The 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 (SRF) 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.

Introduction

Actin is one of the most abundant proteins in eukaryotic cells, and is best known for its role as a cytoskeletal component. Within cells, actin exists either in a monomeric form (globular actin, or G-actin) or in a filamentous form (F-actin). The process by which G-actin is assembled into F-actin and F-actin is depolymerized into G-actin, which is referred to as “actin dynamics”, is tightly regulated and responds to extracellular signals. Remodeling of the actin cytoskeleton through actin dynamics is essential for numerous basic biological processes, including cell polarization, contractile force generation, cell migration, cell division, endocytosis and exocytosis (5, 7, 39). In addition to its structural role, the status of the actin cytoskeleton has been shown to influence a diverse array of intracellular signaling processes. For example, the disruption of proper actin dynamics has been shown to result in the altered expression of cell surface molecules (34, 42), loss of Cyclin D expression (10), and changes in histone acetylation (25). Moreover, recent research has provided convincing evidence that cytoskeletal actin dynamics also plays a direct role in the control of gene expression (33). Although the role of actin dynamics in cell signaling and transcriptional regulation has been investigated in vitro using the cell culture system, it has not been thoroughly explored in vivo.

It has been shown that members of the actin depolymerizing factor (ADF)/cofilin family of proteins are the primary regulators of actin dynamics in vivo (11, 23, 41). These proteins, which include destrin (DSTN; also known as ADF), cofilin 1 (CFL1), and cofilin 2 (CFL2) in mammals, enhance the depolymerization of F-actin into its monomeric form and promote filament severing (11, 28). DSTN shows the strongest depolymerization activity out of all family members and is expressed in epithelial and endothelial cells of multiple tissues (55, 57). CFL1 is ubiquitously expressed, while CFL2 is a muscle specific isoform with the weakest depolymerization activity (55). Animal models with functional mutations in the ADF/cofilin genes have provided opportunities to examine the effect of actin cytoskeletal abnormalities in vivo. Mice homozygous for a targeted null allele of Cfl1 display an embryonic lethal phenotype with defective neural crest cell migration and a lack of neural tube closure (17). Neuronal cell specific targeting of Cfl1 further revealed its function in neuronal migration and cell cycle control in the cerebral cortex (8). Corneal disease 1 (corn1) mice are homozygous for a spontaneous null allele of the Dstn gene and show corneal abnormalities including epithelial cell hyperproliferation and neovascularization in the stroma (19, 46). An allelic missense mutation in Dstn, Dstn^corn1-2J, was also discovered which causes milder corneal epithelial hyperproliferation without corneal neovascularization (19). Although DSTN is expressed in many tissues, the phenotypes in Dstn mutant mice appear to be restricted to the cornea, where the main ADF/cofilin molecule expressed is DSTN (19). In other tissues, the loss of DSTN is likely compensated by other ADF/cofilin family members (19). The cornea of Dstn mutant mice, therefore, presents a unique model in which the effect of the loss of DSTN can be investigated.

In this study, we first examined the state of actin in the cornea of Dstn mutant mice and show that actin dynamics are affected in these mice. Then, we sought to determine the possible cellular mechanisms affected by Dstn mutations and resultant actin dynamics abnormalities in vivo through the identification of differentially expressed genes. We also compared the effects of allelic Dstn mutations on the gene expression profile. Allelic mutants show different levels of F-actin accumulation and gene expression changes, indicating that the level of F-actin may correlate with the level of change in gene expression patterns.

Materials and Methods

Mice

A. BY H2^bc H2-T18^f/SnJ (A. BY wild-type), A. BY H2^bc H2-T18^f/SnJ-Dstn^corn1/J (Dstn^corn1), C57BL/6J (B6 wild-type), and C57BL/6JSmn-Dstn^corn1-2J/J (Dstn^corn1-2J) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in the animal facility at the University of Wisconsin-Madison. All mouse procedures were performed in accordance with the protocols approved by the Animal Care and Use Committee at the University of Wisconsin-Madison, and conform to the ARVO statement for the use of animals in Ophthalmic and Vision Research and APS’s Guiding Principals in the Care and Use of Animals.

Analysis of F/G-actin ratio

The ratio of F-actin and G-actin in the cornea was analyzed using an F-actin/G-actin in vivo assay kit (Cytoskeleton Inc., Denver, CO) based on the manufacturer’s protocol. Briefly, pooled corneas from 5 mice of each genotype were homogenized in 500μl F-actin stabilization buffer [50mM PIPES pH 6.9, 50mM KCL, 5mM MgCl₂, 5mM EGTA, 5% (v/v) Glycerol, 0.1% Nonidet P40, 0.1% Triton X-100, 0.1% Tween 20, 0.1% 2-mercapto-ethanol, 0.001% AntifoamC, 1mM ATP, .004mM Tosyl arginine methyl ester, .015mM Leupeptin, .01mM Pepstatin A, and .01M Benzamidine]. To pellet unbroken cells, lysates were centrifuged at 2,000 rpm for 5 min at 37°C and supernatants were collected. To separate the F-actin from the G-actin pool, supernatants were centrifuged at 100,000 g for 1 hr at 37°C. Supernatants were immediately collected, while pellets were resuspended in ice-cold molecular grade H₂O plus 1μM cytochalasin D, and incubated on ice for 1 hr to dissociate F-actin. The resuspended pellets were gently mixed every 15 min. To measure F/G-actin ratio, equal amounts of both the supernatant (G-actin) and the resuspended pellet (F-actin) for each genotype were subjected to immunoblot analysis with the use of an actin antibody (Cytoskeleton Inc.). Fractionation was performed for three separate groups for each genotype. The F/G-actin ratio was determined by scanning densitometry using ImageJ software (http://rsb.info.nih.gov/ij). Two-tailed, un-paired t-tests were performed comparing mutant and wild-type ratio values.

