Genomic loci modulating the retinal transcriptome in wound healing

Abstract

Purpose: The present study predicts and tests genetic networks that modulate gene expression during the retinal wound-healing response.

Methods: Upstream modulators and target genes were defined using meta-analysis and bioinformatic approaches. Quantitative trait loci (QTLs) for retinal acute phase genes (Vazquez-Chona et al. 2005) were defined using QTL analysis of CNS gene expression (Chesler et al. 2005). Candidate modulators were defined using computational analysis of gene and motif sequences. The effect of candidate genes on wound healing was tested using animal models of gene expression.

Results: A network of early wound-healing genes is modulated by a locus on chromosome 12. The genetic background of the locus altered the wound-healing response of the retina. The C57BL/6 allele conferred enhanced expression of neuronal marker Thy1 and heat-shock-like crystallins, whereas the DBA/2J allele correlated with greater levels of the classic marker of retinal stress, glial fibrillary acidic protein (GFAP). Id2 and Lpin1 are candidate upstream modulators as they strongly correlated with the segregation of DBA/2J and C57BL/6 alleles, and their dosage levels correlated with the enhanced expression of survival genes (Thy1 and crystallin genes).

Conclusion: We defined a genetic network associated with the retinal acute injury response. Using genetic linkage analysis of natural transcript variation, we identified regulatory loci and can didate modulators that control transcript levels of acute phase genes. Our results support the convergence of gene expression profiling, QTL analysis, and bioinformatics as a rational approach to discover molecular pathways controlling retinal wound healing.

Keywords: CNS degeneration; QTL analysis; genetic networks; microarray; retinal degeneration.

Figure 1

Chromosome 12 locus modulates expression of wound-healing genes. Quantitative trait locus (QTL) analysis maps the regulation of gene expression in BXD recombinant inbred (RI) strains. This regulation is based on the genetic correlation of expression (individual rows on the y-axis) to genomic markers across the mouse genome (x-axis). Blue hues represent correlations for elevated expression in mice with the C57BL/6 allele at a given locus, and orange hues represent correlations for elevated expression in mice with the DBA/2J allele. A: Expression of wound-healing genes in mouse forebrains is controlled by three eQTLs on chromosomes 6, 12, and 14. B: Synaptic-related genes in mouse forebrain are also controlled by eQTLs on chromosomes 6 and 14. C: Wound-healing genes shared no eQTLs in hematopoietic stem cells. D: Published data from phenotypes in BXD RI mouse strains further support that chromosome 12 locus also associates with neurological phenotypes. Affymetrix probe set identifiers and BXD Phenotype identifiers are listed in Supplementary Table 1. *Probe set for Lpin1 is not available in Affy U34 chip, however, post meta-analysis predicted and experimental models of gene expression confirmed the role of Lpin1 as a wound healing gene (see Figs. 4 and 5; and Supplementary Fig. 2).

Figure 2

Chromosome 12 locus modulates transcription, differentiation, proliferation, and apoptotic mechanisms. A: Genetic networks were derived from transcripts sharing eQTLs as shown in Figure 1. Blue lines connecting specific genes to the locus represent correlations for elevated gene expression in mice with the C57BL/6 allele, and orange lines represent correlation for elevated gene expression in mice with the DBA/2J allele. Genes located within the eQTLs (cis-eQTLs) are indicated with a two-arrow line. B: The major functional themes described by the network’s gene functions are the regulation of transcription, differentiation, proliferation, and cell death. A nonbiased, statistical approach to defining the function of the network (n = 44 genes) is to compare the observed number of regulated genes as compared to the expected number in a population belonging to a particular functional category. For the chromosome 12 network, we observed 32% (14 out of 44 genes) of genes to be related to the regulation of neural development and differentiation. This percentage is higher than the percentage (7%) observed among the total population of retinal reactive transcripts and much higher than the percentage of expected genes in the entire genome. C: We queried the biological literature using text-mining tools to illustrate networks within the transcripts grouped into the neuro-genesis category (Pax6, Neurod1, Neurod2, Id2, Nfib, Egr1, Hes3, Bcl2, Robo1, Ets1, Sox11, Casp3, Itgb1, and Sdc1). The literature search documents the number of known molecular interactions of these genes, including activation and inhibition, that occur during neurogenesis. *Probe set for Lpin1 is not available in Affy U34 chip, however, post meta-analysis predicted and experimental models of gene expression confirmed the role of Lpin1 as a wound healing gene (see Figs. 4 and 5; and Supplementary Fig. 2).

