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Stage-dependent modes of Pax6-Sox2 epistasis regulate lens development and eye morphogenesis April N. Smith, Leigh-Anne Miller, Glenn Radice, Ruth Ashery-Padan and Richard A. Lang There was an error published in Development 136, 2977-2985. The acknowledgements should have mentioned the Binational Science Foundation. The corrected acknowledgements section appears in full below. The authors apologise to readers for this mistake. We thank Mr Paul Speeg for excellent technical assistance. We are indebted to Dr Hans Arnheiter for providing the anti-Mitf antibodies. This work was supported by NIH RO1s EY10559, EY15766, EY16241 and EY17848, and by funds from the Abrahamson Pediatric Eye Institute Endowment at Children’s Hospital Medical Center of Cincinnati (R.A.L.). Research in the laboratories of R.A.L. and R.A.-P. is supported by the Binational Science Foundation. Development 136, 3377 (2009) doi:10.1242/dev.043802 CORRIGENDUM

Stage-dependent modes of Pax6-Sox2 epistasis …...Stage-dependent modes of Pax6-Sox2 epistasis regulate lens development and eye morphogenesis April N. Smith 1, Leigh-Anne Miller

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Page 1: Stage-dependent modes of Pax6-Sox2 epistasis …...Stage-dependent modes of Pax6-Sox2 epistasis regulate lens development and eye morphogenesis April N. Smith 1, Leigh-Anne Miller

Stage-dependent modes of Pax6-Sox2 epistasis regulate lens development and eyemorphogenesisApril N. Smith, Leigh-Anne Miller, Glenn Radice, Ruth Ashery-Padan and Richard A. Lang

There was an error published in Development 136, 2977-2985.

The acknowledgements should have mentioned the Binational Science Foundation. The corrected acknowledgements section appears in

full below.

The authors apologise to readers for this mistake.

We thank Mr Paul Speeg for excellent technical assistance. We are indebted to Dr Hans Arnheiter for providing the anti-Mitf antibodies. This work was supported

by NIH RO1s EY10559, EY15766, EY16241 and EY17848, and by funds from the Abrahamson Pediatric Eye Institute Endowment at Children’s Hospital Medical

Center of Cincinnati (R.A.L.). Research in the laboratories of R.A.L. and R.A.-P. is supported by the Binational Science Foundation.

Development 136, 3377 (2009) doi:10.1242/dev.043802

CORRIGENDUM

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2977RESEARCH ARTICLE

INTRODUCTIONThe Pax6 and Sox2 genes are both known to have major roles in eyedevelopment (Callaerts et al., 1997; Chow and Lang, 2001;Treisman, 2004; Kondoh, 2008). Pax6 is a paired domain andhomeodomain-containing transcription factor that is essential foreye development in both invertebrates and vertebrates (Quiring etal., 1994). It also has the remarkable ability to induce ectopic eyes,both in flies and frogs, when misexpressed (Halder et al., 1995;Chow et al., 1999). Pax6 is expressed in the presumptive lens in aregion that includes the lens placode and adjacent ectoderm(Grindley et al., 1997; Furuta and Hogan, 1998; Ashery-Padan et al.,2000; Dimanlig et al., 2001). According to tissue recombinationexperiments (Fujiwara et al., 1994), the generation of Pax6 mutantchimeric mice (Collinson et al., 2000), lineage-traced ectopic lenses(Chow et al., 1999) and Pax6 conditional deletion (Ashery-Padan etal., 2000), Pax6 has an autonomous role in lens development. WhenPax6 is misexpressed in the frog, it has been observed that ectopiclenses can form in isolation, whereas ectopic retina development isalways accompanied by adjacent ectopic lens tissue (Chow et al.,1999). This has suggested that the developing lens may provideimportant signals for formation of the retina.

Sox2 is one member of the larger family of high mobility group(HMG) domain transcription factors (Kamachi et al., 2000).Members of the Sox family have diverse tissue-specific expressionpatterns throughout early development and have been implicated in

cell fate decisions in numerous processes (Uwanogho et al., 1995).Sox1, Sox2 and Sox3 are all expressed in the lens (Kamachi et al.,1998). Sox2 and Sox3 expression in the early lens placode isdependent on the optic vesicle and this implies that they areresponsive to inductive signals (Kamachi et al., 1998). Sox1 is firstexpressed later during invagination of the lens placode, and alsoduring lens morphogenesis in lens fiber cells (Kamachi et al., 1998).Sox2 has been implicated in lens development through its regulationof the δ1-crystallin gene in the chick (Kamachi et al., 2001) and,more recently, of N-cadherin (Matsumata et al., 2005), an adhesionmolecule known to be required for normal lens morphogenesis andthe differentiation of lens fiber cells (Pontoriero et al., 2009). It hasalso been proposed that the Sox2 gene is regulated by the Sox2protein product in combination with Pax6 through the N-3 enhancerthat is active in the presumptive lens (Inoue et al., 2007). Consistentwith an important role for Sox2 in eye development, it was recentlyshown that, in humans, heterozygous Sox2 mutation can result inanophthalmia-esophageal-genital (AEG) syndrome (Fantes et al.,2003; Taranova et al., 2006; Bakrania et al., 2007).

