NOTCH2 REGULATES BMP4 AND MORPHOGENESIS IN THE DEVELOPING MOUSE CILIARY BODY

Abstract

Notch is an evolutionarily conserved cell-signaling pathway that is ubiquitous throughout development from Drosophila to humans. Defects in the Notch pathway result in several human genetic diseases and are associated with acquired disease states including cancer. Using the mouse eye as a model, we have discovered a new role for Notch2 in development and a novel interaction between the Notch2 and BMP4 pathways. The ciliary body is an important part of the anterior eye which secretes aqueous humor and controls lens accommodation. The ciliary body also modulates the trabecular meshwork which maintains proper intraocular pressure by regulating its outflow. Using conditional Cre-loxP knockout technology, we selectively removed the Notch2 receptor from the pigmented outer ciliary epithelium in mouse. The loss of Notch signaling resulted in severe dysmorphogenesis of the ciliary body while sparing the iris. Contributing to this lack of morphogenesis is a significant reduction in proliferation across multiple time points. Cell-adhesion both dorsally and ventrally involving N-cadherin, α-catenin, and β-catenin is also disrupted. Thirdly, a ventral specific reduction of pSMAD1/5/8 was detected. Using a GFP reporter line which identified Cre expression, we determined that Notch2 affects N-cadherin and cell adhesion in a cell autonomous manner, while pSMAD1/5/8 is regulated both cell autonomously and non-cell autonomously only on the ventral side of the eye. This is a novel interaction between the Notch and BMP pathways. To further define the roles of Notch2 versus BMP4, we crossed the same Cre-line with Alk2/Alk3 mice to conditionally remove the BMPR1 receptors. While previous work showed that BMP4 heterozygotes displayed ciliary body dysmorphogenesis, our new cross demonstrates that BMP4 signaling in the outer ciliary epithelium is necessary for ciliary body morphogenesis. In the Alk2/Alk3 mice, N-cadherin, α-catenin, and β-catenin were all present, indicating that they are downstream of Notch2 and not BMP4. This is consistent with our Notch2 knockout studies. Notch2 staining is also retained in the DM61/Alk2fx/fx/Alk3fx/fx mice, further confirming that Notch2 is upstream of BMP4. Finally, to confirm the role of adhesion in the ciliary body, we crossed our Cre-line with an N-cadherin floxed line. The DM61/N-cadherinfx/fx mice recapitulated the Notch2 phenotype and confirmed the necessity of N-cadherin in ciliary body morphogenesis. Histologic analysis suggest a positive feedback loop between N-cadherin and Notch2, as Notch2 was reciprocally downregulated in the DM61/N-cadherinfx/fx mice. BMP4 signaling was also downregulated. Taken together, we have discovered a new role for Notch2 in eye development. Notch2 is a necessary signal in regulating the morphogenesis of ciliary body development. It does this, at least in part, through a concerted program of proper proliferation and adhesion. In parallel with regulating adhesion and possibly upstream of proliferation, Notch2 regulates BMP4 signaling through a novel mechanism. This study is the first to provide any evidence of signaling pathway integration in anterior eye development. Proper development of the anterior eye is significant in several human diseases, including primary congenital glaucoma. These findings may have far-reaching implications across both development and disease, especially in Notch biology.