RESEARCH ARTICLE

Conditional deletion of AP-2 in mouse cranial neural crest resultsin anterior segment dysgenesis and early-onset glaucomaVanessa B. Martino1, Thomas Sabljic1, Paula Deschamps1, Rebecca M. Green2,3, Monica Akula1,Erica Peacock1, Alexander Ball1, Trevor Williams2,3,* and Judith A. West-Mays1,*

ABSTRACTAnterior segment dysgenesis (ASD) encompasses a group ofdevelopmental disorders in which a closed angle phenotype in theanterior chamber of the eye can occur and 50% of patients developglaucoma. Many ASDs are thought to involve an inappropriatepatterning andmigration of the periocular mesenchyme (POM), whichis derived from cranial neural crest cells (NCCs) and mesoderm.Although, the mechanism of this disruption is not well understood, anumber of transcriptional regulatory molecules have previously beenimplicated in ASDs. Here, we investigate the function of thetranscription factor AP-2, encoded by Tfap2b, which is expressedin NCCs and their derivatives. Wnt1-Cre-mediated conditionaldeletion of Tfap2b in NCCs resulted in post-natal ocular defectstypified by opacity. Histological data revealed that the conditional AP-2 NCC knockout (KO) mutants exhibited dysgenesis of multiplestructures in the anterior segment of the eye including defects in thecorneal endothelium, corneal stroma, ciliary body and disruption inthe iridocorneal angle with adherence of the iris to the cornea. Wefurther show that this phenotype leads to a significant increase inintraocular pressure and a subsequent loss of retinal ganglion cellsand optic nerve degeneration, features indicative of glaucoma.Overall, our findings demonstrate that AP-2 is required in the POMfor normal development of the anterior segment of the eye and thatthe AP-2 NCC KO mice might serve as a new and exciting model ofASD and glaucoma that is fully penetrant and with early post-natalonset.

KEY WORDS: Anterior segment dysgenesis, Glaucoma, AP-2

INTRODUCTIONGlaucoma is the current leading cause of irreversible blindnessworldwide, and due to the rapidly aging population it is predicted toaffect over 70.6 million people by 2020 and 111.8 million people by2040 globally, with a disproportionate prevalence seen in Asian and

African countries (Quigley and Broman, 2006; Tham et al., 2014). Itis a multifactorial disease process that is characterized by a loss ofretinal ganglion cells (RGCs), excavation of the optic nerve head(ONH) and defects of specific visual fields. Elevation in intraocularpressure (IOP) is a major risk factor and, currently, it is the onlyclinically treatable feature of the disease (Kass et al., 2002). Thereare many forms of glaucoma, with primary open angle (POAG)being the most prevalent, but angle closure glaucomas are alsowidespread, particularly in individuals of Asian descent (Quigleyand Broman, 2006).

Closed angle glaucomas often arise due to congenital disordersand are classified as anterior segment dysgenesis (ASD) (Ito andWalter, 2014). The anterior segment of the eye including thecornea, iris, lens, ciliary body and ocular drainage structures such asthe trabecular meshwork (TM) and Schlemms canal has acomplex developmental origin involving cross-talk between thesurface ectoderm, optic cup, mesoderm and neural crest (Ito andWalter, 2014). The latter two tissues form the periocularmesenchyme (POM) that eventually gives rise to the maturecorneal stroma and endothelium, ciliary body stroma and muscle,anterior iris stroma, iridocorneal angle and drainage structures. Incontrast, the lens and corneal epithelium are derived from thesurface ectoderm. Although the mechanisms underlying theformation of ASDs are not well understood, many are thought toinvolve an inappropriate patterning and migration of the POM.Ultimately, failure of normal POM development can lead toanatomical and/or functional defects in the drainage structuresresponsible for aqueous humor production and outflow.Impediments in aqueous outflow subsequently lead to anincrease in IOP, creating an environment favorable to thedevelopment of glaucoma (Kupfer and Kaiser-Kupfer, 1978,1979; Shields, 1987).

Mutations in a number of genes are known to cause human ASD,including PAX6, PITX2, PITX3, FOXC1, FOXE3, EYA1, CYP1B1,LMX1B and MAF (Cvekl and Tamm, 2004; Gage and Zacharias,2009; Gould et al., 2004; Ito and Walter, 2014; Sowden, 2007).These mutations can result in a spectrum of ocular disorders, whichare classified based on the tissues involved (Sowden, 2007). One ofthese disorders is AxenfeldRieger syndrome (ARS) in whichpatients exhibit abnormalities that are more confined to tissuesaffecting the angle, as opposed to other ASDs that involve the lens.Features of ARS include adhesions between the iris and cornea, irishypoplasia, holes in the iris and abnormal chamber angle tissue(Mears et al., 1998; Semina et al., 1996). The most common geneticcauses of ARS are mutations in the transcription factors PITX2 andFOXC1 (Gould et al., 2004). Both of these regulators arespecifically expressed in the developing POM and are thought tobe part of a similar regulatory network. Further analysis of these twotranscription factors using gene knockout (KO) approaches in micehas demonstrated that the loss of either Pitx2 or Foxc1 results inReceived 4 March 2016; Accepted 21 June 2016

1Department of Pathology and Molecular Medicine, McMaster University HealthScience Centre, Room 4N65, 1200 Main St. West, Hamilton, Ontario, Canada L8N3Z5. 2Department of Craniofacial Biology, University of Colorado Denver, AnschutzMedical Campus, Mailstop 8120, RC-1 South Building, 11th Floor, Room 111,12801 East 17th Ave. P.O., Aurora, CO 80045, USA. 3Department of Cell andDevelopmental Biology, University of Colorado Denver, Anschutz MedicalCampus, Mailstop 8120, RC-1 South Building, 11th Floor, Room 111, 12801 East17th Ave. P.O., Aurora, CO 80045, USA.

