Expression Patterns and Potential Biological Roles of Dip2a

RESEARCH ARTICLE Expression Patterns and Potential Biological Roles of Dip2a Luqing Zhang1,2☯, Humphrey A. Mabwi1☯, Norberto J. Palange1, Ruirui Jia1, Jun Ma1, Fatoumata Binta Bah1, Rajiv Kumar Sah1, Dan Li1, Daji Wang1, Fatoumata Binta Maci Bah1, Jacques Togo1, Honghong Jin1, Luying Ban1, Xuechao Feng2*, Yaowu Zheng1,2* 1 Transgenic Research Center, School of Life Sciences, Northeast Normal University, Changchun, China, 2 Key Laboratory of Molecular Epigenetics of Ministry of Education, Northeast Normal University, Changchun, China ☯ These authors contributed equally to this work. * (XCF); (YWZ) Abstract OPEN ACCESS Citation: Zhang L, Mabwi HA, Palange NJ, Jia R, Ma J, Bah FB, et al. (2015) Expression Patterns and Potential Biological Roles of Dip2a. PLoS ONE 10 (11): e0143284. doi:10.1371/journal.pone.0143284 Editor: Austin John Cooney, University of Texas at Austin Dell Medical School, UNITED STATES Received: July 24, 2015 Accepted: November 3, 2015 Published: November 25, 2015 Copyright: © 2015 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Disconnected (disco)-interacting protein 2 homolog A is a member of the DIP2 protein family encoded by Dip2a gene. Dip2a expression pattern has never been systematically studied. Functions of Dip2a in embryonic development and adult are not known. To investigate Dip2a gene expression and function in embryo and adult, a Dip2a-LacZ mouse model was generated by insertion of β-Gal cDNA after Dip2a promoter using CRISPR/Cas9 technology. Dip2a-LacZ mouse was designed to be a lacZ reporter mouse as well as a Dip2a knockout mouse. Heterozygous mice were used to study endogenous Dip2a expression and homozygotes to study DIP2A-associated structure and function. LacZ staining indicated that Dip2a is broadly expressed in neuronal, reproductive and vascular tissues, as well as in heart, kidney, liver and lung. Results demonstrate that Dip2a is expressed in ectoderm-derived tissues in developing embryos. Adult tissues showed rich staining in neurons, mesenchymal, endothelial, smooth muscle cells and cardiomyocytes by cell types. The expression pattern highly overlaps with FSTL1 and supports previous report that DIP2A to be potential receptor of FSTL1 and its protective roles of cardiomyocytes. Broad and intense embryonic and adult expression of Dip2a has implied their multiple structural and physiological roles. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work is supported in whole or in part by National Natural Science Foundation of China (31301189 and 81270953), Research Funds for Central Universities (12QNJJ015 and 10JCXK001), Research Fund for Doctoral Program of Higher Education of China (20130043120010), ScienceTechnology Foundation for Young Scientist of Jilin Province (20130522003JH) and Program of International S&T cooperation (2015 DFA31580). The funders had no role in study design, data collection and analysis or decision to publish. Introduction DIP2A is a member of Disconnected (disco)-interacting protein 2 (DIP2) family with other two isoforms, DIP2B and DIP2C. Bioinformatic analysis using Predict Protein and Homolo Gene suggested that DIP2A is a type I receptor molecule with DMAP, CaiC and AMP-binding domains [1]. Mukhopadhyay reported that DIP2 homologs are evolutionarily conserved in organisms from C. elegans to humans. DIP2A proteins may exert their signaling roles as receptors in prokaryotes and eukaryotes and may provide positional cues for axon path finding and patterning [2]. Although expression pattern and physiological function of DIP2A are currently PLOS ONE | DOI:10.1371/journal.pone.0143284 November 25, 2015 1 / 16 Dip2a Expression Patterns Competing Interests: The authors have declared that no competing interests exist. unknown, previous studies have indicated that Dip2a expression is restricted to brain in mouse embryos, including neocortex, striatum and thalamus using Northern and in situ hybridization. LacZ enzyme activity can be easily visualized by X-Gal substrate staining [3]. Transgenic expression of β-Galactosidase gene (LacZ) has been widely used to trace endogenous gene expression and to predict potential biologic functions. In order to systematically investigate Dip2a gene expression in embryonic development and in adult tissues, we generated Dip2aLacZ mouse using CRISPR/Cas9 system [4]. Embryos from E9.5, E11.5, E12.5, E15.5 and adult tissues from Dip2aLacZ/+ and Dip2aLacZ/LacZ mouse were harvested. To identify specific Dip2a expression, whole-mount and frozen sections were stained with X-gal in parallel with wild type littermate controls. Materials and Methods All the general chemicals were from Sigma, USA and enzymes from Takara, Dalian, China. Animals All animals were maintained in a clean facility in Northeast Normal University. Mice were kept in IVC cages (5 per cage) with free access of food and water, at 20°C and 50 ± 20% relative humidity under a 12:12h light:dark cycles and pathogen free conditions. Mice were anesthetized before sacrificing with 1% pentobarbital at a dose of 10 mg/kg. All procedures were based on Guide for Care and Use of Laboratory Animals of National Institutes of Health and approved by Institutional Animal Care and Use Committee of Northeast Normal University (NENU/IACUC, AP2013011). C57BL/6J mice were purchased from Vital River (Beijing, China). Dip2a-LacZ mice were generated using CRISPR/Cas9 technology as described previously [4]. B6J.129S4/SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym/JNju were obtained from Nanjing Biomedical Research Institute of Nanjing University, China. Genotyping Embryos, pups and adult mice were genotyped for LacZ insertion by PCR. Genomic DNA was extracted from yolk sacks, embryonic limbs and tail tips. Samples were digested with GNT-K buffer at 55°C overnight [5]. Tail lysates were diluted and boiled for 15min. PCR was performed at 94°C for 2min followed by 30 cycles of denaturation at 94°C for 30sec, annealing at 60°C for 30sec and extension at 72°C for 30sec. Final extension was at 72°C for 5min and hold at 4°C. PCR product of 250bp was identified on 0.8% agarose gel. The primer sequences used for LacZ allele were ZF5'-ACCACACCTCCTGCTGTATAC-3' and ZR5'-ACGACGGGAT CATCGCGAGCCAT-3'. Primers WF5'-GGGTCACCTGGGCGACATTGA-3' and WR5'TCACCTTCG-GACAGCTCCAGCT-3' were used for wild type allele using GC-rich buffer I (Takara biotechnology) and slow down PCR program [6]. Neo gene was PCR amplified using primers NF5'-AGCTGGGGCTCGACTAGAGCTT-3', NR5'-TCACCTTCGGACA-GCTC CAGCT-3' and Flipase gene using primers FF5'-AAAGCATCTGGGAGAT-CACTGAG-3' and FR5'-TATACAAGTGGATCGATCCTAC-3' respectively. Whole mount embryos and adult tissues collection and LacZ staining Embryos and adult tissues (...truncated)