A mouse model of aniridia reveals the in vivo

Biochimica et Biophysica Acta 1863 (2017) 60–67

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A mouse model of aniridia reveals the in vivo downstream targets of Pax6 driving iris and ciliary body development in the eye Xia Wang, Xianghong Shan, Cheryl Y. Gregory-Evans ⁎ Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, BC, Canada

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Article history: Received 8 July 2016 Received in revised form 13 October 2016 Accepted 18 October 2016 Available online 20 October 2016 Keywords: Pax6 Aniridia Iris Transcription factor Downstream target

a b s t r a c t The Pax6 transcription factor is essential for development of the brain, eye, olfactory and endocrine systems. Haploinsufficiency of PAX6 in humans and mice causes the congenital condition aniridia, with defects in each of these organs and systems. Identification of the PAX6 transcription networks driving normal development is therefore critical in understanding the pathophysiology observed with loss-of-function defects. Here we have focused on identification of the downstream targets for Pax6 in the developing iris and ciliary body, where we used laser capture microdissection in mouse eyes from E12.5–E16.5, followed by chromatin immunoprecipitation, promoter-reporter assays and immunohistochemistry. We identified 6 differentially expressed genes between wildtype and Pax6 heterozygous mouse tissues and demonstrated that Bmp4, Tgfβ2, and Foxc1 were direct downstream targets of Pax6 in developing iris/ciliary body. These results improve our understanding of how mutations in Bmp4, Tgfβ2, and Foxc1 result in phenocopies of the aniridic eye disease and provide possible targets for therapeutic intervention. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Pax6 is an evolutionarily conserved transcription factor that has a critical role in the development of the visual system, the CNS, olfactory bulb, pituitary and pineal glands and pancreatic endocrine function [1–5]. In the visual system Pax6 is necessary and sufficient for eye formation from flies to humans [6–8]. However, at later stages of development Pax6 is also required for differentiation and maintenance of the retina, lens, anterior segment and lacrimal gland [9–12]. Studies in naturally occurring rodent models with heterozygous Pax6 mutations [13, 14], in homozygous Pax6 mutants [15], and in transgenic over expression models [16] have revealed that the eye is particularly sensitive to Pax6 dosage. For instance, differentiation of the lens, cornea, iris and ciliary body are specifically affected by Pax6 levels [17–19]. Identification of the genes directly regulated by Pax6 is fundamental in decoding the transcriptional networks that explain tissue sensitivity to Pax6 dosage. Several high throughput studies in the brain and lens have identified a number of Pax6-regulated genes [20–22]. However, the targets of Pax6 in the developing iris and ciliary body have not

Abbreviations: CNS, central nervous system; E14.5, embryonic day 14.5; PCR, polymerase chain reaction; TBE, Tris-boric acid-EDTA; OCT, optical cutting temperature; PBS, phosphate buffered saline; ORF, open reading frame; DMEM, Dulbecco's modified eagle's medium; BSA, bovine serum albumin; DAPI, 4′,6-diamidino-2-phenylindole. ⁎ Corresponding author at: Dept of Ophthalmology and Visual Sciences, University of British Columbia, 2550 Willow Street, Vancouver, BC V5Z 3N9, Canada. E-mail address: [email protected] (C.Y. Gregory-Evans).

http://dx.doi.org/10.1016/j.bbadis.2016.10.018 0925-4439/© 2016 Elsevier B.V. All rights reserved.

been identified. This is particularly important because this would give further insight into understanding the pathophysiology of iris and ciliary body defects in human aniridia patients with PAX6 gene defects [23–25]. Although patients frequently exhibit pan-ocular defects, the main diagnostic feature at birth is complete or partial absence of iris tissue [26,27]. The mechanism that leads to variable iris tissue loss is related to Pax6 dosage requirements [18], but recent work from a number of laboratories suggests that genetic factors other than Pax6 may play a role [28–32]. During embryogenesis the iris starts to develop from the margins of the double-layered optic cup at around E14.5. At this stage the margins contain non-neuronal progenitors of the iris and ciliary body, separate from the adjacent neuronal retinal progenitor cells [33]. At around E16.5 the margins begin to extend and begin to express smooth muscle markers, which will provide the contractile function to the iris for regulating light entry into the eye. Cells from the periocular mesenchyme migrate along the iris epithelium and differentiate into the iris stroma [34]. Maturation of the iris continues postnatally in mice and is complete by the time the eyes open at P14. Although Pax6 is expressed in the iris throughout its development and in adult tissue, a gradient of expression is evident with highest levels in the non-neuronal progenitor pool destined for iris specification [35]. When Pax6 dosage is specifically reduced in the optic cup margin, it results in a decrease in the size of progenitor pool, delays the muscle-specific marker expression, results in a decrease in the length of the iris (60–80% of normal) and affects maturation of the muscle sphincter [18]. However, the downstream targets of Pax6 that mediate this iris hypoplasia remain to be determined.

