Decipher the Transcriptome during Mouse Liver Development by RNA-Seq

During development, fetal liver mainly functions as a hematopoietic organ. After birth, profound changes occur in hepatic gene expression when liver rapidly transitions into the major organ for xenobiotic metabolism and nutrient homeostasis. Although much is known in adults regarding the regulation of drug-processing genes in liver, very little is known during the pediatric period, placing newborns and children at a higher risk of adverse drug reactions than adults. More importantly, recent studies demonstrate that many genes have multiple mRNA isoforms that may lead to different translation potential and protein activity. However, there is limited knowledge regarding age-specific mRNA isoforms of drug-processing genes during liver development that may influence the capacity of drug biotransformation. RNA-Seq allows unbiased detection of novel isoforms on a transcriptome-wide scale. The purpose of this study was to determine the ontogeny of hepatic transcriptome by RNA-Seq, and unveil novel mRNA isoforms of essential drug-processing genes during liver development. Male C57BL/6 mouse livers were collected at 12 ages (prenatal: GD17.5; neonatal: Day1, 3, 5, and 10; adolescent: Day15, 20, 25, and 30; adult: Day45 and Day60), and mRNA transcriptome was determined by RNA-Seq on Illumina HiSeq2000 with 200 cycles paired-end (n=3 per age). RNA-Seq generated an average of 160-180 million reads per sample which were aligned to the mouse reference genome NCBI37/mm9. More than 60% of reads were mappable by TopHat. The abundance of genes and transcripts was estimated by Cufflinks. 18,622 genes had a positive FPKM (Fragments per kilobase of exon per million fragments mapped) value within at least one age during development. Among all 12 ages, a histogram showed that the expression of genes centered on a FPKM value of 5. A heatmap of the whole transcriptome showed that more mRNAs appear to be produced at perinatal ages (between GD17.5 and Day5) than at later developmental ages (Day10 and after). Correlation analysis showed three distinct age clusters, with high correlation of gene expression within each cluster: cluster 1 (Day-2 and Day0), cluster 2 (Day1, 3 and 5), as well as cluster 3 (Day10 to 60). The mRNAs from a total of 3852 genes were significantly altered during development (p<0.05). Ingenuity Pathway Analysis showed that compared to Day60 adult age, genes that were significantly higher at perinatal and neonatal ages are mainly important in pathways such as cell cycle, amino acid and carbohydrate metabolism, protein synthesis, and RNA post-transcriptional modifications; whereas at adult age, genes that were significantly increased are involved in lipid metabolism and xenobiotic metabolism. Cufflinks identified 9331 novel isoforms within the 3 ages examined, corresponding to 5217 genes in liver. Among 238 critical drug-processing genes, novel mRNA isoforms were observed for 42 phase-I enzymes, 15 phase-II enzymes, and 22 transporters. In conclusion, the present study decoded age-specific transcriptome signatures with broader coverage across multiple ages during liver development. This work has also unveiled distinct expression patterns of novel isoforms for critical drug-processing genes, paving the path for further understanding mechanisms of adverse drug reactions in developing liver (Supported by NIH ES-019487 and RR-021940.)