RNA isolation from cornea

Total RNA was isolated from corneas with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) and further purified with an RNeasy kit (Qiagen, Valencia, CA). For each biological replicate, 20 corneas (10 mice) were pooled for microarray analysis and 2 corneas (1 mouse) were used for quantitative real-time PCR (qPCR). Total RNA quality was assessed on the basis of the A260/A280 ratio, the integrity of 18S and 28S rRNA, and chromatograms from the Agilent 2100 Bioanalyzer RNA Nanochip. Samples that had A260/A280 ratios between 1.9–2.1, gel profiles exhibiting distinct bands for 18S and 28S rRNA, and no obvious degradation as observed by Bioanalyzer were used for microarray and qPCR analyses.

Microarray analysis

Microarray experiments were designed to comply with minimum information about microarray experiment (MIAME) guidelines. Microarray analysis for each genotype was performed with three individually derived RNA samples, each being hybridized to one Affymetrix MG 430 2.0 array (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions (1). Briefly, 5.5 μg of total RNA sample was used to synthesize double stranded cDNA with the Super Script II double-stranded cDNA synthesis kit (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Labeled cRNA was synthesized using the Affymetrix IVT amplification and labeling kit (Affymetrix, Santa Clara, CA). Ten μg of purified, fragmented labeled cRNA was hybridized with an array at 45°C for 16 hours in an Affymetrix 640 hybridization oven. The post hybridization process was performed in an Affymetrix 450 fluidic station according to the manufacturer’s instructions. All gene chips were scanned on an Affymetrix GC3000 G7 scanner and data were extracted from scanned images using AFX GCOS 1.4 software. All technical microarray procedures were carried out by the Gene Expression Center at the University of Wisconsin-Madison Biotechnology Center (UWBC).

Microarray data analysis

Background intensities were adjusted and normalized using GCRMA (version 2.0.0) (56) implemented in R (version 2.4.1). After the normalization process, only those probe sets for which at least one gene chip for each genotype had a “P (present)” call according to the Affymetrix detection algorithm, and had sequences unique to single genes were included for further analysis (2). Two-tailed, un-paired t-tests were performed comparing mutant (n=3) and wild-type (n=3) log₂ expression values. The probe sets with a p-value of less than 0.01 were considered differentially expressed. Functional annotation was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) Functional Annotation Tool (http://david.abcc.ncifcrf.gov/) (13) with all probe sets for the mouse 430 2.0 chip as the background. This tool allowed us to identify Gene Ontology (GO) terms (4) that are associated with differentially expressed genes, measure over-representation of GO terms in differentially expressed gene lists using a modified Fisher Exact Test, and calculate the p-value. The p-value is referred to as the Expression Analysis Systematic Explorer (EASE) score (18). Euclidean distance complete linkage clustering was performed for probe sets commonly up- and down-regulated between Dstn^corn1 and Dstn^corn1-2J using Genesis (version 1.7.2) (14, 50). The complete data set generated from this study is available from NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) with GEO Accession number GSE9743.

Quantitative real-time PCR (qPCR) analysis

Total RNA was extracted as described above. For each biological replicate, 1 animal (2 cornea) was analyzed. Twenty ng of total RNA was converted to cDNA using Superscript III first-strand synthesis system (Invitrogen Inc, Carlsbad, CA) according to the manufacturer’s instructions. qPCR reactions (20 μL) contained 2 μL of template cDNA, 10 μM of each primer, 1x Takara Premix Ex Taq (Takara Bio Inc, Madison, WI), 1x ROX reference dye, and 5% dimethyl sulfoxide. Amplification was performed using an ABI Prism 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA). Each cDNA sample was assayed in triplicate. The primer sequences used are listed in Supplemental Table S1. Thermal cycle conditions consisted of an initial denaturation step at 95°C for 10 seconds, followed by 40 cycles of a denaturing step at 95°C for 5 seconds and an annealing/extension step at 60°C for 45 seconds. Fluorescence was measured at the beginning of each annealing/extension step. To check the specificity of each primer pair, the predicted PCR amplicon Tm (melting temperature) was confirmed by a dissociation curve analysis. PCR products were denatured at 95°C for 60 seconds and gradually re-annealed while the temperature was adjusted to 60°C. The fluorescence was measured at every 0.5°C interval. Amplicon size was determined by electrophoresis on an agarose gel (1.5% [w/v]). All PCR amplicons were sequenced at the UWBC and the correct products were confirmed for all the primer pairs.

Quantitative real-time PCR (qPCR) data analysis

DART-PCR software (version 1.0) (http://www.gene-quantification.de/DART_PCR_version_1.0.xls) (37) was used to compute the amplification efficiency for each primer set and to compute the relative expression value. The amplification efficiency of each primer set is listed in Supplemental Table S2. Relative expression values for genes of interest were normalized to the geometric mean of the relative expression values of the two most stable reference genes (Fmr and Ubc). Two-tailed, un-paired t-tests were performed comparing mutant and wild-type relative expression values. Reference genes were selected by the following procedure. Five genes stably expressed in both mutant and wild-type mice were selected as candidate reference genes based on our microarray data (Cat, Fmr, Rgs10, Gapdh and Ubc). The relative expression values of these genes were analyzed by geNorm (version 3.4) (53). After the analysis, a gene with the highest M value (a stability measure) was eliminated. This procedure was repeated until elimination of the gene with the highest M value had no significant effect on the newly calculated normalization factor.

