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Human Molecular Genetics Advance Access originally published online on November 9, 2007
Human Molecular Genetics 2008 17(4):490-505; doi:10.1093/hmg/ddm326
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

FOXC1 is required for cell viability and resistance to oxidative stress in the eye through the transcriptional regulation of FOXO1A

Fred B. Berry1,*,{dagger}, Jonathan M. Skarie2,{dagger}, Farideh Mirzayans1, Yannick Fortin3, Thomas J. Hudson3, Vincent Raymond4, Brian A. Link2 and Michael A. Walter1

1 Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada 2 Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA 3 McGill University and Genome Quebec Innovation Centre, Montreal, PQ, Canada 4 Laboratory of Ocular Genetics and Genomics, Molecular Endocrinology and Oncology Research Center, Laval University Hospital (CHUL) Research Center, Québec City,PQ, Canada

* To whom correspondence should be addressed at: Department of Medical Genetics, University of Alberta, 832 Medical Sciences Building, Edmonton, AB, Canada, T6G 2H7. Tel: +1 7804923028; Fax: +1 7804926934; Email: fberry{at}ualberta.ca

Received October 12, 2007; Revised October 12, 2007; Accepted November 6, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Mutations in the human FOXC1 transcription factor gene underlie Axenfeld–Rieger (AR) syndrome, a disorder characterized by anterior segment malformations in the eye and glaucoma. Through the use of an inducible FOXC1 protein, along with an intermediate protein synthesis blocker, we have determined direct targets of FOXC1 transcriptional regulation. FOXC1 regulates the expression of FOXO1A and binds to a conserved element in the FOXO1A promoter in vivo. The zebrafish foxO1a orthologs exhibit a robust expression pattern in the periocular mesenchyme. Furthermore, FOXO1A expression is reduced in cultured human trabecular meshwork (TM) cells and in the zebrafish developing eye when FOXC1 expression is knocked down by siRNAs and morpholino antisense oliognucleotides, respectively. We also demonstrate that reduced FOXC1 expression increases cell death in cultured TM cells in response to oxidative stress, and increases cell death in the developing zebrafish eye. These studies have uncovered a novel role for FOXC1 as an essential mediator of cellular homeostasis in the eye and indicate that a decreased resistance to oxidative stress may underlie AR–glaucoma pathogenesis. Given that FOXO1A influences cellular homeostasis when positively or negatively regulated; the dysregulation of FOXO1A activities in the eye through FOXC1 loss of function mutations and FOXC1 gene duplications provides an explanation into how seemingly similar human disorders can arise from both increases and decreases in FOXC1 gene dose.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Glaucoma is a leading cause of irreversible blindness, with estimates that 60 million cases will be diagnosed worldwide by 2010 (1). The disease is characterized by degeneration of retinal ganglion cells (RGCs), whose axons form the optic nerve, resulting in a cupping of the optic nerve head. A leading risk factor for glaucoma is elevated intraocular pressure (IOP). Structures in the anterior segment of the eye such as the trabecular meshwork (TM) and Schlemm’s canal are important constituents in the aqueous humor outflow pathway that maintains IOP homeostasis. Cellular dysfunction in this system can lead to an imbalance between aqueous humor production and drainage that may lead to elevated IOP. If diagnosed early, glaucoma can be effectively managed and vision loss can be minimized. However, the detection of presymptomatic glaucomatous conditions can be difficult. To improve these diagnostic capabilities, along with furthering therapeutic interventions, increasing research efforts have focused on identifying the underlying genetic basis for glaucoma in order to understand disease pathogenesis.

Axenfeld–Rieger (AR) syndrome is a disorder inherited as an autosomal dominant trait and is characterized by a spectrum of malformations in the anterior segment of the eye (2,3). Typically, AR manifests as iridocorneal angle malformations and iris hypoplasia. The most deleterious consequence of AR is a significantly increased risk of developing glaucoma, with over half of affected individuals diagnosed with this progressively blinding disorder in their third or fourth decade of life (3,4). To date, AR malformations have been mapped to three chromosomal loci 4q25, 6p25 and 13q14. Although the gene at 13q14 has yet to be identified (5), two known AR causative genes have been identified: FOXC1 at 6p25 (6,7) and PITX2 at 4q25 (8). However, as mutations in either FOXC1 or PITX2 account for only 40% of AR, additional disease causing genes remain to be found.