It has been recognized, based on the analysis of cis-actingregulatory elements, that Pax6 and Sox2 are likely to be cross-regulated during development (Kondoh et al., 2004; Hever et al.,2006; Inoue et al., 2007). Despite this, to date, an assessment of thegenetic and functional interactions between Pax6 and Sox2 has notbeen performed. In the current study, we generated a Sox2conditional allele in the mouse and, in combination with the existingPax6 conditional allele (Ashery-Padan et al., 2000), formally testedthe genetic and functional relationships between Pax6 and Sox2 inthe lens. This showed that in pre-placodal ectoderm, Pax6 and Sox2expression is not inter-dependent, but that the two proteins cooperatefunctionally in the very early steps of eye development.Unexpectedly, we show that various combinations Pax6 and Sox2deletion in the pre-placodal ectoderm have profound effects on themorphogenesis of the eye. This suggests that Pax6 and Sox2 arerequired for the production of signals that initiate eyemorphogenesis. We also show that, in pre-placodal ectoderm, N-cadherin is dependent on Sox2, but not Pax6. After the lens placode

Stage-dependent modes of Pax6-Sox2 epistasis regulate lensdevelopment and eye morphogenesisApril N. Smith1, Leigh-Anne Miller1, Glenn Radice2,*, Ruth Ashery-Padan3 and Richard A. Lang1,4,5,†

The transcription factors Pax6 and Sox2 have been implicated in early events in lens induction and have been proposed tocooperate functionally. Here, we investigated the activity of Sox2 in lens induction and its genetic relationship to Pax6 in the mouse.Conditional deletion of Sox2 in the lens placode arrests lens development at the pit stage. As previously shown, conditionaldeletion of Pax6 in the placode eliminates placodal thickening and lens pit invagination. The cooperative activity of Sox2 and Pax6is illustrated by the dramatic failure of lens and eye development in presumptive lens conditional, compound Sox2, Pax6heterozygotes. The resulting phenotype resembles that of germ line Pax6 inactivation, and the failure of optic cup morphogenesisindicates the importance of ectoderm-derived signals for all aspects of eye development. We further assessed whether Sox2 andPax6 were required for N-cadherin expression at different stages of lens development. N-cadherin was lost in Sox2-deficient but notPax6-deficient pre-placodal ectoderm. By contrast, after the lens pit has formed, N-cadherin expression is dependent on Pax6. Thesedata support a model in which the mode of Pax6-Sox2 inter-regulation is stage-dependent and suggest an underlying mechanism inwhich DNA binding site availability is regulated.

KEY WORDS: Pax6, Sox2, Development, Eye, Lens, Morphogenesis, Mouse

Development 136, 2977-2985 (2009) doi:10.1242/dev.037341

1Division of Pediatric Ophthalmology and 4Division of Developmental Biology,Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH 45229, USA.2Center for Research on Reproduction and Women’s Health, University ofPennsylvania School of Medicine, Philadelphia, PA 19104, USA. 3Sackler Faculty ofMedicine, Department of Human Molecular Genetics and Biochemistry, Tel AvivUniversity, Tel Aviv, Israel. 5Department of Ophthalmology, University of Cincinnati,Cincinnati, OH 45229, USA.

*Present address: Center for Translational Medicine, College of Graduate Studies,Thomas Jefferson University, Philadelphia, PA 19107, USA†Author for correspondence ([email protected])

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has formed, Pax6 and Sox2 change to a different geneticrelationship, whereby Sox2 expression is dependent on Pax6. N-cadherin expression at this later stage is dependent on Pax6. Thesedata support a model in which the mode of Pax6-Sox2 inter-regulation is stage dependent, and point to an underlying mechanismin which DNA binding site availability is regulated.

MATERIALS AND METHODSAnimal maintenance and useAnimals were housed in a pathogen-free vivarium in accordance withinstitutional policies. Gestational age was determined through detection ofa vaginal plug. At specific gestational ages, fetuses were removed byhysterectomy after the dams had been anesthetized with isoflurane. In thisanalysis, eight to 20 embryonic eyes were analyzed for each genotype andstage of development.

Generation of the Sox2flox alleleThe Sox2flox allele was generated using conventional gene-targetingmethods. A 5� loxP site was placed in the 5� untranslated region of Sox2 anda 3� loxP site downstream of the single Sox2 exon (Fig. 1A). The Frt site-flanked neo gene of the targeting vector was excised in vivo using a flippase-expressing mouse line (Rodriguez et al., 2000). A similar allele of mouseSox2 has been generated by others (Miyagi et al., 2008).

Mouse linesThe following transgenic and gene-targeted mice were used in this study:Le-cre (Ashery-Padan et al., 2000), AP2α-cre (Macatee et al., 2003), Pax6flox

(Ashery-Padan et al., 2000), Pax6sey (Hill et al., 1991), N-cadherinLacz

(Radice et al., 1997), N-cadherinflox (Kostetskii et al., 2005) and αMHC-Ecad (Luo et al., 2001). All mouse lines used in this study were genotypedby PCR using primers and protocols described previously. Yolk sacs fromstaged embryos or tail tips were digested overnight at 55°C in lysis bufferand genomic DNA extracted using a Kingfisher 96 Magnetic ParticleProcessor. Primers for genotyping of the Sox2flox allele were as follows:VS635, TGGAATCAGGCTGCCGAGAATCC; VS636, TCGTTCTGG -CAACAAGTGCTAAAGC; and VS369, CTGC CATAGC CA CT CG -AGAAG. The PCR protocol used was 95°C for 4 minutes followed by 25cycles of 94°C for 30 seconds, 58°C for 30 seconds and 72°C for 30 seconds,with a final extension period of 72°C for 7 minutes. These primersproduce bands of 421 bp for the wild-type allele and 546 bp for the targetedallele.

