Biochemistry and Molecular Biology
Mouse Conjunctival Forniceal Gene Expression during Postnatal Development and Its Regulation by Krüppel-like Factor 4 Divya Gupta,1 Stephen A. K. Harvey,1 Naftali Kaminski,2 and Shivalingappa K. Swamynathan1,3,4 PURPOSE. To identify the changes in postnatal mouse conjunctival forniceal gene expression and their regulation by Klf4 during the eye-opening stage when the goblet cells first appear. METHODS. Laser microdissection (LMD) was used to collect conjunctival forniceal cells from postnatal (PN) day 9, PN14 and PN20 wild-type (WT), and PN14 Klf4-conditional null (Klf4CN) mice, in which goblet cells are absent, developing, present, and missing, respectively. Microarrays were used to compare gene expression among these groups. Expression of selected genes was validated by quantitative RT-PCR, and spatiotemporal expression was assessed by in situ hybridization. RESULTS. This study identified 668, 251, 1160, and 139 transcripts that were increased and 492, 377, 1419, and 57 transcripts that were decreased between PN9 and PN14, PN14 and PN20, PN9 and PN20, and PN14 WT and Klf4CN conjunctiva, respectively. Transcripts encoding transcription factors Spdef, FoxA1, and FoxA3 that regulate goblet cell development in other mucosal epithelia, and epithelium-specific Ets (ESE) transcription factor family members were increased during conjunctival development. Components of pathways related to the mesenchymal– epithelial transition, glycoprotein biosynthesis, mucosal immunity, signaling, and endocytic and neural regulation were increased during conjunctival development. Conjunctival Klf4 target genes differed significantly from the previously identified corneal Klf4 target genes, implying tissue-dependent regulatory targets for Klf4. CONCLUSIONS. The changes in gene expression accompanying mouse conjunctival development were identified, and the role of Klf4 in this process was determined. This study provides new probes for examining conjunctival development and function and reveals that the gene regulatory network necessary for goblet cell development is conserved across different mucosal epithelia. (In-
From the Departments of 1Ophthalmology and 3Cell Biology and Physiology, the 2Dorothy P. and Richard P. Simmons Center for Interstitial Lung Disease, Pulmonary, Allergy and Critical Care Medicine, and the 4McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania. Supported by the National Eye Institute (NEI) K22 Career Development Award EY016875 (SKS), startup funds from the Department of Ophthalmology, Core Grant 5P30 EY08098-19 for vision research, Research to Prevent Blindness, the Eye and Ear Foundation Pittsburgh, and the NEI Intramural Research Program. Submitted for publication December 15, 2010; revised February 14, 2011; accepted February 24, 2011. Disclosure: D. Gupta, None; S.A.K. Harvey, None; N. Kaminski, None; S.K. Swamynathan, None Corresponding author: Shivalingappa K. Swamynathan, University of Pittsburgh School of Medicine, Eye and Ear Institute, 203 Lothrop Street, Room 1025, Pittsburgh PA 15213;
[email protected]. Investigative Ophthalmology & Visual Science, July 2011, Vol. 52, No. 8 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.
vest Ophthalmol Vis Sci. 2011;52:4951– 4962) DOI:10.1167/ iovs.10-7068
T
he health of the transparent cornea, which accounts for more than 70% of the refractive power of the eye, is dependent on other components of the ocular surface, including the conjunctiva, lacrimal and accessory lacrimal glands, and meibomian glands.1–5 Different components of the ocular surface are connected by the tear film, a complex structure consisting of an outermost lipid layer secreted by the meibomian glands, central aqueous layer secreted by the lacrimal glands and the corneal and conjunctival epithelial cells, and an inner glycocalyx layer of the membrane-bound mucins on the superficial epithelial cells.5,6 Additional components of the aqueous include soluble mucins, antimicrobial peptides, antibodies, and different solutes that are secreted by conjunctival goblet cells and conjunctival and corneal epithelial cells, or are derived by diffusion from the conjunctival vasculature.5– 8 The conjunctiva consists of the basal collagenous lamina propria covered with an epithelium comprising four different kinds of stratified squamous cells, including apical granule-rich cells, endoplasmic reticulum-rich cells, Golgi-rich cells and mitochondria-rich cells interspersed with mucin-secreting goblet cells.9 In addition, the conjunctiva is highly innervated, with parasympathetic nerves regulating the goblet cell secretions and sympathetic nerves regulating the stratified squamous cell secretions.10,11 The conjunctival goblet cells play a critical role in ocular surface health by producing and secreting mucins, trefoil factors, and other components of the tear film by an apocrine mechanism, in which all the secretory granules in the cell are emptied on stimulation. Loss of the conjunctival goblet cells is associated with severe ocular surface disorders, such as dry eye, ocular cicatricial pemphigoid (OCP), and Sjo ¨ gren’s syndrome.10,12–15 Developmental studies of the ocular surface have mostly focused on the cornea, resulting in characterization of the involvement of the transcription factors Pax6, Klf4, Klf6, E2F, AP1, AP2␣, Sp1, Sp3, Sp6, Shh, Cited2, and IB in embryonic development, postnatal maturation, and maintenance of the cornea.16 –30 Despite its critical contributions to the homeostasis of the ocular surface, development of the conjunctiva has been relatively understudied. In the mouse, initial conjunctival epithelial stratification and goblet cell development occur around eye opening, when the conjunctiva is first exposed to photo, oxidative, and environmental stresses. The conjunctival goblet cells share similarities in their structure and function with the intestinal, colonic, and airway mucosal epithelial goblet cells.31,32 In these tissues, goblet cell development is regulated by genetic programs involving the Notch pathway; transcription factors Hnf4␣, Hnf1␣, HNF1, Klf4, Klf5, FoxA1, FoxA2, FoxA3, Spdef, and IB; and increased cytokine levels.31,33– 48 Similar studies of conjunctival goblet cells are lim4951
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ited,10,27,29,49,50 resulting in sparse information on developmental changes in conjunctival gene expression. Microarrays have been used successfully in the study of developmental changes in gene expression51–53 and for comparative analysis of gene expression in diverse organs.49,54 In this study, we catalogued the changes in gene expression accompanying postnatal development of conjunctival forniceal cells by microarray-based gene expression profiling at postnatal day (PN) 9, PN14, and PN20, when goblet cells are absent, developing, and present, respectively, and identified the conjunctival Klf4 target genes by comparing the gene expression patterns between PN14 WT and Klf4-conditional null (Klf4CN) conjunctivas lacking goblet cells.27 These results provide the first detailed description of the dynamic changes in gene expression accompanying postnatal conjunctival maturation involving epithelial cell stratification and goblet cell development and define the role of Klf4 in this process.