FOXC1 is a member of the Forkhead Box or FOX class of transcription factors. These proteins are characterized by a conserved 110 amino acid DNA-binding domain, known as a forkhead domain (FHD). FOX proteins are key regulators of diverse cellular functions including the development of many organ systems, energy homeostasis and oncogenesis (9–11). Mouse genetic studies have shown that Foxc1 plays critical roles in the normal development of ocular, skeletal, cardiovascular and urogenital systems (12–17). In particular, Foxc1–/– mice display a number of ocular phenotypes that include open eyelids, a failure of the anterior chamber to form, iris hypoplasia, attachment of the lens to the cornea, a thickening of the corneal epithelium, an absence of the corneal endothelium and a disorganized corneal stroma (12,13). Biochemical analysis of AR-causing FOXC1 missense mutations reveals defects in protein stability, nuclear localization, DNA-binding specificity and, ultimately, impaired transcriptional regulation of FOXC1-target genes (18,19). Moreover, interstitial duplications of the FOXC1 gene can also lead to anterior segment dysgenesis and glaucoma (20,21). These data indicate the necessity of a stringent control of FOXC1 levels and activities for normal development and function of the anterior segment of the eye. To understand the contribution that FOXC1 makes to normal eye function and how FOXC1 dysregulation causes glaucoma, it is crucial to identify targets of FOXC1 transcriptional regulation. We utilized a caged FOXC1 protein that can be activated in the presence of protein synthesis inhibitors, coupled with microarray experiments, to identify genes directly regulated by the FOXC1 transcription factor. We report that FOXC1 regulates the expression of FOXO1A, a key protein in cellular stress response and apoptotic pathways. Furthermore, we demonstrate that a reduction in FOXC1 expression leads to a decrease in FOXO1A expression in cultured human TM cells and that expression of zebrafish FOXO1A orthologs are decreased when FoxC1 is knocked down by morpholino antisense oliognucleotides. We also demonstrate that a reduction in FOXC1 leads to an increase in apoptosis in cultured TM cells in response to oxidative stress and an increase in cell death in the eye of the developing zebrafish. These studies have uncovered a novel role for FOXC1 as an essential mediator of cellular homeostasis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Identification and validation of FOXC1 target genes
To identify genes directly regulated by the forkhead box transcription factor FOXC1, we created a caged or inducible FOXC1 protein. This was achieved through replacement of the FOXC1 inhibitory domain (amino acid residues 215–366) with a truncated human progesterone ligand binding domain (FOXC1-HR; Fig. 1A). Such an insertion rendered FOXC1 inactive in the absence of the progesterone analog mifepristone (Fig. 1B). Transcriptional regulatory activity of FOXC1-HR was stimulated upon the administration of 10–8 M mifepristone for 12 h. To systematically isolate genes directly regulated by FOXC1 in ocular tissues, we transfected human non-pigmented ciliary epithelial (NPCE) cells with FOXC1-HR. The cells were treated with 100 mg/ml of cyclohexamide (CHX) to block intermediate protein synthesis. FOXC1-HR was induced by mifepristone (10–8 M) and RNA was isolated for microarray analysis. The prevention of new protein synthesis by CHX provides for an enrichment of mRNAs expressed in direct response to FOXC1-HR activation. We compared FOXC1-HR RNA with RNA harvested from cells transfected with empty pcDNA4 vector, and also treated with CHX and mifepristone to control for changes in gene expression in response to these drugs. RNA from three independent hormone induction and CHX-treatment regimens was used to generate probes for hybridization to Affymentrix U133A arrays. Probe sets displaying at least a 2-fold differential expression between control and FOXC1-HR expression cells were identified by dCHIP analysis. Expression of 674 known genes was altered in response to FOXC1-HR activation (Supplementary Material, Table S1). Differentially regulated genes were selected for further analysis based on three criteria: (i) expression within the eye, (ii) expression in similar non-ocular tissues as FOXC1 and (iii) the presence of a core FOXC1 DNA-binding consensus sequence (TAAAYA) within 3 kb of the most 5' exon (Table 1). We used Gene Ontology (GO) classifications to categorize the FOXC1-HR-responsive genes by function. Genes differentially regulated by FOXC1 were identified to be involved in diverse biological functions, including regulation of apoptosis, cell growth, cell differentiation, regulation of transcription, metabolism and responses to cellular stress (Table 1). FOXC1-induced regulation of these selected genes was analyzed by northern blotting of RNA isolated from NPCE cells transfected with either FOXC1-HR or empty expression vector and treated with mifepristone and CHX. As illustrated in Figure 2A, expression of candidate target genes was differentially regulated in response to FOXC1-HR induction. Notably, Notch2, FOXO1A, ING2, Heat shock protein 70 (HSPA6), chondroitin sulfate proteoglycan 5 (CSPG5), Rab3-GTPase activating protein (RAB3GAP) and G-protein receptor 39 (GPR39) mRNAs were upregulated by FOXC1-HR. Expression of hepatoma-derived growth factor (HDGF) and collagen IV {alpha}-3 (COL4A3) mRNAs was downregulated by FOXC1 in northern blots, whereas dynein light chain (DNLC) expression was unchanged. The relative changes in expression of these genes determined by northern blotting between empty vector and FOXC1-HR were quantitated after normalization of with beta-actin expression (Fig. 2B).


Figure 1
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  		Figure 1. Hormone inducible FOXC1. (A) Outline of the strategy used to identify mRNAs regulated in direct response to FOXC1. (B) Insertion of the progesterone receptor ligand-binding domain creates an inducible FOXC1 protein (FOXC1-HR). NPCE cells were transfected with FOXC1-HR or empty pcDNA4 expression vector along with 6xFOXC1BS-TK-luciferase reporter. Cells were treated with mifepristone (10–8 M) or ethanol (vehicle) 12 h prior to harvesting. Data are representative of a single experiment transfected in triplicate. The error bars correspond to the standard error of the mean (SEM).

 


Figure 2
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  		Figure 2. Northern blot validation of genes differentially regulated by FOXC1. Expression of genes differentially regulated by FOXC1 in microarray experiments were analyzed by northern blotting. RNA was harvested from NPCE cells transfected with FOXC1 HR (F) or empy pcDNA4 expression vector (V), subjected to a 12 h treatment with cyclohexamide and mifepristone. Probes for NOTCH-2, FOXO1A, ING2, RAB3 GTPase activating protein (RAB3GAP), chondroitin sulfate proteoglycan 5(CSPG5), nuclear factor Y, subunit B (NF-YB), G-protein receptor 39 (GPR39), Heat Shock Protein 70 (HSPA6), HDGF, Collagen IV {alpha}-3, (COL4A3), dynein light chain (DNLC), and β-actin were prepared from plasmid DNA by random prime labelling and hybridized to northern blots. Fold changes of gene expression between vector and FOXC1 HR transfected cells are presented after normalization to β-actin levels.

 


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  		Table 1. Selected genes differentially regulated by FOXC1-HR induction

