Modeling the neurovascular niche: unbiased transcriptome analysis of the murine subventricular zone in response to hypoxic insult

Abstract

Premature infants often experience chronic hypoxia, resulting in cognitive & motor neurodevelopmental handicaps. These sometimes devastating handicaps are thought to be caused by compromised neural precursor cell (NPC) repair/recovery resulting in variable central nervous system (CNS) repair/recovery. We have identified differential responses of two mouse strains (C57BL/6 & CD1) to chronic hypoxia that span the range of responsiveness noted in the premature human population. We previously correlated several CNS tissue and cellular behaviors with the different behavioral parameters manifested by these two strains. In this report, we use unbiased array technology to interrogate the transcriptome of the subventricular zone (SVZ) in these strains. Our results illustrate differences in mRNA expression in the SVZ of both C57BL/6 and CD1 mice following hypoxia as well as differences between C57BL/6 and CD1 SVZ under both normoxic and hypoxic conditions. Differences in expression were found in gene sets associated with Sox10-mediated neural functions that explain, in part, the differential cognitive and motor responsiveness to hypoxic insult. This may shed additional light on our understanding of the variable responses noted in the human premature infant population and facilitate early intervention approaches. Further interrogation of the differentially expressed gene sets will provide a more complete understanding of the differential responses to, and recovery from, hypoxic insult allowing for more informed modeling of the ranges of disease severity observed in the very premature human population.

Figure 1. Schematic diagram depicting the experimental conditions under which the mouse pups were maintained and the areas of the brain (SVZ) isolated (dashed boxes).

Nx = 20% O₂, Hx = 10% O₂, SVZ = subventricular zone, CC = corpus callosum, LV = Lateral Ventricle.

Figure 2. Down- and up-regulated gene probesets of SVZ tissues harvested from P11 C57 hypoxic reared (C57 Hx) compared to C57 normoxic reared (C57 Hx) pups (adjusted p-value <0.01, log fold-change >1).

Analysis of these genes revealed that many were associated with aspects of neural processes and several were validated by quantitative PCR (# denotes qRT-PCR validation; @ denotes neural process related genes; * denotes GSEA Enrichment identification).

Figure 3. GO terms and genes common to them identified in topGO analysis illustrating down-regulated gene probesets in C57 Hx compared to C57 Nx samples ((adjusted p-value <0.01, log-fold change >1).

Figure 4. A. Heat map illustrating the triplicate, differentially expressed mRNA expression levels of SVZ tissues harvested from P11 CD1 SVZ tissues from pups held under normoxic (20% O₂) (CD1 Nx) and hypoxic (10% O₂) (CD1 Hx) conditions as described.

Heatmap values correspond to normalized and log-transformed expression data. B. Down- and up-regulated gene probesets in the SVZ of CD1 hypoxic reared compared to CD1 normoxic reared pups (adjusted p-value <0.01, log fold-change >1). # denotes qRT-PCR validation.

Figure 5. Statistically significant gene probesets from differential contrast: differentially regulated genes in SVZ of [C57 normoxic reared compared to C57 hypoxic reared P11 pups] versus [CD1 normoxic reared compared to CD1 hypoxic reared P11 pups].

(Adjusted p-value <0.05, log fold-change >1.5 or <−1.5). (# denotes qRT-PCR validation; Asterisks denote neural process related genes).

Figure 6. Summary of the genes associated with aspects of neural processes (*) including genes associated with: ensheathment of axons, regulation of action potential in neurons, myelin sheath, response to toxin, myelination, structural molecule activity, regulation of membrane potential, wide pore channel activity, and responses to toxin.

These genes were validated by quantitative PCR and were found to be common in C57 Nx vs. C57 Hx (left column) and found to be differentially regulated in the differential contrast samples (right column). (# denotes qRT-PCR validation; Asterisks denote neural process related genes).

Figure 7. Leading edge genesets (Transmission of Nerve Impulse, Neurological System Process, System Process and Synaptic Transmission) for C57 Nx vs. C57 Hx identified by GSEA analysis.

This figure illustrates genes common to these genesets that were decreased following hypoxia in C57 SVZ. The genes, which are associated with neural processes, common to these gene subsets include MAL, PLP1, CNP, MBP, SCN2B, SCN1B, SYN1, APBA1, DLG4, GHRL, SLC1A2, CPLX1 and CARTPT.

Figure 8. GeneMANIA-generated network of the queried genes (down-regulated gene probesets in C57 Hx compared to C57 Nx, listed in Table S2) illustrating the relationships of these genes with Sox10.

The black circles denote the subset of queried genes that are known to exhibit co-expression with Sox10 (gray lines),. The grey circles denote other genes that are known to exhibit these relationships with Sox10 including Olig2, which also exhibits co-expression with several of the genes in our query set. Genes in our query set which did not exhibit co-expression with Sox10 or Olig2 are represented without any connecting gray lines.

Figure 9. Differential expression of Sox family members and Sox10 in C57 and CD1 under normoxic and hypoxic conditions.

A. Down- and up-regulated Sox gene family probesets in C57 Hx compared to C57 Nx (white bars) and CD1 Hx compared to CD1 Nx (shaded bars) (adjusted p-value <0.01, log fold-change >1) SVZ. Of the Sox family member genes analyzed only Sox10 mRNA was significantly decreased in C57 SVZ samples. B. Transcript levels of Sox10 were evaluated by qRT-PCR in normoxic and hypoxic treated C57 and CD1 pups (SVZ), and cultured NSC and BEC. In response to Hx, Sox10 mRNA expression decreased in C57 SVZ and NSC and increased CD1 SVZ and NSC, n = 3. C. A ΔΔCt method was used to determine fold changes between C57 and CD1 normoxic and hypoxic treated SVZ, NSC and BEC Sox10 transcript levels. This analysis illustrated decreased Sox10 mRNA expression in Hx C57 SVZ and NSC and increased expression in CD1 SVZ and NSC. n = 3. D. Western blot analysis of Sox10 protein levels in normoxic and hypoxic treated C57 and CD1 pups (SVZ), and cultured NSC and BEC revealed decreased Sox10 protein in Hx C57 SVZ and increased Sox10 protein in Hx CD1 SVZ, NSC and BEC. n = 3.