Abstract
Purpose: :
Wnt signaling plays pivotal roles in tissue morphogenesis. In this study, attmepts were made to examine the role of Wnt/β-catenin pathway in eyelid morphogenesis.
Methods: :
RT-PCR and immunofluoresecne staining were employed to determine the expression of Wnts and cellular localization of Ctnnb1 in eyelid during embryonic development. The driver Kera-rtTA (KR), tet-O-Cre (TC) and β-catenin flox (Ctnnb1f/f) mice were crossed to obtain triple transgenic mice KR/TC/ Ctnnbf/f in which Ctnnb1 was ablated by feeding pregnant dams with Dox-chow commenced at the beginning of mating (E0) continued to various gestation stages e.g., E14.5, E15.5, E16.5 and postnatal day 1 when embryos and neonates were collected. The specimens were then subjected to histology and immunohistochemistry examination.
Results: :
Wnt ligands including Wnt3, Wnt5a, Wnt8a, Wnt9a, Wnt9b, Wnt11 and Wnt16 were expressed by eyelid mesenchymal cells as detected by RT-PCR. Immunofluorescence staining indicated that there was a translocation of Ctnnb 1 from plasmic membrane at E13.5 to nuclei at E15.5. The ablation of β-catenin in eyelid mesenchymal cells caused anomaly of the formation of bilateral concaved eyelids at birth. Histological examinations showed that eyelid epithelium of both wild-type and β-catenin lof-mutant (lost of function) mice began to migrate at E14.5. However, the concurrent growth of eyelid stroma and subsequent eyelid closure were disrupted in β-catenin lof mutant, which normally take place at E16.5. Lof mutants also had thinner corneal stroma than that of wild type mice. It was noteworthy that number of BrdU-labeled eyelid mesenchymal cells in lof mutant is 5-fold less than that of wild type, while TUNEL assay did not show any noticeable difference. Immunohistochemistry showed that there was a upregulation of phospho-p38 but phospho-ERK was not affected in mesenchymal cells of lof mutant at E15.5.
Conclusions: :
These findings suggested that the eyelid malformation caused by ablation of Ctnnb1 may be due to reduced proliferation and/or perturbed migration of periocular mesenchymal cell of neural crest origin. Canonical Wnt/β-catenin signaling in periocular mesenchymal cells plays a pivotal role for the eyelid morphogenesis during mouse embryonic development.
Keywords: development • signal transduction • transgenics/knock-outs