Notch2 regulates BMP signaling and epithelial morphogenesis in the ciliary body of the mouse eye

Abstract

The ciliary body (CB) of the mammalian eye is responsible for secreting aqueous humor to maintain intraocular pressure, which is elevated in the eyes of glaucoma patients. It contains a folded two-layered epithelial structure comprising the nonpigmented inner ciliary epithelium (ICE), the pigmented outer ciliary epithelium (OCE), and the underlying stroma. Although the CB has an important function in the eye, its morphogenesis remains poorly studied. In this study, we show that conditional inactivation of the Jagged 1 (Jag1)-Notch2 signaling pathway in the developing CB abolishes its morphogenesis. Notch2 is expressed in the OCE of the CB, whereas Jag1 is expressed in the ICE. Conditional inactivation of Jag1 in the ICE or Notch2 in the OCE disrupts CB morphogenesis, but neither affects the specification of the CB region. Notch2 signaling in the OCE is required for promoting cell proliferation and maintaining bone morphogenetic protein (BMP) signaling, both of which have been suggested to be important for CB morphogenesis. Although Notch and BMP signaling pathways are known to crosstalk via the interaction between their downstream transcriptional factors, this study suggests that Notch2 maintains BMP signaling in the OCE possibly by repressing expression of secreted BMP inhibitors. Based on our findings, we propose that Jag1-Notch2 signaling controls CB morphogenesis at least in part by regulating cell proliferation and BMP signaling.T he mammalian eye is composed of the anterior segment, the posterior retina and the vitreous. The anterior segment consists of cornea, lens, and ciliary body (CB), whereas the posterior retina contains six types of retinal neurons and Müller glial cells, which are organized into three distinct cell layers. The light passes through the cornea and is then focused by the lens and detected by photoreceptors in the retina. Both the aqueous humor anteriorly and the vitreous humor posteriorly function together to sustain intraocular pressure (IOP) in the eye and thereby maintain its shape. Pressure regulation is particularly important because abnormally high IOP is a major risk factor for glaucoma (1). In the eye, the CB is responsible for producing aqueous humor (2). Although high IOP is often attributed to the blockage of the drainage system for the vitreous, known as the trabecular meshwork, abnormal CB function might also contribute to high IOP formation because of excess aqueous humor production. Finally, contraction of the muscles in the CB controls lens accommodation for near versus far vision. Despite its important biological functions and potential medical significance, the formation and development of the CB remain poorly studied.The CB contains two layers of apically adhered epithelial sheets, the pigmented outer ciliary epithelium (OCE) and the nonpigmented inner ciliary epithelium (ICE), and the underlying stroma (2). It forms at the periphery of the developing optic cup and first segregates from the retina and then from the iris. One previous study suggested that blood vesse...

Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation

“…The activity of Mart1::Cre in the eye is restricted to the pigmented epithelial cells of the iris, CB, and RPE (Figure S1), thus allowing deletion of Notch1, Notch2, and RBP‐Jκ specifically in these structures. Consistent with a previous report, loss of Notch2 in pigmented epithelial cells resulted in CB aplasia (Zhou et al., ), a phenotype that was recapitulated in RBP‐Jκ ∆/∆ mice (Figure A). Further histological analysis indicated that other pigmented eye structures such as iris and RPE were normal in RBP‐Jκ ∆/∆ mutants (Figure A and Figure S2), indicating that the CB is the only pigmented structure that requires Notch signaling.…”

“…This revealed a profound increase in actively cycling pigmented epithelial cells (MITF + ) in the CB (Figure E), indicating that the hyperplastic CB results at least in part from hyperproliferation. This is consistent with the growth promoting and oncogenic functions of excessive Notch signaling reported in other tissues (Koch and Radtke, ), and is in accordance with the reduced proliferation observed in Notch2 null CB (Zhou et al., ). Proliferation was also analyzed during development, when patterning and specification of the CB and iris is occurring.…”

“…The data obtained from the Notch loss‐of‐function experiments demonstrate that canonical Notch signaling is essential for CB morphogenesis but is dispensable for iris development. Similar results have been reported by a recent study, which also demonstrated that conditional ablation of Notch2 in the pigmented epithelium resulted in CB aplasia (Zhou et al., ). It was also shown that Notch signaling is induced in the presumptive CB by Jagged‐1 expression in the non‐pigmented, inner ciliary epithelium, which is an extension of the neural retina.…”

Using genetic mouse models to gain insight into glaucoma: Past results and future possibilities

“…Cloning of ASD mutations in mice has led to identifying ASD genes in humans (Kuo et al, 2012; McKeone et al, 2011). Additionally, many phenotypes characteristic of ASD—ciliary body and trabecular meshwork hypoplasia, peripheral iridocorneal adhesion, buphthalmos, and elevated IOP—have been found in both spontaneous and genetically modified mouse mutants (Chang et al, 2001; Gould et al, 2007; Kroeber et al, 2010; Libby et al, 2003; Mao et al, 2011; Sarode et al, 2014; Smith et al, 2000; Sowden, 2007; Weng et al, 2008; Zhou et al, 2013). Mutations in transcription factors ( PITX2 , FOXC1 , FOXF2 , LMX1B , PAX6 ) are responsible for numerous cases of ASD and PCG (Gould et al, 2004b) and mouse mutants have been used to study the cell biology of these genes in the eye.…”

“…Recent work with mice carrying mutations in various cell-cell signaling components is beginning to give us insight into the inductive events that drive anterior segment development. For instance, BMP4, a secreted molecule belonging to the TGFβ superfamily, is believed to be necessary for proper formation of both the ciliary body and the iridocorneal angle (Chang et al, 2001; Zhou et al, 2013). Additionally, mutations in several genes influencing the extracellular matrix (eg.…”