MATERIALS
AND
METHODS
Conditional Deletion of the Klf4 Gene Klf4CN mice were generated by mating Klf4loxP/loxP, Le-Cre/⫺ mice with Klf4loxP/loxP mice and genotyped as described earlier.27,33,55 All procedures described herein were approved by the Institutional Animal Care and Use committee of the University of Pittsburgh and Division of Laboratory Animal Resources (DLAR) and conformed to the ARVO Statement for the use of Animals in Ophthalmic and Vision Research.
Staining for Goblet Cells, Laser Microdissection, and RNA Isolation Goblet cells were visualized by staining 8-m sections from paraformaldehyde-fixed, paraffin-embedded PN9, PN14, and PN20 WT and PN14 Klf4CN head tissue with periodic acid-Schiff’s (PAS) reagent, as described earlier.27 A microscope (model BX60; Olympus America Inc., Lake Success, NY) equipped with a digital camera (Spot; Diagnostic Instruments Inc., Sterling Heights, MI) was used for light microscopy. For LMD, tissue blocks comprising the whole eye, eyelids, and conjunctivas were dissected and incubated in an RNA stabilizer (RNA-Later; Ambion, Austin, TX) for 15 minutes at room temperature, fixed in 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) for 15 minutes, and incubated in 30% sucrose solution for 1 hour.56 Fixed tissue blocks were embedded in OCT compound on dry ice and 8-m-thick sections were cut on a cryotome (model CM3050Sl; Leica Microsystems, Nussloch, Germany). The sections were transferred to membrane slides (PEN; Leica Microsystems), and the conjunctival forniceal region was harvested by LMD (DMLA microscope and LMD software version 4.4; Leica).57,58 We directed the laser beam used for LMD through the conjunctival stroma, to avoid UV laser burns of our target tissue, the conjunctival epithelium. Even though this method yielded relatively undamaged conjunctival epithelial cells, it also resulted in minor contamination with the underlying stromal cells. Considering that a eukaryotic cell contains ⬃20 pg total RNA, we collected approximately 10,000 cells from each stage, enough to isolate ⬃100 ng total RNA for each sample. Microdissected cells were collected in sterile RNase-free microtubes containing lysis buffer, and total RNA was isolated (RNeasy-Micro kit; Qiagen, Germantown, MD). Each of these total RNA preparations was then analyzed (2100 bioanalyzer, with an RNA 6000 Pico chip kit; Agilent Technologies, Santa Clara, CA). Only high-quality RNA with an RNA integrity number (RIN) ⬎ 5 was used for microarray and quantitative RT-PCR analyses.
IOVS, July 2011, Vol. 52, No. 8 mouse genome microarray (430 2.0; Affymetrix, Santa Clara, CA). The microarrays were washed, developed, and scanned using standard protocols. The raw data were processed and analyzed using commercially available (Gene Chip Operating Software [GCOS] ver. 1.4 Affymetrix]), pathway analysis tool (Ingenuity Systems, Redwood, CA) and free (BRB ArrayTools [www.nci.nih.gov/brb-arraytools.html/]) software packages. Differentially expressed genes were categorized into various biological processes using the Panther analysis tool (www.pantherdb.org). Microarray data have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc⫽GSE26076). Detailed protocols for sample preparation, microarray hybridization and analysis of the microarray data are provided as Supplementary Methods (http://www. iovs.org/lookup/suppl/doi:10.1167/iovs.10-7068/-/DCSupplemental).
Validation of Microarray Results RNA samples (100 ng) were amplified as before and 1 g amplified RNA was used for cDNA synthesis (Superscript VILO cDNA synthesis kit; Invitrogen, Carlsbad, CA). qRT-PCR assays were performed with SYBR green reagent (Super-Array Biosystems, Frederick, MD), using prestandardized, gene-specific primers in a commercial PCR system (StepOne Plus Real Time PCR system; Applied Biosystems, Inc. [ABI], Foster City, CA) with GAPDH as the endogenous control and were analyzed with allied software (StepOne software ver. 2.1; ABI). The sequence of primers used for qRT-PCR is provided in Supplementary Table S1 (http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.107068/-/DCSupplemental).
In Situ Hybridization In situ hybridization was performed with 12-m sections cut from fresh frozen tissue in optimal cutting temperature (OCT) compound (Sakura Finetec, Torrance, CA) that were fixed in 4% paraformaldehyde, treated with proteinase K (0.2 g/mL PBS) for 5 minutes, and processed as described earlier.30 Riboprobes were synthesized using a digoxigenin (DIG) RNA labeling kit (Sp6/T7; Roche Molecular Biochemicals, Indianapolis, IN) with linearized plasmid cDNA templates for the respective genes. The color development reaction was allowed to proceed for 60 minutes, after which the reactions for both the sense and antisense riboprobes were terminated at the same time.
RESULTS Development of Goblet Cells and Spatiotemporal Expression of Klf4 in the Mouse Conjunctiva Developing mouse conjunctival sections stained by PAS reagent revealed that the mouse conjunctival goblet cells were absent at PN9, began to first appear at PN14 around the time of eyelid opening, and are well formed by PN20 (Fig. 1). The oneto two-cell layered PN9 conjunctival epithelium stratified and formed a three- to four- and five- to six-cell–layered squamous epithelium by PN14 and PN20, respectively (Fig. 1). In view of the significant role of Klf4 in conjunctival goblet cell development,27 we examined the spatiotemporal expression patterns of Klf4 in the developing mouse conjunctiva by in situ hybridization. Klf4 expression was low at PN9, moderate at PN14 and highest at PN20 in the WT conjunctiva (Fig. 2A–C). In contrast, it was faint in the PN14 Klf4CN conjunctiva, consistent with its successful disruption (Fig. 2D). Similarly processed PN20 WT sections hybridized with a Klf4 sense strand probe served as the negative control (Fig. 2E).