*Authors for correspondence (Trevor.Williams@ucdenver.edu;westmayj@mcmaster.ca)

T.W., 0000-0001-9204-2731; J.A.W., 0000-0003-4293-9131

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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mailto:Trevor.Williams@ucdenver.edumailto:westmayj@mcmaster.cahttp://orcid.org/0000-0001-9204-2731http://orcid.org/0000-0003-4293-9131http://creativecommons.org/licenses/by/3.0http://creativecommons.org/licenses/by/3.0

profound failure in anterior segment differentiation, indicating aconserved role in both mouse and human (Gage et al., 1999; Luet al., 1999). However, these null mice, as well as neural crest cell(NCC)-specific KO mice for either of these two genes, are eitherembryonic lethal or exhibit such severe eye defects, that it is notfeasible to use these models to study anterior segment developmentbeyond birth (Gage et al., 2014, 2005; Kume et al., 1998). Atemporal gene KO approach that was employed to delete Pitx2expression in mice at a later time point during development did,however, reveal that Pitx2 is required for maintaining specificlineages within the developing cornea and in preventingangiogenesis (Gage et al., 2014). Despite the importance of Pitx2and Foxc1 for development of the eye, it is clear that otherregulatory genes responsible for ASDs remain to be identified.The AP-2 transcription factor family, consisting of five genes

encoding the proteins AP-2, AP-2, AP-2, AP-2 and AP-2,respectively, is postulated to be a crucial component of the generegulatory network responsible for the evolution and function of theneural crest in vertebrates (Jin et al., 2015). Moreover, previousstudies have demonstrated that members of the AP-2 family,particularly AP-2 and AP-2, can regulate several aspects of eyedevelopment through their function in the surface ectoderm orneural retina (Bassett et al., 2012, 2007, 2010; Dwivedi et al., 2005;Pontoriero et al., 2008; West-Mays et al., 2002, 2003, 1999). Ofnote, AP-2 and AP-2 function together in vivo to controldevelopment of horizontal and amacrine cell populations in theembryonic retina and potentially in the post-natal eye (Bassett et al.,2012; Jin et al., 2015). Therefore, we were interested in ascertainingwhether the individual AP-2 proteins, or particular combinations ofthese proteins, might also function in the POM to control eyemorphogenesis and function. Here, we focus on AP-2, encoded byTfap2b, which has not been studied in detail in this tissue. Given thatTfap2b-null mice die perinatally (Moser et al., 1997), we utilizedCre-LoxP technology to remove AP-2 specifically from theembryonic neural crest and its derivatives so that we could assesshow loss of this gene impacts upon post-natal development of theanterior eye structures. Our findings indicate that AP-2 expressionin the POM is crucial for anterior segment development andfunction and, moreover, that its absence from this tissue generates avaluable new model of human closed angle glaucoma.

RESULTSAP-2 NCC KO mutants exhibit multiple defects in theanterior segment of the eyeMice lacking Tfap2b die in the early post-natal period, and wesought to determine whether the expression of AP-2 in the neuralcrest was crucial for viability by specifically targeting a floxed alleleof this gene with a Wnt1-Cre transgene. Animals lacking Tfap2b inthe neural crest, hereafter termed AP-2 NCC KO, were viable afterbirth and initially had a similar appearance to control littermates.However, after eyelid opening, mutant animals were readilyidentified by the presence of ocular opacity that persisted as theanimals aged (Fig. 1A,B). These observations were consistent withprevious reports regarding the expression pattern of AP-2 in theNCC-derived POM in mice, which begins by embryonic day (E)9.5(Bassett et al., 2010) and can be detected in a substantial number ofcells populating the developing cornea and angle tissue by E13.5(Bassett et al., 2012). However, given that expression at laterembryonic timepoints has not been examined, we utilizedimmunohistochemistry to analyze the distribution of AP-2protein in the anterior segment at E15.5 and E18.5 in both controland mutant samples (Fig. 1C-F). In E15.5 control animals, AP-2

expression was detected in the POM, the developing retina, thecorneal stroma, corneal epithelium and eyelid epidermis (Fig. 1C).Subsequently, by E18.5, control embryos also exhibited positiveAP-2 staining in the POM, the corneal epithelium, endotheliumand stroma as well as the developing retina (Fig. 1E). In contrast,examination of E15.5 and E18.5 AP-2 NCC KO samplesdemonstrated that expression was absent from the POM, thecorneal endothelium and the stroma, which are NCC in origin(Fig. 1D,F). By contrast, the previously reported pattern of AP-2expression in the developing retina and corneal epithelium (Bassettet al., 2012; Trimarchi et al., 2007; West-Mays et al., 2003), oculartissues that are not of NCC origin, was retained (Fig. 1D,F).Similarly, the ectodermally derived lens, which showed increasedAP-2 expression between E15.5 and E18.5, produced equivalentstaining in AP-2 NCC KO samples (Fig. 1; Fig. S1). Takentogether, these observations are consistent with a role for AP-2 indevelopment of specific anterior segment structures from the POM.

Next, to determine the onset and progression of the ocularpathology in AP-2 NCC KO mutants, we performed a detailedhistological analysis beginning at E10.5, as this marks the first NCCmigration into the presumptive anterior chamber (Gage et al., 2005).The phenotype observed at E10.5, just after the lens vesicle haspinched off from the overlying surface ectoderm and the POM ismigrating into the presumptive anterior segment, was remarkablysimilar for both mutants and control littermates, with no suggestionof abnormal development due to the conditional deletion (n=4)(Fig. 2A,B). By contrast at E15.5, once the cornea had formed fromthe POM and the second wave of mesenchyme had reached theiridocorneal angle, the mutant cornea was less compact, with largegaps within the stroma. Moreover, in stark contrast to the clearseparation between the cornea and lens seen in control samples(Fig. 2C), the cornea and lens adhered to one another in the mutant(n=2) (Fig. 2D, arrowheads).

At the adult stage (2-3 months old), multiple malformations ofthe anterior chamber were apparent in AP-2 NCC KO mutant eyescompared to controls (n=8). The most striking was the disruption ofthe iridocorneal angle with the iris adherent to the cornea, creating afully penetrant closed angle phenotype in the eyes examined (n=8)(Fig. 2E,F). The ciliary body was also malformed and potentiallyrudimentary in the mutants, as it lacked its normally convoluted andlobulated structure (n=8) (Fig. 2E,F). Histological analysis in AP-2NCC KO mice also indicated multiple defects of the corneal layersderived from the POM, including the lack of a clear endotheliallayer and a less-cohesive corneal stroma compared with controls(Fig. 2G,H). Pathological changes were also seen in tissues that arenot neural crest in origin, including a corneolenticular adhesion andthe appearance of anterior subcapsular cataracts, as well as reducedstratification of the corneal epithelial layer (Fig. 2G,H). These latterconditions are presumably secondary defects arising from changesin POM derivatives within the cornea and angle tissue.

