Analysis of Dip2B Expression in Adult Mouse Tissues Using the LacZ Reporter Gene.

Disconnected (disco)-interacting protein 2 homolog B (Dip2B) is a member of the Dip2 superfamily and plays an essential role in axonal outgrowth during embryogenesis. In adults, Dip2B is highly expressed in different brain regions, as shown by in situ analysis, and may have a role in axon guidance. However, the expression and biological role of Dip2B in other somatic tissues remain unknown. To better visualize Dip2B expression and to provide insight into the roles of Dip2B during postnatal development, we used a Dip2btm1a(wtsi)komp knock-in mouse model, in which a LacZ-Neo fusion protein is expressed under Dip2b promoter and allowed Dip2B expression to be analyzed by X-gal staining. qPCR analyses showed that Dip2b mRNA was expressed in a variety of somatic tissues, including lung and kidney, in addition to brain. LacZ staining indicated that Dip2B is broadly expressed in neuronal, reproductive, and vascular tissues as well as in the kidneys, heart, liver, and lungs. Moreover, neurons and epithelial cells showed rich staining. The broad and intense patterns of Dip2B expression in adult mice provide evidence of the distribution of Dip2B in multiple locations and, thereby, its implication in numerous physiological roles.

CDK4/6 inhibitor-induced colitis: a case report and review of the literature.

A 72-year-old woman with metastatic ER/PR-positive breast cancer who had been on ribociclib and letrozole for 1 year developed severe life-threatening colitis. She presented to emergency department with features of acute abdomen and diarrhoea. The diagnosis of colitis was confirmed radiologically as well as by histopathological examination of the biopsy specimen and the patient clinically improved after withholding ribociclib and receiving corticosteroids compatible with ribociclib-induced colitis. The mechanism of injury in CDK 4/6 inhibitor-induced colitis is unknown but may be related to recruitment of inflammatory cells. Whether the development of colitis is associated with tumour response is an interesting and unanswered question.

Effect of low VEGF on lung development and function.

Vascular endothelial growth factor (VEGF) is important for lung development and function but ideal mouse models are limited to decipher the quantitative relationship between VEGF expression levels and its proper development and pathogenesis. Human SPC promoter has been used to faithfully express genes or cDNAs in the pulmonary epithelium in many transgenic mouse models. In the study, a mouse model of lung-specific and reversible VEGF repression (hspc-rtTRtg/+/VegftetO/tetO) was generated. Human SPC promoter was used to drive lung-specific rtTR expression, a cDNA coding for doxycycline-regulated transcription repression protein. By crossing with VegftetO/tetO mice, that has tetracycline operator sequences insertion in 5'-UTR region, it allows us to reversibly inhibit lung VEGF transcription from its endogenous level through doxycycline food, water or injection. The tissue-specific inhibition of VEGF is used to mimic abnormal expression levels of VEGF in lung. Reduced VEGF expression in lung is confirmed by quantitative real time PCR and immunoblotting. Lung development and structure was analyzed by histology analysis and found significantly affected under low VEGF. The pulmonary epithelium and alveolarization are found abnormal with swelling alveolar septum and enlargement of air space. Genome-wide gene expression analysis identified that immune activities are involved in the VEGF-regulated lung functions. The transgenic mouse model can be used to mimic human pulmonary diseases. The mouse model confirms the important regulatory roles of epithelial expressed VEGF in lung development and function. This mouse model is valuable for studying VEGF-regulated lung development, pathogenesis and drug screening under low VEGF expression.

Global transcriptome study of Dip2B-deficient mouse embryonic lung fibroblast reveals its important roles in cell proliferation and development.

Disco-interacting protein 2 homolog B (Dip2B) is a member of Dip2 family encoded by Dip2b gene. Dip2B has been reported to regulate murine epithelial KIT+ progenitor cell expansion and differentiation epigenetically via exosomal miRNA targeting during salivary gland organogenesis. However, its molecular functions, cellular activities and biological process remain unstudied. Here, we investigated the transcriptome of Dip2B-deficient mouse embryonic lung fibroblasts (MELFs) isolated from E14.5 embryos by RNA-Seq. Expression profiling identified 1369 and 1104 differentially expressed genes (DEGs) from Dip2b-/- and Dip2b+/- MELFs in comparisons to wild-type (Dip2b+/+ ). Functional clustering of DEGs revealed that many gene ontology terms belong to membrane activities such as 'integral component of plasma membrane', and 'ion channel activity', suggesting possible roles of Dip2B in membrane integrity and membrane function. KEGG pathway analysis revealed that multiple metabolic pathways are affected in Dip2b- / - and Dip2b +/ - when compared to Dip2b +/+ MELFs. These include 'protein digestion and absorption', 'pancreatic secretion' and 'steroid hormone synthesis pathway'. These results suggest that Dip2B may play important roles in metabolism. Molecular function analysis shows transcription factors including Hox-genes, bHLH-genes, and Forkhead-genes are significantly down-regulated in Dip2b- / - MELFs. These genes are critical in embryo development and cell differentiation. In addition, Dip2B-deficient MELFs demonstrated a reduction in cell proliferation and migration, and an increase in apoptosis. All results indicate that Dip2B plays multiple roles in cell proliferation, migration and apoptosis during embryogenesis and may participate in control of metabolism. This study provides valuable information for further understanding of the function and regulatory mechanisms of Dip2B.

Brain transcriptome study through CRISPR/Cas9 mediated mouse Dip2c gene knock-out.

Dip2C is highly expressed in brain and many other tissues but its biological functions are still not clear. Genes regulated by Dip2C in brain have never been studied. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, adaptive immune systems of bacteria and archaea, have been recently developed and broadly used in genome editing. Here, we describe targeted gene deletions of Dip2c gene in mice via CRISPR/Cas9 system and study of brain transcriptome under Dip2C regulation. The CRISPR/Cas9 system effectively generated targeted deletions of Dip2c by pronuclei injection of plasmids that express Cas9 protein and two sgRNAs. We achieved targeted large fragment deletion with efficiencies at 14.3% (1/7), 66.7% (2/3) and 20% (1/5) respectively in 3 independent experiments, averaging 26.7%. The large deletion DNA segments are 160.4 kb (Dip2CΔ160kb), spanning from end of exon 4 to mid of exon 38. A mouse with two base pair deletion was generated from a single sgRNA targeting in exon 4 (Dip2cΔ2bp) by non-homologous end joining (NHEJ). Loss of gene expression for Dip2c mRNA was confirmed by quantitative real-time PCR (qPCR). Dip2C-regulated genes and pathways in brain were investigated through RNAseq of Dip2cΔ2bp. In total, 838 genes were found differentially regulated, with 252 up and 586 down. Gene ontology (GO) analysis indicated that DEGs in brain are enriched in neurological functions including 'memory', 'neuropeptide signaling pathway', and 'response to amphetamine' while KEGG analysis shows that 'neuroactive ligand-receptor interaction pathway' is the most significantly enriched. DEGs Grid2ip, Grin2a, Grin2c, Grm4, Gabbr2, Gabra5, Gabre, Gabrq, Gabra6 and Gabrr2 are among the highly regulated genes by Dip2C. Results confirm Dip2C may play important roles in brain development and function.

Correction: Transcriptome profiling of mouse brain and lung under Dip2a regulation using RNA-sequencing.

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

Transcriptome profiling of mouse brain and lung under Dip2a regulation using RNA-sequencing.

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.