X. Wang et al. / Biochimica et Biophysica Acta 1863 (2017) 60–67

In this study we have taken advantage of differential gene expression in the developing iris/ciliary body of Pax6-deficient and wildtype mouse embryos to identify direct downstream targets of the Pax6 transcription factor. Using laser capture microdissection, chromatin immunoprecipitation, promoter-reporter assays and immunohistochemistry we now demonstrate that Bmp4, Tgfβ2, and Foxc1 are direct targets of Pax6 in iris/ciliary body development. This Pax6 gene network now sheds new light on the regulation of anterior segment development, improves on our understanding of the underlying molecular causes of iris tissue loss, and provides potential avenues for re-programming iris growth in the postnatal eye.

2. Material and methods 2.1. Animals The Pax6Sey-1Neu/+ mouse mutant is a semi-dominant allele caused by a point mutation in the donor splice site junction of intron 10 of the Pax6 gene [1]. The mice were maintained on a C57BL/6 background for N 8 generations. Male and female Pax6Sey-Neu/+ offspring were identified phenotypically by the reduced size of their eyes and the presence of cataracts [15], and subsequently confirmed as Pax6 mutant alleles by PCR of genomic DNA. Embryos were obtained at specific days of development from timed-pregnant dams housed on a 12:12 light-dark cycle with access to food and water ad libitum. Noon of the day on which the plug was observed was designated to be 0.5 days post-coitum. Research was carried in accordance with protocols compliant to the Canadian Council on Animal Care with the approval of the Animal Care Committee at the University of British Columbia and with the ARVO statement for the use of animals in vision research.

2.2. Genotyping Pax6 alleles were genotyped using REDExtract-N-Amp Tissue PCR kit (Sigma-Aldrich, Oakville, ON, Canada) prepared from 5 to 10 mg of either tail or mouse embryonic tissues. Each 50 μl PCR reaction contained 1 X PCR master mix (Promega, Madison WI, USA), 400 nM of each primer and 4 μl extracted DNA tissue mix. PCR was amplified for 35 cycles of 30 s at 95 °C, 1 min at 58 °C, and 1 min at 72 °C. Primers were: forward, 5′-CCAGTGTCTACCAGCCAATC-3′; reverse 5′-ACTGTACGTGTTGGTG AGGG-3′. PCR amplified DNA was digested overnight separately with HpaI at 37 °C and BstNI (New England Biolabs, Whitby, ON, Canada) at 60 °C, and then analyzed on a 2.5% TBE agarose gel. The 220 bp wildtype allele was digested by BstNI, and the Pax6Sey-1Neu/+ allele was digested by HpaI.

2.3. Laser capture microdissection Fresh mouse embryonic heads at E12.5, E14.5 and E16.5 were immediately snap frozen in isopentane/dry ice bath in a plastic mold filled with Tissue-Tek OCT (Cedarlane, Burlington, ON, Canada). All cryoblocks were stored at −80 °C until further processing. From frozen tissue 16 μm cryosections of the eye were obtained and mounted on a slide pre-covered with a polyethylene terephthalate membrane (Zeiss, North York, ON, Canada). Specific iris regions were collected by laser microdissection and pressure catapulting using a PALM Laser Capture Microdissection system (PALM-Zeiss, North York, ON, Canada) comprising a PALM MicroBeam and a PALM RoboStage. The tissue area of interest was positioned and cut out using a focused pulsed laser beam under a 10× ocular lens. Dissected iris cells were collected in the cap of 500 μl AdhesiveCap microcentrifuge tube via laser pressure catapulting. Tubes were stored at −80 °C until required for further analysis.

