Detailed Analysis of Dorsal-Ventral Gradients of Gene Expression in the Hippocampus of Adult Rats

Abstract

We performed RNA sequencing of the dorsal and ventral parts of the hippocampus and compared it with previously published data to determine the differences in the dorsoventral gradients of gene expression that may result from biological or technical variability. Our data suggest that the dorsal and ventral parts of the hippocampus differ in the expression of genes related to signaling pathways mediated by classical neurotransmitters (glutamate, GABA, monoamines, etc.) as well as peptide and Wnt ligands. These hippocampal parts also diverge in the expression of axon-guiding molecules (both receptors and ligands) and splice isoforms of genes associated with intercellular signaling and cell adhesion. Furthermore, analysis of differential expressions of genes specific for astrocytes, microglia, oligodendrocytes, and vascular cells suggests that non-neuronal cells may also differ in the characteristics between hippocampal parts. Analysis of expression of transposable elements showed that depletion of ribosomal RNA strongly increased the representation of transposable elements in the RNA libraries and helped to detect a weak predominance of expression of these elements in the ventral hippocampus. Our data revealed new molecular dimensions of functional differences between the dorsal and ventral hippocampus and points to possible cascades that may be involved in the longitudinal organization of the hippocampus.

Keywords: RNAseq; dorsal hippocampus; ribosomal RNA depletion; splicing; transposable elements; ventral hippocampus.

Figure 1

(A) Intersection between genes differentially expressed (dorsal vs. ventral hippocampus (DH vs. VH)) in PolyA and RBD datasets. (B) Top 10 DH- and VH-dominant (blue, red) gene ontology categories for PolyA/RBD intersection gene set. X axis represents ontology category; Y axis, log₂ fold change, averaged across all genes in the group. (C) Plot showing top 10 most significant KEGG categories for PolyA/RBD intersection gene set (4275). (D) Plot showing statistical comparison of KEGG category enrichment in Ventral (VH) vs. Dorsal (DH) gene lists.

Figure 2

Heatmap showing 10 most DE genes for 5 KEGG categories, related to classical neurotransmitter signaling. Colors represent variation around the mean, red color signifies higher expression values. Average (gradation of green) represents mean expression across all datasets. We discretized average values as follows: Very Low, <50 normalized counts; Low, 50–300; Middle, 300–1000; High, 1000–5000; Very High, >5000. Log₂FC (gradations of brown) is absolute log₂ fold change with sign depending on heatmap color. Log₂FC values were discretized as follows: Very Low, <0.5; Low, 0.5–1; Middle, 1–2; High, >2. Dpmn, dopamine.

Figure 3

Heatmap showing 10 most DE genes for 5 KEGG categories related to calcium signaling and axon guidance cues. Colors represent variation around the mean, red color signifies higher expression values. All designations are identical to Figure 2.

Figure 4

Heatmap showing DE genes, present in lists of top 30 most expressed cell-type-specific genes from [19]. Ast, astrocytes; V, vascular cells; M, microglia. All other designations are identical to Figure 2.

Figure 5

(A) Intersection between genes differentially expressed (VH vs. DH) in polyA/RBD conserved gene set (4275) and hippocampal development dataset from [14]. (B,C) Heatmaps for genes from intersection between our dataset and datasets for P45 from [14].

Figure 5

(A) Intersection between genes differentially expressed (VH vs. DH) in polyA/RBD conserved gene set (4275) and hippocampal development dataset from [14]. (B,C) Heatmaps for genes from intersection between our dataset and datasets for P45 from [14].

Figure 6

(A) Intersection between genes differentially expressed (VH vs. DH) in PolyA/RBD conserved gene set (4275) (green), our previously published data [13] (yellow), and hippocampal development dataset from [14] (purple). (B) KEGG plot for 544 genes that fall into intersection. (C,D) Heatmaps showing neurotransmitter and calcium/axon guidance KEGG category genes from 544 ‘core’ set.

Figure 7

(A) Intersection between genes undergoing differential transcript usage (DTU) in PolyA (VH vs. DH) and RBD (VH vs. DH) datasets. (B) Top 10 KEGG categories for genes that fell into intersection from (A). (C) Comparison of mean exon numbers between DTU and non-DTU genes. ***, result is significant with p < 0.05 (Mann-Whitney test).

Figure 8

(A) Normalized read count per repeat family, summarized by number of different genic/intergenic positioned loci in each. (B) Normalized read count per genic/genomic position, summarized by repeat class. (C) Heatmap showing z-scored repeat-expression difference between four datasets (left), read number averaged across all datasets (middle), and relevant log₂ fold change (right). All changes were significant for RBD dataset with p. adj < 0.1. (D) Heatmap with similar measurements for developmental dataset from [14].