Immunohistochemistry

For immunohistochemistry on frozen sections, mice at postnatal day (P) 14 of age were sacrificed by decapitation and the eyes were immediately removed and immersion fixed in 4% paraformaldehyde (PFA) for 2 hours at 4°C, then cryoprotected at 4°C in a graded series of sucrose. Eyes were embedded in optimal temperature cutting compound (OCT) and sectioned at 12 μm thickness. Sections were blocked in phosphate buffered saline (PBS) with 0.5 % Triton X-100 and 2% normal donkey serum for 20 minutes at room temperature. Sections were, then, incubated at 4°C overnight with primary antibody against SRF (1:400; Santa Cruz Biotechnology, Santa Cruz, CA). Sections were rinsed in PBS, and incubated with Alexa Fluor 488 conjugated secondary antibody (1:400, Invitrogen) and Alexa Fluor 568 conjugated phalloidin (1:50; Invitrogen) for 45 minutes at room temperature. Slides were counterstained with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, 1:200; Sigma-Aldrich, Inc., St. Louis, MO).

For whole mount immunohistochemistry, mice at P14 of age were sacrificed by decapitation and the eyes were immediately removed, placed in PBS, and the corneas were isolated. Corneas were fixed overnight in 4% PFA at 4°C. The next day, corneas were rinsed twice in PBS for 10 minutes, post-fixed in 100% acetone for 20 minutes, and rinsed twice in PBS for 10 minutes. Corneas were blocked overnight in PBS with 0.8% Triton X-100 and 2% normal donkey serum, transferred to block solution with beta actin (1:50; Cell Signaling Technology, Danvers, MA) or MAL (MRTF-A) (1:250; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies and incubated overnight. Corneas were rinsed in PBS six times at 30 minutes each. They were transferred to block solution containing phalloidin (1:50; Invitrogen) and an Alexa Fluor 488 conjugated secondary antibody (1:400; Invitrogen) and incubated overnight. The following day, corneas were rinsed in PBS six times at 30 minutes each and counterstained with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, 1:200; Sigma-Aldrich, Inc., St. Louis, MO). All steps following fixation in 4% PFA were performed at room temperature.

Images were captured on a Zeiss 510 Confocal Laser Scanning System and Axio Imager Microscope using LSM 510 Software (release 4.2) (Carl Zeiss MicroImaging, Inc., Thornwood, NY). Immunohistochemistry was performed on Dstn^corn1 and Dstn^corn1-2J mice, as well as on wild-type controls with matched genetic background (A.BY and B6, respectively).

Results

Pertubation of actin dynamics in Dstn mutant mice

We previously reported the increase of phalloidin signals in the corneal epithelium of Dstn^corn1 and Dstn^corn1-2J mice (19). Here, we further tested whether this increase in phalloidin signals reflects the increase of F-actin. We first performed double labeling of the whole mount cornea from mice at P14 of age using phalloidin and an antibody for beta actin. Consistent with our previous data, phalloidin produced increased signals in the corneal epithelial cells of Dstn^corn1 and Dstn^corn1-2J mice (Fig. 1). The signal was much higher in Dstn^corn1 mice compared to Dstn^corn1-2J mice (Fig. 1). In both mutants, we observed overlapping of phalloidin and beta actin signals (Fig. 1), suggesting that the increase of phalloidin signals in Dstn mutants indeed reflects the increase of F-actin. We further examined actin dynamics in these mutants by a biochemical analysis. Corneal lysates from Dstn mutant and wild-type control mice at P14 of age were subjected to ultracentrifugation, and fractionated cell extracts containing free G-actin and F-actin were analyzed using Western blotting. As shown in Fig. 2, there is a highly significant difference in the F/G-actin ratio between the Dstn^corn1 mutant and A.BY wild-type control (.796 ± .032 versus .271 ± .018, respectively; p=0.0001), and a significant difference between the Dstn^corn1-2J mutant and B6 wild-type control (.422 ± .096 versus .117 ± .006, respectively; p=0.0333). These results are consistent with our data for the phalloidin/beta actin staining in Fig. 1. It is also to note that the F-actin level in the A.BY wild-type cornea is considerably higher than that in the B6 wild-type cornea, as shown by both phalloidin staining (Fig. 1) and the fractionation experiment (Fig. 2). Our results demonstrate that Dstn mutations cause actin dynamics abnormalities in the corneal epithelial cells in the physiological setting, and highlight the functional importance of DSTN 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).

Identification of genes differentially expressed in the cornea of Dstn mutant mice

To examine the effect of the Dstn mutation on the gene expression profile, we first performed a microarray analysis using Dstn^corn1 and A.BY wild-type cornea isolated from mice at P14. After normalization of the data, our analysis identified 1,226 annotated genes that are differentially expressed (p<0.01) out of more than 14,500 genes represented on the array. Of these genes, 599 genes were up-regulated and 627 genes were down-regulated in the Dstn^corn1 cornea.

In order to compare the effect of allelic Dstn mutations with different severity (null in Dstn^corn1, missense in Dstn^corn1-2J), we also performed a microarray analysis of Dstn^corn1-2J and B6 wild-type cornea at P14. We identified 202 annotated genes differentially expressed in Dstn^corn1-2J mice compared with B6 wild-type mice (p<0.01), out of which 150 genes are up-regulated and 52 genes are down-regulated. As shown in the Venn diagrams in Fig. 3, a significant portion of these 202 genes (60 up-regulated and 21 down-regulated genes) is also differentially expressed in the Dstn^corn1 cornea.

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.