ImmunofluorescenceImmunofluorescence labeling for cryosections was performed as previouslydescribed (Smith et al., 2005). All sections were permeabilized with freshlyprepared ice-cold 0.5% Triton X-100, 1% sodium citrate in PBS for 5minutes and then washed three times in 0.1% Tween in PBS prior toblocking. Primary antibody dilutions were as follows: rabbit polyclonal anti-N-cadherin antibody (ABCAM, ab18203), 1:300; sheep polyclonal CHX10(Exalpha, X1180P), 1:1000; rabbit polyclonal anti-Pax6 (Covance, PRB-278P), 1:2000; rabbit polyclonal anti-Sox2 (Chemicon, AB5603), 1:1000;goat polyclonal anti-P-cadherin (R&D Systems, AF761), 1:100; polyclonalrabbit anti-β-crystallin (generated in our laboratory), 1:5000; and rabbitpolyclonal Mitf-1 (a gift from H. Arnheiter, National Institute ofNeurological Disorders and Stroke, NIH, USA), 1:2500. Alexa Fluorsecondary antibodies and Alexa phalloidins were obtained from Invitrogenor Molecular Probes and used at a dilution of 1:5000 (A-11072, A-11070,A-11016, A11058, A11015, A11055, A-12379). All sections werecounterstained with Hoechst 33342 (Sigma, B-2261) for the visualization ofnuclei.

RESULTSGeneration of the Sox2 conditional alleleIn order to effectively study the cooperative roles of Pax6 and Sox2in the developing mouse lens, we generated a conditional Sox2flox

allele (see also Miyagi et al., 2008) using conventional genetargeting in ES cells. The mouse Sox2 gene has a single exon and soloxP sites were placed in the 5� untranslated region and downstreamof the 3� UTR (Fig. 1A). A Pax6flox allele was generated previously(Ashery-Padan et al., 2000).

To assess the functions of Pax6 and Sox2 in the developinglens, we took advantage of two Cre-expressing drivers. The Le-cre transgenic mouse line (Ashery-Padan et al., 2000) uses thePax6 ectoderm enhancer (EE) (Williams et al., 1998; Kammandelet al., 1999; Xu et al., 1999) to drive Cre and GFP expression inthe developing lens from the placode stage (approximately E9.0)onwards (Fig. 1B). At later stages of development, Le-cre isexpressed in the periocular surface ectoderm that includes thepresumptive conjunctiva, the corneal ectoderm and the perioculargland epithelia (Williams et al., 1998; Kammandel et al., 1999;Xu et al., 1999; Ashery-Padan et al., 2000; Smith et al., 2005).The AP2α-cre mouse line was generated by inserting the Crerecombinase-coding region into the 3� untranslated region ofthe AP2α gene (Tcfap2a – Mouse Genome Informatics) usinggene-targeting methods (Macatee et al., 2003). This results ina Cre expression domain that includes the dorsal neural tube,neural crest-derived periocular mesenchyme and the headsurface ectoderm that includes the presumptive lens at a pre-placodal stage (Fig. 1B). A comparison of the consequencesof AP2α-cre and Le-cre mediated gene deletion can beinformative (Song et al., 2007), as they represent geneticallydistinct phases of lens development (Grindley et al., 1995; Lang,2004).

RESEARCH ARTICLE Development 136 (17)

Fig. 1. The Sox2 allele and pattern of AP2α-cre and Le-creexpression. (A) Schematic showing the design of the Sox2 targetingvector and the final Sox2flox conditional allele. The positive selectablemarker neo was removed by crossing the Sox2floxNeo allele with agermline flippase mouse line. (B) Expression patterns (green regions) ofAP2α-cre and Le-cre in the eye region at E8.5 and E9.5. The dashedline shows the approximate boundary of the lens placode. hse, headsurface ectoderm; pom, periocular mesechyme; ov, optic vesicle; op,optic pit. D

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After lens placode formation, Pax6 is upstream ofSox2To understand the roles of Pax6 and Sox2 at the placodal stage oflens development, we first performed deletions of each geneseparately using Le-cre. Le-cre-mediated deletion of Pax6confirmed (Ashery-Padan et al., 2000) that Pax6 immunoreactivityin the lens ectoderm was reduced at E9.5 (Fig. 2B) and absent atE10.5 (Fig. 2E). Pax6 labeling in the presumptive retina of Le-cre;Pax6FL/FL embryos was unchanged (Fig. 2B,E) and served toillustrate the specificity of the Le-cre driver. Phenotypically, Pax6deletion in the presumptive lens resulted in a failure of lensdevelopment from the placode stage onwards (Fig. 2B,E,H,K), aswould be anticipated (Ashery-Padan et al., 2000). In addition, theoptic vesicle failed to undergo normal morphogenesis; although theexpected thickness differential was sometimes observed, thepresumptive retinal and retinal pigmented epithelia were notapposed or cupped (Fig. 2E,K). When we labeled for Sox2 in Le-

cre; Pax6FL/FL embryos, there was little change apparent at E9.5(Fig. 2H) but dramatically reduced immunoreactivity by E10.5(Fig. 2K). This indicated that at the pit stage of lens development,Sox2 expression is dependent on Pax6.

Conditional deletion of Sox2FL/FL with Le-cre gave reliablydiminished Sox2 immunoreactivity in the presumptive lens of E9.5embryos (Fig. 2I) and an absence at E10.5 (Fig. 2L). The phenotypicconsequences of Sox2 deletion were milder than in the Pax6 mutant:some placodal thickening was apparent (e.g. Fig. 2C) and a lens pit,albeit shallow compared with that of wild type (Fig. 2J), wasobserved (Fig. 2L). Furthermore, the optic cup of Le-cre; Sox2FL/FL

embryos was formed fairly normally with apposed presumptiveretina and retinal pigment epithelial layers of appropriate thickness.Immunolabeling for Pax6 in Le-cre; Sox2FL/FL embryos did notdetect any changes in Pax6 levels either at E9.5 (Fig. 2C) or at E10.5(Fig. 2F), indicating that Pax6 expression is not dependent on Sox2.When combined with the above data showing the dependence ofSox2 expression on Pax6, this suggests that after the placodal stageof lens development there is a simple linear genetic pathway withPax6 upstream of Sox2.