We further examined pathological changes in the adult corneausing immunohistochemistry for the endothelial marker N-cadherin(n=8) (Reneker et al., 2000). In control samples, a discrete cornealendothelial layer expressed N-cadherin, but this staining was absentin AP-2 NCC KO mice (Fig. 2I,J). Similarly, N-cadherin stainingrevealed that the corneal epithelial layer was substantially reduced inthickness in the mutants and that the lens epithelium wasdisorganized and multi-layered, characteristic of a cataract(Fig. 2J, large white arrow). Next, we used a combination ofhistology and N-cadherin immunohistochemistry to determinewhether abnormalities in the corneal endothelium and angle tissuecould be detected prior to birth when AP-2 was specifically

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removed from the POM (Fig. 3A-F). A comparison of mutant andcontrol littermates (n=6) for N-cadherin staining demonstrated thatdefects in the angle tissue and the corneal endothelium were presentat E18.5, showing that these phenotypes resulted from an earlydevelopmental defect (Fig. 3E,F). Staining of N-cadherin was alsomore limited in the mutant corneal epithelium at E18.5, and this wasalso apparent for AP-2 expression at E18.5, consistent with thereduction of epithelial stratification in the mutant (see Fig. 1E,F andFig. 2G-J). Histological examination of E18.5 samples furtherrevealed the presence of red blood cells in the corneal stroma of theAP-2 NCC KO mice, but not in the controls (n=8; Fig. 3B,D, seearrowheads). Given that the cornea is not normally vascularized, wenext examined adult eyes in detail for evidence of blood vesselswithin this anterior structure by staining for endomucin, asialomucin specifically expressed by the endothelium of bloodvessels. Whereas no endomucin staining was observed in controllittermates, clear reactivity was seen in the corneal stroma of AP-2NCC KO mice at 2 months of age (Fig. S2), and evidence of bloodvessels in the cornea could also be seen at the level of grossmorphology. Further support for the presence of vessels in thestroma of AP-2 NCC KO mice was obtained using opticalcoherence tomography (OCT). A comparison between the cornealstroma of the control and mutant mice indicated that, whereas theformer had a uniform appearance, the latter showed clear areas

indicative of holes or vessels (Fig. 4B, arrowheads). Taken together,these findings provide strong evidence of vascularization of thecornea, indicating a loss of the angiogenic privilege associated withremoval of AP-2 from the POM.

Increased IOP in AP-2 NCC KO mutants relative to controllittermatesObstructions of the aqueous outflow pathway are known to lead toelevations in the IOP in a variety of inducible and spontaneousmouse models of glaucoma (McKinnon et al., 2009). The adhesionbetween the iris and cornea observed in the post-natal fixed AP-2NCC KO samples was striking and we, therefore, used OCT todeterminewhether this defect was also present in vivo at 2 months ofage. In control mice, OCT showed a clear separation of the iris andcornea whereas AP-2 NCC KO mutants had a consistent adhesionbetween the cornea and iris, which was detected 360 around the eye(n=6) (Fig. 4B). These findings confirm the presence of a severe andfully penetrant closed angle phenotype that develops prior to2 months of age in AP-2 NCC KO mutants.

In order to determine whether the closed angle phenotype of theAP-2 NCC KO mutants resulted in elevated IOP, reboundtonometry was used on a cohort of AP-2 NCC KO mutants andtheir control littermates. A significant 3-fold elevation of IOP wasreadily apparent in 3-month-old AP-2NCC KOmutants (Fig. 4C).

Fig. 1. Image of control and AP-2 NCC KOmutant eye and analysis of AP-2 expressionin embryonic eyes. Image of control (A) andWnt1-Cre Tfap2b-targeted (AP-2 NCC KO)(B) adult mice at 9 months of age.Immunofluorescent detection of AP-2 at E15.5(C,D) and E18.5 (E,F) in coronal sections oflittermate control (C,E) and NCC-specificTfap2b-deleted samples (D,F). CEp, cornealepithelium; CEn, corneal endothelium;CS, corneal stroma; dR, developing retina(arrowheads show expression); EE, eyelidepidermis; L, lens; POM, periocularmesenchyme. Note that AP-2 expressionnormally seen in the NCC-derived POM andcorneal stroma and endothelium is lost in theAP-2 NCC KO mice, but expression isobserved in structures including the retina,corneal epithelium and eyelid ectoderm, whichare derived from other tissue layers, illustratingthe specificity of the gene targeting. Scale bars:100 m.

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The control littermatemice had an IOPmeasuring 9.761.88 mmHg(n=12), a value consistent with previous measures of noninvasiveIOP readings in 3-month-old C57Bl/6 background strains (Kipfer-Kauer et al., 2010). In contrast, AP-2 NCC KO mutants had asignificant elevation of IOPmeasuring 28.875.19 mmHg (n=12), avalue that is similar to IOP measurements reported in the neemutantmousemodel of glaucoma (Mao et al., 2011). These data support theidea that the closed angle phenotype observed in theAP-2NCCKO

mutants results in disruption of the aqueous outflow pathwayleading to an increase in IOP. Such an environment is favorable tothe development of glaucomatous retinal damage.

Decreased retinal thickness in AP-2 NCC KO mutantsA comparison between H&E-stained sections of control and mutantretinae at 2-3 months of age indicated that the overall thickness ofthis structure was decreased when AP-2 was removed from the

Fig. 2. Aberrant development of the anterior segment inAP-2 NCC KO mutants. H&E-stained coronal or sagittalsections of E10.5 (A,B), E15.5 (C,D) and 2- to 3-month-old(E-H) control littermates (A,C,E,G) and AP-2 NCC KOsamples (B,D,F,H). In D arrowheads indicate thejuxtaposition of the cornea and lens compared to controllittermates. In F, the blue arrow indicates a closed angle withadherence between the iris and cornea and the blue circleshows hypoplastic ciliary body. In H, the white asteriskindicates the location of the anterior subcapsular cataractsand also note adherence of lens and cornea, as well asthinner corneal epithelium. Immunofluorescent detection ofN-cadherin in 2- to 3-month-old sagittal sections of controllittermate (I) and an AP-2 NCC KO mutant (J). In J, theasterisk indicates the absence of corneal endotheliumstaining and the large white arrow points to the subcapsularcataract region of the lens. CB, ciliary body; CEn, cornealendothelium; CEp, corneal epithelium; CS, corneal stroma;dR, developing retina; IC, iridocorneal angle; LEp, lensepithelium; LV, lens vesicle; OC, optic cup; POM, periocularmesenchyme. Scale bars: 100 m.

