Molecular Vision 2007; 13:1215-1225 <>
Received 26 April 2007 | Accepted 10 July 2007 | Published 19 July 2007

Genetic analysis indicates that transcription factors AP-2α and Pax6 cooperate in the normal patterning and morphogenesis of the lens

Leila F. Makhani,1 Trevor Williams,2 Judith A. West-Mays1

1Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada; 2Departments of CFB and CSB, University of Colorado Health Sciences Center, Denver, CO

Correspondence to: Judith A. West-Mays, Ph.D., Department of Pathology and Molecular Medicine, McMaster University, Health Sciences Centre, Room 1R10, Hamilton, ON, Canada L8N 3Z5; Phone: (905) 525-9140 ext. 26237; FAX: (905) 525-7400; email:


Purpose: The similar lens phenotypes observed in mice with mutations in the genes encoding either Pax6 or AP-2α suggested that these transcription factors work together to regulate specific signaling cascades during lens development. In this study we examined the overlapping expression patterns of Pax6 and AP-2α in the developing mouse lens and further investigated their potential cooperative roles through the creation of double heterozygote mice.

Methods: Colocalization of Pax6 and AP-2α expression patterns were performed on sections of mouse embryos at embryonic days 9.5, 10.5, 13.5, and 16 as well as on adult sections using immunofluorescence. To test the potential cooperation between these two transcription factors, two mouse strains heterozygous for the genes encoding either Pax6 or AP-2α were bred together to produce double heterozygous Pax6+/lacZ/AP-2α+/- mice. Histological examination was then performed on both embryonic and post-natal sections in order to compare double heterozygous Pax6+/lacZ/AP-2α+/- eyes to single heterozygote and wildtype eyes.

Results: Examination of the developmental stages showed distinct colocalization of Pax6 and AP-2α protein in the anterior lens epithelium. However, Pax6 expression continued further into the transitional zone of the lens whereas AP-2α expression ceased just prior to the region where epithelial cells differentiate into fiber cells. Histological investigation of embryonic and post-natal mutant mouse eyes showed that while single Pax6 heterozygote mice exhibited remnants of a corneal-lenticular adhesion, the lens and cornea were physically separated. In contrast, the Pax6+/lacZ/AP-2α+/- double heterozygotes displayed a distinct lens stalk, which protruded towards the surface of the cornea, creating a direct corneal-lenticular attachment.

Conclusions: Colocalization of Pax6 and AP-2α was mainly observed in the proliferating central lens epithelium, the same region in which the lens stalk phenotype was observed in the double heterozygous Pax6+/lacZ/AP-2α+/- eyes. The more severe phenotype observed in these double heterozygous mice, as compared to the single heterozygotes, suggests that Pax6 and AP-2α may work synergistically to control lens development.


Development of the ocular lens is a complex and tightly regulated process. Lens formation begins as a thickening of the surface ectoderm over top of the optic vesicle. The thickened ectoderm forms the lens placode, which subsequently invaginates into a lens pit, then pinches off to form the premature lens and corneal epithelium [1]. The embryonic lens develops its polarity through the formation of an epithelial monolayer on its anterior aspect. As the lens continues to develop, these epithelial cells have the ability to proliferate and contribute to the germinative zone of the lens. With age, proliferating cells from the germinative zone are displaced towards the lens equator and into the lens transitional zone where they differentiate into secondary fiber cells [2]. These events involve a complex network of interacting transcription factors, which regulate multiple aspects of gene expression.

One such important transcription factor is the homeobox containing Pax6 gene. Pax6 is an important regulator of normal lens development [3-5] and is thought to lie near the top of the hierarchies governing multiple signaling cascades in ocular morphogenesis [6,7]. In humans, rare homozygous mutations in Pax6 give rise to anophthalmia (no eyes) whereas heterozygous mutations in Pax6 are thought to be responsible for aniridia, a congenital disorder associated with severe ocular abnormalities [8]. Mutations in the Pax6 gene are implicated in Peter's anomaly, a disease involving defective corneal endothelia, cataractous lenses, and a corneal-lenticular adhesion [9]. Homozygous Pax6-/- mouse embryos lack eyes entirely and do not survive post-natally while heterozygous Pax6+/-, also known as Sey+/-, mice exhibit microphthalmia (small eyes), demonstrate a corneal-lenticular adhesion (lens stalk), and are at risk for corneal opacities, lens cataracts, and lens deterioration [10,11]. Pax6 is thus essential for lens placode formation as well as normal lens cell proliferation and differentiation.

Another transcription factor which plays an important role during development is AP-2α (Activating Protein-2), encoded by the gene Tcfap2a. Tcfap2a belongs to a family of retinoic acid-responsive genes and is a critical regulator of early lens morphogenesis [12]. Previous studies on AP-2α-/- mice showed that they exhibit multiple ocular defects including a corneal-lenticular adhesion [12,13]. Le-AP-2α mutant mice generated in our lab, in which Tcfap2a is conditionally deleted in the lens placode derivatives, similarly demonstrate numerous corneal and lens defects including a peripheral lens stalk [14,15]. Together, these results highlight the intrinsic requirements of AP-2α in lens vesicle development and in the maintenance of the lens epithelial cell phenotype.