Microarray Analysis Microarray Analysis All analyses reported here were performed with three independent samples in each group. Total RNA isolated from the LMD samples was subjected to two rounds of amplification, labeled, and hybridized to a
We collected the PN9, PN14, and PN20 conjunctival forniceal cells by LMD, isolated good-quality total RNA (Fig. 3), and analyzed their gene expression patterns by microarray, as described in Supplementary Methods (http://www.iovs.org/
Gene Expression during Conjunctival Forniceal Development
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FIGURE 1. Timeline of conjunctival goblet cell development. PAS-stained sections from (A) WT PN9, (B) WT PN14, (C) WT PN20, and (D) Klf4CN PN14 conjunctival fornices show that the goblet cells first appeared in the conjunctival fornix at eyelid opening at PN14 (arrows), increased in number at PN20, and were absent from the Klf4CN conjunctiva at PN14 (n ⫽ 3).
lookup/suppl/doi:10.1167/iovs.10-7068/-/DCSupplemental). Systemic variation in microarray-analyzed gene expression was examined by using redundant panels targeting the housekeeping genes GAPDH and pyruvate carboxylase. The mean coefficient of variation across the 12 samples (four groups with three independent samples in each) was 28%, so that a twofold increase from the mean is equivalent to 3.5 SD. Mean correlation among replicates was high within each group and lower across different groups, indicating the repeatability and reliability of the microarray data (Supplementary Fig. S1, http:// www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-7068/-/ DCSupplemental). Microarray data are available at http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc⫽GSE26076.
Changes in Gene Expression Profiles in the Developing WT and Klf4CN Conjunctiva The numbers of increased and decreased transcripts during conjunctival development and in the Klf4CN compared with
FIGURE 2. Developmental expression of Klf4 in WT and Klf4CN conjunctiva. The expression of Klf4 mRNA was examined by in situ hybridization with Klf4-specific antisense probe in (A) PN9 WT, (B) PN14 WT, (C) PN20, and (D) PN14 Klf4CN conjunctiva. (E) Similar hybridization with a Klf4 sense strand probe at PN20 served as the negative control. A–E, arrow; Conjunctival forniceal epithelium, n ⫽ 3.
WT conjunctiva, detected by pair-wise comparison of microarray results, are shown by Venn diagram (Fig. 4A). The complete list of genes falling in each Venn area is provided in the Supplementary Data, http://www.iovs.org/lookup/suppl/doi: 10.1167/iovs.10-7068/-/DCSupplemental). There were 668, 251, and 1160 transcripts that were increased and 492, 377, and 1419 transcripts that were decreased between PN9 and PN14, PN14 and PN20, and PN9 and PN20 WT conjunctivas, respectively. Comparison of the PN14 WT and Klf4CN conjunctival gene expression patterns revealed 139 increased and 57 decreased transcripts, among which 80 transcripts were increased and 19 decreased during different stages of conjunctival development (Fig. 4A; Supplementary Table S2, http:// www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-7068/-/ DCSupplemental). Venn area A represents the rapid early changes (139 increased and 145 decreased transcripts; Supplementary Table S3, http://www.iovs.org/lookup/suppl/doi: 10.1167/iovs.10-7068/-/DCSupplemental) identified only in the
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7068/-/DCSupplemental) representing the late changes, suggesting that the eyelid-opening stage (around PN12) is an actively morphing period when gene expression patterns are rapidly changing in the conjunctiva (Fig. 4A). Finally, Venn area E contains transcripts that are progressive (i.e., show a twofold change between PN9 and PN14 and a subsequent twofold change in PN14 versus PN20 comparison; Supplementary Table S9, http://www.iovs.org/lookup/suppl/doi:10.1167/ iovs.10-7068/-/DCSupplemental). Categorization of the affected genes according to their biological functions showed consistent changes in PN14 versus PN9, PN20 versus PN14, PN20 versus PN9, and Klf4CN versus WT comparisons (Fig. 4B). Metabolic processes, cellular pro-
After LMD
Area of Cells Captured by LMD
A. Valid 2-fold increases and decreases (with Klf4CN Vs WT numbers in parentheses) PN14 vs PN9 668(40) 492 (10)
47 (0) 16 (4)
A 139 (2) 145 (3)
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F 97 (16) 190 (4)
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Affected in Klf4CN but not during development (59) (38)
581 (11) 898 (2)
PN20 vs PN9 1160 (65) 1419 (9)
B. Categorizaon of the affected genes according to their biological funcons 6 4 11
4
7 17
FIGURE 3. Collection of conjunctival forniceal cells and quality of the total RNA isolated. (A) LMD of conjunctival forniceal cells. PAS-stained WT PN14 sections before and after LMD. Arrows, right: the denuded areas from where the cells have been collected. Roughly 40% of the distance from the fornix to the eyelid junction was collected. Bottom: conjunctival fornix before and after LMD at higher magnification, illustrating that, along with most of the forniceal epithelial cells, some underlying stromal cells were also collected. (B) Quality of the total RNA isolated from the LMD samples. Representative gel image of the total RNA isolated from LMD-harvested PN14 WT and Klf4CN conjunctival forniceal epithelial cells. The RNA integrity number (RIN) is shown, along with their usage status for each of the samples.
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PN9 versus PN14 comparison, and Venn area C represents the rapid late changes (75 increased and 149 decreased transcripts; Supplementary Table S4, http://www.iovs.org/lookup/suppl/ doi:10.1167/iovs.10-7068/-/DCSupplemental) detected only in the PN14 versus PN20 comparison (Fig. 4A). Venn area B contains transient changes (i.e., rapid early changes in the PN9 versus PN14 comparison that were definitively reversed in the PN14 versus PN20 comparison and are listed in Supplementary Table S5, http://www.iovs.org/lookup/suppl/doi:10.1167/ iovs.10-7068/-/DCSupplemental). Venn area G represents the gradual changes spanning the PN9 to PN20 period (581 increased and 898 decreased transcripts; top 10% of transcripts are listed in Supplementary Table S6, http://www.iovs.org/ lookup/suppl/doi:10.1167/iovs.10-7068/-/DCSupplemental) identified only in the PN9 versus PN20 comparison (Fig. 4A). Venn area D (450 increased and 309 decreased; top 50 transcripts are listed in Supplementary Table S7, http://www. iovs.org/lookup/suppl/doi:10.1167/iovs.10-7068/-/DC Supplemental), representing the early changes, contained 2.6 times as many genes as Venn area F (97 increased and 190 decreased; top 50 transcripts are listed in Supplementary Table S8, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-
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Legend Cell communicaon (GO:0007154) Cellular process (GO:0009987) Localizaon (GO:0051179) Transport (GO:0006810) Cellular component organizaon (GO:0016043) Apoptosis (GO:0006915) System process (GO:0003008) Reproducon (GO:0000003) Response to smulus (GO:0050896)
9 1
8 5 2 9
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PN14 Klf4CN Vs WT Regulaon of biological process (GO:0050789) Homeostac process (GO:0042592) Developmental process (GO:0032502) Generaon of precursor metabolites and energy (GO:0006091) Metabolic process (GO:0008152) Cell cycle (GO:0007049) Immune system process (GO:0002376) Cell adhesion (GO:0007155)
FIGURE 4. Microarray comparison of developmental changes in gene expression. (A) Representation of the microarray data in a Venn diagram. Within each Venn area, the number of increased transcripts is shown in red and the number of decreased transcripts in green. Values in parentheses give the number of transcripts modulated in the Klf4CN versus WT comparison. Please see the Supplementary Tables (http:// www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-7068/-/DCSupplemental) for the full list of transcripts in each Venn area. (B) Categorization of the affected transcripts according to their biological functions. Significantly affected transcripts are categorized into various biological processes using the Panther analysis tool (www.pantherdb.org). Corresponding gene ontology (GO) terms are shown in parentheses in the legend.