 
Next, we investigated whether expression of these candidate target genes were regulated by FOXC1 in vivo. We utilized morpholino-antisense oligonucleotide (MO) knockdown of foxC1 in zebrafish to examine the expression of orthologous FOXC1 target genes by RT–qPCR. The zebrafish foxC1 has been duplicated and both foxC1.1 and foxC1.2 transcripts are expressed within the periocular mesenchyme (POM) and developing anterior segment structures (22). MO knockdown of the foxC1 genes, along with the analysis of MO specificity and resulting early somite defects, has been described previously (23). Knockdown of foxC1.1 by either of two independent MOs resulted in a strong phenotypes by 32-48hfp including a small eye, ocular anterior segment dismorphogenesis, hemorrhagic hydrocephaly and cardiovascular defects. These phenotypes are consistent with phenotypes found with loss of foxC1 in other species, including mouse (12–17). Knockdown of foxC1.2 by either of the two independent translation-inhibiting MOs resulted in a mild phenotype, consistent with the previously reported results (23). To address specificity of these phenotypes, zebrafish foxC1.1 RNA not targeted by the MO designed against the 5'-untranslated region was co-injected with foxC1.1 MOs. As previously reported in Topczewska et al. (23), zebrafish embryo is very sensitive to overexpression of foxC1 and injection of >50 pg of foxC1.1 RNA in our experiments caused severe morphological defects (data not shown). Additionally, protein produced from an injected YFP-tagged foxC1.1 RNA was found to be depleted before 22 hpf, indicating that foxC1.1 RNA is degraded during early development (data not shown). The degradation of foxC1.1 RNA and overexpression phenotypes made analysis of post-24 hpf phenotype recovery not possible, but independent MOs and consistency of phenotypes across species provide strong evidence for specificity of foxC1 knockdown phenotypes. To study the expression of all possible foxC1 targets, embryos were injected with a combination of MOs against both foxC1.1 and foxC1.2, creating zebrafish embryos with depleted foxC1 activity (foxC1dMO). Knockdown of both genes by a combination of MOs targeting foxC1.1 and foxC1.2 resulted in a phenotype similar to, but stronger than, knockdown of foxC1.1 alone.

As indicated in Table 2, we observed a significant reduction in expression of zebrafish foxO1a.1 (–3.77-fold), foxO1a.2 (–2.28-fold), notch2 (–1.46-fold), cspg5 (–2.7-fold) and rab3gap (–1.84-fold) mRNAs isolated from ocular tissue when FoxC1 levels were suppressed, indicating that the zebrafish Foxc1 proteins act to regulate positively the expression of these genes in zebrafish. No significant changes in expression were observed for ing2, nf-yB and hdgf.


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  		Table 2. Changes in gene expression in foxC1 knockdown embryos

 
Finally, we tested whether FOXC1 was able to bind the promoter regions of these target genes in vivo through chromatin immunoprecipitation (ChIP) assays in ocular cell lines. Potential FOXC1-binding sites were identified in 5' region using an FOXC1 DNA-binding site matrix and the program Possum (http://zlab.bu.edu/~mfrith/possum/). The specificity of the antibodies used for the immunoprecipitation of FOXC1 in the context of chromatin has been described previously (24). As a negative control, we attempted to amplify a region of the β-actin promoter. Expression of β-actin was not activated by FOXC1 in our microarray analysis, but is actively expressed in NPCE cells. As illustrated in Figure 3, this promoter element was not recovered by FOXC1 ChIP experiments, but was recovered in histone H3-acetylated lysine 9 ChIP products (H3-AcK9), as would be expected for a gene actively expressed, but not regulated, by FOXC1. Upstream DNA sequences containing FOXC1-binding sites were recovered from the NOTCH-2, FOXO1A, ING2, RAB3GAP, CSPG5 NFYB and HDGF genes following ChIP with FOXC1 and H3-AcK9 antibodies. These data indicate that FOXC1 occupies the promoter regions of the genes, in vivo, that were differentially expressed in response to FOXC1.


Figure 3
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  		Figure 3. Occupancy of promoter regions by FOXC1 in vivo. Chromatin from NPCE cells was cross-linked and was immunoprecipitated with antibodies against GFP, FOXC1 or acetylated lysine 9 of Histone 3 (H3-AcK9). Input represents of 1:40 000 dilution of cross-linked chromatin that was not immunoprecipitated. gDNA, genomic DNA. Immunoprecipitated DNA was amplified by PCR using primers designed to amplify regions flanking putative FOXC1 binding sites in the promoters of the NOTCH2, FOXO1A, ING2, RAB3GAP, CPSG5 and NFYB and β-actin genes.

 
FOXC1 is a crucial mediator of cellular homeostasis through the transcriptional regulation of FOXO1A
There is a critical balance in FOXC1 gene dose required for normal development and function of the eye. Paradoxically, strikingly similar disorders result in humans when FOXC1 gene activity is reduced through loss of function mutation as well as when an extra copy of FOXC1 arises through gene duplication (6,7,20,21). In both situations, patients present with malformations of the anterior segment of the eye and are at a heightened risk to develop glaucoma. Our data thus far have determined that the forkhead box transcription factor gene, FOXO1A, is a target of FOXC1 transcriptional regulation. As FOXO1A can positively and negatively regulate cell survival (25), it was an attractive gene to explain why similar disorders arise from an increase or decrease in FOXC1 gene dosage. Thus, we will focus on the regulation of FOXO1A expression by FOXC1 and the biological significance of this regulation for the remainder of the paper.

A perfect consensus FOXC1 DNA-binding site (GTAAACAAA) was found in the proximal promoter region in the human FOXO1A gene that was conserved in the 5' upstream regions of several mammalian FOXO1A genes, suggesting it is of important functional consequence (Fig. 4A). ChIP revealed that FOXC1 did indeed bind to this site in the human FOXO1A gene in vivo (Fig. 3). We subsequently cloned a region of the FOXO1A gene. The sequences 580 bp upstream and 250 bp downstream (–580/+250) of the predicted transcription start site were positioned in front of a luciferase reporter to test for the activation of the FOXO1A promoter by FOXC1. As indicated in Figure 4B, promoter activity was enhanced in HeLa cells in a FOXC1-dependant manner. We created deletions in the FOXO1A promoter to eliminate the conserved FOXC1-binding site as well as two other potential elements that loosely fit the FOXC1-binding consensus. The removal of all three sites (–172/+250) completely abolished FOXC1 activation of this reporter. Finally, activation of the –580/+250 FOXO1A luciferase reporter is diminished when disease-causing S131L FOXC1 alleles are transfected into cells (Fig. 4C).


