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Disconnected interacting protein 2 homolog A (DIP2A) is highly expressed in nervous system and respiratory system of developing embryos. However, genes regulated by Dip2a in developing brain and lung have not been systematically studied. Transcriptome of brain and lung in embryonic 19.5 day (E19.5) were compared between wild type and Dip2a-/- mice. An average of 50 million reads per sample was mapped to the reference sequence. A total of 214 DEGs were detected in brain (82 up and 132 down) and 1900 DEGs in lung (1259 up and 641 down). GO enrichment analysis indicated that DEGs in both Brain and Lung were mainly enriched in biological processes 'DNA-templated transcription and Transcription from RNA polymerase II promoter', 'multicellular organism development', 'cell differentiation' and 'apoptotic process'. In addition, COG classification showed that both were mostly involved in 'Replication, Recombination, and Repair', 'Signal transduction and mechanism', 'Translation, Ribosomal structure and Biogenesis' and 'Transcription'. KEGG enrichment analysis showed that brain was mainly enriched in 'Thyroid cancer' pathway whereas lung in 'Complement and Coagulation Cascades' pathway. Transcription factor (TF) annotation analysis identified Zinc finger domain containing (ZF) proteins were mostly regulated in lung and brain. Interestingly, study identified genes Skor2, Gpr3711, Runx1, Erbb3, Frmd7, Fut10, Sox11, Hapln1, Tfap2c and Plxnb3 from brain that play important roles in neuronal cell maturation, differentiation, and survival; genes Hoxa5, Eya1, Errfi1, Sox11, Shh, Igf1, Ccbe1, Crh, Fgf9, Lama5, Pdgfra, Ptn, Rbp4 and Wnt7a from lung are important in lung development. Expression levels of the candidate genes were validated by qRT-PCR. Genome wide transcriptional analysis using wild type and Dip2a knockout mice in brain and lung at embryonic day 19.5 (E19.5) provided a genetic basis of molecular function of these genes.

[This corrects the article DOI: 10.1371/journal.pone.0213702.].

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.

ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and β-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies.

Uterus development during pre-implantation stage affects implantation process and embryo growth. Aberrant uterus development is associated with many human reproductive diseases. Among the factors regulating uterus development, vascular remodeling promoters are critical for uterus function and fertility. Vascular endothelial growth factor (VEGF), as one of the major members, has been found to be important in endothelial cell growth and blood vessel development, as well as in non-endothelial cells. VEGF mediation in reproduction has been broadly studied, but VEGF-induced transcriptional machinery during implantation window has not been systematically studied. In this study, a genetically repressed VEGF mouse model was used to analyze uterus transcriptome at gestation 2.5 (G2.5) by Solexa/Illumina's digital gene expression (DGE) system. A number of 831 uterus-specific and 2398 VEGF-regulated genes were identified. Gene ontology (GO) analysis indicated that genes actively involved in uterus development were members of collagen biosynthesis, cell proliferation and cell apoptosis. Uterus-specific genes were enriched in activities of phosphatidyl inositol phosphate kinase, histone H3-K36 demethylation and protein acetylation. Among VEGF-regulated genes, up-regulated were associated with RNA polymerase III activity while down-regulated were strongly related with muscle development. Comparable numbers of antisense transcripts were identified. Expression levels of the antisense transcripts were found tightly correlated with their sense expression levels, an indication of possibly non-specific transcripts generated around the active promoters and enhancers. The antisense transcripts with exceptionally high or low expression levels and the antisense transcripts under VEGF regulation were also identified. These transcripts may be important candidates in regulation of uterus development. This study provides a global survey on genes and antisense transcripts regulated by VEGF in the pre-implantation stage. Results will contribute to further study the candidate genes and pathways in regulating implantation process and related diseases.

CD97 is over-expressed in the majority of gastric adenocarcinomas and is associated with its dedifferentiation and aggressiveness. Our previous results demonstrated that out of three CD97 isoforms tested, only the small one was able to promote increased invasiveness in vitro. Based on these data we further aimed to investigate the role of CD97 small isoform in gastric cancer progression in vivo by employing the cells with a stable CD97 small isoform knock-down and an orthotopic gastric cancer mouse model. We could demonstrate that the knock down of CD97/EGF1,2,5, led to a significant decrease in the number of cells penetrating the gelatin coated membrane as compared with control cells. In the gastric cancer mouse model, both the hypodermic and the orthotopic yielded tumor masses of the CD97/EGF1,2,5kd group and were significantly smaller than the control. Metastatic tumor cell number in early metastatic regional lymph nodes on post-operative day 42 was distinctly decreased in the CD97/EGF1,2,5kd group as compared with the SGC-NS group, and was accompanied with the downregulation of CD44, VEGFR, CD31 and CD97. We concluded in this study that CD97 small isoform not only supported gastric cancer local growth, but also promoted metastatic spread in orthotopically implanted mouse model suggesting involvement of the CD97 small isoform in the preparation of (pre)metastatic niche.