At the pre-placodal stage of lens development,Pax6 and Sox2 function in parallelAP2α-cre (Macatee et al., 2003) is expressed earlier than Le-cre andis active at E8.5 in the head surface ectoderm that encompasses thepre-placodal presumptive lens. Because Pax6 and Sox2 are notexpressed in the crest-derived periocular mesenchyme where AP2α-cre is also active, this component of driver activity is not of concern.To determine the function of Pax6 and Sox2 and to examine theirepistatic relationship at pre-placodal stages, we performedconditional deletion of each individual gene with AP2α-cre.

2979RESEARCH ARTICLEPax6 and Sox2 in lens and eye development

Fig. 2. Placodal deletion of Pax6 and Sox2 produce distinctconsequences for lens and eye development. (A-L) Cryosections ofthe indicated embryonic stage (left) and genotype (top) showingimmunofluorescence signal for either Pax6 or Sox2 (red) and nuclei(blue). For clearer examination of areas of targeted deletion (whitebrackets), red channels are magnified and shown separately, eitherbelow (A-C,G-I) or above (D-F,J-L) the parent panel. lp, lens placode; lv,lens vesicle; pr, presumptive retina; prpe, presumptive retinal pigmentedepithelium; ple, presumptive lens ectoderm; pi, lens pit; ov, opticvesicle.

Fig. 3. Pre-placodal Pax6 is not dependent on Sox2.(A-I) Cryosections of the indicated embryonic stage (left) and genotype(top), showing immunofluorescence signal for Pax6 (red) and nuclei(blue). For clearer examination of areas of targeted deletion (whitebrackets), red channels are magnified and shown separately, eitherbelow (A-C) or above (D-F). lp, lens placode; lv, lens vesicle; pr,presumptive retina; prpe, presumptive retinal pigmented epithelium;ple, presumptive lens ectoderm; ov, optic vesicle. D

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In AP2α-cre; Pax6FL/FL embryos, typical nuclear Pax6immunoreactivity was largely lost at E8.5 in the surface ectoderm(Fig. 3B), but was somewhat patchy, presumably because some late-deleting cells retained residual Pax6. By E9.5 (Fig. 3E) and E10.5(Fig. 3H) the presumptive lens ectoderm of AP2α-cre; Pax6FL/FL

embryos showed no Pax6 immunoreactivity. We were not able todiscern any change in Pax6 immunoreactivity in the optic vesicle atany stage in any (n=20) of the AP2α-cre; Pax6Fl/Fl embryosexamined. A phenotypic response to the absence of pre-placodalPax6 did not become apparent until E9.5 and beyond but manifestedas an absence of placodal thickening (Fig. 3E) and a complete failureof eye morphogenesis (Fig. 3H) that was more severe than when Le-cre was used to delete Pax6FL (Fig. 2). Indeed, the phenotype ofAP2α-cre; Pax6FL/FL embryos most closely resembles that ofPax6Sey homozygotes in which the lens placode does not thicken,there is no invagination of either lens pit or optic cup and the opticstalk fails to constrict in the proximal eye region (Grindley et al.,1995). In AP2α-cre; Sox2FL/FL embryos (see Fig. 4 for confirmationof deletion) there was no impact on Pax6 immunoreactivity in thepresumptive lens from E8.5 to E10.5 (Fig. 3C,F,I). This indicatesthat during these stages of lens development, Sox2 is not upstreamof Pax6.

As expected, AP2α-cre; Sox2FL/FL resulted in the absence of Sox2immunoreactivity in the surface ectoderm and presumptive lens fromE8.5-E10.5 (Fig. 4C,F,I). The phenotypic consequence of this pre-placodal Sox2 deletion was similar to the consequence of placodaldeletion with Le-cre, in that the presumptive lens and retina underwenta modest invagination that arrested at the equivalent of E10.0 (Fig. 4I).However, there were also distinctions between AP2α-cre and Le-cremediated Sox2 deletion. Unlike Le-cre mediated Sox2 deletion wherethe optic cup layers were well formed, AP2α-cre-mediated Sox2deletion resulted in a failure of the presumptive retina and the RPE toform nested cups. Instead, the RPE was widely separated from thepresumptive retina (Fig. 4I) and transitioned to an optic stalk regionthat, as in AP2α-cre; Pax6FL/FL embryos, showed no proximalconstriction. This suggests that the early phase of Sox2 expression inthe surface ectoderm is required for the production of signals thatregulate some aspects of optic cup morphogenesis.

Sox2 labeling of AP2α-cre; Pax6FL/FL embryos revealed thatimmunoreactivity was retained from E8.5-E10.5 (Fig. 4B,E,H). Atfirst glance, this might appear surprising given the loss of Sox2 inthe presumptive lens of Le-cre; Pax6FL/FL embryos at E10.5 (Fig.2K), but is likely explained by arrested lens and eye development inthis genotype. In other words, the eye of the E10.5 AP2α-cre;Pax6FL/FL embryo is developmentally equivalent to an E9.0 eye, andat this pre-placodal stage, Sox2 expression is independent of Pax6.The notion that this represents a developmental arrest is reinforcedby the pattern of Chx10 labeling in presumptive retina of AP2α-cre;Pax6FL/FL embryos. Normally, at E9.0, Chx10 is expressed at a lowlevel in a small domain of the central presumptive retina. This regionexpands to encompass the entire presumptive retina by E9.5 (Fig.6K) and E10.5 (Burmeister et al., 1996) (Fig. 6C). By contrast, inE10.5 AP2α-cre; Pax6FL/FL embryos, Chx10 was observed in acentral domain of presumptive retina at the low expression levels(Fig. 4L) characteristic of the E9.0 eye.