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neural crest. Measurement of the combined retinal layers revealedthat the mutant retinae were significantly thinner relative to that oftheir age-matched littermates (Fig. 5). Subsequent separatemeasurements of the outer nuclear layer (ONL), inner nuclearlayer (INL) and inner plexiform layer (IPL) indicated that the overallreduction in retinal thickness in the mutants was mainly due tothinning of the IPL, the layer that contains the dendrites of RGCs(Fig. 5). In comparison, the ONL and INL, the layers that containthe nuclei of the photoreceptors and the nuclei of amacrine, bipolarand horizontal cells respectively, exhibited no significant differencein thickness between the mutant and control littermates. Thisfinding suggests that the thinning of the mutant retinae at 2-3 months of age results from a loss of RGCs as the layer thatcontains their dendrites is significantly reduced.

Loss of RGCs in 2-month-old AP-2 NCC KO mutant retinaerelative to control littermatesTo determine whether the changes in the anterior segment associatedwith the increase in IOP in AP-2NCCKOmutants resulted in a lossof RGCs, the retinal cell type affected in glaucoma, a neuronal tracermethod for assessing RGC number was employed (Pang and Wu,2011). The neuronal tracer Neurobiotin (NB) was placed upon theoptic nerve (ON) stump to be taken up and transported retrogradelyby the RGC axons to the cell bodies. Fluorescence microscopy ofNB-labeled RGCs in retinal whole mounts showed that there was astark contrast between AP-2 NCC KO mutants and controllittermates, with significantly fewer RGCs and their axons in themutant group (Fig. 6A,B). It was interesting to note that the loss of

RGC axonswas not uniform, but occurred in a segmental, fan-shapedmanner, a phenotype that is similar to what has been observed in themost well-characterized mouse model of glaucoma, the DBA/2Jinbred line (Jakobs et al., 2005). Manual counting of DAPI-labelednuclei and NB-labeled RGC somas was performed and revealed thatcontrol littermates had on average 10,1631603 nuclei/mm2 of which4244356 nuclei/mm2, 42%, were from RGCs (means.d., n=4retinae). These values are consistent with total neuronal counts in theganglion cell layer (GCL) and the calculation that 43% of cells in thislayer are RGCs (Jeon et al., 1998). Cell counts of AP-2 NCC KOmutant retinae showed significantly fewer cells, with 6505511nuclei/mm2 and 2769462 RGC/mm2 (43%; n=4 retinae) (Fig. 6C,D,G). These data demonstrate that, by two to three months of age, theAP-2 NCC KO mutants exhibited a substantial loss of RGCs.However, the RGCs are only one of the two major cell types in theGCL of the retina, the other being displaced amacrine cells, (dACs).The dACs are interneurons that play an important role in inner retinalvisual processing and account for 57% of the cells in the GCL.Therefore, from the previous quantifications of cells in the GCL(above), the number of dACs was calculable by subtracting thenumber of RGCs from the total number of nuclei present in the GCL.This revealed that the number of dACs in wild-type littermates was57741178 dACs/mm2 and that of AP-2 NCC KO mutant retinaewas 3641755 dACs/mm2, which is significantly lower (n=4 retinae,P=0.0225, two-tailed Students t-test). Thus, these data suggest thatadditional cells within this layer, particularly displaced amacrine cells(dAC), must be lost to a similar extent by 2 months of age in thismouse model (Fig. 6G).

Fig. 3. Embryonic corneal defects in E18.5AP-2 NCC KO mutant embryos. Coronalsections of E18.5 anterior segments stained usingeither H&E (A-D) or immunofluorescence forN-cadherin expression (E,F) for controls (A,C,E) orAP-2 NCC KO mutants (B,D,F). Boxed regions inA and B are shown in greater detail in C and D. Redarrow in F shows that corneal endothelium doesnot stain for endothelial cell marker N-cadherin inabsence of Tfap2b. Blue arrowheads in B and Dindicate presence of red blood cells in mutantcorneal stroma. CEn, corneal endothelium; aCEn,absent corneal endothelium; CEp, cornealepithelium; CS, corneal stroma; L, lens; Re, retina.Scale bars: 100 m.

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Loss of retinal ganglion cell axons in ONsTo test for further evidence of glaucomatous damage in the AP-2 NCC KO model, cross-sections of ONs from 2-month-oldmutant and control mice were assessed by electron microscopyto examine the axons of the RGC bodies. The ONs of thecontrol littermates had a normal appearance with many axons ofvarious sizes closely compacted and surrounded by myelinsheaths (Fig. 7A). The ONs of the AP-2 NCC KO mutants,however, had a decreased number of myelinated axons, thepresence of degenerating axons (Fig. 7A, arrow) and areas ofsevere atrophy (Fig. 7A, asterisk). Quantification of the total

number of axons also revealed a significant reduction in thenumber of ON axons in the AP-2 NCC KO mutants whencompared to control littermates (Fig. 7B). Presumably, as aconsequence of the loss of axons, the cross-sectional area of themutant ONs was also significantly reduced (Fig. 7C), by 2-fold relative to their control littermates, further supporting thatthe AP-2 NCC KO mutant ONs have undergone severeglaucomatous progression. RGC axon loss in the mutants wasalso associated with damage of the ON head, consistent withexcavation or cupping, further indicative of glaucomatouspathology (Fig. S3).

