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Chia-Yang Liu, Yujin Zhang, Winston W. Kao; Notch Activation Downregulates Foxl2, Leading To Congenital Blepharophimosis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1108.
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© ARVO (1962-2015); The Authors (2016-present)
To understand Notch signaling in the normal eyelid morphogenesis and the pathogenesis of human congenital blephrophimosis syndrome (BPES), we studied the effects of aberrant Notch activation in neural crest-derived periocular mesenchymal cells (POMCs), which contribute to the formation of corneal and eyelid stroma.
Notch 1 intracellular domain (N1-ICD) was conditionally over-expressed in peri-ocular mesenchymal cells of KerartTA/tetO-Cre/RosaN1-ICD (KR/TC/RosaN1-ICD) triple transgenic mice induced by doxycycline (Dox, 80 mg/kg body weight).The phenotypes were examined by histology and immunohistochemistry. Chromatin immunoprecipitation (ChIP) and FoxL2 and Acta-2 promoter-luciferase assays in cell cultures were employed to investigate the mechanism of how the Notch1 activation may serve as the upstream control of expression of FoxL2 by peri-ocular mesenchyma which in turn controlling eyelid levator smooth muscle formation
Transgenic mice over-expressing Notch 1 intracellular domain (N1-ICD) in the POMCs (hereafter abbreviated as POMCN1-ICD) showed delayed and incompletely eyelid closure at embryonic (E) day 15.5 and birth, respectively. POMCN1-ICD had little effect to the cornea, but it increased cell apoptosis and decreased cell proliferation during eyelid morphogenesis. POMCN1-ICD impaired eyelid levator smooth (Müeller) muscle formation due to down-regulation of FoxL2,resembling human type II BPES (Blepharophimosis, Ptosis, and Epicanthus inversus Syndrome). ChIP assay demonstrated that Notch-RBPJ complex directly bound to FoxL2 promoter. Hes-1 transcriptional suppressor also bound to 3 of 4 N-Box motifs in the FoxL2 promoter. Promoter assays showed that low dose CMV-N1-ICD up-regulated but high dose CMV-N1-ICD down-regulated FoxL2 promoter activity via Hes-1 activation. Moreover, FoxL2 could direct bind to Acta-2 promoter in vivo and transfection of CMV-FoxL2 wild-type but not mutant enhanced Acta-2 promoter activity.
These data strongly implicated that physiologically low level of Notch has critical role to control proper FoxL2 expression in the POMCs, which is essential for Müeller muscle formation and normal eyelid development. In contrast, POMCN1-ICD inhibits FoxL2 expression and impairs Müeller muscle formation, leading to eyelid malformation and BPES-like phenotypes.
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