Transcriptome of embryonic and neonatal mouse cortex by high-throughput RNA sequencing

Abstract

Brain structure and function experience dramatic changes from embryonic to postnatal development. Microarray analyses have detected differential gene expression at different stages and in disease models, but gene expression information during early brain development is limited. We have generated >27 million reads to identify mRNAs from the mouse cortex for >16,000 genes at either embryonic day 18 (E18) or postnatal day 7 (P7), a period of significant synaptogenesis for neural circuit formation. In addition, we devised strategies to detect alternative splice forms and uncovered more splice variants. We observed differential expression of 3,758 genes between the 2 stages, many with known functions or predicted to be important for neural development. Neurogenesis-related genes, such as those encoding Sox4, Sox11, and zinc-finger proteins, were more highly expressed at E18 than at P7. In contrast, the genes encoding synaptic proteins such as synaptotagmin, complexin 2, and syntaxin were up-regulated from E18 to P7. We also found that several neurological disorder-related genes were highly expressed at E18. Our transcriptome analysis may serve as a blueprint for gene expression pattern and provide functional clues of previously unknown genes and disease-related genes during early brain development.

Fig. 1.

An example of mapping reads to a gene. The UCSC gene/cluster 11631 has 2 transcripts, A and B. Transcript A has exons 1, 2, 3, 5, 6, and 7. Transcript B has exons 4, 5, 6, and 7. Thirteen paired-end reads (red) mapped to this gene at E18 and 65 paired-end reads (green) mapped to this gene at P7. At P7 stage, some reads mapped to transcript-specific exons 1 and 2, providing evidence for the expression of transcript A. Four reads mapped to an unknown junction (the red line) between exons 2 and 4, indicating a possibly unreported transcript at P7 stage. Because the cDNA synthesis was polyT primed, there were more reads in 3′ region than 5′.

Fig. 2.

A comparison between 2 biological replicates and among all datasets. (A) The comparison of reads per gene between the first and second paired-end data in E18. Since the read number per gene ranges from 0 to >10,000, the read numbers adding 1 were transformed by log₁₀. There is a good correlation between the first and second paired (R = 0.97). (B) The comparison of reads per gene between the first and second paired-end data in P7 (R = 0.96). (C) The line chart showing the distribution of genes with different reads. The y axis is the number of genes. The x axis is different intervals of read number. The number of genes at E18 and P7 showed parallel changes in both single-end and paired-end analysis. Each biological replicate at each stage contained the empbryos of pups from a single female mouse; these embryos/pups were probably more similar than embryos/pups from different litters. It is possible that potential maternal effects, such as age and other variability, might contribute to the results, which nevertheless are highly reproducible between 2 different mothers for each stage.

Fig. 3.

Venn diagrams showing the number of expressed genes. (A) The number of expressed genes in 3 analyses, with a total of 16,083. (B) The number of highly expressed genes. The top 5% genes were regarded as highly expressed ones. The 2 paired-end data were summed. (C) The number of differentially expressed genes between E18 and P7. There were a substantial number of differentially expressed genes (3, 758) supported by both paired-end analyses.