Clustering analysis of differentially expressed genes

The overlapping but less severe effect of the Dstn^corn1-2J mutation could be further visualized when hierarchical clustering analysis was performed using the expression data for 81 genes (83 probe sets) identified as differentially expressed (up-regulated or down-regulated) in both Dstn^corn1 and Dstn^corn1-2J mice. In Fig. 4, the dendrogram showing clustering of the arrays (Fig. 4, top) indicates that arrays were successfully grouped by the genotype. Clustering of the probe sets based on the similarity of their expression patterns (dendrogram shown on left) yielded 5 distinct clusters (A–E). A box plot representation of log₂ fold changes between mutant and wild-type cornea was calculated for each cluster (Fig. 4, right). Cluster A includes genes that are highly up-regulated in both Dstn^corn1 and Dstn^corn1-2J cornea. The box plot shows that up-regulation of these genes is more pronounced in the Dstn^corn1 cornea. As observed in the heat map, most genes in clusters B and C are also up-regulated to a greater extent in Dstn^corn1 compared to Dstn^corn1-2J mice. We also observed that most genes in clusters D and E are down-regulated to a greater extent in Dstn^corn1 compared to Dstn^corn1-2J mice. In summary, for the majority of genes that are commonly disregulated in both Dstn mutants, the level of change (up-regulation or down-regulation) tends to be greater in Dstn^corn1 mice compared to Dstn^corn1-2J mice.

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).

Identification of significantly affected functional gene classes

In an attempt to determine which functional categories of genes are significantly affected by the Dstn^corn1 and Dstn^corn1-2J mutations, we performed functional annotation of the differentially expressed genes using The Database for Annotation, Visualization and Integrated Discovery (DAVID) Functional Annotation Tool (http://david.abcc.ncifcrf.gov/) (13). Through this analysis, 67 Gene Ontology (GO) terms were found to be significantly (EASE Score<0.01) over-represented (enriched) (Table 1) in the list of genes up-regulated in the Dstn^corn1 cornea, which included 599 genes. Among these, one of the most significantly over-represented terms was “cytoskeleton” which included 55 genes (EASE Score=2.4×10⁻⁷, Table 2). Six other terms (“actin cytoskeleton”, “cytoskeletal protein binding”, “actin binding”, “cytoskeletal organization and biogenesis”, “actin filament-based process”, and “actin cytoskeletal organization and biogenesis”) are also associated with actin and/or cytoskeleton (Table 1). Consistent with the epithelial hyperproliferation phenotype caused by the Dstn^corn1 mutation, cell cycle associated terms (“cell cycle”, “regulation of progression through cell cycle”, “regulation of cell cycle”, “cell cycle regulator”, “mitotic cell cycle”, “mitosis”, “M phase of mitotic cell cycle”, and “cell division”) are also over-represented. We also observed that inflammation associated terms (“chemokine receptor binding”, “chemokine activity”, “response to biotic stimulus” and “myeloid cell differentiation”) are over-represented.

Table 1.

List of GO terms over-represented (EASE score<0.01) in the up-regulated gene list for Dstn^corn1mice

GO Term-Biological Process	# genes	GO Term-Cellular Component	# genes
cell cycle	46	actin cytoskeleton	24
regulation of protein metabolism	22	cytoskeleton	55
regulation of protein biosynthesis	14	adherens junction	11
regulation of biosynthesis	15	cell junction	16
regulation of progression through cell cycle	28	non-membrane-bound organelle	65
regulation of cell cycle	28	intracellular non-membrane-bound organelle	65
positive regulation of cellular process	40	cytoplasm	145
cytoskeleton organization and biogenesis	31	cell-cell adherens junction	5
regulation of cellular biosynthesis	14	Golgi apparatus	27
cytokine metabolism	10
cytokine biosynthesis	10
biosynthesis	62	GO Term-Molecular Function	# genes
positive regulation of cellular physiological process	36	cytoskeletal protein binding	29
apoptosis	34	actin binding	23
smooth muscle contraction	6	protein binding	181
macromolecule biosynthesis	39	interleukin-1 receptor binding	5
positive regulation of physiological process	37	cytokine activity	19
positive regulation of biological process	43	cell cycle regulator	4
programmed cell death	34	chemokine receptor binding	7
translational initiation	8	chemokine activity	7
protein biosynthesis	35	G-protein-coupled receptor binding	8
cell death	35	phosphotransferase activity, alcohol group as acceptor	38
death	35	growth factor activity	14
cytokine production	10
actin filament-based process	14
response to biotic stimulus	43
glycolysis	8
mitotic cell cycle	16
positive regulation of programmed cell death	14
muscle contraction	9
actin cytoskeleton organization and biogenesis	13
cellular biosynthesis	52
tissue development	17
myeloid cell differentiation	9
protein modification	71
mitosis	12
M phase of mitotic cell cycle	12
cell division	15
glucose catabolism	8
monosaccharide catabolism	8
hexose catabolism	8
positive regulation of apoptosis	13
alcohol catabolism	8
cellular carbohydrate catabolism	9
carbohydrate catabolism	9
glucose metabolism	10
immune response	34

GO terms are listed in the order of significance within each ontology category (Biological Process, Cellular Component, Molecular Function).

Table 2.

Genes that are associated with the GO term “cytoskeleton” in the up-regulated gene list for Dstncorn1 mice

