Abstract
Purpose:
Lacrimal, Harderian and Meibomian glands develop from the prospective conjunctival and eyelid epithelia by branching morphogenesis and produce secretions that lubricate and protect the ocular surface. To better understand the mechanisms responsible for their formation, we identified genes expressed selectively in the prospective conjunctiva and tested the role of one of them, Sox9, in the formation of the mouse ocular glands.
Methods:
Microarray analysis was used to identify transcripts that were differentially expressed in the prospective cornea, conjunctiva and eyelid margin at E12.5. Laser microdissected tissues were isolated, total RNA extracted, reverse transcribed and amplified using the NuGEN Pico kit and hybridized to Illumina Mouse6 whole genome bead arrays. Antibodies to Sox9 were used to map its expression from E10.5 through birth. Transgenic mice expressing Cre recombinase in the ocular surface epithelia (LeCre) were mated to mice carrying floxed alleles of Sox9, Fgfr2 or Smad4. Embryos and postnatal mice were fixed and processed for standard histology, immunohistochemistry staining, in situ hybridization.
Results:
Sox9 transcripts and protein were initially expressed throughout the ocular surface epithelium at E10.5, but by E12.5 were preferentially localized to the prospective conjunctival epithelium. Sox9 expression decreased after E15.5, becoming undetectable in the conjunctiva at birth. However, Sox9 expression remained in the epithelium of the lacrimal/Harderian glands. Sox9 conditional knockout mice lacked lacrimal/Harderian glands and had reduced numbers of Meibomian glands in the lower eyelid. Mice heterozygous for Sox9 had variable degrees of defective lacrimal gland morphogenesis, including absence of the extraorbital (lacrimal) lobe (62.5%) and small or ectopic branches (9.4%). Although the epithelial component of the lacrimal/Harderian glands was absent in Sox9-/- mice, Fgf10, which is required for lacrimal gland formation, was still expressed in the adjacent mesenchyme. Sox9 expression was decreased at E14.5 in Fgfr2 and Smad4 conditional knockout mice, in which the lacrimal/Harderian glands do not form.
Conclusions:
Sox9 is required for the formation of lacrimal/Harderian glands and contributes to Meibomian gland formation. Sox9 expression in the prospective conjunctiva appears to be regulated by FGF and BMP signaling. Loss of one Sox9 allele reduced branching of the lacrimal gland.
Keywords: 576 lacrimal gland •
474 conjunctiva •
497 development