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2.4. RNA isolation and quantitative real-time RT-PCR Total RNA was isolated from captured iris cells using Arcturus PicoPure RNA isolation kit (Applied Biosystems, Toronto, ON, Canada) according to the manufacturer's instructions, including on-column DNase treatment (Qiagen, Toronto, ON, Canada). The RNA quality (A260/A280 ratio) and concentration were measured with a NanoDrop spectrophotometer and the samples were stored at −80 °C until further processing. From each sample, 50 ng of total RNA was reversetranscribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen, Toronto, ON, Canada) as per the manufacturer's protocol. Gene expression was quantified using the TaqMan primer/labeled probe system and the 7500 Fast Real-Time PCR system (Applied Biosystems, Toronto, ON, Canada). All reactions were performed using the TaqMan Fast Advanced Master Mix (2 ×), FAM-labeled TaqMan pre-validated gene expression assays for FoxC1, Igf2, Zic2, Tgfb2, Pitx2 and Bmp4, VIC-labelled TaqMan endogenous control Gapdh, and 5 ng of cDNA. All reactions were run in replicates of 3, with n = 3. Thermocycling parameters were as follows: 2 min at 50 °C, 20 s at 95 °C, 40 cycles of 1 s at 95 °C, plus 20 s at 60 °C. Real-time PCR data were analyzed by the comparative CT method [36].

2.5. In vivo chromatin immunoprecipitation (ChIP) assay The ChIP procedure was carried out using the ChIP assay kit (EMD Millipore, Etobicoke, ON, Canada) and protocol as previously described [37]. Mouse embryo eye tissue (10 eyes) was dissected out, rinsed in cold PBS and then incubated with 400 μl of PBS containing 1% formaldehyde at 37 °C for 15 min. After three washes with cold PBS containing protease inhibitors (1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml pepstatin), tissue was homogenized in 200–400 μl of SDS lysis buffer. The lysates were sonicated using a Diagenode Bioruptor Sonicator with NESLAB RTE-7 Digital One Refrigerated Bath (ThermoScientific, Vancouver, BC, Canada) at on/off 30 s each for 45 cycles at 4 °C to obtain DNA in size range of 200 to 1000 bp. Fragmented chromatin sizes were confirmed by agarose gel electrophoresis, and then sheared chromatin was diluted ten-fold in ChIP buffer to achieve 0.2 U A260 before immunoprecipitation. An aliquot of the sonicated lysate (40–50 μl) without the antibody was used as input DNA to quantitate the total amount of DNA present in different sample extracts before immunoprecipitation. The positive control used was RNA pol II and the negative control was using EF1alpha primer, according to manufacturer's instructions (ChIP-IT Control Kit from Active Motif,Carlsbad, CA, USA). Typically, 5 μg of a Pax6 antibody (BioLegend® [catalog number 901,301], San Diego, CA, USA) was used for immunoprecipitation at 4 °C overnight. Immune complexes were then precipitated with protein A agarose provided in the kit, and washed sequentially with low salt, high salt immune complex wash, LiCl immune complex wash and TE buffer. The immune complex was then eluted from agarose beads with elution buffer (1% SDS, 0.1 M NaHCO 3). At the end of the ChIP procedure, both the input DNA and the protein/DNA cross-linked chromatin complex immunoprecipitated with specific antibodies were reversed cross-linked with 5 M NaCl at a final concentration of 200 mM at 65 °C for 8–12 h. Samples were then treated with proteinase K (20 μg) for 1 h at 45 °C. Protein-free DNA was extracted in phenol/chloroform, precipitated and washed in ethanol and re-dissolved in nuclease-free water. This was repeated for three independent mouse embryo tissue samples. An aliquot (1 μl) of the extracted DNA was used for PCR amplification of the Pax6 promoter covering putative binding sites for FoxC1 (2 sites), Igf2 (1 site), Tgfβ2 (2 sites), Pitx2 (3 sites) and Bmp4 (3 sites). PCR primers for each site are available in the Appendix Table A1. PCR products were confirmed using a 2% agarose gel and visualized with ethidium bromide staining.