Unigene ID	Gene symbol	Gene name	FC
Mm.329515	Abl2	V-abl Abelson murine leukemia viral oncogene 2 (arg, Abelson-related gene)	0.54
Mm.292865	Actg2	actin, gamma 2, smooth muscle, enteric	5.8
Mm.253564	Actn1	actinin, alpha 1	4.56
Mm.235960	Ank3	ankyrin 3, epithelial	2.38
Mm.197387	Bicd2	bicaudal d homolog 2 (drosophila)	0.87
Mm.308134	Cald1	caldesmon 1	3.81
Mm.378893	Capza1	capping protein (actin filament) muscle z-line, alpha 1	0.28
Mm.140601	Cdc42ep3	Cdc42ep3, CDC42 effector protein (Rho GTPase binding) 3	2.31
Mm.161470	Cenpe	centromere protein E	1.34
Mm.329655	Cfl1	cofilin 1, non-muscle	1.56
Mm.138740	Clasp1	CLIP associating protein 1	1.46
Mm.241109	Clip1	CAP-GLY domain containing linker protein 1	1.07
Mm.196484	Csrp1	cysteine and glycine-rich protein 1	4.58
Mm.389224	Enah	enabled homolog (drosophila)	1.8
Mm.223980	Farp1	FERM, RhoGEF (Arhgef) and pleckstrin domain protein 1 (chondrocyte-derived)	1.12
Mm.295533	Flna	filamin, alpha	3.06
Mm.28095	Flnb	filamin, beta	3.07
Mm.286536	Fblim1	filamin binding LIM protein 1	4.24
Mm.44591	Gas2l1	growth arrest-specific 2 like 1	1.21
Mm.289377	Krt7	keratin 7	3.86
Mm.358618	Krt8	keratin 8	4.86
Mm.439898	Krt14	keratin 14	2.21
Mm.14046	Krt17	keratin 17	7.04
Mm.22479	Krt18	keratin 18	6.56
Mm.290657	Krt23	keratin 23	2.1
Mm.106868	Krt75	keratin 75	6.27
Mm.153911	Lcp1	lymphocyte cytosolic protein 1	3.36
Mm.33207	Lima1	LIM domain and actin binding 1	1.62
Mm.243014	Lmna	lamin A	1.18
Mm.402299	Macf1	microtubule-actin crosslinking factor 1	0.75
Mm.138876	Msn	moesin	4.17
Mm.2444	Myc	myelocytomatosis oncogene	1.46
Mm.29677	Myh9	myosin, heavy polypeptide 9, non-muscle	2.5
Mm.337074	Myl6	myosin, light polypeptide 6, alkali, smooth muscle and non-muscle	1.57
Mm.46514	Myl7	myosin, light polypeptide 7, regulatory	4.43
Mm.271770	Myl9	myosin, light polypeptide 9, regulatory	2.41
Mm.3390	Myo1b	myosin IB	3.13
Mm.234502	Myo1c	myosin IC	0.92
Mm.288980	Nedd9	neural precursor cell expressed, developmentally down-regulated gene 9	2.5
Mm.6343	Npm1	nucleophosmin 1	0.76
Mm.397111	Pafah1b1	platelet-activating factor acetylhydrolase, isoform 1b, beta1 subunit	0.8
Mm.29933	Palld	palladin, cytoskeletal associated protein	2.03
Mm.275648	Pdlim7	PDZ and LIM domain 7	3.99
Mm.2252	Pkp2	plakophilin 2	3.63
Mm.465244	Rcc2	regulator of chromosome condensation 2	1.4
Mm.46014	Shroom3	shroom family member 3	2.36
Mm.322121	Stk38l	serine/threonine kinase 38 like	1.57
Mm.121878	Tpm1	tropomyosin 1, alpha	3.14
Mm.646	Tpm2	tropomyosin 2, beta	1.86
Mm.295124	Tpm4	tropomyosin 4	1.71
Mm.33909	Ttl	tubulin tyrosine ligase	0.92
Mm.379227	Tubb2b	tubulin, beta 2b	1.55
Mm.181860	Tubb6	tubulin, beta 6	3.03
Mm.309867	Twf1	twinfilin, actin-binding protein, homolog 1 (Drosophila)	0.36
Mm.279361	Vcl	vinculin	2.71

FC: log₂ fold change of gene expression value compared to A.BY wild-type. SRF target genes are highlighted in bold.

The same analysis performed on the microarray data from Dstn^corn1-2J and B6 wild-type cornea yielded 20 GO terms over-represented in the up-regulated gene list which included 150 genes (Table 3). Interestingly, the most significantly over-represented term was also “cytoskeleton”, which included 22 genes for this mutant (EASE Score=4.3×10⁻⁶, Table 4). Other GO terms associated with actin and/or cytoskeleton were also identified (Table 3). In contrast, GO terms associated with cell cycle progression and inflammation were not identified as over-represented in the up-regulated gene list for the Dstn^corn1-2J cornea. These data suggest the existence of a threshold for the induction of gene expression due to actin perturbation. The increased expression of genes associated with actin and/or cytoskeleton is observed in both Dstn mutants, which shows that genes falling into these categories may be up-regulated due to actin perturbation occurring even at mild levels.

Table 3.

List of GO terms over-represented (EASE score<0.01) in the up-regulated gene list for Dstn^corn1-2J mice

GO Term-Biological Process	# genes	GO Term-Cellular Component	# genes
cytoskeleton organization and biogenesis	15	cytoskeleton	22
muscle contraction	5	actin cytoskeleton	10
DNA replication	6	adherens junction	6
actin cytoskeleton organization and biogenesis	6	cell junction	9
morphogenesis	17	cortical actin cytoskeleton	3
organelle organization and biogenesis	16	non-membrane-bound organelle	23
		intracellular non-membrane-bound organelle	23
		cortical cytoskeleton	3
		intracellular organelle	71
		intercellular junction	6
		organelle	71
		GO Term-Molecular Function	# genes
		actin binding	11
		cytoskeletal protein binding	13
		protein binding	52

GO terms are listed in the order of significance within each ontology category (Biological Process, Cellular Component, Molecular Function).

Table 4.

