Effect of destrin mutations on the gene expression profile in vivo

Abstract

Remodeling of the actin cytoskeleton through actin dynamics (assembly and disassembly of filamentous actin) is known to be essential for numerous basic biological processes. In addition, recent studies have provided evidence that actin dynamics participate in the control of gene expression. A spontaneous mouse mutant, corneal disease 1 (corn1), is deficient for a regulator of actin dynamics, destrin (DSTN, also known as ADF), which causes epithelial hyperproliferation and neovascularization in the cornea. Dstn(corn1) mice exhibit an actin dynamics defect in the corneal epithelial cells, offering an in vivo model to investigate cellular mechanisms affected by the Dstn mutation and resultant actin dynamics abnormalities. To examine the effect of the Dstn(corn1) mutation on the gene expression profile, we performed a microarray analysis using the cornea from Dstn(corn1) and wild-type mice. A dramatic alteration of the gene expression profile was observed in the Dstn(corn1) cornea, with 1,226 annotated genes differentially expressed. Functional annotation of these genes revealed that the most significantly enriched functional categories are associated with actin and/or cytoskeleton. Among genes that belong to these categories, a considerable number of serum response factor target genes were found, indicating the possible existence of an actin-SRF pathway of transcriptional regulation in vivo. A comparative study using an allelic mutant strain with milder corneal phenotypes suggested that the level of filamentous actin may correlate with the level of gene expression changes. Our study shows that Dstn mutations and resultant actin dynamics abnormalities have a strong impact on the gene expression profile in vivo.

Fig. 1

Immunohistochemical staining for beta actin and phalloidin in the corneal epithelial cells of wild-type and Dstn mutant mice. Single slice confocal images of beta actin (green) and phalloidin (red) demonstrate the colocalization of beta actin with F-actin only in Dstn mutant cornea, albeit to a significantly greater level in Dstn^corn1 mice. Corneas were counterstained with DAPI (blue). The scale bar represents 20 μm.

Fig. 2

Analysis of actin polymerization in the cornea of wild-type and Dstn mutant mice. The F/G-actin ratio is significantly higher in Dstn mutant mice as compared to wild-type controls, demonstrating an increase in the amount of polymerized actin in these mice (Top). Error bars represent SEM. * denotes statistical significance by t-test. * p<0.05, ** p<0.01, *** p<0.001. A representative immunoblot image of relative F-actin and G-actin levels in wild-type and Dstn mutant mice is shown (Bottom).

Figure 3

Venn diagrams showing the number of genes significantly up- or down-regulated (p<0.01) in the cornea of Dstn^corn1 and Dstn^corn1-2J mice. Sixty genes were up-regulated in both Dstn^corn1 and Dstn^corn1-2J mice, while 21 genes were down-regulated in both mutants.

Figure 4

Hierarchical clustering of the 83 probe sets (81 genes) that are significantly up- or down-regulated (p<0.01) in both Dstn^corn1 and Dstn^corn1-2J mice. All of the probe sets are grouped into 5 distinct clusters (labeled A–E) based on their expression patterns. Each row corresponds to a single probe set, and each column to a single array. Affymetrix Probe IDs and gene symbols are indicated at right. Relative gene expression signal levels with wild-type as the baseline are indicated by color as shown in a scale at the top of the figure (green represents decreased expression and red represents increased expression). Box plots at right show the average log₂ fold changes (y axis) for probe sets in the corresponding cluster using wild-type as the baseline for each mutant. The dendrogram at the top of the figure shows clustering of the experiments (arrays).

Fig. 5

Expression of SRF, its target genes, and coactivator MAL in the Dstn^corn1 cornea. (A): Quantitative real-time PCR (qPCR) analysis demonstrates a significantly higher mRNA level for Srf in the Dstn^corn1 cornea as compared to A.BY wild-type (WT). (B): Immunohistochemistry shows the nuclear accumulation of SRF in Dstn^corn1 but not in WT cornea. SRF expression (green) is detectable in the nuclei (DAPI, blue) of the superficial layers in mutant cornea, where F-actin (phalloidin, red) accumulation occurs. The scale bar represents 20 μm. (C): qPCR analysis of SRF target genes confirms their up-regulation in the Dstn^corn1 cornea. Relative expression values for Tpm2 are not observable for WT and mutant due to the scale used, but are .0317 ± .0024 and .0641 ± .0098, respectively. Error bars represent SEM. * denotes statistical significance by t-test. * p<0.05, ** p<0.01, *** p<0.001. (D): Immunohistochemical staining for MAL and Phalloidin in the corneal epithelial cells of wild-type and Dstn^corn1 mice. Single slice confocal images of MAL (green), phalloidin (red), and DAPI (blue) demonstrate an increase in the MAL signal in the cytoplasm and nucleus of Dstn^corn1 mice as compared to wild-type. The scale bar represents 20 μm