Defective eyelid leading edge cell migration in C57BL/6-corneal opacity mice with an “eye open at birth” phenotype L.C. Wu1, C. Liu2, M.R. Jiang1, Y.M. Jiang1, Q.H. Wang1, Z.Y. Lu1, S.J. Wang1, W.L. Yang1 and Y.X. Shao1,2 1Laboratory Animals Center, Nantong University, Nantong, China 2Institute of Comparative Medicine, Nantong University, Nantong, China Corresponding author: Y.X. Shao E-mail: shaoyx@ ntu.edu.cn Genet. Mol. Res. 15 (3): gmr.15036741 Received May 7, 2015 Accepted May 25, 2016 Published August 26, 2016 DOI http://dx.doi.org/10.4238/gmr.15036741 Copyright © 2016 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution ShareAlike (CC BY-SA) 4.0 License. ABSTRACT. Development of the eyelid requires coordination of the cellular processes involved in proliferation, cell size alteration, migration, and cell death. C57BL/6J-corneal opacity (B6-Co) mice are mutant mice generated by the administration of N-ethyl- N-nitrosourea (100 mg/kg). They exhibit the eyelids open at birth phenotype, abnormal round cell shape from tightened F-actin bundles in leading edge keratinocytes at E16.5, and gradual corneal opacity with neovessels. The tip of the leading edge in B6-Co mice did not move forward, and demonstrated a sharp peak shape without obvious directionality. Analysis of the biological characteristics of B6-Co mice demonstrated that abnormal migration of keratinocytes could affect eyelid development, but proliferation and apoptosis in B6-Co mice had no effect. Mutant gene mapping and sequence analysis demonstrated that in B6-Co mice, adenosine was inserted into the untranslated regions, Genetics and Molecular Research 15 (3): gmr.15036741 L.C. Wu et al. 2 between 3030 and 3031, in the mRNA 3'-terminal of Fgf10. In addition, guanine 7112 was substituted by adenine in the Mtap1B mRNA, and an A2333T mutation was identified in Mtap1B. Quantitative real-time polymerase chain reaction analysis showed that expression of the Hbegf gene was significantly down-regulated in the eyelids of B6- Co mice at E16.5, compared to B6 mice. However, the expression of Rock1, Map3k1, and Jnk1 genes did not show any significant changes. Abnormal keratinocyte migration and down-regulated expression of the Hbegf gene might be associated with impaired eyelid development in B6-Co mice. Key words: B6-Co mouse; Leading edge; Keratinocyte migration; Eyelid development; Gene expression INTRODUCTION During normal development in mammals, the eyelids grow across the eye, fuse, and subsequently reopen. In mice, the periderm forms an outer layer of the epidermis on days 10 and 11 of gestation (E10 and E11). It is one-cell thick and covers the outer surface of the embryo from day E12 until it is shed on day E17. During this period the underlying epidermis rapidly proliferates and differentiates to become a stratified, keratinized epithelium. The upper and lower eyelids are fused to each other on day E16.5 and reopen on about postnatal day (P) 14 (Harris and Juriloff, 1986). The gaping lids (gp) or lidgap-Gates (lgGa) mutations, which cause the eyelids open at birth phenotype in mice, were discovered in 1961. They exhibit autosomal recessive inheritance spontaneously developed from C57BL/6-ax stain mice with 100% penetrance (Ricardo and Miller, 1967). The mutant gene was mapped to a chromosome between D13Mit76 (62.35 cM) and D13Mit53 (63.73 cM) using microsatellite genetic markers. There is a 27.5-kb deletion including exons 2-9 in the Map3k1 gene of lgGa mice (Juriloff et al., 2005). Mice with eyelids open at birth are generally referred to as having an eye open at birth (EOB) phenotype. EOB mice gradually develop corneal disease, which mainly presents as keratitis and corneal opacity. The developmental process of this phenotype mimics relevant diseases in humans. Therefore, the EOB mouse model is very useful for characterization of the molecular mechanisms underlying the process of eyelid closure and fusion. In our previous study, heredity C57BL/6J-corneal opacity (B6-Co) mice were generated by administering N-ethyl-N-nitrosourea (ENU) (100 mg/kg) to wild-type C57BL/6 mice (Shao et al., 2006). The eyes of these mice were open at birth, and the cornea gradually became opaque. The mutant gene was located between 112,546,283 and 113,397,654 bp on chromosome 13, determined by single nucleotide polymorphism (SNP) fine mapping (Jiang et al., 2010). In the present study, histochemical analysis was conducted to phenotypically characterize B6-Co mice at different embryonic stages. The wound healing assay, 5-bromo-2-deoxyuridine (BrdU) staining, and TUNEL assay were used to investigate migration, proliferation, and apoptosis, respectively, of keratinocytes in the eyelids of B6-Co mice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to