Reiterative FGF signaling determines the identity and morphology of the lacrimal gland

Abstract

The lacrimal gland plays an essential role in protection of the ocular surface by secreting the aqueous component of the tear film. Deficiency in the lacrimal gland is the main cause of dry eye disease, but existing treatments only alleviate the symptoms without curing the underlying disease. To develop curative measures, a thorough understanding of lacrimal gland development is needed. Lacrimal gland is formed as a result of interaction between the neural crest-derived mesenchyme and the conjunctival epithelium. The mesenchyme secretes the chemo-attractive signal of Fgf10, which binds to epithelial Fgfr2b and co-receptor heparan sulphate proteoglycans, to promote budding and branching morphogenesis of the lacrimal gland. However, the mechanism by which Fgf10 expression is regulated within the neural crest and the direct downstream targets of Fgf signaling in the epithelium are currently unknown. In this study, we show that FGF signaling mediated by protein phosphatase Shp2 is required for the proper patterning and differentiation of the neural crest-derived mesenchyme to produce Fgf10. Genetic evidence further demonstrates that Shp2 is recruited by Frs2α to activate Ras-MAPK signaling downstream to Fgfr1 and Fgfr2 but not to Pdgfrα in the neural crest. By differential gene expression analysis, we identified homeodomain transcription factor Alx4 as the key effector of Shp2 signaling to control expression of Fgf10 in the periocular mesenchyme. Loss of function ALX4/Alx4 mutation disrupted lacrimal gland development in both human and mouse. Our results reveal a FGF-Shp2-Alx4-Fgf10 axis in regulating neural crests during lacrimal gland development. In addition, we also show that Fgf signaling cascade mediated by Pea3 family of transcription factors are critical for lacrimal gland duct elongation and branching. High-throughput gene expression analysis revealed that Pea3 genes were important for establishing the tissue identity of the lacrimal gland. Loss of Pea3 resulted in upregulation of Notch signaling with the concomitant loss in the expression of the members of Six family of transcription factors and a switch of cell fate to the epidermal skin-like cells. These findings show that Fgf signaling is used reiteratively to establish the identity of both the epithelium and mesenchyme of the lacrimal gland.