Genes that are associated with the GO term “cytoskeleton” in the up-regulated gene list for Dstncorn^1-2J mice

Unigene ID	Gene symbol	Gene name	FC
Mm.329515	Abl2	V-abl Abelson murine leukemia viral oncogene 2 (arg, Abelson-related gene)	0.43
Mm.214950	Acta1	actin, alpha 1, skeletal muscle	8.27
Mm.197387	Bicd2	bicaudal d homolog 2 (drosophila)	0.49
Mm.308134	Cald1	caldesmon 1	1.99
Mm.276826	Cfl2	cofilin 2, muscle	0.45
Mm.196484	Csrp1	cysteine and glycine-rich protein 1	3.16
Mm.28919	Dstn	destrin	0.58
Mm.29755	Incenp	inner centromere protein	0.75
Mm.439898	Krt14	keratin 14	1.2
Mm.14046	Krt17	keratin 17	5.12
Mm.290657	Krt23	keratin 23	2.98
Mm.271967	Lasp1	LIM and SH3 protein 1	0.14
Mm.138876	Msn	moesin	0.83
Mm.29677	Myh9	myosin, heavy polypeptide 9, non-muscle	0.87
Mm.397111	Pafah1b1	platelet-activating factor acetylhydrolase, isoform 1b, beta1 subunit	0.45
Mm.5567	Pdlim1	PDZ and LIM domain 1 (elfin)	0.27
Mm.2252	Pkp2	plakophilin 2	1.24
Mm.148781	Ranbp9	RAN binding protein 9	0.42
Mm.252321	Synpo	synaptopodin	0.57
Mm.646	Tpm2	tropomyosin 2, beta	1.25
Mm.181860	Tubb6	tubulin, beta 6	2.2
Mm.279361	Vcl	vinculin	1.38

FC: log₂ fold change of gene expression value compared to B6 wild-type. SRF target genes are highlighted in bold.

Functional annotation of the down-regulated gene list for Dstn^corn1 mice (627 genes) identified 10 over-represented GO terms (Table 5) including “GTPase regulator activity”, “guanyl-nucleotide exchange factor activity”, “oxidoreductase activity” and “alcohol dehydrogenase activity”. Just two were identified from analysis of the list of genes down-regulated in Dstn^corn1-2J (52 genes), and these were “small GTPase mediated signal transduction” and “guanyl-nucleotide exchange factor activity” (Table 6).

Table 5.

List of GO terms (EASE score<0.01) over-represented in the down-regulated gene list for Dstn^corn1 mice

GO Term-Cellular Component	# genes	GO Term-Molecular Function	# genes
Intracellular	254	guanyl-nucleotide exchange factor activity	12
cellular component unknown	49	oxidoreductase activity, acting on the CH-OH group	11
Chromatin	11	of donors, NAD or NADP as acceptor
		GTPase regulator activity	19
		oxidoreductase activity, acting on CH-OH group	11
		of donors
		small GTPase regulator activity	14
		alcohol dehydrogenase activity	4
		2′-5′-oligoadenylate synthetase activity	3

GO terms are listed in the order of significance within each ontology category (Cellular Component, Molecular Function).

Table 6.

List of GO terms over-represented (EASE score<0.01) in the down-regulated gene list for Dstn^corn1-2J mice

GO Term-Biological Process	# genes	GO Term-Molecular Function	# genes
small GTPase mediated signal transduction	6	guanyl-nucleotide exchange factor activity	4

Expression of SRF, its target genes and coactivator MAL

Out of 55 genes identified as up-regulated in the cornea of Dstn^corn1 mice that are associated with the GO term “cytoskeleton” (Table 2), 24 of them are known downstream targets of a transcription factor, serum response factor (SRF) (6, 29, 38, 45, 52, 58). This phenomenon is of particular interest since it has been shown that the activity of SRF is influenced by actin dynamics in cultured cells (47). Moreover, Srf itself is one of the genes included in the list of differentially expressed genes in Dstn^corn1 mice. By real-time PCR using the cDNA of P14 Dstn^corn1 and A.BY wild-type cornea, we verified that the expression of Srf is significantly up-regulated at the mRNA level (Fig. 5A). To examine the spatial expression pattern of the SRF protein in the Dstn^corn1 cornea, we performed immunohistochemistry. The SRF signal was detected in the nuclei of corneal epithelial cells in Dstn^corn1, but not in A.BY wild-type mice, at P14 (Fig. 5B), demonstrating the presence of SRF at the site of its potential activity in the Dstn^corn1 cornea. SRF expression was observed primarily in the superficial layers of the corneal epithelium, where strong accumulation of F-actin is also observed (Fig. 5B).

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

We expanded our analysis and examined the expression data for ~400 known SRF target genes ((6, 29, 38, 45, 52, 58); 180 validated, 212 hypothetical; a list compiled by Miano et al. (30)) from Dstn^corn1 and A.BY wild-type controls, and found that 53 of them are up-regulated in Dstn^corn1 at a statistically significant level (p<0.01, Supplemental Table S3). To confirm the up-regulation of these SRF target genes in the Dstn^corn1 cornea, we performed real-time PCR analyses for 17 genes shown in Fig. 5C. All of the genes tested are up-regulated in Dstn^corn1 mice, validating the microarray result. We also found 24 SRF target genes in the up-regulated gene list for Dstn^corn1-2J mice (Supplemental Table S4), which include the Dstn gene. The microarray experiment did not detect Srf signals above the baseline in either Dstn^corn1-2J or B6 wild-type cornea (no present [P] call in any of the chips). Consistent with the microarray data, the real-time PCR analysis showed that the Srf expression level was extremely low in both Dstn^corn1-2J and B6 wild-type cornea without a statistically significant difference between them (data not shown). The SRF protein was not detected in the Dstn^corn1-2J cornea by immunohistochemistry (data not shown).

Since in vitro studies have shown that actin dynamics control SRF activity by regulating the subcellular localization of myocardin-related SRF coactivator, MAL, we examined whether the expression pattern of the MAL protein is affected in the Dstn^corn1 cornea. The wholemount cornea from Dstn^corn1 and A.BY wild-type mice were subjected to immunohistochemistry using an anti-MAL antibody. The MAL signal was detected in both the cytoplasm and nucleus in the corneal epithelial cells of A.BY wild-type mice (Fig. 5D). In the cornea of Dstn^corn1 mice, the increased level of MAL signal was observed both in the cytoplasm and nucleus (Fig. 5D). The MAL signal in B6 wild-type and Dstn^corn1-2J cornea was similar to that observed in A.BY wild-type cornea (data not shown). These results show that, while its subcellular localization pattern was not affected, the protein expression level of MAL is higher in the Dstn^corn1 cornea compared to that in the A.BY wild-type cornea.