Gene Expression during Conjunctival Forniceal Development
Validation of Microarray Results Selected transcripts that were unaffected, increased, or decreased in each of these comparisons were measured by real time qRT-PCR, using cDNA generated with three different independent samples of total RNA than the ones used for microarray analyses. These qRT-PCR results were largely similar to those obtained from microarray analyses, thus validating the results obtained through microarray analyses (Fig. 5).
Klf4 Target Genes in the Mouse Conjunctival Fornix Comparison of the PN14 WT and Klf4CN conjunctival forniceal gene expression patterns identified 139 and 57 transcripts increased and decreased, respectively, in the Klf4CN conjunctiva (Fig. 4A). A list of the top 20 most increased and decreased transcripts between PN14 WT and Klf4CN conjunctival cells is provided in Table 2 (complete list in Supplementary Table S2, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.107068/-/DCSupplemental). Among these, Muc5Ac, Gcnt3, and Slc5a1 were also present in the list of most highly increased transcripts in the course of conjunctival development between PN9 and PN20 (Tables 1, 2). Transcripts encoding four other mucins (Muc5b, Muc4, Muc16, and Muc20) and Lypd2, a member of the Ly6 family of proteins, were significantly decreased in the Klf4CN conjunctiva (Table 2). Transcripts encoding the goblet cell markers Muc5Ac, Lman1l, and Gcnt3; transcription factor Spdef, which regulates goblet cell development in airway and colonic epithelial tissues; the mucosal epithelium enriched serine protease Prss22; and the solute carrier family member Slc5a1 were significantly decreased in the Klf4CN conjunctiva and increased during conjunctival forniceal development between PN9 and PN20.14,36,37,41,65,66 Comparison of the conjunctival Klf4 target genes with the previously identified corneal Klf4 target genes67 yielded a surprisingly small list of common genes. Most of these changes were concordant between the cornea and the conjunctiva, with only seven genes showing discordant changes (Table 3). Thus, Klf4 appears to perform distinct, nonoverlapping functions in these two physically connected components of the ocular surface. These results also show that Klf4 has fewer target genes in the conjunctiva than in the cornea.67 A reason for this could be that in the case of the cornea, RNA was isolated from the whole cornea consisting of several cell
Notch3
Igf1
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Col12A1 Ptges3
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0.1
Relative Expression
FIGURE 5. Validation of microarray results. Real-time qRT-PCR was used to compare the expression levels of selected transcripts in (A) PN14 WT and Klf4CN, (B) PN9 and PN14, (C) PN14 and PN20, and (D) PN9 and PN20 conjunctival forniceal cells harvested by LMD. Relative expression levels were calculated from three independent samples, each with two replicates. Mean relative expression levels detected by qRT-PCR (red bars) are compared with the values derived from the microarray data (blue bars). Logarithmic scale is used on the y-axis axis. Error bars, SE (n ⫽ 3).
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A list of the top 20 most increased and decreased transcripts between PN9 and PN20 is provided in Table 1. The zymogen granule membrane protein GP2 that binds enterobacterial type 1 fimbria FimH59,60 was the most increased transcript between PN9 and PN20 suggesting that the conjunctival mucosal immune system develops around this time. Consistent with this, two members of the tetraspanin family with putative roles in mucosal immunity,61 CD274 (PD-L1)62 and polymeric immunoglobulin receptor (Pigr),63 also were significantly increased during this time period. Other significantly increased transcripts relevant to conjunctival physiology include the goblet cell marker Muc5Ac and glucosaminyl transferase Gcnt3, required for glycosylation of mucins (Table 1). Expression of several mucins and glycosylation-related genes, although increased in the course of conjunctival development, was decreased in the Klf4CN conjunctiva, consistent with the appearance of goblet cells during this period and the absence of goblet cells in the Klf4CN conjunctiva (Supplementary Table S10, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.107068/-/DCSupplemental). Expression of a large number of solute carrier family members that play important roles in different mucosal epithelial tissues was increased during conjunctival development (Supplementary Table S11, http:// www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-7068/-/DCSupplemental). Several members of the Wnt signaling pathway were significantly decreased during conjunctival development (Sup-
Relative Expression
Significant Changes in Gene Expression during Conjunctival Development
10
4955
plementary Table S12, http://www.iovs.org/lookup/suppl/ doi:10.1167/iovs.10-7068/-/DCSupplemental), consistent with the lack of involvement of Wnt pathway in enteroendocrine cell differentiation.64 The cytokines Il15 and Il33, associated with goblet cell hyperproliferation and development,48 and Jak2 and Stat1, members of the Jak-STAT pathway through which these cytokines exert their influence, were altered in the developing WT and the Klf4CN conjunctiva.
cesses, cell communication, developmental processes, and transport were the most affected biological processes in both the developing and the Klf4CN conjunctiva. Other important processes affected included immune system process, response to stimulus, cell adhesion, and cell cycle (Fig. 4B).
