mRNA expression, splicing and editing in the embryonic and adult mouse cerebral cortex

Abstract

The complexity of the adult brain is a result of both developmental processes and experience-dependent circuit formation. One way to look at the differences between embryonic and adult brain is to examine gene expression. Previous studies have used microarrays to address this in a global manner. However, the transcriptome is more complex than gene expression levels alone, as alternative splicing and RNA editing generate a diverse set of mature transcripts. Here we report a high-resolution transcriptome data set of mouse cerebral cortex at embryonic and adult stages using RNA sequencing (RNA-Seq). We found many differences in gene expression, splicing and RNA editing between embryonic and adult cerebral cortex. Each data set was validated technically and biologically, and in each case we found our RNA-Seq observations to have predictive validity. We provide this data set and analysis as a resource for understanding gene expression in the embryonic and adult cerebral cortex.

Figure 1

Gene expression (a) Expression Heatmap. A heatmap of sample-to-sample distances on the matrix of variance stabilized data for overall gene expression is shown where darker red colors indicate more similar expression, as shown on the color key to the upper right of the panel. Clustering, drawn above the heatmap, demonstrates that the adult samples are very similar to each other, but show complete separation from the embryonic samples. (b) Differential expression. Plot of differential gene expression with fold difference of log₂ normalized expression in adult cerebral cortex (n=3) versus embryonic cerebral cortex (n=4) on the x-axis and −log₁₀ adjusted p-value on the y-axis. Each gene is colored based on the log₁₀ base mean expression, i.e. more highly expressed genes are in darker colors. (c) Technical validation of differential expression. The x-axis shows the log₂ fold expression (Adult/Embryonic) using the RNA-Seq data compared to qRT-PCR data for eight genes (y axis). Ppid was used as the normalization gene. The size of each point represents the base mean expression level from the RNA-Seq data as in (b). The grey shaded area indicates the 95% confidence interval for the regression. (d) Boxplots showing biological validation of differential expression. The same eight genes were used for validation with a new set of animals and an expanded set of developmental stages. On the y-axes are the log₂ expression values normalized to E15 measured with qRT-PCR with Ppid used as the normalization gene. The x-axes show each developmental stage with E15 (n=4), E17 (n=5), P0 (n=6), P14 (n=4), P28 (n=4), and adult (n=4). The boxes represent the range between first and third quartiles and whiskers indicate highest value and lowest values within 1.5 multiples of the inter-quartile range; outliers from this range are plotted as individual dots.

Figure 2

Alternative Exon Utilization (a) Differential alternative event ratios. A graph of the proportion of exon inclusion (counts per exon/counts per gene) with adult ratios (n=3) on the x-axis and embryonic ratios (n=4) on the y-axis. The values are colored based on the type of event, and sized according to the −log₁₀ of the adjusted p-value. For clarity, only events that were significantly different between groups (FDR adjusted p<0.05) are shown. (b) Abr alternative 5′UTR. Plot of RNA-Seq reads generated using the UCSC genome browser covering an alternative 5′UTR of the Abr gene, boxed in red; note that Abr is on the negative DNA strand and the 5′UTR is on the right of the plot. (c) Validation of Abr alternative isoform using RT-PCR of technical replicates. Using PCR, primers for the alternative UTR only amplified the product in the embryonic tissue. Reactions are representative of triplicate biological samples. (d) Abr alternative 5′UTR biological replication. Using RT-PCR, primers for the alternative UTR amplified the product in the developmental stages E15, E17, and P0. In contrast, there is no detectable product at P14, P28, and adult. Gel is representative of duplicate biological replicates. (e) Exon inclusion in Kif1b. The adult brain had a relatively higher proportion of an inclusion sequence expressed after exon 12 (boxed in red) compared to embryonic brain. (f) Kif1b exon inclusion technical replication. RT-PCR with gene specific primers confirmed the inclusion of a novel sequence shown by a shift in expected size from embryo to adult brain using duplicate biological replicates. (g) Kif1b exon inclusion biological replication. A similar RT-PCR approach in independent biological samples shows the same pattern of inclusion of the 42bp sequence towards adult animals. Gels are representative of three reactions with independent biological replicates.

Figure 3

Adenosine to Inosine RNA editing (a) Adenosine to Inosine editing. Average percent edited in adult mouse cerebral cortex (n=3) on the x-axis and average percent editing in the embryonic mouse cerebral cortex (n=4) on the y-axis. Yellow indicates the edit is found in the UTR, green indicates the edit results in a non-synonymous amino acid changes, and purple indicates the edit results in no change in the amino acid sequence. (b, c) Technical validation of RNA editing. Eleven sites, both coding and noncoding, were chosen for validation. The x-axis shows the total average percent of the edited site using the RNA-Seq data compared to validation using cloning and sequencing on the y-axis for embryonic samples (b) and adult data (c). The grey shaded areas indicate the 95% confidence interval for the regression. (d-i) Biological validation of RNA editing. We took sites in Cyfip2 (d), a non-synonymous amino acid change (e) and a synonymous edit (f) in Son and sequenced both cDNA and genomic DNA clones for each site. For each panel in d-f an example of edited and non-edited cDNA clones are shown in the top two chromatograms and the genomic sequence is shown in the lowest chromatogram. We then quantified the percentage of cDNA clones that showed editing for Cyfip2 (g), the non-synonymous amino acid change (h) and synonymous edit (i) in Son across different developmental stages as indicated on the x-axes for the proportion of clones with the edited sequence (y axis, error bars indicate range across replicates). (j,k) Expression of genes coding for ADAR enzymes. Expression of Adar (j) and Adar1b (k) was estimated by qRT-PCR with Ppid as a reference gene using the biological replicates at the indicated developmental stages on the x-axis. Note that both y-axes are on log₂ scales but that the proportional increase in expression is higher for Adar1b than for Adar. The boxes represent the range between first and third quartiles and whiskers indicate highest value and lowest values within 1.5 multiples of the inter-quartile range; outliers from this range are plotted as individual dots.