Discussion

Dstn mutations lead to dramatic alteration of the gene expression profile in vivo

In this study, we demonstrated that mutations in the Dstn gene, a regulator of actin dynamics, leads to dramatic alteration of the gene expression profile in the cornea of Dstn^corn1 and Dstn^corn1-2J mice. DSTN is known to regulate actin dynamics by enhancing the depolymerization of F-actin into G-actin and promoting filament severing (11, 28). This is accomplished by binding of DSTN to actin subunits in F-actin, which results in the conformational change of F-actin (27). This conformational change is believed to be responsible for the increase in the dissociation rate for actin subunits from the pointed (minus) end of F-actin and for the weak filament-severing activity. Through these functions, DSTN regulates the balance between two pools of cytoplasmic actin, F-actin and G-actin. Consistent with this notion, the F/G-actin ratio is markedly increased in the corneal epithelium of Dstn mutant mice (Fig. 2). Our results suggest that actin dynamics and the status of actin (G- or F-actin) have a strong influence on the gene expression profile in vivo.

Allelic Dstn mutants show different levels of F-actin accumulation and gene expression changes

We previously identified that Dstn^corn1 is a null mutation with complete deletion of the entire coding sequence of the Dstn gene, while Dstn^corn1-2J is a missense mutation that results in a Proline to Serine amino acid change (19). In the current study, we showed that these 2 mutations both lead to the increase of F-actin. However, the level of F-actin is much higher in Dstn^corn1 mice compared to Dstn^corn1-2J mice (Fig. 1 and 2). There are two possibilities underlying this difference. First, Dstn^corn1-2J may be a hypomorphic allele with reduced DSTN activity, thus causing milder F-actin accumulation. The Dstn^corn1-2J mutation occurred at a Proline residue that is conserved across species (7, 26) and is located in the region implicated in both F- and G-actin binding (24). It is possible that the Dstn^corn1-2J mutation reduces the binding of DSTN to F- and G-actin, but does not abolish DSTN activity completely. Alternatively, the difference in the level of F-actin may be due to the genetic background difference between Dstn^corn1 and Dstn^corn1-2J. In double labeling and actin fractionation experiments (Fig. 1 and 2), we noted that the F-actin level in the A.BY wild-type cornea is significantly higher compared to that of the B6 wild-type cornea. It is possible that this basal difference in the F-actin level between inbred strains is reflected to the F-actin levels in Dstn^corn1 and Dstn^corn1-2J mice.

The current microarray study showed that the number of differentially expressed genes identified in the Dstn^corn1 cornea (1,226 genes) is much larger than that in the Dstn^corn1-2J cornea (202 genes). Moreover, the level of change for commonly disregulated genes tends to be higher in the Dstn^corn1 cornea (Fig. 4). These findings indicate that the level of F-actin may correlate with the level of change in the gene expression profile.

Pertubation of SRF target genes by Dstn mutations in vivo

The functional gene categories that are most significantly affected by the Dstn^corn1 mutation are those associated with the cytoskeleton, which included many SRF target genes. Further analysis of our microarray data identified 53 SRF target genes to be up-regulated (p<0.01) in the Dstn^corn1 cornea, strongly suggesting that SRF-dependent transcription is activated in this mutant. Through studies using the cell culture system, SRF has been found as a transcription factor that links actin dynamics and gene expression. It was shown that depletion of G-actin is required for the activation of SRF and transcription of its target genes (40, 47). In the proposed model, cytoplasmic G-actin binds to myocardin-related SRF coactivator, MAL, and prevents its nuclear translocation (32). A most recent study showed that nuclear G-actin also plays an important role in this mechanism by promoting actin-dependent nuclear export of MAL, and by preventing SRF activation (54) through its binding to MAL. In response to actin polymerization, depletion of cytoplasmic G-actin promotes nuclear import of MAL, and depletion of nuclear G-actin (and therefore, diminished interaction between nuclear G-actin and MAL) results in decreased nuclear export of MAL and allows SRF activation (54). Based on this model, the observed accumulation of F-actin with concomitant depletion of cellular G-actin level that is expected to occur in the Dstn^corn1 cornea may activate this pathway and promotes SRF-dependent gene transcription. However, the result of our immunohistichemical analysis for MAL indicated that the molecular mechanism of the phenomenon observed in the Dstn^corn1 cornea may not be as simple. While we did not observe the nuclear translocation of MAL in the Dstn^corn1 cornea, a significant increase in the MAL signal was observed in both the cytoplasm and nucleus. More MAL proteins are available in the nucleus of Dstn^corn1 corneal epithelial cells compared to that in the wild-type mice, which could account for the up-regulation of SRF-dependent transcription. It remains to be investigated how the Dstn^corn1 mutation leads to the observed increase of MAL, and whether this increase is solely responsible for the activation of SRF-dependent transcription. Nevertheless, our results strongly suggest that the link between actin dynamics and SRF-dependent transcription exists in vivo.

In contrast to the Dstn^corn1 cornea where up-regulation of Srf and nuclear localization of SRF are observed, the expression level of Srf was very low and the SRF protein was not detectable by immunohistochemistry in the Dstn^corn1-2J cornea. Nevertheless, 24 SRF target genes are up-regulated in the Dstn^corn1-2J cornea (p<0.01), indicating that SRF-dependent transcription may be activated in this mutant as well. These results suggest that a relatively low level of SRF expression is sufficient for the transcription of its target genes to be activated, although the level of activation appears to be much higher (53 vs 24 SRF target genes up-regulated in Dstn^corn1 and Dstn^corn1-2J cornea, respectively) with increased SRF expression.