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TABLE 1. Top 20 Most Increased and Decreased Transcripts in the PN20 Compared with PN9 Conjunctiva along with Changes in Other Developmental Stages and in the Klf4CN Conjunctiva Gene Symbol
Gene Name
Most Increased Transcripts Gp2 Glycoprotein 2 (zymogen granule membrane) Slc5a1 Solute carrier family 5, member 1 Apbh Androgen-binding protein eta Muc5ac Mucin 5, subtypes A and C, tracheobronchial/gastric Gcnt3 Glucosaminyl (N-acetyl) transferase 3, mucin type Reg3g Regenerating islet-derived 3 gamma Gnat1 Guanine nucleotide binding protein, ␣ transducing 1 Aqp4 Aquaporin 4 Elovl4 Elongation of very long chain fatty acids-like 4 Slc44a4 Solute carrier family 44, member 4 Asb11 Ankyrin repeat and SOCS box-containing 11 Olfm4 Olfactomedin 4 Cd274 CD274 antigen Pigr Polymeric immunoglobulin receptor Tspan1 Tetraspanin 1 Slc24a1 Solute carrier family 24, member 1 Cldn2 Claudin 2 Tspan8 Tetraspanin 8 Clic5 Chloride intracellular channel 5 Slc6a14 Solute carrier family 6, member 14 Most Decreased Transcripts Igf2 Insulin-like growth factor 2 Fbn2 Fibrillin 2 Acan Aggrecan Lrrc15 Leucine rich repeat containing 15 Gjc1 Gap junction protein, gamma 1 Agtr2 Angiotensin II receptor, type 2 Col11a1 Collagen, type XI, alpha 1 Mdk Midkine Smoc2 SPARC related modular calcium binding 2 Gpx7 Glutathione peroxidase 7 Tchh Trichohyalin Upk3b Uroplakin 3B Podxl2 Podocalyxin-like 2 Actg2 Actin, gamma 2, smooth muscle, enteric Lgals1 Lectin, galactose binding, soluble 1 Meg3 Maternally expressed 3 Aebp1 AE binding protein 1 Col9a3 Collagen, type IX, alpha 3 Loxl1 Lysyl oxidase-like 1 Selm Selenoprotein M
types,67 whereas here we used LMD to collect conjunctival forniceal cells consisting mostly of epithelial cells mixed with a few underlying stromal cells.
Transcription Factors Affected in the Developing WT and the Klf4CN Conjunctiva Families of transcription factors significantly affected during the postnatal development of the mouse conjunctiva between PN9 and PN20 are listed in Table 4, with the complete list of increased and decreased transcription factors provided in Supplementary Table S13 (http://www.iovs.org/lookup/suppl/doi: 10.1167/iovs.10-7068/-/DCSupplemental). Prominent among them are the members of the forkhead box (Fox) and SRYrelated HMG box (Sox), Ets, and Krüppel-like family members. Foxa3, Foxa1, Foxk1, Foxo3, and Foxp1 were increased, and Foxp2 was decreased during conjunctival development (Supplementary Table S13, http://www.iovs.org/lookup/suppl/doi: 10.1167/iovs.10-7068/-/DCSupplemental). Sox4, Sox11, and Sox12, members of the group C Sry-related HMG box proteins co-expressed in embryonic neuronal progenitors and in mesenchymal cells in many developing organs,68,69 were all decreased between PN9 and PN20 (Table 4; Supplementary Table
PN 20/PN9
PN14/PN9
PN20/PN14
Klf4CN/WT
87.04 56.37 49.75 38.25 29.21 26.50 26.33 20.32 19.78 19.62 17.55 17.31 17.10 16.69 15.74 15.19 15.18 14.96 14.69 14.25
25.55 30.10 5.30 7.16 6.66 21.22 33.64 12.23 25.24 9.99 13.07 9.64 11.36 14.42 8.43 16.24 5.63 2.30 1.51 7.09
3.25 1.87 24.16 3.59 4.47 6.27 1.42 1.68 0.87 1.93 1.24 1.79 1.50 1.22 1.69 1.80 2.80 14.84 6.14 1.99
0.91 0.26 133.20 0.00 0.15 3.46 1.30 0.63 2.48 0.60 0.61 1.79 0.47 0.93 0.57 0.98 1.11 0.92 0.40 1.54
0.22 0.24 0.21 0.44 0.34 0.49 0.38 0.27 0.21 0.50 0.28 0.20 0.43 0.49 0.39 0.33 0.37 0.30 0.39 0.38
0.28 0.30 0.43 0.23 0.18 0.21 0.26 0.40 0.55 0.22 0.21 0.59 0.31 0.27 0.34 0.39 0.35 0.15 0.34 0.37
0.76 0.47 1.39 1.01 1.51 1.15 0.97 1.22 0.67 0.65 0.18 0.41 0.62 0.79 0.75 0.53 0.57 0.12 0.77 0.73
0.063 0.069 0.088 0.092 0.103 0.107 0.110 0.115 0.115 0.121 0.124 0.126 0.130 0.134 0.134 0.134 0.135 0.139 0.140 0.144
S13, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.107068/-/DCSupplemental). In contrast, Sox21, which counteracts the Sox1, Sox2, and Sox3 group required for stem-cell maintenance,70 was increased between PN14 and PN20, favoring differentiation. These changes in Sox family transcripts indicate a gradual tapering off of support for progenitor cell maintenance, promoting their differentiation into the stratified squamous epithelial cells. The epithelial-specific Ets (ESE) transcription factors Spdef, Elf3, Ehf, and Elf5, with critical roles in airway and intestinal epithelial development,36 –38,71,72 were significantly increased during conjunctival development (Table 4; Supplementary Table S13, http://www.iovs.org/lookup/suppl/doi: 10.1167/iovs.10-7068/-/DCSupplemental). In contrast, Ets1 was the most significantly decreased transcription factor during conjunctival development. Transcripts encoding several transcription factors that favor epithelial–mesenchymal transition (EMT),73 including two Twist family members (Twist1 and Twist2), two ZEB family members (Zeb1 and Zeb2), and Snail family member Snai2/Slug were decreased during conjunctival development (Table 4; Supplementary Table S13. http://www.iovs.org/lookup/suppl/doi:10.1167/
Gene Expression during Conjunctival Forniceal Development
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TABLE 2. Top 20 Most Significantly Increased and Decreased Transcripts in the Klf4CN Conjunctiva Fold Change Gene Symbol
Description
