determined" by="by" the="the" sage2000="sage2000" analysis="analysis" software.="software." p="p"></_0.01> 
Supplemental Table 3B - (21.0 KB) Genes Differentially Up-Regulated in Adult Cornea 
Tag counts were not normalized. The fold difference was at least 4.0 and p <_0.01 p-chance="p-chance" as="as" determined="determined" by="by" the="the" sage2000="sage2000" analysis="analysis" software.="software." p="p"></_0.01> 
Supplemental Table 4 - (27.0 KB) Transcriptional Regulators 
The authors thank Seth Blackshaw for advice on the construction of the SAGE libraries; Richard B. Hough, Shivalingappe Swamynathan, and David Nees for helpful discussions of the manuscript; and Thomas Miele, Michael Coury, and William Lanahan from Beckman Coulter for advice on sequencing and annotation; and Patrick Gilles from Invitrogen Inc., who provided advice on library construction. 
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Figure 1.
 
Functional categories of the cornea enriched genes. A total of 339 adult sequence tags were considered to be enriched in the cornea (defined as five times more frequent in the corneal library than in eight other mouse SAGE libraries). Of the enriched genes, 58% were placed in the unknown function category, which contains tags that correspond to named genes with unknown function, ESTs, and novel tags. Genes encoding proteins with functions relating to barrier, HMT, transcription, or redox activities represent 17%, 17%, 4%, and 4%, of the remaining enriched genes, respectively.
Figure 1.
 
Functional categories of the cornea enriched genes. A total of 339 adult sequence tags were considered to be enriched in the cornea (defined as five times more frequent in the corneal library than in eight other mouse SAGE libraries). Of the enriched genes, 58% were placed in the unknown function category, which contains tags that correspond to named genes with unknown function, ESTs, and novel tags. Genes encoding proteins with functions relating to barrier, HMT, transcription, or redox activities represent 17%, 17%, 4%, and 4%, of the remaining enriched genes, respectively.
Figure 2.
 
Localization of abundant transcripts in the cornea by in situ hybridization. Expression of several abundant tags was investigated in adult mouse corneas by in situ hybridization using an antisense and sense riboprobe for each transcript. SLURP1 (A) and PSCA (B) mRNAs were detected in all cells of the cornea. Calcyclin (C) and calpactin (D) mRNAs were limited to the corneal epithelial cells. mRNAs for aquaporin 5 (E), EMP1 (F), GST ω-1 (G), and GST α-4 (H) were preferentially expressed in distinct corneal epithelial layers. No signal was detected when a sense PSCA riboprobe (I) was hybridized with adult cornea. No hybridization signal was observed for any of the other sense riboprobes (data not shown). ce, corneal epithelium; s, stroma; cn, corneal endothelium.
Figure 2.
 
Localization of abundant transcripts in the cornea by in situ hybridization. Expression of several abundant tags was investigated in adult mouse corneas by in situ hybridization using an antisense and sense riboprobe for each transcript. SLURP1 (A) and PSCA (B) mRNAs were detected in all cells of the cornea. Calcyclin (C) and calpactin (D) mRNAs were limited to the corneal epithelial cells. mRNAs for aquaporin 5 (E), EMP1 (F), GST ω-1 (G), and GST α-4 (H) were preferentially expressed in distinct corneal epithelial layers. No signal was detected when a sense PSCA riboprobe (I) was hybridized with adult cornea. No hybridization signal was observed for any of the other sense riboprobes (data not shown). ce, corneal epithelium; s, stroma; cn, corneal endothelium.
Figure 3.
 
Overlapping and nonoverlapping transcripts. Collectively, the two libraries identified 8929 unique (n > 1) sequence tags. Whereas 3483 (39%) of the 8929 unique tags were observed at both developmental stages, 3052 (34%) and 2394 (27%) were identified exclusively in PN9 and adult corneas, respectively.
Figure 3.
 
Overlapping and nonoverlapping transcripts. Collectively, the two libraries identified 8929 unique (n > 1) sequence tags. Whereas 3483 (39%) of the 8929 unique tags were observed at both developmental stages, 3052 (34%) and 2394 (27%) were identified exclusively in PN9 and adult corneas, respectively.
Table 1.
 
Summary of SAGE Libraries
Table 1.
 
Summary of SAGE Libraries
Total Tags Unique Tags Unique Tags (n > 1)
PN9 64,272 19,544 6,535
ADULT 62,206 18,509 5,877
Table 2A.
 
Most Abundant Genes in PN9 Cornea
Table 2A.
 
Most Abundant Genes in PN9 Cornea
  Most Abundant Genes in PN9 Cornea
Table 2B.
 
Most Abundant Genes in Adult Cornea
Table 2B.
 
Most Abundant Genes in Adult Cornea
  Most Abundant Genes in Adult Cornea
Table 3.
 
Transcription Factor Families Expressed in Mouse Cornea
Table 3.
 