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2.6. Luciferase promoter reporter assays To assess whether those downstream target genes (FoxC1, Pitx2, Tgfb2, Bmp4) were directly regulated by Pax6, an in vitro luciferase promoter assay was performed. The promoter region containing Pax6 binding sites of the downstream target genes were PCR amplified with primers containing restriction sites using mouse genomic DNA as the template (see Table A2 for primer sequences). PCR products were then gel purified and constructed by the insertion of multiple cloning sites of pGL4.23[luc2/minP] vector (Promega, Madison, WI, USA). Sitedirected mutagenesis (Table A2 for primer sequences and mutation sites) were used to introduce the mutant bases into wild-type pGL4.23-(FoxC1, Pitx2, Tgfb2, Bmp4) constructs using the QuikChange Lightning Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA). Gateway pDEST26 vector carrying the mouse Pax6 ORF sequence was cloned from Pax6 ORF sequence in pENTR221 vector using Gateway LR Clonase II Enzyme Mix (Invitrogen, Burlington, ON, Canada). Plasmid constructs for transfection were purified with PureLink HiPure Plasmid FP Maxiprep kit (Invitrogen, Burlington, ON, Canada) and quantitated with Nanodrop spectroscopy. COS7 cells were maintained in DMEM containing 10% fetal calf serum and 10,000 U penicillin/streptomycin, in 5% CO2. Liposome-mediated co-transfections of each gene promoter construct with the Pax6 expression vector into COS7 cells were performed in six-well plates according to the manufacturer's protocol. Renilla luciferase (pGL4.74[hRluc/TK) was co-transfected (0.3 or 0.5 μg/well) as a control for efficiency of transfection. Cells were harvested at 48 h. Dual luciferase activity was measured in cell lysates using a GloMax luminometer (Promega, Madison, WI, USA). Each report construct was transfected three times and mean values taken from three independent transfections. All results are expressed as the mean ± SEM.

Fig. 1. Differential gene expression in developing mouse iris/ciliary body tissue. (A) Cryosection images of developing iris tissue at E16.5 taken before and after laser capture microdissection (LCM). (B) Real-time mRNA expression levels for 6 genes at 3 embryonic ages (E12.5, E14.5 and E16.5) in iris/ciliary body tissue from Pax6heterozygous mouse eyes relative to wildtype controls (mRNA level = 1.0). For each gene data are present as mean ± SEM from 3 independent samples each run in triplicate. * P b 0.05.

2.7. Immunohistochemistry Immunohistochemical studies were performed on 4% paraformaldehyde-fixed cryosectioned tissues (10 μm). Sections were rinsed 3 times for 3 min each in PBS-0.1% Tween 20, and then blocked in serum blocking solution containing 5% goat or donkey serum, 2% of BSA and 0.25% of TritionX-100 for 1 h at RT. The sections were then incubated with the mixture of Pax6 antibody (rabbit polyclonal, 1:500; BioLegend® [catalog number 901301], San Diego, CA, USA) and one of downstream primary antibodies: Bmp4 (mouse monoclonal, 1:300; Antibodies Online, West Lebanon, NH, USA), Foxc1 (goat polyclonal, 1:50; Novus Biologicals, Littleton, CO, USA), Tgfβ2 (monoclonal, 1:500; Abcam, Eugene, OR, USA) at 4 °C overnight. After rinsing off the primary antibodies with PBS-0.1% Tween 20 (3 × 3 min washes), the sections were incubated with a mixture of secondary antibodies (1:500) for 1 h at RT: goat anti-rabbit Alexa Fluor 594 (for Pax6), with donkey anti-goat Alexa Fluor 488 (for Foxc1) or goat anti-mouse Alexa Fluor 488 (for Bmp4 and Tgfβ2). All secondary antibodies were obtained from Invitrogen, Burlington, ON, Canada. Nuclei were counterstained with DAPI. The images were visualized with Zeiss LSM 510 Meta Confocal microscope. 3. Results 3.1. Identification of genes that are differentially expressed in developing iris/ciliary body For expression profiling in the developing iris/ciliary body we used quantitative real-time PCR from Pax6 heterozygous and wildtype optic cup margin tissue at E12.5 (prior to iris progenitor specification), E14.5 (when iris progenitor pool is specified) and E16.5 (the beginning of optic cup margin extension). Embryos were genotyped to establish Pax6 status and RNA was isolated from tissue that had been obtained