The stage dependence of the severity of the Sox2 mutantphenotype is nicely illustrated by the expression of β-crystallin inboth conditional mutants (Fig. 5A-C). In control embryos (Fig.5A), the lower half of the lens vesicle expressed β-crystallin atE11.5. With AP2α-cre-mediated deletion, only a fewdifferentiated cells could be detected (Fig. 5C), whereas Le-cre-mediated deletion gave an intermediate-sized β-crystallin-expressing region (Fig. 5B). Thus, the phenotype is more severewhen Sox2 is deleted earlier.

In some AP2α-cre; Sox2FL/FL embryos, lenses of a reasonable sizebut abnormal morphology were observed later in development. AtE17.5, control eyes showed robust β-crystallin and Prox1 labeling(Fig. 5D,G). By contrast, most AP2α-cre; Sox2FL/FL eyes (n=6/10)had no morphologically recognizable lenses but did have anoccasional ectopic β-crystallin-positive cell (Fig. 5E,H). Theremaining embryos of this genotype (n=4/10) had lenses, albeit ofabnormal morphology, that expressed both β-crystallin and Prox1(Fig. 5F,I). In Pax6 conditional mutants generated using either Credriver, neither morphologically recognizable lenses nor β-crystallinimmunoreactivity was ever detected (data not shown) (Ashery-Padan et al., 2000). This is consistent with the idea that Pax6 has themore upstream role in lens development.

Unchanged Sox2 immunoreactivity in the head surface ectodermof AP2α-cre; Pax6FL/FL embryos (Fig. 4B) is perhaps in contrastwith earlier data examining Pax6Sey/Sey mutants (Furuta and Hogan,1998), suggesting that Sox2 expression in this location is dependenton Pax6. This information emerged from Sox2 in situ hybridizationstudies performed on Pax6Sey-Neu/Sey-Neu embryos at 27 somites(approximately E10). To examine this issue further, we performed

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Fig. 4. Pre-placodal expression of Sox2 is not dependent on Pax6.(A-I) Cryosections of the indicated embryonic stage (left) and genotype(top), showing immunofluorescence signal for Sox2 (red) and nuclei(blue). For clearer examination of areas of targeted deletion (whitebrackets), red channels are magnified and shown separately, eitherbelow (A-C) or above (D-F). (J-L) Immunofluorescence signal for nuclei(blue), F-actin (green) and Chx10 (red) in wild-type (J) and AP2α-cre;Pax6Fl/Fl-deleted embryos (K,L) at E10.5. se, surface ectoderm; lp, lensplacode; lv, lens vesicle; pr, presumptive retina; prpe; presumptiveretinal pigmented epithelium; ple, presumptive lens ectoderm; pi, lenspit; ov, optic vesicle. D

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immunolabeling on Pax6Sey/Sey embryos. We confirmed that, at E9.5,Pax6Sey/Sey embryos showed no nuclear Pax6 immunoreactivity (Fig.5K; cytoplasmic immunoreactivity was frequently detected but wasnot greater than background levels). Furthermore, Pax6Sey/Sey

embryos showed Sox2 immunoreactivity at apparently normallevels despite obvious morphological defects (Fig. 5M). Thus, bothgermline (Pax6Sey/Sey) and conditional (AP2α-cre; Pax6FL/FL) Pax6deletion suggests that prior to E9.5, Sox2 expression in the surfaceectoderm and presumptive lens is independent of Pax6.

Ectodermal Pax6 and Sox2 cooperate in lensdevelopment and eye morphogenesisThe possibility that Pax6 and Sox2 cooperate developmentally israised by their co-expression, by their ability to form a transcriptionregulation complex (Kamachi et al., 2001) and by the identificationof potential binding sites in cis-elements that might mediate cross-regulation (Kondoh et al., 2004; Hever et al., 2006; Inoue et al.,2007). To assess the possibility of Pax6 and Sox2 cooperation in lensdevelopment, we generated conditional compound heterozygotesusing Le-cre and assessed the phenotypic consequences. In E10.5Le-cre; Pax6+/Fl; Sox2+/Fl embryos, we observed phenotypicvariation that ranged from a small but otherwise normal eye(n=10/20; data not shown) to an eye that showed arrested

development at an early stage (n=10/20; Fig. 6E-H). Compared withnormal E10.5 eyes that showed a lens pit or lens vesicle (Fig. 4A-D), conditional compound heterozygotes that were severely affectedshowed no placodal thickening and no lens pit or optic cupinvagination. The morphology of these mutant eyes most closelyresembled that of the Pax6Sey/Sey (Grindley et al., 1995) or the AP2α-cre; Pax6Fl/Fl mutants (Figs 3, 4).