Figure 3

Candidate genes for chromosome 12 network. A candidate gene must have genetic polymorphisms that result in the expression variability of its own transcript (cis-eQTL). In addition, the candidate gene’s function must be consistent with molecular events that occur during retinal wound healing. A: Within the locus there are over 50 polymorphic genes. Within the enlarged interval, bars represent genes, and their spacing represents approximate location. Red bars represent genes that meet criteria. B: Transcript abundance variability in normal forebrain of BXD RI mouse strains is due to genetic polymorphisms between the parental C57BL/6 and DBA/2J mouse strains. The graph illustrates transcript abundance variability (y-axis) for genes (dots) within the 10- to 30-Mb interval of chromosome 12 (x-axis). Lpin1 and Id2 are polymorphic genes that displayed significant transcript variability and cis-eQTLs in normal forebrain of BXD mouse strains. C: We identified genes whose expression patterns in forebrain, cerebellum, and striatum of BXD mouse strains map to their gene locations. Within the interval, we identified in red the genes that display eQTLs in at least two brain regions. Linkage maps were generated using the Interval Mapping and Cluster Tree tools at GeneNetwork (GeneNetwork). D: The structure of the Id2 gene illustrates an SNP at the promoter region and four SNPs within the second intron. The SNP within the promoter region (Ensembl SNPView ID rs4229289 and Celera SNP ID mC22302957) is located within a highly conserved region and is adjacent to a nuclear transcription factor Y (NF-Y) binding site (TRANSFAC ID M00185). In the diagram, filled and open boxes represent translated and untranslated regions E: We determined genes that are differentially expressed during retinal development and retinal healing. AL024210 is highly homologous to human MTCBP1 (NP_060739) and rat Alp1 (NP_954528). Affymetrix probe set identifiers and BXD Phenotype identifiers are listed in Supplementary Table 1.

Figure 4

Expression of Id2 and Lpin1 correlates with the segregation of chromosome 12 locus. To test the hypothesis that Id2, Lpin1, or Sox11 modulates the eQTL, we used the natural range of expression in BXD RI strains. A: We compared the wound-healing response of retinas in strains with the C57BL/6 allele (C57BL/6 and BXD38) to strains with the DBA/2J allele (DBA/2J and BXD60). The BXD60 strain has an additional recombination between genetic markers at 22 and 30 Mb. B, C, D: Acute and subacute expression changes for Lpin1, Id2, and Sox11 were measured in these strains. Fold changes represent expression differences between normal and injured conditions (log₂ scale). Averages are expressed as the mean ± SEM. Significance was measured using Student’s t-test.

Figure 5

Higher levels of Id2 and Lpin1 (C57BL/6 allele) correlated with higher levels of survival markers. To investigate the role of chromosome 12 locus during retinal injury, we examined the wound-healing response of retinas that expressed high levels of Id2/Lpin1 (strains with the C57BL/6 allele: C57BL/6 and BXD38) and compared this to low levels of Id2/Lpin1 (strains with the DBA/2J allele: DBA/2J and BXD60). Acute-phase and subacute-phase mRNA levels were measured 6 h and 3 d after an optic nerve crush. A: Clustered dendogram shows the correlation across experiments (columns) and across genes (rows) in terms of distance (1 — correlation). B: Retinas with the C57BL/6 allele expressed higher fold changes of Thy1 and Cryab than did retinas with the DBA/2J allele. Fold changes represent expression differences between normal and injured conditions (log₂ scale). Averages are expressed as the mean ± SEM. Significance was measured using Student’s t-test. C: Genetic-based differences in GFAP immunoreactivity. Six days after an optic nerve crush, we measured stress levels using the classic stress marker GFAP, a cytoskeletal protein normally expressed by astrocytes and the end-feet of Muller cells.