Fig. 4. OCT and IOP analysis of 2-3-month-old AP-2NCC KOmutants.OCT images of the anterior chamber ofa control littermate (A) and an AP-2NCCKOmutant (B) at2 to 3 months of age.White arrows in A and B show relativepositions of the cornea and iris, with adherence of the iris tothe cornea in B. The presence of round foramina in thecorneal stroma of the mutant is shown by redarrowheads (B). (C) A comparison of IOP between3-month-old AP-2 NCC KO mutants and their controllittermates. Individual measurements are overlaid on bargraph comparing IOP between control mice and AP-2NCC KO mutant mice. Mutant mice have increased IOP(28.87 mmHg5.19, n=12) relative to their controllittermates (9.76 mmHg1.88, n=12). Measurementstaken with a Rebound Tonometer (TonoLab Vantaa,Finland). Results are means.d. *P

Increased glial reactivity of the retinaFollowing central nervous system (CNS) injury and disease,corresponding to the retinal damage in glaucoma, hypertrophyand proliferation of glial cells such asMller glia and astrocytes willoccur, which is also known as gliosis. A hallmark of gliosis is theupregulation of intermediate filaments, such as glial fibrillary acidicprotein (GFAP), that are in contact with the cytoskeleton andextracellular matrix and will facilitate the rapid and long-termremodeling of tissue structure. In the control littermates, normalGFAP staining of astrocytes was seen in the GCL and fiber layersextending from the GCL, with no staining present in Mller glia inthe other retinal layers as expected (Fig. 8A) (Bjorklund et al., 1985;Sarthy et al., 1991). AP-2NCCKOmutants, however, exhibited anupregulation in expression of GFAP in the Mller glia (Fig. 8B),similar towhat has been previously observed in other animal models

of glaucoma (Tanihara et al., 1997; Wang et al., 2000). Takentogether, these data indicate that the absence of AP-2 from thePOM results in significant changes in the retina and ON that areconsistent with glaucomatous pathology.

DISCUSSIONGlaucoma is a major cause of blindness worldwide and is frequentlyassociated with increased IOP that can damage the retina and ON.There are clear genetic links to human glaucoma, but the underlyingmechanisms of disease progression remain unclear, which presents asignificant barrier to early detection and treatment. This issue iscompounded by the relative lack of animal models that developglaucoma consistently and with a short latency. The increase in IOPfound in glaucoma is often thought to arise from defects in thedrainage system of the eye present at the iridocorneal boundary. In

Fig. 6. Decreased number of RGCs, total nuclei anddACs in 2-month-old AP-2 NCC KO mutants.(A,B) Immunofluorescent frontal view of flat mount retinafrom control (A) or AP-2 NCC KO mutants (B) followingretrograde labeling with neurobiotin. (C,D) Detailed viewsof images shown in A and B showing reduced numbers ofretinal ganglion cell bodies (white arrow) and axons (whitearrowhead). (E,F) Flat mount preparation showing theoverall decreased number of DAPI-labeled nuclei in theGCL of AP-2 NCC KO mutant retinas as compared tocontrol littermates. (G) AP-2 NCC KO mutants havesignificantly decreased numbers of RGCs, totalnuclei and dACs (displaced amacrine cells) per mm2

(2768461, 6505511, 3663755, n=3, respectively)relative to their control littermates (4244365,10,1631603, 57741178, n=3). Results are means.d.*P

open angle glaucoma, these defects are not readily apparent usingstandard diagnostic or histological approaches; however, in closedangle glaucoma there are clear structural defects associated with theanterior segment that prevent normal drainage of the aqueoushumor. In this respect, mutations in FOXC1, PITX2, SH3PXD2Band LMX1B, among others, cause various ASDs and have asignificantly increased risk of early onset glaucoma. SH3PXD2B,mutated in glaucoma-associated FrankTerHaar syndrome, encodesa cytoskeletal adaptor protein involved in podosome formation thatis widely expressed in the eye (Iqbal et al., 2010) although itsprimary site of action in anterior segment development remains to bedetermined. In contrast, FOXC1 and PITX2, which are mutated inboth AxenfeldRieger syndrome and Iridogoniodysgenesis, aretranscription factors that function in the NCC-derived POM toregulate development of the anterior segment (Gage et al., 1999,2005, 2014; Kume et al., 1998; Seo et al., 2012). Lmx1b is anadditional transcription factor expressed in the POM and itsassociated derivatives, which is linked to eye defects in bothmouse and human. Lmx1b-null mice as well as mice lacking Lmx1bin the neural crest have a variety of ocular defects including smallereyes, hypoplasia of the iris and ciliary body, and the keratocytes of

the corneal stroma less densely packed (Jin et al., 2015; Pressmanet al., 2000), and conditional mutant mice also exhibit post-natalanterior segment defects. In humans, mutations in LMX1B havebeen linked to nail-patella syndrome, which has been associatedwith glaucoma (Knoers et al., 2000; Lichter et al., 1997).

Here, we have identified AP-2 as a further transcription factor inthe gene regulatory network involved in development of the anteriorsegment from the NCC-derived POM. Our previous studies havedemonstrated roles for the AP-2 transcription factors indevelopment and differentiation of multiple ocular tissues,including those of the anterior eye segment (Bassett et al., 2012,2010; Dwivedi et al., 2005; Pontoriero et al., 2008). These studieshave focused mainly on Tfap2a because homozygous (global)deletion of AP-2 in mice results in a number of ocular phenotypes(West-Mays et al., 1999). In contrast, overt eye defects were notobserved in mouse models targeting other members of the AP-2gene family, including AP-2 (Moser et al., 1997). However, oneunderlying reason for this is that the Tfap2b-null mice die in theperinatal period from presumptive neural transmitter and/or heartdefects preventing analysis of the crucial period of anterior segmentdevelopment that occurs in the weeks immediately after birth (Zhao

Fig. 7. ON defects in two-month-old AP-2 NCC KOmutants. (A) Transmission electron micrographs of crosssections of adult ONs from control and AP-2 NCC KOmutants. Degenerating axons (arrow) and areas of severeatrophy (asterisk) are evident. Scale bars: 2 m. (B) Bar graphcomparing axon numbers from cross sections of the ONbetween control and AP-2NCCKOmutants. AP-2NCCKOmutants display a significantly decreased number ofmyelinated RGC axons (63654284, n=4) relative to theircontrol littermates (36,1437276, n=4). Results aremeans.d. *P

et al., 2011). Therefore, in this study, we used a new Tfap2b floxedallele in combination with the Wnt1-Cre transgene to examine theconsequences of loss of AP-2 specifically within the NCCpopulation. Our findings reveal a crucial role for AP-2 inregulating anterior segment development from the POM, andfurther demonstrate that loss of AP-2 leads to the typical features ofprimary closed angle glaucoma with increased IOP and significantearly post-natal defects impacting upon the retina and ON.The development of the anterior chamber of the eye has been