Figure 4
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  		Figure 4. FOXC1 regulates expression of FOXO1A. (A) Alignment of the consensus FOXC1 binding sequence present in the promoter regions of mammalian FOXO1A genes. Potential FOXC1 binding sites are present in the promoters of zebrafish Foxo1a.1 and Foxo1a.2 genes. (B) Activation of the FOXO1A promoter by FOXC1. A region of the human FOXO1A gene corresponding to 580 bp upstream and 250 bp downstream from the putative transcriptional start site (–580/+250) was cloned and inserted in front of a luciferase reporter. This region contains 3 putative FOXC1 binding elements (FOXBS) with the conserved element indicated in bold. Promoter reporter constructs were transfected into HeLa cells along with WT FOXC1 for transactivation assays. (C) The –580/+250-FOXO1A promoter luciferase reporter was co-transfected with WT and S131L FOXC1 alleles. Data presented are from three independent transfections performed in triplicate. Error bars indicated the standard error of the mean (SEM).

 
Foxo1A is expressed in the developing POM
In the mammalian eye, FOXC1 is critical for the development of POM into structures of the anterior segment of the eye. The zebrafish eye also requires Foxc1 for correct formation of the anterior segment, and the gene is expressed within the POM in developing embryos. The expression and role of FOXO1A in the eye has not been examined thoroughly. To determine whether FOXO1A is expressed in relevant anterior segment structures of the eye, we examined the expression of zebrafish FOXO1A orthologs by in situ hybridization analysis. Expression of foxO1a.1 was robust within the POM, in the branchial arch region and in the developing pancreas (Fig. 5). FoxO1a.2 was expressed at very low levels throughout the eye, but was detected at high levels in the branchial arch region and within the nasal epithelium (Fig. 5). In situ hybridization analysis was also carried out in foxC1dMO zebrafish to determine whether foxO1a gene levels were reduced in the regions of co-expression with the foxC1 genes. foxC1dMos are identifiable by a small eye phenotype, which can be observed in the sections shown in Figure 5. A clear reduction in foxO1a.1 was found within the POM in morphant embryos, consistent with the results obtained by RT–qPCR (Fig. 5). As foxO1a.2 was not found in levels clearly detectable by in situ hybridization within the eye, no clear reduction was found in foxC1dMOs.


Figure 5
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  		Figure 5. Expression of zebrafish foxO1a genes. Expression of zebrafish foxO1a.1 and foxO1a.2 was assessed by whole mount in situ hybridization. (A) Side and ventral views of whole mount foxO1a.1 expression in 48 hpf WT embryos. The gene is enriched within the POM, in the branchial arch region and along the developing GI tract. Frozen section of 48 hpf WT (B) and foxC1dMO (C) show robust expression within the POM (arrows) of WT embryos, and a clear reduction in morphants. (D) At 24 hpf, strong expression is found within the pancreas (arrows) of a WT embryo, which is also evident by section at 48 hpf in WT. No difference in pancreas expression was found in foxC1dMO. (E) foxO1a.2 whole body expression. Transcripts are found at low levels throughout the head, with enrichment within the nasal epithelium (arrows). By section, lower levels are found throughout the eye in 48 hpf WT embryos (G), with enrichment in the branchial arch region (arrow). No clear differences within ocular structures were found in foxC1dMO embryos by section (F).

 
FOXC1 is required for the survival of trabecular meshwork cells in response to oxidative stress
We subsequently pursued the biological significance of FOXC1-mediated regulation of FOXO1A expression with respect to AR-associated glaucoma. There is mounting evidence suggesting a role for oxidative damage in the trabecular meshwork of glaucomatous eyes (26,27). Given that FOXO1A has an important role in the resistance to oxidative stress (28–30), we sought to determine whether a loss of FOXC1 expression reduced FOXO1A expression and impaired cellular oxidative stress responses. We reduced the expression of FOXC1 in human trabecular meshwork (HTM) cells by siRNA transfection. As illustrated in Figure 6A, we observed a robust decrease in the levels of FOXC1 protein in nuclear extracts of HTM cells transfected with 50 nM of FOXC1-specific siRNAs. The reduction of FOXC1 expression by siRNAs also led to a reduction in the protein levels of FOXO1A in HTM cells (Fig. 6B). FOXO1A protein expression was estimated to be about 2.4-fold lower when FOXC1 levels were reduced by siRNA. Next, we investigated whether the silencing of FOXC1 expression influenced cell survival and response to oxidative stress. When HTM cells were transfected with FOXC1 siRNA, we observed a reduction in cell viability compared with cells treated with a non-specific siRNA molecule. Treatment with 500 µM H2O2 also resulted in reduced cell viability of FOXC1 siRNA transfected cells compared with control transfected cells (Supplementary Material, Fig. S1). To determine whether apoptosis contributed to this increase in cell death, we examined caspase 7 cleavage in cells transfected with FOXC1 siRNAs, with and without the addition of H2O2. Reducing FOXC1 expression by siRNA transfection resulted in an increase in caspase 7 cleavage and this increase was augmented when cells were treated with 500 µM H2O2 for 2 h (Fig. 6C–E). Through immunoblot analysis, we observed a decrease in FOXO1A expression when cells were treated with FOXC1 siRNAs and an increase in the cleavage of Caspase 7, which was again elevated when cells were treated with H2O2 (Fig. 6C).


Figure 6
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  		Figure 6. FOXC1 is required for cellular resistance to oxidative stress. (A) HTM cells were transfected with siRNAs specific to FOXC1 or with an siRNA molecular that does not target any known gene. Mock-transfected cells underwent the transfection procedure, but did not receive any siRNA molecule. Levels of FOXC1 protein expression was monitored by immunoblot experiments with nuclear extracts harvested 3 days after transfection. Antibodies to TFII-D were used as a loading control. (B) Reduction in FOXC1 levels by siRNA reduces FOXO1A protein levels. HTM whole-cell extracts from FOXC1 siRNA transfected cells were analyzed by immunoblot experiments using antibodies against FOXO1A and ERK1/2 (loading control). (C) HTM cells transfected with non-targeting siRNA or FOXC1-specific siRNAs were treated with 500 µM H2O2 for 2 h and subjected to immunofluorescence with antibodies recognizing cleaved caspase 7. (D) Quantification of cells immunoreactive for caspase 7 following transfection with non-targeting (Ctrl) or FOXC1-specific siRNAs treated with or with 500 µM H2O2. Means from nine transfections with at least 500 cells scored per transfection are presented. The error bars represent the SEM. (E) Immunoblot analysis of FOXO1A protein levels and cleavage of caspase 7 in HTM cells following siRNA transfection and H2O2 treatment. Erk1/2 antibodies were used as a loading control.

