AIM To explore the molecular mechanisms in lens development and the

AIM To explore the molecular mechanisms in lens development and the pathogenesis of Peters anomaly in Smad4 defective mice. Smad4 defective mice and control mice at E16.5. Statistical evaluations were performed using the unpaired Student’s em t /em -test (two-tailed) by SPSS 11.0 software. RESULTS Conditional deletion of Smad4 on eye surface ectoderm resulted in corneal dysplasia, iridocorneal angle closure, corneolenticular adhesions and cataract resembling Peters anomaly. Loss of Smad4 function inhibited E-cadherin expression in the lens epithelium cells and corneal epithelium cells in Smad4 defective eye. Expression of N-cadherin was up-regulated in corneal epithelium and corneal stroma. Both E-cadherin and N-cadherin were down-regulated at the future trabecular meshwork region in mutant eye. The qPCR results showed that the expression of Twist2 was increased significantly in the mutant lens ( em P /em 0.01). CONCLUSION Smad4 is essential to eye development and likely a candidate pathogenic gene to Ctgf Peters anomaly by regulating epithelial-mesenchymal transition. Twist2 can be regulated by Smad4 and plays an essential role in lens development. strong class=”kwd-title” Keywords: Peters anomaly, anterior segment dysgenesis, Smad4, N-cadherin, Twist2 INTRODUCTION Peters anomaly Verteporfin manufacturer is referred to a range of a congenital abnormality of the anterior segment of the eye, such as corneal opacity, shallow anterior chamber, corneolenticular adhesions, cataract and so on[1]C[2]. Over 50% of Peters anomaly cases have glaucoma and more than 15% of cases accompanied lenticular malformations[1]C[2]. Right now there is no effective treatment for the disease, and visual loss is inevitable. Although surgical techniques have been constantly improved to cure the disease, the rate of surgical success still remains low. Organogenesis Verteporfin manufacturer of the eye is a complicated process. The surface ectoderm becomes thickened and invaginates to form the lens vesicle. The lens vesicle gradually develops into the mature lens, while the remained surface ectoderm develops into corneal epithelium. The cranial paraxial mesoderm and mesenchymal cells of neural crest origin migrate into the space between the lens Verteporfin manufacturer vesicle and the remained surface ectoderm, and give rise to corneal stroma, corneal endothelium, ciliary muscle as well as the trabecular meshwork. It has been proposed that abnormal development of surface ectoderm and disturbed neural crest cells migration during eye development are responsible for Peters anomaly, but the precise pathogenesis still remains unknown[1]C[2]. Verteporfin manufacturer Smad4 is a key intracellular effector of the transforming growth factor (TGF-) superfamily of secreted ligands, which plays an essential role in organogenesis and tissue homeostasis during developmental process. Previous studies have shown that Smad4 is expressed in both the lens vesicle and presumptive corneal ectoderm, and conditional deletion of Smad4 in the eye surface ectoderm leads to severe abnormality in the anterior segment [3]C[5]. However, the precise role of Smad4 in anterior segment development and the underling mechanism are still unclear. Here we present data that Smad4 in the ocular surface ectoderm is required for cornea, lens and anterior chamber angle development. Conditional deletion of Smad4 on eye surface ectoderm resulted in corneal dysplasia, iridocorneal angle closure, corneolenticular adhesions and cataract resembling Peters anomaly. Mechanistically, Smad4 in the eye surface ectoderm affected the epithelial-mesenchymal transition, and regulated the expression of Twist2. MATERIALS AND METHODS Animals All animal experiments followed the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Le-Cre transgenic mice [6] and mice carrying floxed Smad4 alleles (Smad4fl/fl)[7] were kindly gifted from Dr. Yi-Hsin Liu (University of Southern California, Los Angeles, USA). The Le-Cre; Smad4fl/fl mice were acquired as the mating picture shown (Figures 1A), and littermate mice carrying Smad4fl/fl or Smad4fl/+ were used as controls. Polymerase chain reaction (PCR) was performed to establish the genotype with the primers as previous described (Figures 1B, ?,1C)1C) [6]C[7]. Open in a separate window Figure 1 Generation of conditional deleted of the Smad4 gene in surface ectodermA: Mice mating procedure to acquire Le-Cre; Smad4fl/fl mice; B, C: The detection of Le-Cre (B) and Smad4 (C) allele by PCR. A fragment of 350 bp indicated the existence of Cre gene. The fragment of 438 bp indicated the Smad4 floxed allele and 385 bp of wild-type Smad4 gene. Hematoxylin and Eosin Staining and Immunohistochemical Staining Pregnant mice were sacrificed to get the embryos. The embryos were fixed in 4% paraformaldehyde overnight at 4C, then dehydrated through graded alcohols and embedded in paraffin. The 4 m sections were cut for hematoxylin and eosin Verteporfin manufacturer (HE) staining and immunohistochemical staining. Immunohistochemical staining was performed as previously described[8]..