extensively studied by fate mapping with tissue derivations of eachstructure being well-defined in both mouse and chick. It is wellestablished that the NCC population, alongside mesoderm, whichtogether comprise the POM, will ultimately contribute to theextraocular muscles, corneal stroma, corneal endothelium, irisstroma, ciliary body muscle, ciliary body stroma and trabecularmeshwork of the anterior eye (Gage et al., 2005). Many of theseanterior segment structures are directly impacted by the loss ofTfap2b in the neural crest, strongly suggesting a cell autonomousrole for AP-2 in the regulation of their development. The moststriking malformation was the fully penetrant closed anglephenotype resulting from synechia, the abnormal adhesionbetween the cornea and iris. Potentially, this defect could occur asa direct result of an aberrant interaction between the iris and corneaduring development or be due to the additional malformationsfound in other anterior structures.The cornea itself performs multiple roles for optimal functioning

of the eye, and the AP-2 NCC KO mutants exhibited a variety ofcorneal phenotypes, affecting all three cell layers of this structure.Specifically, the corneal endothelium was missing, there wasdisorganization and vascularization of the corneal stroma, andreduced stratification of the corneal epithelium. With respect to theabsence of the corneal endothelium, we postulate that this is a directresult of AP-2 removal from the NCC population because thisinnermost corneal layer is derived from the POM and was alsoabsent in the mutants beginning in embryogenesis. Loss of thecorneal endothelium would be expected to impact upon two crucialaspects of corneal function based on previous studies. With respect

to the observed synechia, it has been hypothesized that this cell layerprovides a barrier between the anterior chamber and the stickyextracellular matrix of the corneal stroma, based in part ontransgenic mouse models in which the endothelium is lost(Reneker et al., 2000; Waring et al., 1982). Therefore, in theabsence of this protective barrier in the AP-2NCCKOmutants, theiris might have adhered to the exposed extracellular matrix of thecorneal stroma resulting in the closed angle phenotype observed. Asimilar mechanism could account for the observed corneallenticular adhesions and subsequent subcapsular cataracts thatoccur in this Tfap2b model. A second major function of the cornealendothelium is to regulate hydration and nutrition of the cornealstroma. For example, a mouse model with a tissue-specific knockoutof N-cadherin, the major cadherin expressed in the cornealendothelium, exhibited disorganization of collagen fibrils in thestroma, accompanied by corneal edema and clouding (Vassilevet al., 2012). It was hypothesized that the phenotypes in this modelwere due to the loss of proper regulation of water andmacromolecules from exiting and entering the corneal stroma.Given that we also see corneal stromal disorganization and cloudingin the Tfap2b NCC KOmutants, these stromal phenotypes might besecondary to loss of AP-2 in the endothelial layer. However, wehypothesize that there is also a direct effect of AP-2 in the POM-derived corneal stroma based upon the observed neovascularizationthat is not seen in other mouse models of endothelial cell loss (Tianet al., 2012). The transparency on the cornea is crucial for optimaloptical performance, and its maintenance of avascularity, recentlytermed angiogenic privilege, is a necessity for proper visualfunction (Azar, 2006). Our data indicate that AP-2 expression inthe NCC population is required in order to establish angiogenicprivilege during the development of the cornea.

Reduced stratification of the corneal epithelium, from five- or six-cell layers to only two, was also observed in the AP-2 NCC KOmutants. Reduced corneal epithelial stratification has also beenobserved in other mouse models lacking a corneal endothelium(Reneker et al., 2000; Vassilev et al., 2012). Interestingly, in somestudies, the corneal epithelium is seen to return to a five- or six-celllayer epithelium in areas of the cornea where the endotheliumremained present (Reneker et al., 2000). Given that the Wnt1-Cretransgene used in the current study did not target the cornealepithelium, the reduced stratification observed is presumed to havebeen a secondary effect of loss of AP-2 in the corneal endothelium.In this context, we have also examined the anterior segment ofTfap2b-null mice just prior to birth to determine whether additionalcorneal defects were apparent at the level of morphology (Chenet al., 2016). These studies indicate that the Tfap2b-null and Tfap2b-NCC knockout mice have very similar phenotypes at E18.5, with noadditional overt pathology observed in other regions of the anteriorsegment, including the corneal epithelium. However, it was notpossible to examine later defects in the Tfap2b-null mice, includingfeatures of glaucoma, due to perinatal lethality. These findingsstrongly suggest that it is the action of AP-2 in the NCC componentof the POM that is responsible for the role of Tfap2b in anteriorsegment development, but further study using additional Crerecombinase transgenic lines will be needed to determine the exactcell autonomy of the defects observed in the various corneal layers.Similarly, whether the closed angle phenotype is solely aconsequence of the absent endothelium, or is due to otheranomalies related to tissues within the anterior angle, or even toloss of Tfap2b earlier in the POM itself, remains to be determined.

In combination, the results from the characterization of the AP-2NCCKOmodel place this transcription factor in a similar regulatory

Fig. 8. Increased retinal glial reactivity in the 2-month-old AP-2 NCC KOmutants. Immunofluorescent detection of GFAP (green) demonstratesincreased expression in Muller glia cells in AP-2 NCC KO mutant retinas(B) as compared to control littermates (A) supporting the hypothesis ofglaucomatous damage. GCL, ganglion cell layer; INL, inner nuclear layer;ONL, outer nuclear layer. Samples were also stained with DAPI to detectnuclei. Scale bars: 100 m.

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network for anterior segment development as Foxc1 and Pitx2.Mouse mutants that have a conditional deletion of Foxc1 or Pitx2 inthe POM or its derivatives also develop neovascularization of thecornea (Gage et al., 2014; Seo et al., 2012). In addition, these mousemodels exhibit similar anterior segment phenotypes as the new AP-2 NCC KO mutants including lack of formation of the cornealendothelium, disorganization of the corneal stroma andabnormalities of the corneal epithelium (Gage et al., 1999;Kidson et al., 1999). The possibility that these three transcriptionfactors might function in the same gene regulatory network fordevelopment of the anterior segment has prompted an analysis ofthe interplay between these genes. Initial studies have focused onPitx2 and AP-2, and have indicated that AP-2 levels aresignificantly downregulated in the absence of Pitx2 in the POM(Chen et al., 2016). Further studies will be needed to determine howAP-2 levels are affected in the absence of Foxc1 and how thesethree genes interact to direct development of the anterior segment.Mutations involving Foxc1 and Pitx2 not only cause eye defects inmice, but are also responsible for several human eye defects,including AxenfeldRieger syndrome. As yet, there is no substantialevidence linking TFAP2B to human eye disease; however, this geneis mutated in Char syndrome, a rare dominantly inherited conditionthat is associated with patent ductus arteriosus, facialdysmorphology and digit defects as well as sleep disorders (Gelband Chung, 2014; Mani et al., 2005). Although reports of eyeexamination are uncommon in Char syndrome patients, it should benoted that several individuals do present with strabismus, and thiscould reflect a function of AP-2 in the NCC-component of theextraocular muscles. In future, it would be potentially important toinclude a detailed examination of the eye in Char syndrome patients.Alternatively, given that the mouse model represents a Tfap2b lossof function, whereas Char syndrome is inherited in a dominantmanner suggestive of TFAP2B haploinsufficiency, these specificgenetic alterations might result in a different spectrum of eyepathology.We have further characterized the post-natal defects in AP-2