 
Increased cell death in the developing eye of zebrafish foxC1dMO embryos
To compare cell viability in foxC1dMOs, control MOs and uninjected zebrafish embryos, the DNA-binding dye, acridine orange (AO) was used to assess the number of dead or dying cells. Within the living embryo, AO is taken up only by cells with compromised plasma membranes, and not by healthy cells. Confocal microscopy visualization of the eyes of 32 hpf embryos showed that cell death is increased in foxC1dMOs compared with both control MOs and uninjected siblings (Fig. 7A). Levels of cell death were quantified by measuring total area of fluorescence within the eyes of five embryos of each group, excluding the lens that normally contains cells with compromised plasma membranes. A statistically significant increase in cell death was found in foxC1dMO embryos compared with uninjected or control MO-injected embryos by Student’s t-test (P ≤ 0.05) (Fig. 7B). In addition, cell death was also observed in the developing central nervous system and somites when foxC1 levels were reduced (data not shown). Overall, these results show a clear reduction in ocular cell viability during development with loss of foxC1.


Figure 7
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  		Figure 7. Cell death in the developing foxC1dMo zebrafish eye. Acridine orange staining was used to assess cell death in the eyes of 32 hpf foxC1dMO, control Mo and uninjected zebrafish embryos. (A) Images representing the mean levels of fluorescence for each group are shown. The foxC1dMOs show an obvious increase in cell death, indicated by an increase in fluorescing cells. (B) The mean percentage of total area fluorescing was quantified for five embryos from each group and were plotted and analyzed by Student’s t-test. The increase in cell death found in foxC1dMOs is significant compared with control MO (P = 0.02) and uninjected embryos (P = 0.05). The error bars correspond to the SEM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Mutations in the FOXC1 transcription factor gene result in AR syndrome, a disorder characterized by a spectrum of malformations in the anterior segment of the eye. Individuals with AR are at a heightened risk to develop glaucoma, a blinding disorder characterized by the degeneration of the optic nerve and RGCs. In order to understand the contribution of FOXC1 in these ocular malformations and glaucoma pathogenesis, it is important to identify genes that are regulated by FOXC1. To date, few direct targets of FOXC1 transcription regulation have been identified. Several studies have examined changes in gene expression in Foxc1 mutant mice in an effort to understand cellular events regulated by FOXC1 (12,31). However, although these studies have identified many genes whose expression was altered when the Foxc1 gene was inactivated, it is unclear whether differential gene expression occurs in direct response to FOXC1 or by indirect mechanisms. Through the use of a hormone-inducible FOXC1 protein coupled with microarray analysis, we have identified a number of genes that are direct targets of FOXC1 transcriptional regulation. We observed an altered expression of target genes in response to FOXC1 activation, a decrease in expression in vivo when foxc1 expression was reduced in zebrafish, and a binding of FOXC1 to the promoter element of these genes by ChIP assays. Together, these data indicate that FOXO1A, NOTCH2, RAB3GAPand CSPG5 are bona fide direct target genes of FOXC1 transcriptional regulation in the eye. The diverse biological roles of the genes identified in this study indicate that FOXC1 targets are involved in regulating developmental processes and their misregulation may contribute to the AR malformations. For example, NOTCH2, a member of the notch signaling pathway, is expressed in the developing mouse tooth epithelium and in the enamel-producing ameloblasts (32). Thus, misregulation of NOTCH2 may contribute to the dental anomalies observed in AR. Moreover, Alagille syndrome, a disorder characterized by hepatic, cardiac, skeletal and ophthalmologic manifestations, can be caused by mutations of the NOTCH2 gene (33). Ocular malformations observed in Alagille syndrome, such as posterior embryotoxon, indicate a developmental role for NOTCH2 in the anterior segment of the eye. It has been determined that Foxc1 and Foxc2 are required for expression of notch-signaling pathways necessary for the correct patterning of somites in mice (31). Our work has established that the NOTCH2 gene is a target of FOXC1 transcriptional regulation in the eye and, therefore, FOXC1 may be a key regulator of expression of additional NOTCH gene members. RAB3GAP is also crucial for the correct development of the eye. Mutations in RAB3GAP underlie Warburg–Micro syndrome (34). Ocular manifestations including micropthalmos, microcornea, congenital cataracts and optic atrophy, along with neurological and urogenital abnormalities, are present in individuals harboring inactivating alleles of RAB3GAP (34). These data place FOXC1 at the top of a gene regulatory hierarchy required for normal development of the eye and mutations of these direct targets of FOXC1 cause anterior segment malformations in humans.

We provide compelling evidence that expression of FOXO1A is directly regulated by FOXC1 in vitro and in vivo. On the basis of the biological functions of FOXO1A, our data are consistent with the notion that misregulation of FOXO1A expression contributes to many of the pathogenic phenotypes observed in patients with FOXC1 mutations. FOXO1A is an important modulator of cell cycle arrest, is required for cellular stress responses and is a key regulator of apoptotic mechanisms (35). AR malformations and the related anterior segment dysgeneses resulting from mutations in FOXC1 in humans and mice result from an impaired differentiation of the POM cells (12,13). Since FOXO1A has a prominent role in cell cycle arrest, a loss of FOXO1A activity through FOXC1 mutations may account for the impaired differentiation in a subset of POM-derived structures in which FOXO1A is normally expressed. Moreover, FOXO1A maps to human chromosome 13q14, a region that has been linked to AR malformations (5) and is an excellent candidate gene for AR and anterior segment dysgenesis.