by laser-capture microdissection (Fig. 1A). We tested candidate genes preferentially expressed in developing optic cup margin [33,38] and genes associated with abnormal iris development in human disease (supplemental Table 1). In Pax6 heterozygous iris/ciliary body tissue we found 6 genes to be differentially expressed compared to wildtype controls. Pitx2 and Igf2 were significantly increased in the Pax6deficient tissue at the different stages of development, whereas Foxc1, Tgfβ2, Zic2 and Bmp4 expression were decreased compared to wildtype (Fig. 1B). 3.2. Target gene promoter binding sites for Pax6 To determine if the 6 differentially expressed genes were direct or indirect downstream targets of Pax6 we carried out a number of in vitro and in vivo assays. Using in silico motif analysis we searched the genomic DNA sequence for Pax6 binding sites [39] immediately upstream of the transcription start sites for each of the genes using MatInspector program (www.genomatix.de). This analysis revealed there were predicted consensus binding sites for Pax6 (Supplementary Fig. S1-S4) in the promoter of Bmp4 (3 sites), Pitx2 (2 sites), Foxc1 (2 sites) and Tgfβ2 (3 sites), but no sites were found for Zic2 or Igf2. This suggested that the decreased expression of Zic2 and increased expression of Igf2 that we observed by real-time PCR was an indirect consequence of decreased Pax6 transcription factor activity. To determine whether endogenous Pax6 protein could bind to the different consensus sites for each gene promoter in vivo, we performed chromatin immunoprecipitation assays (ChIP-PCR) from embryonic eye tissue at the stage where there was greatest differential gene expression (e.g., for Pitx2, Foxc1 and Tgfβ2 we used E12.5; for Bmp4 we used E16.5). Genomic PCR fragments for all 3 sites in the Bmp4 promoter were amplified from Pax6 immunoprecipitates (Fig. 2A), whereas only 1 site in the Pitx2 promoter was amplified. ChIP-PCR also demonstrated that Foxc1 and Tgfβ2 promoters each had 1 in vivo binding site for Pax6.


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Fig. 2. In vivo ChIP-PCR using Pax6 antibody for 4 different gene promoters. Each gene promoter has a different number of predicted Pax6 binding sites. Site 1 in each gene promoter is furthest away from the ATG start site, whereas sites 2 and 3 are closer to the ATG, respectively. (A) Pax6 binds to all three putative Pax6 binding sites in the Bmp4 promoter; to site 1 in the Pitx2 promoter; to site 2 in the Foxc1 promoter; to site 2 in the Tgfβ2 promoter. +C, positive control (RNA pol II EF1-alpha); In, Input DNA (EF1-alpha); −C, negative control (no band for IgG EF1-alpha). Marker, HyperLadder IV; 200 bp and 100 bp. (B) 36 bp extended Pax6 consensus binding site with major residues in bold type. Alternate residues at a particular site are shown above and below the main sequence. The boxed area is the main binding site compared to the promoter sites identified by ChIP. Matching nucleotides are in bold.

Fig. 3. Pax6 responsive elements in different gene promoter regions. Diagram at top depicts the relative position of putative Pax6 binding sites (white ovals) in the reporter constructs. Hatched box, exon 1 of the gene. ATG site denoted by arrow. L, luciferase gene. Reporter constructs were co-transfected with a Pax6 expression vector into COS7 cells (this cell line does not express Pax6 endogenously). The relative luciferase activity for each wildtype construct was set at 100%.The filled ovals represents mutation of the specific Pax6 site so it is no longer functional. The activity of the last construct relates to the Foxc1 promoter construct that is transfected without Pax6 expression vector, showing specificity of transcriptional activation. Relative luciferase activity is presented as mean ± SEM, n = 9. *, P b 0.01.