Immunolabeling of severely affected Le-cre; Pax6+/Fl; Sox2+/Fl

embryos revealed that at E10.5, both Pax6 and Sox2 were absentfrom the surface ectoderm (Fig. 6E,F). Because these embryos areconditional mutants in which Pax6 and Sox2 heterozygote deletiontook place approximately one day earlier, the complete loss of Pax6and Sox2 immunoreactivity probably represents a secondaryconsequence of loss of the entire lens development program. Chx10and Mitf are markers for presumptive retina and RPE, respectively,that reveal whether the optic cup has been patterned (Nguyen andArnheiter, 2000; Horsford et al., 2005). Despite the absence of eyemorphogenesis, Chx10 was expressed in central presumptive retina(Fig. 6G, compare with E9.5 control, Fig. 6K), and Mitf inpresumptive RPE (Fig. 6H, compare with E9.5 control, Fig. 6L).This indicates that the first steps of optic cup patterning haveoccurred in Le-cre; Pax6+/Fl; Sox2+/Fl embryos, and suggests that amajor cooperative function of Pax6 and Sox2 is to signal optic cupmorphogenesis.

Sox2 regulates N-cadherin expressionThe adhesion molecule N-cadherin has a complex pattern ofexpression in the epithelia of the developing eye. At E8.5, N-cadherin was expressed in both the optic pit and the surface

2981RESEARCH ARTICLEPax6 and Sox2 in lens and eye development

Fig. 5. Sox2 has an important role in lens development.(A-M) Cryosections from embryos of the indicated age (left) andgenotypes (above) showing immunofluorescence signal for nuclei(blue), β-crystallin (A-C, red; D-F, green) Prox1 (G-I, red), Pax6 (J,K, red)and Sox2 (L,M, red). lp, lens placode; lv, lens vesicle; pr, presumptiveretina; ple, presumptive lens ectoderm; pi, lens pit; ov, optic vesicle; r,retina; m, mesenchyme; l, lens.

Fig. 6. Ectodermal Pax6 and Sox2 cooperate in lens and eyedevelopment. (A-L) Cryosections from embryos of the indicated agesand genotypes labeled for F-actin (A-H, green), nuclei (I-L, blue), orPax6, Sox2, Chx10 or Mitf (red) as indicated at left. Asterisk in Gindicates a retinal fold that has been grazed in the plane of section.lp, lens placode; lv, lens vesicle; pr, presumptive retina; prpe,presumptive retinal pigmented epithelium; ple, presumptive lensectoderm; ov, optic vesicle; os, optic stalk.

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ectoderm of the head fold (Fig. 7A). N-cadherin expression wasmaintained in the optic vesicle at E9.5 but was downregulated in thelens placode region of the surface ectoderm (Fig. 7B). N-cadherinexpression was further retained in the presumptive retina and theRPE of E10.5 embryos and was upregulated in the lens pit duringinvagination (Fig. 7C). Because it has been suggested that N-cadherin is regulated by Sox family members (Matsumata et al.,2005) or Pax6 (van Raamsdonk and Tilghman, 2000), we assessedthis possibility using the conditional mutants.

AP2α-cre; Pax6Fl/Fl embryos at E8.5 showed a wild-typedistribution of N-cadherin immunoreactivity (Fig. 7E). By contrast,AP2α-cre; Sox2Fl/Fl embryos at E8.5 had lost N-cadherin expressionfrom the surface ectoderm (Fig. 7F). Even though the N-cadherinsignal was not high at this stage of development, this change wasconsistently detected in an analysis of six AP2α-cre; Sox2Fl/Fl

embryos. Previously, it has been shown that Pax6Sey heterozygoteshave reduced N-cadherin transcript levels in the lens pit (vanRaamsdonk and Tilghman, 2000). Because neither the Pax6 nor theSox2 homozygous Le-cre conditional mutants developed a lens pit(Fig. 2), it was not possible to assess N-cadherin expression at thisstage in these genotypes. However, we have confirmed that in Le-cre and AP2α-cre conditional Pax6 heterozygotes (Fig. 7H,K), N-cadherin levels were reduced in the lens pit. In the case of AP2α-cre;Pax6Fl/Fl embryos, we quantified this by measuring N-cadherinimmunoreactivity and expressing the data as the ratio ofpresumptive retina to lens pit signal intensity (Fig. 7M); the N-cadherin signal was significantly reduced in the Pax6 mutant(control, n=4; AP2α-cre; Pax6+/Fl, n=9; P=0.00004). A similaranalysis for Sox2 conditional heterozygotes suggested a reduced N-cadherin signal in the lens pit according to observation of

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Fig. 7. N-cadherin expression is Sox2 dependentin the pre-placode, but Pax6 dependentsubsequently. (A-C) Expression pattern of N-cadherin in the developing wild-type lens from E8.5to E10.5. (D-L) Cryosections from embryos of theindicated ages and genotypes labeled for the color-coded markers shown on the left. (D-F) The redchannel that represents labeling for N-cadherin isshown below the parent panel. (M) Quantification ofN-cadherin immunolabeling for control (C), AP2α-cre; Pax6FL/FL (P) and AP2α-cre; Sox2FL/FL (S) E10.5eyes expressed as the ratio of retina/lens intensity.Significance values according to one-way ANOVA areas indicated. (N-S) Cryosections from embryos of theindicated ages and genotypes labeled for the color-coded markers shown on the left. lp, lens placode;lv, lens vesicle; pi, lens pit; pr, presumptive retina;prpe, presumptive retinal pigmented epithelium; op,optic pit; ov, optic vesicle.

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micrographs (Fig. 7I,L), but quantification (Fig. 7M) produced onlya trend of reduced N-cadherin signal (control, n=4; AP2α-cre;Sox2+/Fl, n=8; P=0.10). These findings confirm that in the lens pitN-cadherin expression is dependent on Pax6, and leave open thepossibility that part of this regulation might be mediated by Sox2(Fig. 8). The latter suggestion would be consistent with theidentification of Sox2-binding sites in N-cadherin enhancers(Matsumata et al., 2005).

