Mouse Conjunctival Forniceal Gene Expression during Postnatal Development and Its Regulation by Kruppel-like Factor 4

Department of Ophthalmology, University of Pittsburgh School of Medicine, Eye and Ear Institute, 203 Lothrop Street, Pittsburgh PA 15213, USA.
Investigative ophthalmology & visual science (Impact Factor: 3.66). 03/2011; 52(8):4951-62. DOI: 10.1167/iovs.10-7068
Source: PubMed

ABSTRACT 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.
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.
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.
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.

1 Follower
  • [Show abstract] [Hide abstract]
    ABSTRACT: The ocular surface epithelia, including the stratified but non-keratinized corneal, limbal and conjunctival epithelium, in concert with the epidermal keratinized eyelid epithelium, function together to maintain eye health and vision. Abnormalities in cellular proliferation or differentiation in any of these surface epithelia are central in the pathogenesis of many ocular surface disorders. Goblet cells are important secretory cell components of various epithelia, including the conjunctiva; however, mechanisms that regulate goblet cell differentiation in the conjunctiva are not well understood. Herein, we report that conditional deletion of transforming growth factor β receptor II (Tgfbr2) in keratin 14-positive stratified epithelia causes ocular surface epithelial hyperplasia and conjunctival goblet cell expansion that invaginates into the subconjunctival stroma in the mouse eye. We found that, in the absence of an external phenotype, the ocular surface epithelium develops properly, but young mice displayed conjunctival goblet cell expansion, demonstrating that TGFβ signaling is required for normal restriction of goblet cells within the conjunctiva. We observed increased expression of SAM-pointed domain containing ETS transcription factor (SPDEF) in stratified conjunctival epithelial cells in Tgfbr2 cKO mice, suggesting that TGFβ restricted goblet cell differentiation directly by repressing Spdef transcription. Gain of function of Spdef in keratin 14-positive epithelia resulted in the ectopic formation of goblet cells in the eyelid and peripheral cornea in adult mice. We found that Smad3 bound two distinct sites on the Spdef promoter and that treatment of keratin 14-positive cells with TGFβ inhibited SPDEF activation, thereby identifying a novel mechanistic role for TGFβ in regulating goblet cell differentiation.
    Development 11/2014; DOI:10.1242/dev.117804 · 6.27 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The cornea is the clear, outer-most portion of the eye composed of three layers: an epithelium that provides a protective barrier while allowing transmission of light into the eye, a collagen-rich stroma, and an endothelium monolayer. How cornea development and aging is controlled is poorly understood. Here we characterize the mouse cornea transcriptome from early embryogenesis through aging, and compare to transcriptomes of other epithelial tissues, identifying cornea-enriched genes, pathways, and transcriptional regulators. Additionally, we profiled corneal epithelium and stroma, defining genes enriched in these layers. Over 10,000 genes are differentially regulated in the mouse cornea across the timecourse, showing dynamic expression during development and modest expression changes in fewer genes during aging. A striking transition time-point for gene expression between postnatal days 14 and 28 corresponds with completion of cornea development. Clustering classifies co-expressed, and potentially co-regulated, genes into biologically informative categories, including groups that exhibit epithelial- or stromal-enriched expression. Based on these findings and through loss of function studies and ChIP-seq we show that the Ets transcription factor EHF promotes cornea epithelial fate through complementary gene activating and repressing activities. Furthermore, we identify potential interactions between EHF, KLF4, and KLF5 in promoting corneal epithelial differentiation. These data provide insights into the mechanisms underlying epithelial development and aging, identifying EHF as a regulator of cornea epithelial identity, and pointing to interactions between Ets and KLF factors in promoting epithelial fate. Furthermore, this comprehensive gene expression dataset for cornea is a powerful tool for discovery of novel cornea regulators and pathways.
    Journal of Biological Chemistry 10/2013; 288(48). DOI:10.1074/jbc.M113.504399 · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Purpose: Recent development of mice null for either Muc5ac or Muc5b mucin allows study of their specific roles at the mouse ocular surface. A recent report indicated that Muc5ac null mice show an ocular surface phenotype, similar to that seen in dry eye syndrome. The purpose of our study was to determine the effect of lack of Muc5ac or Muc5b on the ocular surface and to determine if environmental desiccating stress exacerbated a phenotype. Methods: Muc5ac null and Muc5b null mice and their wild-type controls were examined for ocular surface defects by fluorescein staining; the number of goblet cells per area of conjunctival epithelium was counted; and levels of mucin gene expression and genes associated with epithelial stress, keratinization, and differentiation, known to be altered in dry eye syndrome, were assayed. To determine if the null mice would respond more to desiccating stress than their wild-type controls, they were challenged in a controlled environment chamber (CEC) and assessed for changes in fluorescein staining, tear volume, and inflammatory cells within the conjunctival and corneal epithelia. Results: Unlike the previous study, we found no ocular surface phenotype in the Muc5ac null mice; even after exposure to desiccating environmental stress. Similarly, no ocular surface phenotype was present in the Muc5b null mice, either before or after exposure to a dry environment in the CEC. Conclusions: Our results indicate that deleting either the Muc5ac or Muc5b gene is insufficient to create an observable dry eye phenotype on the ocular surface of these mice.
    Investigative ophthalmology & visual science 12/2013; 55(1). DOI:10.1167/iovs.13-13194 · 3.66 Impact Factor

Full-text (2 Sources)

Available from
May 31, 2014