NCC KO and shown that the presence of ASDs is accompanied bythe hallmarks of congenital closed angle glaucoma. First, the AP-2NCC KO mutants exhibited significantly higher IOP levels thantheir control littermates. This presumably results from the closedangle phenotype that blocked access to both the trabecularmeshwork and Schlemms canal as well as the uveoscleralpathway. Rebound tonometry was used in this study to measureIOP and this non-invasive method is reliant upon the characteristicsof the cornea to calculate the IOP. Thus, it is possible that thecorneal phenotype present in these mutants in some way impactedupon the measurements recorded, although we do not favor thishypothesis given the highly penetrant closed angle phenotype.Along with the raised IOP, there was also a loss of RGCs as early assix weeks of age, with a significant loss calculated at two months ofage, as well as ON cupping. Retinal defects were apparent throughthinning of the IPL, retrograde labeling, and ON counts and cross-sectional area measurements. It should also be reiterated that the fan-shaped sectorial loss of RGCs observed in the mutants is consistentwith damage to bundles of axons at the ONH as a result of increasedIOP, creating a pattern of loss that is similar to that found in thehuman condition of glaucoma (Jakobs et al., 2005).Mouse models of glaucoma have proven to be extremely useful in

not only helping to determine the molecular and genetic interactionsthat occur during the disease process but also in providing an avenuefor testing future therapeutic treatments to arrest and potentiallyreverse retinal damage. A variety of mouse models of glaucoma

exist, including for primary closed angle, secondary closed angle,primary open angle and normal tension. The AP-2NCCKOmodelrepresents a new fully penetrant congenital primary closed anglemodel. Of the few spontaneous or genetic mouse models that areused, such as the well-established DBA/2J inbred strain, our modelprovides some advantages. The first is the timeline at which ourmutants begin to lose RGCs. Spontaneous models such as thewidely used DBA/2J inbred strain, and myocilin and 1 subunit ofcollagen type I transgenic models do not begin to experience loss ofRGCs until 6-12 months of age with only a modest elevation in IOP(Aihara et al., 2003; Libby et al., 2005; Senatorov et al., 2006),whereas in our model these changes occur more quickly. Second,the AP-2 NCC KO mutants also exhibited these glaucomatousfeatures in a fully penetrant manner whereas the DBA/2J inbredstrain has highly variable phenotypes with varying loss of RGCstherefore necessitating a large cohort of animals to be examined inorder to gain significant data (Libby et al., 2005). In this context, thephenotype of the AP-2NCCKOmutants more closely mimics thatof the genetic Sh3pxd2b (nee) mouse model, which exhibit aconsiderable increase of IOP and early loss of RGCs (Mao et al.,2011). This likely has to do with the similarity in the closed anglephenotype exhibited in each of these models.

Glaucoma is typically diagnosed based on observations ofexcavation of the ONH and thinning of the nerve fiber layers thathouse the nuclei and dendrites of the RGCs, respectively (Mouraet al., 2012), features we see in the AP-2 NCC KO model. Thethinning of these layers indicates RGC loss, whereas the remaininglayers of the retina typically retain their thickness. It was initiallythought that only RGCs were damaged in glaucoma. However,several recent studies in humans and animal models have suggestedthat amacrine cells might also be lost (Cooper et al., 2008). Thus,future studies to understand the progressive loss of the differentretinal cell types in our model will be important in understandinghow IOP influences retinal degeneration and how this compares tothe human condition of glaucoma.

In summary, the current study demonstrates that AP-2 has acrucial autonomous function in the NCC-derived POM fordevelopment of the cornea and angle tissue. Previous experimentshave also shown that AP-2 can act in the retina in conjunction withAP-2 to regulate the development of specific neuronal cell types(Bassett et al., 2012). Therefore, it is now apparent that Tfap2b hasan extensive influence on multiple aspects of eye development,serving as an important component of the gene regulatory networkresponsible for anterior segment formation. Finally, because theAP-2 NCC KO mice exhibit a significant elevation in IOP and asubsequent loss of RGCs accompanied by ON degeneration, theymight serve as a valuable new model of human closed angleglaucoma.

MATERIALS AND METHODSGeneration of AP-2 NCC KO mutantsAll animal procedures were performed in accordance with the ARVOStatement for the Use of Animals in Ophthalmic and Vision Research.Conditional deletion of Tfap2b from NCCs was achieved by breedingWnt1-Cre mice heterozygous for a Tfap2b-null allele (Moser et al., 1997;Danielian et al., 1998) to homozygous floxed Tfap2b mice (T.W.,unpublished). Genotyping for Tfap2b null, Tfap2b flox and Wnt1-Crealleles was performed by PCR using DNA extracted from ear clippingsusing the EZNA Tissue DNA Kit (Omega Bio-Tek) with relevant primersequences shown in Table S1. Mice that were Wnt1-Cre Tfap2bnull/flox,hereafter termed AP-2 NCC KO animals, were gender matched to controllittermates that still contained at least one functional copy of Tfap2b in theNCC.