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For each gene promoter we compared the ChIP-positive binding sites to the extended Pax6 consensus site [40] (Fig. 2B). To verify Pax6 responsive elements in the gene promoters, the Pax6 in vivo binding sites were mutated in promoter constructs and then tested using dual luciferase reporter assays (Fig. 3) in COS-7 cells. Sitedirected mutagenesis of Pax6 binding site 1 in the Bmp4 promoter construct reduced luciferase reporter expression by 74% ± 8%, but mutagenesis of the other two in vivo Pax6 binding sites had no effect on luciferase expression. Similarly, mutation of site 3 in the Tgfβ2 construct reduced luciferase expression to only 19% ± 10% of wildtype construct and mutagenesis of site 1 in the Foxc1 promoter construct reduced luciferase expression by 53% ± 14%. Mutation of the Pax6 binding site in the Pitx2 construct had no effect on luciferase reporter activity. Specificity of transcriptional activation was verified for the promoter constructs when transfected alone without the Pax6 expression vector into COS-7 cells (e.g. see Foxc1 construct in Fig. 3), as this cell line does not express Pax6 endogenously [41]. Overall these results demonstrate that Bmp4, Tgfβ2 and Foxc1 genes are direct downstream targets of Pax6 in the developing iris/ciliary body.

3.3. Spatiotemporal expression of validated Pax6 downstream targets Although the gene expression patterns of Pax6, Bmp4, Tgfβ2 and Foxc1 have been well documented in the developing anterior segment of the eye [11,33,42–44], the protein expression patterns in developing tissues are less well defined. To determine the protein expression patterns of the Pax6 direct targets we performed immunohistochemistry in the developing mouse iris/ciliary body (Fig. 4A). At E16.5 in wildtype eyes, Bmp4 protein was detected in a sub-population of cells at the optic cup margin destined to become the ciliary body and iris epithelium and this overlapped with Pax6 protein expression (Fig. 4B,C) and with Bmp4 gene expression [45]. In the Pax6 heterozygous mutant eye, the expression of Bmp4 protein in Pax6 positive cells was reduced suggesting there was a deficit of Bmp4-expressing iris/ciliary body progenitor cells. There also appeared to be a reduction in Pax6 protein expression

compared to the wildtype optic cup margin as would be expected in a haploinsufficient eye. High levels of Tgfβ2 protein were detected in the optic cup margins of wildtype eyes at E16.5 that overlapped with Pax6 protein expression, and this was markedly reduced in the Pax6+/− mutant eye (Fig. 4E–F). The expression of Tgfβ2 protein in the optic cup margin is consistent with expression of the Tgfβr2 receptor that it binds to [42]. We observed Foxc1 protein expression in the periocular mesenchyme (POM) at E16.5, and in a few cells in the optic cup margin of wildtype eyes (Fig. 4G). In the Pax6+/− mutant, although the optic cup margin is smaller and is becoming disorganized in structure at this stage of development, there are still some cells expressing Foxc1 protein (Fig. 4H). This is consistent with Foxc1 gene expression in POM [43] and in non-pigmented ciliary epithelium [46].

4. Discussion Although it has been known for more than two decades that PAX6 deficiency in humans causes partial or complete absence of iris tissue [23], the direct targets of Pax6 regulating normal iris development have remained elusive. In this study, we demonstrate by both in vitro and in vivo experiments that the Foxc1, Tgfβ2, and Bmp4 genes are direct downstream targets of the Pax6 transcription factor in the developing iris/ciliary body. Although PAX6 gene mutations have been synonymous with the diagnosis of aniridia, there are cases of classic aniridia that do not have PAX6 coding sequence or intragenic mutations, nor chromosomal defects that involve PAX6 or its regulatory elements, suggesting that mutations in other genes could be the cause of aniridia [47,48]. Thus, mutations in the Pax6 downstream targets that we have identified could also explain cases of aniridia or cases of isolated iris hypoplasia. BMP4 is a growth factor belonging to the TGFβ superfamily and is expressed in the optic cup margins, developing ciliary body and adult iris [49,50]. Suppression of BMP4 expression by the BMP inhibitor noggin results in the inner epithelial layer of the iris being thinner and the outer layer disorganized, in addition to complete absence of the ciliary body [50]. In Bmp4 heterozygous mice the iris is hypoplastic, there is a