Based on the possible difference in SRF activation between allelic Dstn mutant mice, SRF target gene expression may contribute to the development and severity of the corneal abnormalities in each mutant. SRF activation has been shown to induce the expression of genes upstream of growth factor and cell cycle genes (15, 20). It has also been shown to mediate angiogenesis (12) as well as cell proliferation (16). Additionally, SRF-dependent transcription is essential for the normal expression of contractile genes (31, 36) and the organization of normal cytoskeleton and contractile generation systems (reviewed in (30)). It will be of benefit to examine the effect of SRF inactivation in the cornea of Dstn mutants, since it may determine the in vivo effects of SRF-dependent transcription as regulated by alterations in actin dynamics.

Affected functional categories and phenotypes

In addition to the molecular mechanisms that are directly affected by Dstn mutations and abnormal actin dynamics, our microarray data should also reflect the physiological changes that have occurred to the corneal tissue originally due to the mutations. Although these changes may not be the direct effect of the mutations on gene expression, they could indicate the molecular pathways through which Dstn mutations lead to observed phenotypes in vivo. Considering the phenotypes observed in Dstn mutant mice, there are some functional categories of genes identified by our analysis that are of particular interest. At the age (P14) used in this study, Dstn^corn1 and Dstn^corn1-2J mice both exhibit an accumulation of F-actin in the superficial layers of the corneal epithelium. Through the microarray analysis, genes that are associated with the GO term “cytoskeleton” and terms related to actin and/or cytoskeleton were idenitified as up-regulated in the Dstn mutants. While the biological significance of this up-regulation is unknown, this does show that a disruption in proper actin dynamics in vivo can modify gene expression to accommodate cellular changes in the structure of the cytoskeleton. Further analyses of the genes associated with cytoskeletal structure are necessary to determine the biological roles for these proteins and whether downstream signaling is possibly affected by their up-regulation.

At P14, Dstn^corn1 mice show epithelial cell hyperproliferation and the neovascularization phenotype is just at the onset. Dstn^corn1-2J mice display a milder hyperproliferation phenotype at this time point and do not display neovascularization at any age. “Cell cycle” associated categories that are enriched in the up-regulated gene list for Dstn^corn1 mice may include genes that are responsible for the hyperproliferation of the corneal epithelial cells. In particular, “M (Mitotic) phase of mitotic cell cycle” is over-represented in Dstn^corn1 mice, indicating that this particular phase of the cell cycle may be affected. While GO terms associated with the cell cycle were not found to be enriched for the Dstn^corn1-2J mutant, this may be due to that only a small number of genes associated with this change are up-regulated in this mutant. Additionally, this may reflect a change occurring at the protein level that contributes to the hyperproliferation phenotype or the fact that genes causing this phenotype may not have been previously associated with an increase in cell proliferation.

Inflammation associated terms were also over-represented in the Dstn^corn1, but not the Dstn^corn1-2J, mutant. Since inflammatory cells and molecules are known to participate in the angiogenic process (35), this group of genes could be involved in the induction of the neovascularization phenotype in Dstn^corn1 mice. The list of genes associated with these functional categories obtained in this study would serve as entry points for further investigation toward the identification of the molecular bases for the Dstn^corn1 and Dstn^corn1-2J phenotypes.

Functional categories of down-regulated genes

One group of GO terms over-represented in the list of genes down-regulated in the Dstn^corn1 and Dstn^corn1-2J cornea is associated with the GTPase regulator activity. Among them, the GO term “guanyl-nucleotide exchange factor (GEF) activity” was enriched in both mutants. This term represents molecules that stimulate the exchange of GDP for GTP in association with the GTPases (http://www.geneontology.org/), which act as binary switches by cycling between an inactive (GDP-bound) and an active (GTP-bound) state (21, 48). GEFs promote the generation of activated forms of GTPases that are capable of recognizing downstream targets or effectors (49). Therefore, down-regulation of GEFs in Dstn mutants likely results in an alteration of cell signaling through GTPases. Particularly in the down-regulated gene list for Dstn^corn1 mice, this GO term category included 3 Rho GEFs. This group was of interest since Rho GEFs stimulate the generation of the activated form of the Rho GTPase (43), which is the upstream negative regulator of DSTN and other ADF/cofilin family members (9, 22, 44, 51). The activated Rho phosphorylates Rho-associated coiled-coil containing protein kinases (ROCKs) (3), which in turn phosphorylates the LIM kinase that phosphorylates ADF/cofilin family members (9, 44). The phosphorylation of ADF/cofilin family members by the LIM kinase prevents them from binding actin. Down-regulation of Rho GEFs in the Dstn^corn1 cornea should decrease the amount of activated Rho (an upstream negative regulator of ADF/cofilins), which would result in the increase of the binding of ADF/cofilins to actin. Therefore, it appears that there may be a feed-back mechanism that detects the loss of DSTN function or the abnormality in actin dynamics, and compensates this by increasing the activity of the ADF/cofilin family of proteins.

Summary

In summary, this study demonstrated that Dstn mutations and resultant aberrant actin dynamics lead to dramatic alterations of the gene expression profile in the cornea. In particular, we have shown that the expression of genes related to the structure of the cytoskeleton are strongly affected. We also have identified genes that may be responsible for the development of Dstn mutant phenotypes, and provided strong evidence that the change in actin dynamics results in the activation of SRF-dependent transcription in vivo. Dstn mutant mice should serve as in vivo models to further investigate cell signaling mechanisms and transcriptional regulation affected by actin dynamics.