Laminar and temporal expression dynamics of coding and noncoding RNAs in the mouse neocortex

Abstract

The hallmark of the cerebral neocortex is its organization into six layers, each containing a characteristic set of cell types and synaptic connections. The transcriptional events involved in laminar development and function still remain elusive. Here, we employed deep sequencing of mRNA and small RNA species to gain insights into transcriptional differences among layers and their temporal dynamics during postnatal development of the mouse primary somatosensory neocortex. We identify a number of coding and noncoding transcripts with specific spatiotemporal expression and splicing patterns. We also identify signature trajectories and gene coexpression networks associated with distinct biological processes and transcriptional overlap between these processes. Finally, we provide data that allow the study of potential miRNA and mRNA interactions. Overall, this study provides an integrated view of the laminar and temporal expression dynamics of coding and noncoding transcripts in the mouse neocortex and a resource for studies of neurodevelopment and transcriptome.

Figure 1. Study Design and Quality Control Measures

(A) Representative sagittal tissue section of the Dcdc2a-Gfp mouse forebrain showing Gfp expression in layer 4 (L4) of the primary somatosensory cortex (S1C). Dashed lines outline the infragranular layers (IgL), L4 and supragranular layers (SgL). HIP, hippocampus; NCX, neocortex. (B) qPCR analysis of the expression of well-established layer-enriched genes. (C) Gfp expression across layers (D) Expression of laminar markers depicted in a heat map of the log ratio RPKM data. (E) Box plots representing uniquely mapped reads for either miRNA or mRNA transcriptomes in each sample. (F) Violin plots representing the distribution of the transcribed ratios of the genome (black) and the transcriptome (grey). Fchr, female chromosome; Mchr, male chromosome; chrM, mitochondrial chromosome. (G) Violin plots representing percentage distribution of smRNA reads across different length of reads. See also Figure S1 and Table S1.

Figure 2. Spatiotemporal Dynamics of Mouse Neocortical Transcriptome

(A and B) Principal component analyses of mRNA (A) and smRNA (B) transcriptomes. (C and D) Venn diagrams representing the number of DEX protein-coding mRNAs (C) and miRNAs (D). See also Figure S2, S3 and Table S2.

Figure 3. Spatiotemporal Dynamics of Alternative Splicing Events

(A) Numbers of known (dark blue) and novel (red) splicing events. B) Reads coverage in the Dlg2 gene region correspondent to exons present in X and Y (isoforms 1 and 3) and 9 and Y (isoform 2). The yellow box highlights the temporal coverage of mRNA-seq reads mapped to exon 9. Black bars/boxes underneath the exonic read distribution indicates exon junctions. Red arrows depict location of exon-specific PCR primers. (C and D) Exon-specific PCR of the cassette exons “X-9-Y” in the mouse IgL (C) and human S1C of equivalent developmental time points (D). Biological replicates indicated as “a” or “b”. See also Figure S3 and Table S4.

Figure 4. Weighted Gene Co-Expression Networks

(A) Heatmap matrix showing modular eigengenes across ages for each layer. (B) Pairwise Pearson correlations among modules. (C) Developmental trajectories of modules M5, M7 (top panels), and proportion of genes reported to be enriched in different neural cell types (bottom bar graphs). See also Figure S4 and Table S5. (D) Proportion of genes reported to be enriched in different neural cell types in spatial clusters II–IV. (E) Proportion of genes reported to be enriched in immature and mature astrocytes in spatial clusters II–IV. See also Figure S5.

Figure 5. Spatial Cluster II Inter- and Intra- Modular Connectivity

(A) Developmental trajectories (left column) and neural cell type enrichment (right column) of cluster II modules. (B) Connectivity of cluster II inter- and intra-modular hub genes. See also Figure S6.

Figure 6. miRNA-mRNA Regulation Prediction

(A) Normalized expression trajectory of miR-92b to the expression profile of its putative target mRNAs (n=119). Data are expressed as mean ± 95% confident intervals for target mRNAs. (B) Developmental trajectories of Foxp2 expression in different layers. (C) miRNAs predicted to regulate Foxp2 mRNA. See also Figure S7 and Table S6.

Figure 7. Transcriptional Correlates of Neurodevelopmental Events

(A) Developmental trajectories of genes associated with select major neurodevelopmental processes in different laminar compartment (SgL, blue; L4, green; IgL, red). (B) Schematic of changes in dendritic morphology of L4 spiny stellate cells (SSCs; dark green) and the formation of L4 barrels (light green) (C) Expression trajectories of genes correlated with developmental changes in dendritic morphology of L4 (green) SSCs.