Most Increased Transcripts Apbh Androgen-binding protein eta Sprr2a Small proline-rich protein 2A Ppbp Pro-platelet basic protein Fam19a2 Family with sequence similarity 19, member A2 Elovl4 Elongation of very long chain fatty acids-like 4 Ms4a4c Membrane-spanning 4-domains, subfamily A, member 4C Alox15 Arachidonate 15-lipoxygenase Siah1a Seven in absentia 1A Umod Uromodulin Expi Extracellular proteinase inhibitor Cyp2d9 Cytochrome P450 family-2, subfamily-d, polypeptide-9 Far2 Fatty acyl coa reductase 2 Soat1 Sterol O-acyltransferase 1 P2ry10 Purinergic receptor P2Y, G-protein coupled 10 Crisp1 Cysteine-rich secretory protein 1 Depdc1b DEP domain containing 1B Krt79 Keratin 79 Retnla Resistin like alpha Cap1 CAP, adenylate cyclase-associated protein 1 Cytip Cytohesin 1 interacting protein Most Decreased Transcripts Muc5ac Mucin 5, subtypes A and C, tracheobronchial/gastric Lypd2 Ly6/Plaur domain containing 2 Muc5b Mucin 5, subtype B, tracheobronchial Krt78 Keratin 78 Muc4 Mucin 4 Gcnt3 Glucosaminyl (N-acetyl) transferase 3, mucin type AW112010 Expressed sequence AW112010 Fam83e Family with sequence similarity 83, member E Avil Advillin Efcab6 EF-hand calcium binding domain 6 Slc28a3 Solute carrier family 28, member 3 Krt12 Keratin 12 5430404G13Rik RIKEN cdna 5430404G13 gene Crygs Crystallin, gamma S Prss32 Protease, serine, 32 Mip Major intrinsic protein of eye lens fiber Upk1b Uroplakin 1B Muc20 Mucin 20 Slc5a1 Solute carrier family 5, member 1
iovs.10-7068/-/DCSupplemental). The concomitant increase of Klf4, an inhibitor of EMT,74 and suppression of the Wnt pathway (Supplementary Table S12, http://www.iovs.org/lookup/suppl/ doi:10.1167/iovs.10-7068/-/DCSupplemental), a promoter of EMT,75 is consistent with changes favoring mesenchymalepithelial transition (MET) during conjunctival development.
Developmental Expression Patterns of Selected Transcripts in the Conjunctiva We examined the developmental expression of the protein disulfide isomerase Agr2 and transcription factors FoxA3, Spdef, and Mitf by in situ hybridization, using cryosections from embryonic day 16.5, PN1, PN9, and PN14. We included Agr2, Spdef, and FoxA3, in view of their known involvement in goblet cell development in other mucosal epithelia, and Mitf because it was the only transcription factor with a known function in eye development76 that was decreased from PN9 to PN14 during conjunctival development. Expression of these transcripts was largely confined to epithelial cells, and followed a similar trend, as detected by microarray analysis (Fig. 6). At E16.5 and PN1, these transcripts were expressed at low levels, more or less uniformly across the conjunctival epithelium.
Klf4CN/WT
PN14/PN9
PN20/PN14
PN20/PN9
13.50 7.41 7.18 6.41 6.25 4.98 4.41 4.27 4.11 4.09 4.03 3.98 3.82 3.81 3.71 3.60 3.49 3.48 3.44 3.30
5.30 0.58 0.71 0.69 25.24 0.41 1.28 0.68 1.39 0.68 0.55 35.18 3.91 1.02 2.15 0.24 15.20 2.95 1.15 0.99
24.16 1.12 0.85 1.03 0.87 1.02 1.12 3.46 0.67 1.80 0.89 0.43 0.93 1.05 1.92 3.54 0.42 0.72 0.90 0.82
128.1 0.65 0.60 0.71 22.06 0.42 1.44 2.37 0.93 1.22 0.49 15.19 3.65 1.07 4.13 0.85 6.32 2.12 1.03 0.81
0.01 0.02 0.10 0.11 0.14 0.15 0.17 0.17 0.19 0.19 0.20 0.20 0.21 0.22 0.22 0.23 0.23 0.23 0.25
7.16 6.34 2.27 4.36 5.31 6.66 8.67 1.77 1.21 1.44 6.07 3.03 1.32 2.19 5.72 1.01 1.69 3.60 30.10
3.59 0.47 1.89 1.88 0.71 4.47 1.73 2.01 2.05 0.84 1.56 0.36 0.32 0.93 1.20 1.35 0.70 0.87 1.87
25.73 2.99 4.31 8.17 3.79 29.74 14.95 3.56 2.49 1.21 9.45 1.09 0.43 2.04 6.87 1.36 1.18 3.13 56.36
Postnatal expression of FoxA3 and Agr2 gradually increased from PN1 to PN9 and PN14, with a noticeable enrichment of the corresponding transcripts in the conjunctival fornix, consistent with the tendency of the mouse conjunctival goblet cells to cluster in this area (Fig. 6). Expression of Spdef also increased gradually from PN1 to PN9 and PN14. However, unlike FoxA3 and Agr2, there was no enrichment of Spdef transcripts in the conjunctival fornix at PN9 or PN14. Expression of Mitf reached a peak on PN9 and displayed relatively decreased expression at PN14, consistent with the microarray results. Sections probed with similarly labeled sense strand riboprobes served as negative controls in these experiments (Fig. 6).
Canonical Pathways Affected in the Course of Conjunctival Goblet Cell Development Pathway analysis of the increased and decreased transcripts between PN9, PN14, and PN20 (1588 panels, targeting 1152 unique characterized genes) identified 84 canonical pathways as significantly (P ⬍ 0.05) enriched in at least one group. Among these, 33 canonical pathways showed higher enrich-
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TABLE 3. Klf4 Target Genes with Concordant and Discordant Changes in the Klf4CN Cornea and Conjunctiva Concordant Increases
Concordant Decreases
Discordant Changes
A130040M12Rik Asrgl1 Car2 Ccl6 Ccnd2 Cebpb Cp Cytip Elavl2 Expi Fabp4 Flt1 H2-Ab1 H2-Eb1 H2-Q7 Il33 Il7r Krt17 Laptm5 Mmp13 Mpeg1 Nlrp10 Ppbp Ptn Pvrl3 Serpina3n Sprr2a1 /// Sprr2a2 Tfrc Tnc Trps1
2200001115Rik Akr1c12/13 Arhgef4 Bnc1 Clic3 Clic5 Cryab Efcab6 Fam184b Jakmip1 Paqr5 Ppp1r3c Prss32 Ptgr1 Rasgrf1 Serpinb8 Slc28a3 Spaca1 Upk1b
Gadd45a* 4930523C07Rik Agr2 AW112010 Pcdhb9 Runx2 Sox4
The filtering criteria used in the present study were applied to the data from our previous report on the cornea, to select 454 and 322 unique characterized corneal Klf4 target genes with increased and decreased expression, respectively. A comparison of those corneal Klf4 target genes with the present conjunctival Klf4 target genes identified 56 genes that appeared in both the data sets. A 2 test (P ⫽ 1.46 ⫻ 10⫺23) revealed that there were more than four times the expected number (based on random chance) of concordant changes. In contrast, the number of discordant changes was at or lower than that predicted by random chance alone. * Gadd45a was increased in Klf4CN conjunctiva, but decreased in Klf4CN cornea. All other discordant changes were decreased in Klf4CN conjunctiva, but increased in Klf4CN cornea.
