Unlike the cornea, relatively few studies have addressed the changes in gene expression in the conjunctiva (). In addition to the epithelial cells, conjunctiva contains goblet cells which produce and secrete mucins to the tear film. Conjunctival goblet cells play an important role in maintaining ocular surface homeostasis by producing and secreting mucins to the tear film [58
]. In spite of their importance, relatively little is known about the factors regulating conjunctival goblet cells. Studies in other tissues have demonstrated the requirement of factors such as Foxa1, Foxa2, Foxa3, and Spdef for colonic and airway epithelial goblet cell development [59
]. Studies in our laboratory have demonstrated that Klf4 and Klf5 are both required for conjunctival goblet cell development () [64
Figure 3 Hierarchical network of transcription factors regulating goblet cell development. Foxa1, Foxa3, and SPDEF, required for goblet cell development in other mucosal epithelia, are downregulated in Klf4CN and Klf5CN conjunctiva , placing Klf4 and Klf5 (more ...)
In order 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, we used Laser microdissection (LMD) to collect conjunctival forniceal cells from PN 9, PN14, and PN20 wildtype (WT) and PN14 Klf4
-conditional null (Klf4
CN) mice, in which goblet cells are absent, developing, present, and missing, respectively. We 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 Klf4
CN conjunctiva, respectively [16
]. Comparison of the conjunctival Klf4-target genes [16
] with the corneal Klf4-target genes [66
] identified a small number of common target genes, suggesting that Klf4 performs distinct functions in different tissues, by regulating a diverse array of targets. This tissue-selective nature of Klf4 is important in view of the widespread expression of Klf4 in several parts of the body. How such tissue-selective nature is achieved remains to be understood.
Klf4 may exert its influence on conjunctival goblet cells directly or by controlling the expression of other transcription factors regulating goblet cell development. By comparing the wild type and Klf4
CN PN14 conjunctival forniceal gene expression, we identified the transcription factors affected in the Klf4
CN conjunctiva. Four among these factors were previously shown to be required for goblet cell development in other tissues such as colon or lung. Transcripts encoding Spdef, Foxa1, and Foxa3 that regulate goblet cell development and epithelium-specific Ets (ESE) transcription factor family members were increased during conjunctival development [16
Sterile alpha motif- (SAM-) pointed domain containing Ets family protein (SPDEF) is a member of the ETS family of transcription factors. SPDEF contains an ETS DNA-binding domain at the C-terminal and a regulatory region consisting of the SAM-pointed domain at the N-terminal [67
]. It preferentially interacts with ETS-binding sites with a core sequence GGAT. Spdef is required for goblet cell development in the intestine [63
] and tracheal/laryngeal submucosal glands as well as the conducting airway epithelium after allergen exposure [61
]. In transient transfection assays, both Klf4 and Klf5 stimulated mouse Spdef promoter activity (Gupta and Swamynathan, unpublished). However, Klf5 had a relatively greater effect, and cotransfection with Klf4 and Klf5 did not have any additional stimulatory effect, suggesting that Klf4 and Klf5 act through the same cis-elements in Spdef promoter (Gupta and Swamynathan, unpublished) ().
Though the studies summarized above have given us a general picture of the goblet cell gene expression patterns, we are yet to understand what goes wrong in pathophysiological conditions which affect goblet cell densities. For example, changes in gene expression associated with goblet cell hyperplasia in allergic conjunctivitis and asthma or, alternatively, their absence in ocular cicatrizing pemphigoids remains to be identified. Additional studies in these directions are necessary to gain a better understanding of the genetic network of transcription factors which regulate goblet cell development and function in healthy and disease conditions.