Generation and analysis of the Rett syndrome-associated MeCP2- null rat model.

MeCP2 mutations are associated with the Rett syndrome (RTT). Currently, there is an urgent need for new animal models for RTT as the existing MeCP2 knockout mouse models fail to fully mimic the pathogenesis and symptoms of RTT patients. In order to investigate the role of MeCP2 in brain development and RTT pathogenesis, we aimed to set up the MeCP2-null rat model using the CRISPR/Cas9 technology. Firstly we constructed the MeCP2 targeting vector and then microinjected Cas9 mRNA and sgRNA mixtures into fertilized ova of SD rats. The sgRNA was designed to target the exon 2 of MeCP2. Next, knockout rats were confirmed using DNA sequencing and Western blotting. Lastly, phenotypes including growth and behaviors of MeCP2 knockout rats were analyzed. The results indicated that the MeCP2 knockout rats showed body weight loss, anxiety tendency and cognitive deficits. The MeCP2-null rat model established in this study recapitulates the major symptoms of RTT patients and provides an alternative tool for future studies of MeCP2 functions.

Whole-exome sequencing implicates UBE3D in age-related macular degeneration in East Asian populations.

Age-related macular degeneration (AMD) is a leading cause of irreversible central blindness among the elderly worldwide. We use exome sequencing to analyse nonsynonymous single-nucleotide variants (SNVs) across the whole genome of 216 neovascular AMD cases and 1,553 controls. As a follow-up validation, we evaluate 3,772 neovascular AMD cases and 6,942 controls from five independent cohorts in the East Asian population. Here we show strong evidence of an association at a novel, missense SNV, rs7739323, which is located in the ubiquitin protein ligase E3D (UBE3D) gene (Pmeta=1.46 × 10(-9), odds ratio (OR)=0.74, 95% confidence interval (CI): 0.63-0.88). Furthermore, ablation of the UBE3D protein lead to an abnormal amount of pigment granules deposited in retinal pigment epithelium microvilli area and an abnormal response on electroretinography (ERG) in UBE3D(+/-) heterozygous mice. Our findings indicate that the ubiquitin-proteasome system may play a role in the pathogenesis of neovascular AMD.

Msx2 plays a critical role in lens epithelium cell cycle control.

AIM: To investigate the effects of Msx2 on lens epithelium cell cycle, and evaluate the changes of the proliferation, apoptosis of lens epithelium cells. METHODS: Mice lens epithelium cells were cultured and transfected with pEGFP-Msx2 and control. Msx2-deficient mice (Msx2(-/-) ) lens tissue were isolated. Lens tissue and transfected cells were prepared for mRNA extraction using Trizol reagent. CyclinD1 and Prox1 expression were evaluated by real-time RT-PCR. BrdU incorporation and apoptosis rate were investigated by immunofluorescence and flow cytometry analysis. RESULTS: After transfected with pEGFP-Msx2, lens epithelium cells failed to incorporate BrdU and anti-phospho-histone-3 immunofluorescence failed to detect cell nuclei which GFP were positive. Msx2 over expression resulted in increasing apoptosis rate in lens epithelium cells. CyclinD1 and Prox1 expression increased significantly in Msx2 knockout mice by real-time RT-PCR quantization and CyclinD1 expression decreased significantly in Msx2 overexpressed cell. CONCLUSION: Msx2 has the effect of inhibiting proliferation and differentiation, triggering apoptosis on mice lens epithelium cells.

Identification of Notch-1 expression in the limbal basal epithelium.

PURPOSE: To determine whether Notch-1, a ligand-activated transmembrane receptor known to maintain cells in an undifferentiated state, primarily progenitor cells in other systems, could be used as a stem cell marker in human limbal epithelium. METHODS: Human corneoscleral tissues obtained from the Doheny Eye & Tissue Transplant Bank were prepared for cross section and whole mount analysis. Tissue for whole mount was incubated in dispase; the epithelial sheet was removed and fixed in 4% paraformaldehyde. Sections and whole mount were stained with antibodies against Notch-1, Notch-2, beta-1 integrin, alpha-6, and the G2 subtype member of the ATP binding cassette transporter (ABCG2). Specificity of the Notch-1 antibody was determined by western blot analysis with Cos-7 cells transfected with Notch-1. Explant culture was performed and only primary cultures were used in this experiment. RESULTS: Notch-1 was found to be expressed in the limbal basal region where stem cells reside. Notch-1 antigenicity was more pronounced in cell clusters, mainly in the palisades of Vogt. The central cornea was almost devoid of Notch-1. The intensity of Notch-1 staining in cultured cells from the limbal explants was high in only a few cells. The Notch-1 signal was diminished in dividing cells. Expression in cultured cells was more cytoplasmic; few cells showed additional nuclear staining. The Notch-1-stained whole mount showed only a few cells in the limbal region. A 300 kDa and a 110 kDa band confirmed the specificity of the antibody in Cos-7 cells transfected with Notch-1. Double staining for ABCG2 and Notch-1 showed some ABCG2-positive cells co-expressing Notch-1 in the limbal basal epithelium, indicating that Notch-1-expressing cells might be a unique subpopulation of cells with stem cell properties. CONCLUSIONS: Immunofluorescence data shows that Notch-1 could be a possible marker for the stem cells in the limbal basal epithelium. Further studies and characterization of the Notch pathway in corneal development will provide valuable clues for the identification of stem cells.

BMP4 activation and secretion are negatively regulated by an intracellular gremlin-BMP4 interaction.

Bone morphogenetic protein 4 (BMP4) is a potent growth factor that is involved in many important biological processes. Regulation of the level of secreted mature BMP4 determines the biological effects of BMP4 on cells in the local microenvironment. Previous studies suggested that Gremlin, a member of DAN family proteins, antagonizes BMP4 activity by sequestering extracellular BMP4. Herein, we report a novel intracellular regulatory mechanism by which Gremlin interacts with BMP4 precursor, prevents secretion of mature BMP4, and therefore inhibits BMP4 activity more efficiently. Furthermore, we also defined a 30-amino acid peptide sequence within the Gremlin DAN domain that is essential for BMP4 interaction. This novel Gremlin-mediated BMP4 posttranslational regulatory mechanism implies that the level of BMP4 mRNA expression does not truly reflect BMP4 activity when Gremlin and BMP4 are coexpressed within the same cell. Similar regulatory mechanisms may be utilized by other DAN family proteins.