Transcription Factor Families Expressed in Mouse Cornea
ETS: ELF3
Ets homologous factor
Ets variant gene 6
ELK3
Ets domain protein
Est 2
HLH: SREBF 1 and 2
HEB
E47
Tcf4
ID 1 and 2
USF2
HES1
Max
Max binding protein
Mad2
Per 1 and 3
Hif 1a
Dec 1 and 2
Lisch7
TFEB
TFE3
Mitf
Zinc finger: KLF 3, 4, 5, 6, 13, 16
Ring finger protein 4
MAZ
mOVO
Odd-skipped related 2
YY1 associated factor 2
Schnurri2
ZNF36
ZFP 62, 100, 103, 106, 144, 162, 185, 207, 216, 238, 276, 319, 326
LIM only 4
CLIM
Four and a half LIM domains
Homeobox: PAX6
ES cell derived homeobox
TGIF
CUX
PHTF1
Hox1.6
IRX3
PRX1
NKX2.3
bZIP: Jun, JunB, JunD
JDP2
Fra2
MafK, G, B, F
NFR1
CREB 2 and 3
ATF 1 and 2
OASIS
Nuclear receptor: Rev-erbA-α
EAR2
RXR, γ
Estrogen related receptor, α
N-CoR 1 and 2
Thyroid hormone receptor α
Nuclear receptor coactivator 1 and 4
CAR
Nur77
SOX: Sox 7, 15
IRF: IRF 1, 3, 6, 7
Table 4.
 
Cornea Enriched Transcriptional Regulators
Table 4.
 
Cornea Enriched Transcriptional Regulators
Adult Count PN9 Count Total Unigene Description
48 33 81 4325 KLF4
51 21 72 1246 IRF1
22 17 39 3963 ELF3
19 10 29 10724 Ets homologous factor
12 11 23 36894 HEB
13 7 20 30262 KLF5
12 8 20 1167 JunB
11 9 20 193097 Rev-erbA-alpha
11 9 20 4179 IRF6
10 8 18 968 Oxysterols receptor LXR-beta
4 13 17 3608 PAX6
11 3 14 162 MafK
13 13 3233 IRF7
13 13 221168 Y-box binding protein (oxyR)
7 4 11 10723 Per3
11 11 3931 Max
6 4 10 1161 ZFP185
10 10 67919 MafB
9 9 31170 Cbp/p300-interacting transactivator 2
4 4 8 176369 SOX15
2 6 8 25552 Grainyheadlike 1
8 8 195896 ES cell derived homeobox
8 8 27887 LAF4
7 7 38016 SREBF2
7 7 34510 Ets-domain protein
7 7 28170 NF-kappa-B, p65
6 6 23095 Chromatin accessibility complex 1
6 6 231867 Retinoblastoma-binding protein 1
4 2 6 2229 Eya2
4 2 6 25762 TFE3
3 3 6 27962 ZFP238
3 3 6 26587 Thyroid hormone receptor alpha
6 6 116802 NFAT1
6 6 3420 NF-kappa-B, p50
5 5 3146 Transcription factor 20
5 5 193526 Cold shock domain protein A
3 2 5 38323 mOVO
3 2 5 27663 Transcription elongation factor B3
2 3 5 334 Sox6 binding protein
5 5 7373 Per1
5 5 4454 ELK3
4 4 103560 JDP2
4 4 89172 Grainyheadlike 2
4 4 34554 E2F4
2 2 4 39039 IRX3
2 2 4 23704 Fra2
4 4 29891 Forkhead box O1
4 4 2032 Interferon dependent 3 gamma
4 4 3879 Hif1a
3 3 20827 Homeodomain-IP kinase 1
3 3 12891 ZFP326
3 3 42162 SOX7
3 3 28269 I-kappa-B kinase beta
3 3 4067 Lisch7
3 3 33797 HDAC 7A
3 3 180561 RBP-J kappa
3 3 10353 Oasis
Table 5A.
 
Transcriptional Regulators Differentially Down-Regulated in Adult Cornea
Table 5A.
 
Transcriptional Regulators Differentially Down-Regulated in Adult Cornea
PN9 Count Adult Count Fold P Unigene Description
12 0 12 0.0003 153618 KOX17
11 0 11 0.0006 3931 Max
10 0 10 0.001 67919 MafB
8 0 8 0.005 21534 Interleukin enhancer binding factor 2
8 0 8 0.005 195896 ES cell derived homeobox
7 0 7 0.008 25703 FUSE binding protein
7 0 7 0.008 34510 Ets-domain protein
7 0 7 0.008 1068 Bcl3
15 3 5 0.005 16421 HMGB1
28 6 4.7 0.0002 3608 PAX6
18 4 4.5 0.003 42240 ZFP162
25 6 4.2 0.0006 911 HMGN2
18 5 3.6 0.007 641 CREB2
Table 5B.
 
Transcriptional Regulators Differentially Up-Regulated in Adult Cornea
Table 5B.
 
Transcriptional Regulators Differentially Up-Regulated in Adult Cornea
PN9 Count Adult Count Fold P Unigene Description
0 10 10 0.0007 20827 Homeodomain-interacting protein kinase 1
0 9 9 0.002 31170 Cbp/p300-interacting transactivator 2
0 8 8 0.003 2436 Dec1
0 8 8 0.003 8884 I-kappa-B, alpha
2 13 6.5 0.003 3233 IRF7
0 6 6 0.01 25877 PHTF1
0 6 6 0.01 20927 TSC22
0 6 6 0.01 28261 Nuclear receptor coactivator 4
0 6 6 0.01 34554 E2F4
3 13 4.3 0.009 22688 Tumor susceptibility gene 101
21 51 2.4 0.0001 1246 IRF1
11 25 2.3 0.01 38016 SREBF2
Supplemental Table 1A
Supplemental Table 1B
Supplemental Table 2A
Supplemental Table 2B
Supplemental Table 3A
Supplemental Table 3B
Supplemental Table 4
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