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

Abstract

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.

Fig 1. Venn diagrams showing overlap and unique unigenes identified within Wild type (WT) brain, Wild type (WT) lung, Dip2a^-/- brain and Dip2a^-/- lung.

(a) In WT group, total 5884 genes were expressed in both samples, 314 genes unique to brain and 257 genes unique to lung (b) In Dip2a^-/- group, total 5908 genes were expressed in both samples, 319genes were unique to brain and 233 unigenes were unique to lung. (C) 5787 genes were expression in all samples, 2 genes were unique to WT brain and 4 genes were unique to WT lung.

Fig 2. Differentially expressed genes volcano map. (a) WT brain vs. Dip2a^-/- brain. (b) WT lung vs. Dip2a^-/- lung.

The red and green dots in the figure represent up-regulated and down-regulated differentially expressed genes respectively.

Fig 3. Gene Ontology (GO) classification of DEGs from WT brain vs. Dip2a^-/- brain (a, c) and WT lung vs. Dip2a^-/- lung (b, d).

(a,b) Histogram of GO annotation was generated by KOBAS (kobas.cbi.pku.edu.cn). The X-axis indicates GO classification, the Y-axis on the left indicates the percentage of genes, and the Y-axis on right indicates the number of genes. One gene could be assigned with more than one GO term. (c,d) Most significant enriched biological terms in brain and lung.

Fig 4. List of DEGs annotated to GO term ‘In-utero embryonic development’.

Fig 5. COG classification of differential expression genes (a) WT brain vs. Dip2a^-/- brain and (b) WT lung vs. Dip2a^-/- lung.

The X-axis indicates content of each category of COG and the Y-axis indicates number of genes annotated in each category.

Fig 6. KEGG pathway enrichment scatter plot of DEGs. (a) WT brain vs. Dip2a^-/- brain and (b) WT lung vs. Dip2a^-/- lung.

Each circle in the figure represents a KEGG pathway. The Y-axis represents name of the pathway and the X-axis indicates Enrichment Factor, indicating the proportion of the annotated genes to all genes in the pathway.

Fig 7. Validation of RNA-Seq results by real-time quantitative PCR (QPCR).