Figure 1.
Experimental design for profiling of the hypoxic seizure model.
A. Factorial table showing the average number of replicates per combination of treatment condition and sampling time (hours, post P10) for each tissue separately, for a total of 144 samples profiled; noHS = no hypoxic seizures induced (baseline condition), HS = hypoxic seizures induced at t = 0 h, HS + NBQX = hypoxic seizures induced at t = 0 h, followed by four IP administrations of NBQX over a 48 h period. Control vehicle injections were performed at the same times over the same 48 h period under the noHS and HS treatments. B. Cartoon indicating the location of the sampling times and the duration of hypoxic shock (gray bar) and NBQX (rose bar) treatments, alongside hypothetical gene expression profiles for a developmentally modulated gene which also shows persistent activation after hypoxia-induced seizures (HS profile) relative to the baseline (noHS profile). Two possible responses to NBQX treatment are indicated (increase or decrease).
Figure 2.
Baseline developmental time courses show tissue-specific as well as common gene expression patterns.
Gene expression profiles are displayed for hippocampus (left) and cortex (right), over the time period P10–17. Clustered expression intensities standardized to Z-scores are shown for the thousand genes with most significant variation across all samples. The color scale saturates at Z = ±2. Cluster A: genes with expression specific to cortex; B: genes with expression specific to hippocampus; C: set of genes repressed in time in both tissues, containing many genes for cellular proliferation control; D: set of genes induced in time in both tissues, containing many genes specifying mature neuronal lineages; E: common set of genes with no significant changes of expression.
Figure 3.
A cellular proliferation gene set shows strong repression under baseline developmental conditions.
A. Log2-ratios of gene expression relative to the 1 h time point, in each tissue separately, are displayed as a heat map, with sampling times post-P10 indicated in hours. Colors saturate for log2-ratio = ±1. Almost all members of this manually curated gene set representing cellular proliferation are repressed over time. B. Detailed intensity profiles for MYCN and cyclin B1 (CCNB1) showing strong repression at t = 1 week. C. KS plot showing the distribution of the log2-ratios at t = 1 week for both tissues. P-value and enrichment are reported for hippocampus only. Hipp = hippocampus, cx = cortex. The arrows point to some of the genes referred to in the main text.
Figure 4.
A gene set containing a set of oligodendrocytic markers is strongly induced under baseline developmental conditions.
Gene expression profiles are shown for a manually curated set of oligodendrocytic markers. A. Heat map of gene profiles, showing very strong induction of several genes, including myelin basic protein (MBP), myelin associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG) and myelin-associated oligodendrocyte basic protein (MOBP). B. Detailed intensity profiles for 2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNP1), MBP and MOBP. C. KS plot showing the distribution of the log2-ratios at t = 1 week for both tissues, with very marked leading edges. D. KS plot for an independent empirically determined gene set of oligodendrocytic markers [21] (Figure S9 in File S9), also showing large positive enrichment. See Figure 3 for key to figure color and scale details.
Figure 5.
Determination of the hypoxic seizure and of the NBQX responsive sets.
A. Hypoxic seizure (HS) response set: Venn diagram showing membership of genes selected separately in the indicated tissues for significance of response to hypoxic seizures (two-way ANOVAs, FDR < = 0.25). A total of 1,399 genes are contained in the HS response set. B. NBQX-responsive set: Venn diagram for the subsets of the HS response which are also NBQX-responsive. The NBQX-responsive group contains 257 genes.
Figure 6.
Heat map of gene expression for the hypoxic seizure response set.
Clustred log2 ratios of gene expression of hypoxic seizure relative to time-matched normoxic controls, are shown for each tissue for all 1,399 genes in the HS response set. Colors saturate for log2-ratio = ±1. Maximum differential regulation is observed at 12 h post-HS.
Figure 7.
Geneset × gene membership matrix for the hypoxic seizure response.
The clustered {geneset × gene} membership matrix for the 37 gene sets and the corresponding 261 leading-edge genes selected by gene set enrichment analysis of the hippocampal hypoxic seizure response at t = 12 hours is shown. Red indicates presence of a gene (column) in given gene set (row), and gray its absence in a given gene set. 9 functional clusters, named on the right-hand-side of the figure, were determined by visual inspection. Note that the IGF-1/PI3K/mTOR “cluster” actually consists of two groups (red lettering).
Table 1.
Gene sets enriched in the response to hypoxic seizures.
Figure 8.
Heat map of response to hypoxic seizures for the genes in the Wnt signaling group.
Log2-ratios of gene expression of HS to time-matched normoxic controls are displayed for the genes in the Wnt signaling group defined by inspection of the enrichment membership matrix of Figure 7. Sampling times post-P10 are indicated in hours. Colors saturate for log2-ratio = ±1.
Figure 9.
Expression ratios as a function of time for four selected Wnt pathway genes.
Gene names are indicated on the top of each figure. In each bar chart, time at the bottom refers to the duration following hypoxic seizures; the left-hand panel (labeled “hyp”) display ratios of hypoxic seizure response to baseline; the right-hand panel (labeled “hyp + NBQX”) displays ratios of response to combined hypoxic seizures and NBQX treatment to baseline. The expression ratio R = 1 (no fold change) is indicated by the black horizontal lines.
Figure 10.
Heat map of response to hypoxic seizures for the genes in the IGF-1/PI3K/mTOR group.
Log2-ratios of gene expression of HS to time-matched normoxic controls are displayed for the genes in the IGF-1/PI3K/mTOR group defined by inspection of the enrichment membership matrix of Figure 7. Sampling times post-P10 are indicated in hours. Colors saturate for log2-ratio = ±1.
Figure 11.
Expression ratios as a function of time for selected IGF-1/PI3K/mTOR pathway genes.
Gene names are indicated on the top of each figure. See Figure 9 legend for details on scale and labels.
Figure 12.
Enrichment membership matrix for the NBQX-responsive gene set.
Clustered {gene set × gene} membership matrix for the 61 gene sets and the corresponding 158 genes selected by gene set enrichment analysis of the NBQX-responsive gene set. Red indicates presence of a gene (column) in given gene set (row), and gray its absence in a given gene set. Visual inspection suggested 2 functional clusters (A and B), named on the right-hand-side of the figure. In cluster A, consistent enrichment of gene sets related to Wnt signaling is due to the presence of the four genes CTNNB1, GSK3B, APC and CSNK1A1 (inset below). Cluster B did not have obvious functional categorization, and is labeled “unknown”.