ment in at least one of the three analyses (P ⬍ 0.001) (Table 5). Functionally related pathways are indicated by similar color code in Table 5. Several pathways related to various forms of signaling, endocytosis (light green), mucosal immunity (light pink), neural development and function (light blue), and retinoid receptor activation (light gray) were significantly affected during conjunctival development.
DISCUSSION By comparing the WT conjunctival forniceal gene expression patterns at PN 9, PN14, and PN20, when goblet cells are absent, developing, and present, respectively, we have catalogued the gene expression changes accompanying conjunctival goblet cell development. Similar comparison of the gene expression patterns of PN14 WT and Klf4CN conjunctival fornices (with and without goblet cells, respectively) identified 139 and 57 conjunctival Klf4 target transcripts increased and decreased by more than twofold, respectively, in the Klf4CN compared to WT conjunctiva. By this approach, we have identified a large number of novel transcripts whose levels are modulated in the developing conjunctiva, suggesting that diverse pathways related to signaling, endocytosis, MET, and neural development and function are modulated at eyelid opening. Several pathways are significantly affected during postnatal conjunctival forniceal development (Table 5). Consistent with the appearance of mucin producing goblet cells during eye opening, O-glycan biosynthesis pathway components were significantly increased during conjunctival development. Endocytosis of various solutes and soluble macromolecules from the ocular surface seem to play a crucial role in the conjunctival epithelial homeostasis, judging by the number of endocytosisrelated pathways whose component genes are increased during conjunctival development (Table 5). The relatively large macropinosomes provide an efficient route for nonselective endocytosis of solute macromolecules, facilitating antigen presentation by dendritic cells. Consistent with this, transcripts involved in dendritic cell maturation pathway, the Fc␥ receptor–mediated phagocytosis pathway, and tight junction signaling were increased, lending support for development of the conjunctival mucosal immune system at eyelid opening (Table 5). Genes representing three different pathways related to retinoid signaling were increased (Table 5), suggesting a role for retinoids in conjunctival development and/or function. Increased expression of genes related to neural development and function (Table 5), although consistent with neural regulation of conjunctival epithelial function,32 was puzzling, considering that no neuron cell bodies are present in the conjunctiva. These transcripts may represent those localized to the axons surrounding developing goblet cells, as described previously,32,77,78 or those expressed in conjunctival cells surrounding the axons. Transport across the conjunctiva plays a critical role in ocular surface homeostasis.32 Our analysis has identified a large list of solute carrier family members whose expression is modulated during conjunctival development (Supplementary Table S11, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.107068/-/DCSupplemental). The solute carrier gene superfamily comprises 55 families, out of which members of 18 different families were increased during conjunctival development, 5 of which were affected in the Klf4CN conjunctiva. Chief among
TABLE 4. Most Significantly Affected Transcription Factor Families Transcription Factor Family Krüppel-like factors Epithelial-specific Ets (ESE) factors Forkhead box family proteins High-mobility group proteins Sox family members Interferon regulatory factors
Increased during Conjunctival Development
Decreased during Conjunctival Development
Klf4, Klf6, Klf9 Spdef, Ehf, Elf3, Elf5
Klf13, Klf7, Klf10, Gli2 Ets1
Foxa3, Foxa1, Foxk1, Foxo3, Foxp1 Hmgb1, Hmgb2 Sox21 Irf1
Foxp2 Hmg20b Sox4, Sox11, Sox12 Irf3
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Agr2
Spdef
4959
Mitf
Sense Control
PN14
PN9
PN1
E16.5
FoxA3
Gene Expression during Conjunctival Forniceal Development
FIGURE 6. Spatiotemporal expression patterns of selected developmentally regulated transcripts in the mouse conjunctiva. Expression of Foxa3, Agr2, Spdef, and Mitf was examined by in situ hybridization at E16.5, PN1, PN9, and PN14 (n ⫽ 3). Sections probed with the similarly labeled corresponding sense strand served as negative control for each gene. Arrow: the conjunctival fornix in each panel.