Fig. 4. Immunohistochemical localization of Pax6 downstream targets at E16.5. (A) Cartoon depicting the location of images in panels B–H. RPE, retinal pigment epithelium; Me periocular mesenchyme. (B) Pax6 protein expression in a wildtype (WT) eye. OCM, optic cup margin delineated by dotted line; lens epithelium (Lens Epith). Size bar = 30 μm and is the same in panels B–H. (C) Double staining in WT eye for Pax6 (red) and Bmp4 (green). (D) Pax6 (red) and Bmp4 (green) protein expression in a Pax6 heterozygous mutant (Pax6+/−) eye. (E) Pax6 (red) and TGFβ2 (green) protein expression in a WT eye. (F) Pax6 (red) and TGFβ2 (green) protein expression in a Pax6+/− eye. (G) Pax6 (red) and Foxc1 (green) protein expression in a WT eye. Sections also stained with DAPI (blue). (H) Pax6 (red) and Foxc1 (green) protein expression in a Pax6+/– eye.


very a small Schlemm's canal and there is hypoplasia of the trabecular meshwork, leading to raised intraocular pressure [45]. These studies provide evidence that Bmp4 signalling has a direct role in iris development and could contribute to glaucoma due to obstruction of the aqueous outflow tract. As a direct downstream target of Pax6 in the developing iris/ciliary body, reduction of Bmp4 expression in the Pax6Sey/+ mouse model may account for some of the anterior segment defects and raised intraocular pressure observed in this mouse. It has been demonstrated that Bmp4 dosage is critical to eye morphogenesis since eye defects occur in 35% of Bmp4 heterozygous mice [51,52]. In addition, haploinsufficiency for BMP4 has also been demonstrated in human disease with anterior segment ocular defects such as Reiger anomaly, iris coloboma, microphthalmia, and congenital glaucoma [53, 54], further supporting correct BMP4 dosage requirements in the developing eye. Defects in TGFβ signalling are associated with neural crest cell abnormalities causing anterior segment dysgenesis in the developing eye. Mice lacking Tgfβ2 [55] have corneal defects, patterning defects in the retina, an underdeveloped iris stroma and changes to the chamber-angle structures controlling intraocular pressure [42] similar to that seen in Pax6 mutant eyes [11,56]. In another perturbation, heparin sulfate deficiency in neural crest cells causes anterior chamber dysgenesis due to loss of Tgfβ2 signalling, and down-regulation of Foxc1 and Pitx2 expression [57]. Tgfβ2 is highly expressed in the lens [42], iris sphincter muscle [58], in the developing human iris-ciliary body complex [59], ciliary body epithelium [60], and developing optic cup margin [61], which overlaps with Pax6 expression domains [33,49,62], suggesting Tgfβ2 has a role in iris and ciliary body development. Interestingly, Cre/loxP-mediated inactivation of one Pax6 allele in the developing distal optic cup was shown to decrease the size of the progenitor pool destined for iris fate resulting in an iris that was approximately 20% shorter than wildtype, and the timing of the iris muscle differentiation was delayed during the early stages of muscle cell formation [35]. These data demonstrate that development of the iris tissue is Pax6 dosage-sensitive. Our data showing that transcription of the Tgfβ2 gene is directly regulated by Pax6 supports the hypothesis that Tgfβ2 has a specific role in iris development via a Pax6-dependent mechanism. Previous studies have identified Tgfβ2 as a direct downstream target of Pax6 in the lens [22], but in this context it is thought that lenssecreted Tgfβ2 drives development of Schlemm's canal and the trabecular meshwork in a non-cell autonomous manner [62]. FOXC1 is a dosage-sensitive member of a superfamily of Forkhead box transcriptions factors with a central role in organogenesis and in adult tissue [63]. In the eye it is expressed in non-pigmented ciliary epithelia cells, ciliary body and iris stroma [46,64]. Several FOXC1 mutations have been identified in patients diagnosed with complete absence of iris tissue segregating with congenital glaucoma and corneal opacity [30,31]. These patients would be considered as having a more severe phenotype than in classical aniridia because the glaucoma and corneal opacities occur from birth whereas in classic aniridia these defects tend to develop later on in life. Patients with ocular anterior segment dysgenesis defects (which include partial iris hypoplasia) segregating with glaucoma have also been found to have FOXC1 mutations [65,66]. Loss-of-function studies of Foxc1 in mice have also demonstrated iris hypoplasia [67] and in zebrafish vascular defects in the developing eye have been demonstrated [68]. Duplications of the FOXC1 gene can also cause iris hypoplasia and anterior segment dysgenesis [69]. Furthermore, FOXC1 mutations are associated with raised intraocular pressure [70] and in mice Foxc1 is necessary to prevent angiogenesis thereby maintaining a clear cornea [71]. This may provide an explanation for pannus (corneal neovascularization) and glaucoma in PAX6-aniridia patients if Foxc1 is also a Pax6 downstream target in the Pax6 dosage-sensitive cornea [62] and trabecular meshwork [11]. This however remains to be determined. PITX2 is a member of a multigene family of homeobox transcription factors with pleiotropic functions and dose-dependent effects [72].