To determine whether N-cadherin was an important componentof Sox2-dependent lens development, we deleted N-cadherin atthe pre-placodal stage by using an existing Ncadflox conditionalallele (Kostetskii et al., 2005; Li et al., 2005). AP2α-cre; NcadFl/Fl

embryos are difficult to produce at lens development stages owingto an early developmental lethality that probably results fromneural tube and heart development defects (Radice et al., 1997).However, in embryos that were viable at E12.5, we could confirmthat the normally robust level of N-cadherin in the lens vesicle(Fig. 7N) was absent in the conditional mutants (Fig. 7O,P), eventhough Pax6 expression (Fig. 7R,S) was retained. Furthermore, inAP2α-cre; NcadFl/Fl embryos, as expected (Pontoriero et al.,2009), we observed a failure of the lens vesicle to separate fromthe surface ectoderm and a persistence of P-cadherin expression(Fig. 7O,P). Even though this phenotype was striking, it wasmilder that that observed when Sox2 was deleted with AP2α-cre(Figs 3, 4) and suggests that N-cadherin is just one of severaldownstream genes that Sox2 regulates during lens development.Furthermore, because this early deletion of N-cadherin resultedin a phenotype that manifested only at E11.5-E12.5, this suggeststhat N-cadherin does not have a crucial function in pre-placodallens ectoderm.

DISCUSSIONIn this analysis, we have assessed the genetic relationship anddevelopmental functions of Pax6 and Sox2 in the early stages of lensdevelopment in the mouse. We show that there is an epistatic

relationship between Pax6 and Sox2, but that this exists only in adefined developmental window. We also show that, when deletedonly in presumptive lens, Pax6 and Sox2 have a cooperative actionthat regulates lens development but that also initiates morphogenesisin the adjacent optic cup. Finally, we show that N-cadherin isregulated by Sox2 and that, during the placodal phase of lensdevelopment when Pax6 regulates Sox2, N-cadherin is alsodependent on Pax6. These data raise a number of questions.

Stage-dependent regulation of Sox2 by Pax6Our data show that during lens development there are dynamicchanges in the genetic relationship between Pax6 and Sox2. In pre-placodal lens ectoderm, even though there is evidence for afunctional cooperation of the gene products, Pax6 and Sox2transcription is regulated independently. After lens placodeformation, the mode of interaction changes to one in which Sox2expression is dependent on Pax6. This changing relationship is alsoillustrated by an assessment of N-cadherin expression. In pre-placodal presumptive lens, N-cadherin expression is dependent onSox2, but not Pax6. After lens placode formation, N-cadherinexpression is dependent on Pax6. These data suggest a model inwhich Pax6 becomes a regulator of Sox2 after lens inductionsignaling has been initiated (Fig. 8). The N-3 enhancer of Sox2(Inoue et al., 2007) is a good candidate for mediating Pax6regulation of Sox2 after placode formation.

In earlier experiments in which Pax6 was conditionally deletedin the presumptive lens with Le-cre (Ashery-Padan et al., 2000),Sox2 immunoreactivity was retained and this contrasts with thecurrent data in which Le-cre deletion of Pax6 results in the loss ofSox2. An explanation for this difference might lie in the genotypeof the experimental animals. Previously, Pax6flox wasconditionally deleted on a Pax6 heterozygous [Pax6lacZ (St-Ongeet al., 1997)] background, whereas here we used the homozygousconditional allele. It might be that the Pax6 heterozygousbackground produces an earlier developmental defect and thatPax6+/lacZ; Le-cre embryos more closely resemble AP2α-cre;Pax6flox/flox embryos, in which there is an early developmentalarrest and in which Sox2 expression is retained. It will be veryinteresting to compare the eye transcriptomes of these mutants tounderstand whether these differences define early steps in lensinduction.

The observation that the Pax6-Sox2 genetic relationship changeswith developmental stage suggests that an additional level oftranscriptional regulation is at play. Specifically, these data indicatethat, regardless of whether regulation is direct or indirect, the Sox2transcriptional control element that mediates Pax6-dependentregulation in the lens pit is inactive at earlier stages. Clearly thereare many mechanisms that could explain this switching. Given thestage of development at which this regulatory switching occurs, itmight be that optic vesicle-dependent inductive signaling can throwthe switch. Switching might be mediated by co-regulator availabilityor perhaps by chromatin remodeling (Li et al., 2007). Furtherinvestigation will be required to gain an understanding of thismechanism.

Pax6 and Sox2 function in lens induction in the context of a largerset of transcription factors and several signaling pathways (Lang,2004; Medina-Martinez and Jamrich, 2007; Kondoh, 2008). Forexample, the Six3 transcription factor is known to be important forthe early stages of lens development (Liu et al., 2006) and is likelyto function in a positive-feedback loop with Pax6 that would resultin the enhanced expression of both (Liu et al., 2006). It has beensuggested (Liu et al., 2006) that the mechanism of Six3 regulation

2983RESEARCH ARTICLEPax6 and Sox2 in lens and eye development

Fig. 8. A model for ectodermal Pax6 and Sox2 function in earlyeye development. The analysis we present indicates that in pre-placodal ectoderm, Pax6 and Sox2 are regulated independently butfunctionally cooperate. By contrast, after the lens placode has formed,Sox2 expression is dependent on Pax6. In an unexpected finding, weshow that Pax6 and Sox2 in the presumptive lens cooperate to providesignals (orange arrow) that are required for morphogenesis of the opticcup. At the pre-placodal stages, N-cadherin is regulated by Sox2. Afterthe lens pit has formed, N-cadherin is regulated by Pax6. It is possiblethat in the lens pit, the dependence of N-cadherin expression on Pax6 isin part mediated by Sox2. D

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of Pax6 is direct binding to the ectoderm enhancer (Williams et al.,1998; Kammandel et al., 1999; Xu et al., 1999). It has also beensuggested that Six3 is upstream of Sox2 (Liu et al., 2006) and that,here too, the mechanism is direct transcriptional regulation, in thiscase, via the N4 enhancer (Uchikawa et al., 2003). The positiveregulation of Sox2 by a positive-feedback loop provides a strongrationale for its upregulation during early lens development.