them are members of the neurotransmitter transporter Slc6 family, consistent with the neural development– and functionrelated pathways modulated during conjunctival development, as described above. In addition, mitochondrial transporter Slc25 family members were significantly affected. The sodium/ glucose co-transporter Slc5a1 increased 30-fold between PN9 and PN14, and decreased by approximately 4-fold in the Klf4CN conjunctiva, raising the possibility that it is directly regulated by Klf4 (Supplementary Table S11, http://www.iovs.org/ lookup/suppl/doi:10.1167/iovs.10-7068/-/DCSupplemental). Even though Notch, Math1, Foxa1, Foxa3, and neurogenin-3 are known to regulate colonic goblet cell development,31 molecular mechanisms regulating conjunctival goblet cell development remain poorly understood.79 – 81 This report on developmental changes in gene expression in the mouse conjunctival fornix where goblet cells tend to congregate serves as a useful reference point for identifying novel therapeutic targets to modulate goblet cell densities in human diseases such as ocular cicatricial pemphigoid (OCP), dry eye, and Sjo ¨ gren’s syndrome, where goblet cell densities are reduced, or allergic conjunctivitis, where goblet cell densities are increased. Considering that the goblet cell densities are affected in gastrointestinal and respiratory epithelial disorders as well, this knowledge is expected to be useful, not only in the ocular surface but
also in other mucosal epithelial tissues, where goblet cells play critical roles. By comparing the WT and Klf4CN PN14 conjunctival forniceal gene expression, we identified a list of transcription factors whose expression was affected in the Klf4CN conjunctiva lacking goblet cells (Table 4; Supplementary Table S13, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.107068/-/DCSupplemental). Among them, factors such as FoxA1, FoxA3, and Spdef have been shown to be essential for goblet cell development in other mucosal epithelia, validating our approach. In addition, our analysis has identified other transcription factors such as Grhl3, Creb3l4, and Bnc1, hitherto not thought to be involved in conjunctival development. It is important to analyze their contributions in conjunctival development and function in the near future. Comparison of the conjunctival Klf4 target genes with those in the cornea yielded a small number of common genes, consistent with significant differences in the function of Klf4 in these two related tissues (Table 3). Cyclin-mediated cell cycle regulation, one of the most affected pathways in the cornea67 was not affected in the conjunctiva, suggesting that Klf4 plays a major role in the rapid renewal of epithelial cells in the cornea but not in the conjunctiva. On the other hand, glycosylation-related transcripts that were decreased in the Klf4CN
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TABLE 5. Canonical Pathways Affected in the Course of Development Valid PN14 vs. PN9 Canonical Pathway Caveolar-mediated endocytosis signaling
Genes 8
2.8E-02
Biosynthesis of steroids
8
1.2E-04
Virus entry via endocyƟc pathways
12
1.3E-03
Dendri c cell matura on
15
4.1E-03
Macropinocytosis signaling
7
O-glycan biosynthesis
5
TREM1 signaling
7
LXR/RXR acƟvaƟon
8
ILK signaling
12
Valid PN20 vs. PN14 Genes
All PN20 vs.PN9
Aggregate Genes
3.0E-04
27
1.1E-04
85
12
1.4E-03
12
7.2E-03
128
4
25
9.1E-04
29
6.0E-04
5
31
1.6E-02
33
4.4E-02
3
17
1.5E-02
19
2.2E-02
74
1.7E-02
2
8
10
4.1E-02
47
1.7E-02
2
13
13
2.0E-02
2
16
4.8E-02
20
1.9E-02
86
3.5E-05
38
1.1E-02
52
1.2E-04
188
52
6.2E-05
204
46
4.2E-04
167
4.2E-02
69
4
14
Calcium signaling
8
3.6E-02
29
Tight juncƟon signaling
9
8.3E-03
36
Acute myeloid leukemia signaling
6
9.3E-03
15
15
5
1.9E-02
15
19
Agrin interac ons at neuromuscular junc on
4
Ephrin receptor signaling
11
Integrin signaling
10
Axonal guidance signaling
20
HepaƟc fibrosis/hepaƟc stellate cell acƟvaƟon
10
Leukocyte extravasaƟon signaling
11
Cardiac hypertrophy signaling Fcγ receptor-mediated phagocytosis in macrophages and monocytes Germ cell–sertoli cell junc on signaling
5.5E-03
98 188
69
84
47
5.5E-05
52
2.0E-04
198
47
3.7E-04
57
6.2E-05
204
83
1.2E-04
93
6.5E-04
407
5
28
2.8E-02
36
4.5E-03
134
6
39
1.0E-02
47
4.7E-03
196
7
7
46
1.6E-02
56
6.3E-03
245
7
3
23
1.0E-02
27
7.2E-03
101
8
5
31
4.5E-02
40
7.8E-03
160
22
1.5E-02
26
1.0E-02
106
37
1.0E-02
43
1.1E-02
181
3
15
1.1E-02
17
1.3E-02
52
Α-adrenergic signaling Rar ac va on
Total Genes
Genes 23
9
Semaphorin signaling in neurons Androgen signaling
7
3
25
3.3E-02
31
1.2E-02
144
Clathrin-mediated endocytosis signaling
13
4
35
1.2E-02
40
1.7E-02
169
VDR/RXR ac va on
5
2
19
2.5E-02
23
1.3E-02
80
4
21
3.5E-02
26
1.4E-02
102
17
2.1E-02
IGF-1 signaling Melatonin signaling Protein kinase A signaling
10
55
20
1.5E-02
77
72
4.3E-03
318
For clarity, P values are omitted if P ⬎ 0.05, although the number of pathway genes is included. Green: endocytosis-related pathways; blue: neuronal development–and/or function–related pathways; gray: retinoid receptor–related pathways; pink: mucosal immunity–related pathways; bold: pathways validated by BRB-ArrayTools.
conjunctiva were relatively unaffected in the Klf4CN cornea.67 Protein disulfide isomerase Agr2, significantly decreased in the Klf4CN conjunctiva, was increased in the cornea.67 These changes in expression may be an indirect outcome of the absence of mucin-producing goblet cells in the Klf4CN conjunctiva or a reflection of the direct involvement of Klf4 in glycoprotein biosynthesis in the conjunctiva, but not the cornea. Although the data provided here are valuable, some limitations remain. First, we focused on the mouse conjunctival fornix, where goblet cells tend to congregate. Thus, the data may not be representative of the events occurring in the palpebral or bulbar conjunctivae, away from the fornix. Second, LMD yielded a mixed population of cells consisting mostly of the conjunctival forniceal epithelial cells and a small but variable fraction of the underlying stromal cells, potentially contributing to some variation in the data. Finally, we have focused on the conjunctival gene expression changes at the level of transcripts. Although the changes in transcript levels detected by the current microarray analyses are important, they may not completely and accurately reflect the changes in the proteome, as has been demonstrated in the yeast.82 Therefore, it is important to examine changes in the proteins of interest in further analyses. In summary, through microarray analysis, we have generated the first catalog of gene expression changes accompanying postnatal conjunctival forniceal development in the mouse. We have also identified several transcriptional regulators dif-
ferentially expressed during conjunctival forniceal development. In addition, by identifying the Klf4 target genes in the mouse conjunctival fornix around the time of initial goblet cell differentiation, we have shown that Klf4 regulates goblet cell development directly, by controlling the expression of genes with critical roles in goblet cell physiology, and indirectly, by controlling the expression of other transcription factors known to influence goblet cell development, such as Spdef, FoxA1, and FoxA3. Thus, we now have a better understanding of the genetic network of transcription factors regulating conjunctival goblet cell development. We anticipate that the information in this report will provide new probes for studying conjunctival development, opening new avenues for understanding the complex genetic programs underlying conjunctival development and the ocular surface disorders associated with defective conjunctiva.
Acknowledgments The authors thank Gloria Limetti and Cindy Stone (Balaban Laboratory, Department of Otolaryngology, University of Pittsburgh) for help with histology and Kira Lathrop (Imaging Core Module, Department of Ophthalmology, University of Pittsburgh) for help with microscopy.
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