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 (). 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 endocytosis-related pathways whose component genes are increased during conjunctival development (). 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
γ receptor–mediated phagocytosis pathway, and tight junction signaling were increased, lending support for development of the conjunctival mucosal immune system at eyelid opening (). Genes representing three different pathways related to retinoid signaling were increased (), suggesting a role for retinoids in conjunctival development and/or function. Increased expression of genes related to neural development and function (), 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.10-7068/-/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 Klf4
CN conjunctiva. Chief among them are members of the neurotransmitter transporter Slc6
family, consistent with the neural development– and function-related 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 Klf4
CN 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 Sjögren'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 Klf4
CN PN14 conjunctival forniceal gene expression, we identified a list of transcription factors whose expression was affected in the Klf4
CN conjunctiva lacking goblet cells (; Supplementary Table S13, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-7068/-/DCSupplemental
). Among them, factors such as FoxA1
, 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
, 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 (). 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 Klf4
CN conjunctiva were relatively unaffected in the Klf4
Protein disulfide isomerase Agr2, significantly decreased in the Klf4
CN 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 Klf4
CN 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 differentially 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.