The most disabling complication of AR is the elevated risk for developing glaucoma. AR-associated glaucoma caused by FOXC1 mutations is early onset rather than congenital, with symptoms developing in the patients 20s or 30s. Furthermore, Foxc1+/– mice develop AR-like anterior segment malformations, under certain genetic backgrounds, but do not develop elevated IOP and glaucoma (16). We propose that a dysfunction of the anterior segment of the mature eye is a contributing factor to the onset of glaucoma in individuals with FOXC1 mutations. In AR-individuals with FOXC1 mutations, the resistance to these cellular stresses may be altered and glaucoma pathogenesis progresses in an accelerated manner. In our studies, increased TM apoptosis and heightened sensitivity to oxidative damage via H2O2 resulting from a reduction in FOXC1 protein levels, along with the reduction of cell viability with loss of FoxC1 in the living zebrafish, agree with this hypothesis. The loss of TM cells may compromise the aqueous humor drainage capacity in the anterior segment of the eye and lead to an elevated IOP. Such a loss may be gradual, leading to the 20 or 30 year latency that AR patients exhibit prior to the diagnosis of glaucoma. In addition, the differential exposure to environmental factors that can contribute to the generation of ROS may explain, in part, the variability in the onset of glaucoma in AR patients and provide exciting insight into gene–environment interactions.

There is a critical threshold of FOXC1 activity required for normal development and function of the eye. Both loss of function mutations in FOXC1 and segmental duplications of 6p25, resulting in an extra copy of FOXC1, result in strikingly similar, ocular phenotypes (6,21). Similarly, a delicate balance of FOXO1A activity may be essential for correct function of the eye. As discussed above, FOXC1 regulation of FOXO1A is required for survival of TM cells in response to oxidative stress. A loss of FOXC1 activity can therefore reduce FOXO1A expression and such a loss may contribute to AR and glaucoma pathogenesis. In addition, overexpression or hyperactivity of FOXO1A is also deleterious. Members of the FOXO family can act as pro-apoptotic factors under a variety of physiological conditions such as DNA damage or serum deprivation (35). Phosphorylation by AKT/protein kinase B (PKB) inactivates FOXO proteins and promotes cell survival (36). Overexpression of a gain-of-function FOXO3A mutant protein induces apoptosis in cerebellar granule cells (36). Thus, increased FOXC1 gene dosage may lead to elevated FOXO1A and compromised cell viability either through activation of pro-apoptotic genes or through inappropriate activation of a stress response pathway. Recently, it has been demonstrated that the chronic activation of PKB in bone marrow cells, normally an antiapoptotic signal, activates cells death through increased Foxo3a expression (37). Likewise, chronic activation of FOXO1A-regulated stress response pathways may be deleterious and underlie disease pathogenesis resulting from increases in FOXC1 gene dosage.

A growing body of evidence suggests a contribution of oxidative stress in glaucoma pathogenesis (26,27). As glaucoma incidence increases with age, it is thought that a prolonged, chronic exposure of cellular stresses such as oxidative damage over the lifetime of an individual may damage and compromise the function of the eye. Reactive oxygen species (ROS) are generated as a consequence of aerobic respiration. Low ROS concentrations are beneficial and essential for normal cellular activities, but exposures to higher concentrations are detrimental. The TM possesses enzymes necessary for the detoxification of ROS, notably members of superoxide dismutase (SOD) family, catalase and those involved in glutathione pathways (26,27). A decrease in the number of TM cells is associated with age and with POAG (38,39). Since the activity in SOD declines with age in the TM, this progressive age-related loss in TM cells in glaucomatous eyes may be attributable to free radical damage accumulated over time (38,39). Our data demonstrating that reduced FOXC1 activity in the TM leads to increased cell death and an increased sensitivity to oxidative stress is consistent with the observed loss of TM cellularity occurring in glaucomatous eyes. Furthermore, in AR patients, the loss of function FOXC1 mutations may lead to an increased sensitivity to ROS and an accelerated degeneration of the TM. Oxidative damage in the TM has been detected in glaucomatous eyes and heightened damage, marked by 8-hydroxy-2'-deoxyguanosine levels, correlates with visual field loss and elevated IOP (40,41). Furthermore, oxidative damage to the TM compromises its function and impedes its drainage capacity (42,43). ELAM-1 expression is activated specifically in the TM of glaucomatous eyes by NF-kB (44). It is thought that this activation occurs in response to sublethal cellular stress and chronic activation of such stress response pathways may further exacerbate disease pathogenesis. Oxidative damage-induced apoptosis is also thought to contribute, in part, to the degeneration of the RGC and optic nerve following axotomy (45). RGC degeneration can be rescued by trophic factors and anti-oxidants in a rat model of elevated IOP (46,47). Finally, disorders affecting the biogenesis of peroxisomes, an organelle key for the detoxification of H2O2, are associated with ocular anomalies including glaucoma (48). ROS themselves may act as secondary messengers to activate additional stress responses and initiate pathological processes. Therefore, there is compelling evidence for a role of oxidative stress in glaucoma pathogenesis. Our data place FOXC1 and its direct target, FOXO1A, as central mediators for oxidative stress resistance and cell viability in the eye.

The identification of FOXC1 target genes provides an important first step to understand AR and glaucoma pathogenesis. These studies have uncovered a novel role for FOXC1 as an important regulator of cellular homeostasis in the eye. Our data are consistent with the hypothesis that in AR individuals with FOXC1 mutations, oxidative stress resistance is impaired. This increased susceptibility to oxidative damage can result in a loss of TM cellularity, which, in turn, prevents aqueous humor uptake and leads to elevated IOP. Furthermore, we provide evidence for the transcriptional regulation of FOXO1A by FOXC1 and indicate a critical balance in the activities of FOXC1, and FOXO1A is required for normal eye function. The dysregulation of FOXO1A activities in the eye through FOXC1 loss of function mutations and FOXC1 gene duplications provides molecular insight into how seemingly similar human disorders can arise from both increases and decreases in FOXC1 gene dosages.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Plasmid construction
A hormone-inducible FOXC1 (FOXC1-HR) protein was constructed by inserting a truncated progesterone ligand-binding domain between the FOXC1 DNA-binding FHD and the C-terminus transactivation domain (amino acid residues 215–366). The removal of this region does not impair FOXC1 transcriptional activation (49). pcDNA4-Xpress FOXC1 has been reported previously (18).