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Although we found that in vivo ChIP-PCR resulted in one binding site for Pax6 in the Pitx2 upstream genomic region, when this site was abolished it had no effect on expression of the promoter-reporter activity. Due to the size of the upstream region (~5000 bp) we only included part of the upstream sequence surrounding the Pax6 binding site in the LUC reporter. In the context of the full genomic sequence it is possible that the Pax6 binding site has a role in regulating Pitx2 expression. There are complex regulatory elements at play at the Pitx2 locus including long-range cis-regulatory elements [73], two promoters and alternatively spliced transcripts coding for three major protein isoforms [74]. There have been reports of isolated iris hypoplasia [65], aniridia [75] and iris stromal defects in Rieger syndrome [76] for confirmed PITX2 mutations, providing evidence that PITX2 is critical to iris development, mostly likely through non-cell autonomous effects [77]. Targeted deletion of Pitx2 in mice results in severe defects to the corneal endothelium and stroma, tissues which are derived from the periocular mesenchyme [77,78]. Conversely, Pitx2 gain-of-function directed to periocular tissues results in irido-corneal adhesions, corneal hypertrophy and glaucoma [79]. Morpholino knockdown of pitx2 in zebrafish causes vascular defects in the anterior segment of the eye [80]. These data suggest that in PAX6-mutant aniridia, some of pan-ocular phenotypes observed could be attributed to a decrease in PITX2 activity via haploinsufficiency of PAX6. Interestingly, in vitro studies have been established that PITX2 negatively regulates FOXC1 though physical interaction of the homeodomain of PITX2 with the c-terminal domain of FOXC1 [80,81]. This could explain why increased FOXC1 dosage produces an iris phenotype resembling PITX2 mutation phenotypes. Several studies have investigated the role of the alternatively spliced Pax6 protein that has an extra exon designated Pax6(5a), which introduces an additional 14 amino acids into the N-terminal region of the paired domain [82,83]. This has the effect of abolishing Pax6(5a) binding to the consensus DNA binding site. Therefore, the Pax6(5a) protein would presumably have different downstream targets. There is a low level of Pax6(5a) expression during development [84], but it is significantly increased in the postnatal period suggesting that there could be a switch in targets for Pax6 at this time. A homozygous knockout of exon 5a results in the absence of eyes, whereas the heterozygous mouse has a grossly normal eyes at birth, but develops iris hypoplasia postnatally [85], suggesting that the Pax6(5a) protein is important to iris development at this stage. Several patients have been identified with mutations in exon 5a (V54D, Q60R, IVS5-3T N C) and their phenotype includes foveal hypoplasia, Peters' anomaly and nystagmus, but a relatively intact iris [86,82]. Although differences in phenotype exist between the human and mouse Pax6(5a) mutations it would nevertheless be very interesting to identify the downstream targets for the alternatively spliced Pax6(5a) protein during postnatal iris tissue maturation.

5. Conclusions Our results provide the first insight into the downstream targets for Pax6 in the developing iris/ciliary body. We analysed differential gene expression in the developing eye of wildtype and heterozygous Pax6 mice and identified Bmp4, Tgfβ2 and Foxc1 as direct downstream targets. This is important as we recently demonstrated that manipulation of Pax6 dosage after birth was able to rescue the aniridic phenotype in mice [87], suggesting that these downstream targets are also active postnatally. These results offer the potential for exploring treatments for aniridia, based on these three Pax6 targets. Transparency document The Transparency document associated with this article can be found, in online version.

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