test the expression of genes involved in eyelid development. B6-Co mice are an excellent model for investigating the mechanisms of eyelid development, and exploring drug targets for treating human cornea matrix denaturalization. Genetics and Molecular Research 15 (3): gmr.15036741 C57BL/6-corneal opacity mouse 3 MATERIAL AND METHODS Laboratory animals B6-Co, a mutant mouse model on a C57BL/6J (B6) background, and wild-type B6 mice were both obtained from the Laboratory Animal Center of Nantong University. Mice were kept in a barrier system at 23° ± 2°C with humidity of 55 ± 5%. The mice were maintained under a 12-h light/12-h dark cycle with free access to radiation-sterilized food (Shuangshi, Suzhou, China) and sterile water. All experimental procedures involving animals were conducted in accordance with the Institutional Animal Care guidelines, and were ethically approved by the administration committee of experimental animals, Jiangsu Province, China. Phenotypic characterization The corneal opacity of B6-Co mice was examined under a slit lamp. Fetuses of E14.5-P0 were sacrificed by decapitation, and heads were collected for phenotypic characterization. The heads of fetuses were fixed in 4% paraformaldehyde overnight at 4°C, dehydrated with a graded sucrose solution series, and embedded in Tissue Freezing Medium (O.C.T). Tissues were then cut into 8-mm thick frozen sections using a freezing microtome of Leica CM1900 (Leica, Solms, Germany). The sections were then subjected to hematoxylin and eosin (H&E) staining before acquiring images using a color digital camera. The tissue sections were incubated with FITC-phalloidin and Hoechst 33342 (Sigma, San Jose, CA, USA) and observed under a fluorescence microscope of Olympus cxk41 (Olympus, Tokyo, Japan). Cell culture Primary mouse epidermal keratinocytes and dermal fibroblasts were prepared from B6 and B6-Co E16.5 fetuses. B6-Co pups displayed the EOB phenotype. Keratinocytes were prepared as previously described (Zhang et al., 2003b) and all cell culture reagents were obtained from Hyclone (Hyclone, Logan, UT, USA). In vitro wound-healing assay Confluent monolayers of B6 and B6-Co epidermal keratinocytes and dermal fibroblasts were starved in growth factor-free media for 24 h before being wounded by scratching the monolayer with a micropipette tip. The wounded cells were then washed, and cultured at 37°C in the appropriate media with or without growth factors. Images of the wounded area were acquired 24 h after wounding. BrdU incorporation The proliferation of keratinocytes was determined using a BrdU labeling/detection kit (Sigma), according to the manufacturer instructions. After BrdU labeling, the keratinocytes were observed under a fluorescent microscope. The percentage of BrdU-positive cells was calculated from 10 random fields in 3 wells. Genetics and Molecular Research 15 (3): gmr.15036741 L.C. Wu et al. 4 In vitro apoptosis assay Keratinocytes isolated from the eyelids of E16.5 mice were used to detect apoptotic cells. A TUNEL assay was performed on samples using the One Step TUNEL Apoptosis Assay Kit, according to the manufacturer instructions (Beyotime, Haimen, China). Sequence analysis and real-time PCR Total RNA was isolated from B6-Co and B6 embryos using the TRIzol kit, according to the manufacturer instructions (Invitrogen, Carlsbad, CA, USA). Full-length cDNA was synthesized using RevertAidTM M-MuLV reverse transcriptase (Fermentas, Burlington, Canada) and was then amplified by PCR using Taq polymerase (Fermentas, Burlington, Canada). The primers were designed using the Primer Prepmer 5.0 software (Premier, Toronto, Canada) (Table 1). The RT-PCR products for Fgf10, and Mtap1B were 1138 and 938 bp, respectively. PCR products were gel purified using the EZ Spin Column DNA Gel Extraction Kit (Sangon, Shanghai, China), and recombinant vectors were transformed in competent cells after DNA fragments were cloned into the pGEM-T vector (Promega, Madison, WI, USA). Positive clones were identified byEco RI restriction endonuclease digestion and sequence analysis (Sangon). Total RNA were extracted from eyelid tissues in B6-Co and B6 embryos for real-time PCR using Fast EvaGreen® qPCR Master Mix (Biotium, Hayward, CA, USA). The primers were designed using the Primer Prepmer 5.0 software (Table 1). The PCR products for Gapdh, Hbegf, Rock1, Map3k1, and Jnk1 were 133, 182, 117, 172, and 162 bp, respectively. The reactions were cycled 45 times under the appropriate parameters for each pair of primers and the fluorescence was measured every 15 s at the end of each cycle to construct the amplification curve. All determinations were performed at least in triplicate. The relative expression of mRNAs was calculated using