Cell culture
Non-pigmented ciliary epithelial cells and HeLa cells were grown in high-glucose DMEM, supplemented with 10% fetal bovine serum (FBS). Immortalized HTM cells were grown in low-glucose DMEM and 10% FBS. For transfection of FOXC1-HR, NPCE cells were plated on to six-well plates at 2.5 x 105 cells per well 24 h prior to transfection. Cells were transfected with 500 ng of FOXC1-HR or empty pcDNA and 2.5 µl of FUGENE6 (Roche, Laval, PQ, CAN) for 48 h. Twelve hours prior to harvesting, cells were treated with CHX (100 µg/ml; Sigma Aldrich, St. Louis, MO, USA) and mifepristone (10–8 M; Invitrogen, Burlington, ON, CAN).

RNA isolation, cDNA synthesis and labeling
RNA was isolated from NPCE cells using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Twenty micrograms of total RNA was used for microarray cDNA probe labeling. cDNA probes were generated from three separate transfection and induction regimens and were hybridized independently to Affymetrix U133A arrays (Affymetrix, Santa Clara, CA, USA). Ten micrograms of total RNA was converted to cDNA using the SuperScript II reverse transcriptase (Invitrogen), which uses T7-oligo-d(T)24 as a primer. Second-strand synthesis was performed using a T4 DNA polymerase and Escherichia Coli DNA ligase, and blunt ended by T4 polynucleotide knase. cDNA was isolated by phenol–chloroform extraction using phase lock gels (Brinkmann, Westburg, NY, USA). cDNA was in vitro transcribed using the T7 BioArray High Yield RNA Transcript Labeling Kit (Enzo Biochem, New York, NY, USA) to produce biotinlated cRNA. Labeled cRNA was isolated by using an RNeasy Mini Kit column (Qiagen). Purified cRNA was fragmented into 200–30mer cRNA using a fragmentation buffer (100 mM potassium acetate–30 mM magnesium acetate–40 mM Tris-acetate, pH 8.1, for 35 min at 94°C). The quality of total RNA, cDNA synthesis, cRNA amplification and cRNA fragmentation was monitored by micro-capillary electrophoresis (Bioanalizer 2100, Agilent Technologies, Santa Clara, CA, USA). The cRNA probes were hybridized to an HG U133A Genechip, 15 µg of fragmented cRNA was hybridized for 16 h at 45°C with constant rotation (60 rpm). Microarrays were processed by using the Affymetrix GeneChip Fluidic Station 400. Staining was performed with streptavidin-conjugated phycoerythrin (SAPE) followed by an amplification with a biotinylated anti-streptavidin antibody and by a second round of SAPE and scanned using a GeneChip Scanner 3000 (Affymetrix). Data were then normalized with GCRMA from Bioconductor.

Microarray analysis
Expression data from Affymetrix microarrays were first log2 transformed and then analyzed with dCHIP software (50,51). Changes in transcript levels were compared among three matched sets of RNA from NPCE cells treated with CHX and mifepristone and transfected with empty vector or FOXC1-HR. A change in expression was determined to have occurred when at least a 2-fold change in expression was detected along with a P-value <0.05 and a ‘Present’ call in two out of three comparisons using Affymetrix MAS5 software.

Northern blotting
Northern blot analysis was performed as described previously (18). Fifteen micrograms of RNA, isolated from CHX-mifepristone inductions independent from the RNA used for microarray analysis, was loaded onto each gel. cDNA fragments corresponding to NOTCH-2, FOXO1A, ING2, RAB3 GTPase activating protein (RAB3GAP), chondroitin sulfate proteoglycan 5(CSPG5), nuclear factor Y, subunit B (NF-YB), G-protein receptor 39 (GPR39), Heat Shock Protein 70 (HSPA6), HDGF, Collagen IV {alpha}-3, (COL4A3), DNLC and β-actin were labeled with 32P-dCTP by random prime labeling (Invitrogen) and hybridized to membranes using ExpressHyb reagent (BD Biosciences, San Jose, CA, USA). Changes in RNA levels were determined using the ImageJ program after normalization with β-actin levels.

Zebrafish MO injection and qPCR
Morpholino injections and RT–qPCR analysis were carried out as described previously (24). Briefly, MOs used for knockdown were ordered from Gene Tools (Corvallis, OR, USA), resuspended in water and injected into 1–2 cell stage embryos. MOs used were (with amounts injected) (Start codon underlined): foxC1.1 MO2 (8 ng) (GeneID 79374): 5'-CCTGCATGACTGCTCTCCAAAACGG-3'; foxC1.2 MO1 (8 ng) (GeneID 79375): 5'-GCATCGTACCCCTTTCTTCGGTACA-3'; Control MO (16 ng): 5'-CCTCTTACCTCAGTTACAATTTATA-3'. foxC1.1 MO2 and foxC1.2 MO1 were injected together to achieve foxC1dMO morphant zebrafish. Other foxC1 MOs used to verify phenotypes were: foxC1.1 MO1 (5–12 ng) 5'- GTCAAGAAGACTGAAGCAATCCACA-3'; foxC1.2 MO1 (5-12 ng): 5'- AAGTGAAATGAAGACTATGCAGACG-3'. Capped and polyA-tailed foxC1.1 mRNA containing no 5'-UTR was made by in vitro transcription from linearized plasmid (mMessage mMachine, Ambion). To analyze gene expression levels, total RNA was isolated from three sets of 100 eyes and associated POM from 48 hpf foxC1dMO MO and three sets of 100 eyes from control MO-injected zebrafish embryos using the QIAGEN RNeasy Plus Mini Kit (Qiagen Inc, Missisauga, ON, CAN). cDNA samples were generated from 0.5 µg of RNA (SuperScript II RT; Invitrogen) and qPCR carried out in triplicate for each primer set using the iQ SYBR Green system (Bio-Rad, Missisauga, ON, CAN). Data were then analyzed using the {Delta}{Delta}Ct method (52). Target genes were identified in the zebrafish by BLAST comparison of human and mouse genes against the whole zebrafish genome (http://www.sanger.ac.uk/Projects/D_rerio/) and submitted EST sequences (http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=7955). All primers were designed using publicly available EST sequence, except for the FOXO1A orthologs, which were both isolated and sequence-confirmed for this study. Predicted protein sequences were further analyzed by alignment with MegAlign software. Primers used were (with GeneID’s): foxO1a.1 (EF421424)—F: 5'-TCAGGATCCAACCAGTCCTC-3', R: 5'-CACTTGCGAAAGTCCTGTTG-3'. foxO1a.2 (EF421425)—F: 5'-GCGCTATCCTCACCTTTGAT-3', R: 5'-ACTCCACCTTGCCCATACAG-3'. rab3gap (EST:EH502918)—F: 5'-TGAGATCACGGATTTCACCA-3', R: 5'-GCCAATCAGCTTCCAATCAT-3'. cspg5 (EST:EH432696)—F: 5'-TTGTCCAGCTCCTCAGACCT-3', R: 5'-TCCTGGAGTTGGAGTGATGA-3'. ing2 (436721)—F: 5'-CAACAGTGAGAGCCGAGACA-3', R: 5'-TCTTCTTCTTGGCCGACTTG-3'. notch2 (58065)—F: 5'-CCAGTGGGACAGGATGACTT-3', R: 5'-TTCTGACCAGTGATGGTTCG-3'. nfyb (503744)—F: 5'-GCCAACGTTGCCAGAATAAT-3', R: 5'-CTCCTTTCATCGCCTCTCTG-3'. hdgf (EST: EH548223)—F: 5'-AGCAGCAATGAAGAGGGAGA-3', R: 5'-CTGATCAGAAACGTCCATGTC-3'.