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http://dmm.biologists.org/lookup/doi/10.1242/dmm.025262.supplemental

Histology and immunofluorescenceWhole eyes of the AP-2 NCC KO mutants were fixed in 10% neutralbuffered formalin for 24 h and then stored in 70% ethanol. Samples wereprocessed (Histology Department, McMaster University) and embedded inparaffin (Paraplast Tissue Embedding Media, Fisher Scientific). Serialsections were cut to 4 m in thickness and used for immunofluorescentanalysis or hematoxylin and eosin (H&E) staining. Paraffin-embeddedsections were deparaffinized in xylene, rehydrated through 100%, 95% and70% ethanol, followed by water, treated with 10 mM sodium citrate buffer(pH 6.0; boiling for 20 min) for antigen retrieval, blocked with normal serumfor 1 h and incubated with primary antibodies overnight at 4C. Indirectimmunofluorescence was performed using the following primary antibodies:1:50 rabbit anti-AP-2 (2509S, Cell Signaling), 1:100 mouse anti-N-cadherin(610920, BD Transduction) and 1:100 rat anti-endomucin (14-5851,eBioscience). Fluorescent secondary antibodies, which were conjugated toeither Alexa Fluor 488 or 568, and of appropriate specificity for the primaryantibody species (1:200; A11029 and A11011, Invitrogen, Molecular Probes)were incubated for 1 h at room temperature. All stains were mounted withProLong Gold antifade reagent containing 4,6-diamino-2-phenylindole(DAPI) (Invitrogen, Molecular Probes). Staining was visualized with amicroscope equipped with an immunofluorescence attachment (Leica), andimages were captured with a high-resolution camera and associated software(Open-Lab; Improvision, Lexington, MA). Images were reproduced forpresentation with image-management software (Photoshop 7.0; AdobeSystems Inc.). Retinal thickness measurements were taken from H&E-stained slides of mice at 2-3 months of age using ImageJ (NIH) forquantification.

Anterior segment imaging with the Phoenix Micron IV imagingmicroscope and OCT attachmentAP-2 NCC KO mutants and their control littermates were weighed andanesthetized with an intraperitoneal (i.p.) injection of 2.5% avertin at0.015 ml/g body weight. Whiskers were trimmed to ensure no obstruction ofthe imaging system and two sets of eye drops applied in order to dilate thepupils (0.5% Tropicamide Ophthalmic Solution and 2.5% PhenylephrineHydrochloride Ophthalmic Solution, AKORN, Lake Forest, IL). Thecorneas were maintained well moistened throughout the procedure withconsistent application of Tear-Gel ophthalmic liquid gel (Alcon Canada,Mississauga, ON, Canada) to prevent drying. The Phoenix Micron IVrodent eye imaging system, with optical coherence tomography (OCT)attachment (Phoenix Research Labs, Pleasanton, CA), was utilized to imagethe corneas and anterior chambers of the AP-2 NCC KO mutants and theircontrol littermates.

IOP measurementsThree-month-old AP-2 NCC KO mutants and their control littermateswere weighed and anesthetized with an i.p. injection of 2.5% avertin at0.015 ml/g body weight. LACRI-LUBE (Allergan Inc., Markham, ON,Canada) was applied to the eyes to maintain a moist tear film and whiskerswere trimmed so there was no impediment of the probe. A validatedcommercial rebound tonometer (TonoLab, Vantaa, Finland) was mountedon a retort stand with the probe aligned horizontally and perpendicularlyto the central cornea, with the tip positioned at 2-3 mm from the eye aspreviously advised (Chatterjee et al., 2013). A mean of six measurementswas determined for each eye and this was repeated ten times in order tocalculate an overall mean. Measurements were only accepted if theyregistered as TonoLab readings with the best reproducibility (no barsymbol) or the next best reproducibility (bar present at the bottom of thescreen) as per the manufacturers manual. All measurements were takenbetween 13:00 to 15:00 in the afternoon and 3 min after injection ofavertin, as this has been previously shown to be a period of stable IOP(Ding et al., 2011).

RGC tracing with NeurobiotinAnimals were deeply anesthetized and the eyes enucleated before animalswere euthanized. The ONs were first removed from each eye to be processedseparately for electron microscopy (EM). The eyes were then placed corneadown in a moist, warm, oxygenated chamber. A piece of gelfoam soaked in

8% Neurobiotin (NB) (Vector Laboratories, Inc.) was placed on the opticstump. The eyes were incubated in the chamber for 1 h, after which they werefixed for 2 h in 4% paraformaldehyde. After washes, the retinaewere removedand incubated in Texas-Red-conjugated Streptavidin (JacksonImmunoResearch Laboratories) overnight at room temperature. The nextmorning the retinae were washed and mounted onto slides with Vectashieldcontaining DAPI (Vector Laboratories). All staining was visualized with amicroscope equipped with an immunofluorescence attachment (Zeiss), andimages were captured with a high-resolution camera and associated software(Zen; Zeiss). Images were reproduced with image-management software(Photoshop 7.0; Adobe Systems Inc.). Retinal whole mounts with DAPI-labeled nuclei and NB-labeled RGCs were sampled four times in the mid-periphery of each petal; the counts of the nuclei were tallied and averaged to beconsidered as one sample. Quantification of the nuclei of RGCs wasperformed using the manual Cell counter plug-in with quantificationsoftware ImageJ (NIH) in order to ensure each soma was counted only onceand an automatic tally was generated.

Electron microscopyFollowing careful dissection of the ONs from the ocular orbit, they wereimmediately immersed in a solution of 4% PFA and 2% glutaraldehydefor 2 h. ONs were then washed in phosphate buffer solution and post fixedin a 1:1 solution of 2% aqueous OsO4 and 0.1 M cacodylate buffer for 1 hat room temperature. Serial dehydration occurred in acetone (20%, 50%,75%, 90% and 100%), and samples were embedded in EPON resin(Canemco and Marivac, QC, Canada). Thin sections of ONs were createdusing a diamond knife with an ultramicrotome (Reichert-Jung Ultracut EMicrotome; American Instruments) and collected on 200 mesh coppergrids (Electron Microscopy Sciences, McMaster University). Sectionswere stained with uranyl acetate and Reynolds lead citrate and viewedusing a JEOL 1200EX electron microscope at an accelerating voltage of80 kV. Images were collected and stored for later analysis andquantification. Five regions of each ON section were sampled in orderto determine axon numbers. The axon numbers were then averaged andconsidered one sample.

Statistical analysesComparisons of IOP measurements, retinal thickness, DAPI and RGCcounts on retinal whole mounts and ON samplings between AP-2NCCKOmutants and control littermates were tested for significance with a Studentstwo-tailed t-test (Prism 6, GraphPad Software). Data were consideredsignificant when P

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