Pax6 and Sox2 in the presumptive lens regulateoptic cup morphogenesisAn unexpected finding from these studies was the absence of opticcup morphogenesis when various combinations of Pax6 and Sox2were deleted in the surface ectoderm. A mild form of thismorphogenesis failure is seen in Le-cre; Pax6flox/flox embryos inwhich the retina becomes convoluted (Ashery-Padan et al., 2000).When Pax6 is deleted earlier in pre-placodal ectoderm with AP2α-cre, eye morphogenesis fails completely and the phenotype mostclosely resembles the changes observed in the Pax6Sey/Sey mice,which are Pax6 germline null (Grindley et al., 1995). This impliesthat Pax6 expression in the surface ectoderm is required for theproduction of signals that initiate optic cup morphogenesis,including the epithelial bending that leads to the formation of nestedcups of retina and RPE. The distances from the surface ectoderm tothe presumptive RPE at the relevant stage of E9.5 are quite large andso, presumably, ectoderm and Pax6-dependent morphogenesissignaling must use a mechanism that can be transmitted or relayedover distance.

The transcription factors Chx10 and Mitf are expressed in thepresumptive retina and the RPE, respectively. It has been shown thatChx10 represses Mitf expression and that this is an importantelement of defining the retinal and RPE territories in the optic cup(Nguyen and Arnheiter, 2000; Horsford et al., 2005). The failure ofChx10 expression to propagate throughout the presumptive retina inAP2α-cre; Pax6flox/flox embryos might provide an explanation for thefailure of optic cup morphogenesis. Specifically, Pax6 in thepresumptive lens appears to be required for the lens-to-retinasignaling that establishes Chx10 expression. In turn, retinal and RPEterritories might remain undefined and this might lead to a failure ofregion-specific morphogenesis. Further analysis will be required toidentify the morphogenesis mechanisms involved.

Our findings were similar when Sox2 was deleted in pre-placodalectoderm, except that the morphogenesis defects were milder. InAP2α-cre; Sox2flox/flox embryos, we typically observed E10.5 eyeswith optic stalk regions that had not constricted, although in othersthere were nested cups of retina and RPE. The difference in theseverity of eye morphogenesis defects following pre-placodaldeletion of Pax6 and Sox2 presumably reflects the degree to whichectodermal morphogenesis signals are dependent on eachtranscription factor. Clearly, the dramatic eye morphogenesis failureapparent in some Pax6, Sox2 conditional heterozygotes nicelyillustrates the functional cooperation of the two transcription factors.

An earlier study (Donner et al., 2007) generated mice that weredouble heterozygotes for Sox2 and Pax6 by using the germlinealleles Sox2βgeo2 (Avilion et al., 2003) and Pax6Sey-Neu (Hill et al.,1991). In contrast to the current data, the analysis of different stagesof eye development in Sox2βgeo2/+; Pax6Sey-Neu/+ embryos revealedno exacerbation of the Pax6Sey-Neu/+ small eye phenotype by Sox2heterozygosity. Although this might seem difficult to reconcile withthe current analysis, there may be explanations. One possibility isthat with Pax6 and Sox2 germline mutations producing a defect veryearly in development, the embryo might have the developmentalplasticity to accommodate the change without major consequences.

The rapid deletion of Pax6 and Sox2 conditional alleles at a laterstage of development, as in this analysis, is unlikely to allowcompensation due to developmental plasticity. There is also thepossibility that the germline and conditional alleles for Sox2 andPax6 do not produce mutations that are functionally equivalent,especially given the complex gene structure and multiple isoformsof Pax6. Further work will be required to better define these issues.

In humans, Sox2 heterozygosity leads to anophthalmia-esophageal-genital (AEG) syndrome (Taranova et al., 2006;Bakrania et al., 2007). This contrasts with findings in the mousewhere Sox2 heterozygosity does not lead to this syndrome or anyobvious eye defects (Avilion et al., 2003). We have also observedthat in AP2α-cre or Le-cre; Sox2 conditional heterozygotes there areno apparent eye defects. However, the dramatic consequences ofconditional Pax6, Sox2 heterozygosity do indicate that, on asensitized background in which there is only half the normal levelof Pax6, Sox2 heterozygosity can lead to anophthalmia. Perhapsindividuals with AEG syndrome arise when the genetic variabilityof the human population provides a sensitized background in whichSox2 heterozygosity can have serious consequences.

AcknowledgementsWe thank Mr Paul Speeg for excellent technical assistance. We are indebted toDr Hans Arnheiter for providing the anti-Mitf antibodies. This work wassupported by NIH RO1s EY10559, EY15766, EY16241 and EY17848, and byfunds from the Abrahamson Pediatric Eye Institute Endowment at Children’sHospital Medical Center of Cincinnati (R.A.L.). Deposited in PMC for releaseafter 12 months.

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2985RESEARCH ARTICLEPax6 and Sox2 in lens and eye development

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