Chromatin immunoprecipitation
ChIP assays were performed as described previously (24). Two micrograms of either {alpha}-GFP (Abcam, Cambridge, MA, USA), {alpha}-FOXC1(Abcam) and {alpha}-Histone H3 acetylated lysine 9 (Cell Signaling Technologies, Danvers, MA, USA) antibodies was used for each immunoprecipitation reaction.

Transactivation assays
Transactivation assays were performed in HeLa cells as described previously (53). A region of the FOXO1A gene corresponding to 580 bp upstream and 250 bp downstream of the putative transcription start site was cloned by PCR and inserted into the pGL3-basic luciferase reporter vector. Deletions of the 5' region of the FOXO1A reporter were generated by restriction digest. Transfections were performed in triplicate and each experiment was performed three times.

Whole mount in situ hybridization
A 654 bp fragment of the zebrafish foxO1a.1 gene and a 472 bp fragment of the foxO1a.2 gene were amplified by PCR with the following primers: foxO1a.1: 5'-GTTTGCCAAGAGCAGAGGAC-3', 5'-AGGAGTCCTGCTGTGCAGTT-3'; foxO1a.2: 5'-GCGCTATCCTCACCTTTGAT-3', 5'-GTGGTTGTGGCTTCATGCTA-3'. These fragments were cloned into the pCRII-TOPO vector (Invitrogen) and used as a template for the generation of a cRNA probe. cRNA probes were produced and whole mount in situ hybridization was conducted as described previously (54) with the addition of a final spin column cRNA probe purification using the ProbeQuant G-50 Micro Column (GE Healthcare, Baie d'Urfé, PQ, CAN). Full details are available from the authors. In each experiment, 10–15 embryos were analyzed per probe. For post-hybridization sectioning, embryos were fixed in 4% paraformaldehyde/PBS and infiltrated with 15% sucrose, 30% sucrose and then 100% Tissue-Tek OCT (Miles Inc., Elkhart, IN, USA). Embryos were oriented in a freezing mold, and then 10 µm sections were cut on a cryostat and mounted on gelatin-coated glass slides.

siRNA transfection
Human FOXC1-specific and non-targeting control siRNAs were purchased from Dharmacon. HTM cells were plated at a density of 5 x 105 cells per 60 mM tissue culture plate or 2 x 105 cells per well of a six-well plate, containing a coverslip, 24 h prior to transfections. Cells were transfected with 50 nM of FOXC1-specific or non-targeting siRNA with Lipofectamine 2000 (Invitrogen) and cultured 48–72 h before harvesting.

Immunoblotting
Whole cell and nuclear extracts were prepared as described previously (53). For the detection of FOXC1 expression, 100 µg of nuclear extracts were resolved on a 10% polyacrylamide gel, transferred to nitrocellulose and probed with anti-FOXC1 antibodies (CeMines, Golden CO) as described previously (53). Antibodies-recognizing transcription factor IID and FOXO1A were purchased from Santa Cruz Biotechnology and used at dilutions of 1:000 and 1:5000, respectively. Antibodies-recognizing Erk1/2 and cleaved caspase 7 were purchased from Cell Signaling Technologie and used at a dilution of 1:1000.

Immunofluorescence
HTM cells grown on coverslips were fixed with 2% paraformaldhyde and permeablized with PBS containing Triton X-100 (0.05%). Cells were incubated with anti-cleaved caspase 7 (1:100 dilution) for 1 h followed by incubation with Cy3-conjugated donkey anti-rabbit IgG secondary antibody. The cells were stained with DAPI and mounted onto microscope slides.

AO staining
A 5 g/ml solution of acridine orange stain was used to detect cell death in 32 hpf foxC1dMO, control MO and uninjected embryos. A total of 10–15 phenolthiolurea (PTU)-treated embryos of each condition were incubated in the AO solution at room temperature for 30 min and then rinsed 3 x 5 min in 0.003% PTU fish water. PTU prevents pigment development to keep embryos transparent. Embryos were then anesthetized with tricaine and visualized by fluorescent confocal microscopy. Z-stacks through the thickness of the eye were compressed for analysis. Cell death was quantified using Meta Morph software assessing total fluorescence within the eye, excluding the lens which normally shows cell death in all developing zebrafish. Areas of fluorescence were characterized by threshold analysis and compared by Student’s t-test.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
This work was supported by grants from the Canadian Institutes for Health Research awarded to M.A.W. and F.B.B., from the National Institutes for Health (R01EY16060) awarded to B.A.L, from the National Institute on Aging (F30AG029763) awarded to J.M.S. and from a National Eye Institute Vision Science Training Grant awarded to J.M.S.


   ACKNOWLEDGEMENTS
 
We thank Ms. May Yu for tissue culture expertise and Dr. M. Coca-Prados for providing us with the NPCE cell line.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

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