Many similar ocular phenotypes are displayed by the Pax6+/-, AP-2α-/-, and conditional AP-2α mutants [8,12,15]. In many cases, these mutants exhibit a small eye phenotype, a lens stalk, and cataract formation as well as corneal epithelial defects. Previous work also revealed overlap in the expression patterns of Pax6 and AP-2α in the corneal epithelium [3]. In addition, Pax6 has been shown to act in conjunction with the AP-2α binding site to direct activity of the gelatinase B (gelB) promoter, a gene encoding matrix metalloproteinase 9 (MMP-9), expressed in the regenerating corneal epithelium [3]. Finally, the corneal defects observed in the Le-AP-2α mutants including abnormal epithelial integrity, irregular basement membrane deposition, and corneal-lenticular adhesion are strikingly similar to those observed in corneas of the Sey+/- mice [3,14,16]. Together these data suggest that Pax6 and AP-2α may share similar signaling pathways in ocular epithelial development and likely coregulate genes within similar cell signaling cascades.

In order to explicitly test the potential cooperative roles of Pax6 and AP-2α during lens morphogenesis, we first examined in detail their overlaying expression patterns during lens and eye development. Next, we created a double heterozygous mouse model. The rationale behind this genetic cross was that double heterozygous mice should exhibit a more severe lens phenotype than the single heterozygous mice, if, in fact, the Pax6 and AP-2α pathways interact to control lens development. Our colocalization studies revealed that in the developing corneal and lens epithelium, Pax6 and AP-2α protein exhibited substantial overlap in expression. Furthermore, double heterozygous mice exhibited more severe lens phenotypes than that of the single heterozygous mice. Together, these data provide evidence that Pax6 and AP-2α cooperate during lens development.


Generation of Pax6+/lacZ/AP-2α+/- mice

All animal procedures were carried out in accordance with the ARVO statement for the use of animals in ophthalmic and vision research. A double heterozygous mouse model for Pax6 and AP-2α was created in the following manner: a portion of the Tcfap2a gene encoding the AP-2α dimerization domain and required for DNA binding, was removed using a targeting vector as described previously [17]. The affected embryonic stem-cells were extracted and injected into male mice. These male chimeric mice were then bred with Black Swiss female mice to produce AP-2α+/- mice. Pax6+/lacZ mice were produced by replacing the Pax6 initiation codon and paired domain with a β-galactosidase (lacZ) neomycin cassette [18]. The AP-2α+/- mice were then bred with the Pax6+/lacZ mice to produce Pax6+/lacZ/AP-2α+/- mice. Day 0.5 of embryogenesis (E0.5) was designated for noon of the day the vaginal plug appeared. DNA from embryonic or adult mouse tail biopsies was extracted using the DNeasy tissue kit (Qiagen, Valencia, CA). PCR analysis was used to identify the genotypes of the offspring. The lacZ primer sequences OLS21 (5'-GAA ATC CGA ATC TCT ATC GTG C-3') and OLS22 (5'-TAC AGA ACT GGC GAT CGT TCG-3'), were used to identify the lacZ knock-in allele (900 base pairs [bp]). PCR analysis was carried out under the following experimental conditions: 120 s at 95 °C, 35 cycles of 95 °C for 45 s, 55 °C for 45 s, 72 °C for 90 s, followed by 72 °C for 10 min. The AP-2α null primer sequences, Alpha3'K0 (5'-CGT GTG GCT GTT GGG GTT GTT GCT GAG GTA C-3'), -3'KO (5'-AAC GCA CGG GTG TTG GGT CGT TTG TTC G-3'), and Alpha6/7F (5'-GAA AGG TGT AGG CAG AAG TTT GTC AGG GC-3'), were used to identify the KO allele (265 bp) and the wildtype allele (500 bp). PCR analysis was carried out under the following experimental conditions: 120 s at 95 °C, 35 cycles of 95 °C for 45 s, 70 °C for 45 s, 72 °C for 60 s, followed by 72 °C for 10 min. Wildtype littermates homozygous for both the normal Pax6 and Tcfap2a alleles were used as controls.


Mice were euthanized by CO2 overdose and embryos or whole eyes were dissected and collected. Tissue was fixed in 10% neutral buffered formalin (Sigma-Aldrich, Oakville, ON) overnight at 4 °C, then processed and embedded in paraffin. Serial sections were cut 4 or 5 μm in thickness and either used for hematoxylin and eosin (H&E) staining, periodic acid schiff (PAS) staining or immunofluorescent analysis.

Immunohistochemical analysis

Paraffin embedded sections were deparaffinized in xylene, rehydrated (through 100%, 95%, and 70% ethanol solutions followed by water), and incubated in a heated sodium citrate buffer (10 mM, pH 6.0) for 20 min in order to retrieve masked antigens. Tissue sections were blocked with normal serum at room temperature (RT) and subsequently incubated with one of the following primary antibodies: mouse monoclonal anti-AP-2α (3B5, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA; used undiluted, overnight at 4 °C); rabbit polyclonal anti-Pax6 (Covance, Princeton, NJ; at a ratio of 1:50, 1 h at RT); and mouse monoclonal anti-E-cadherin (BD Transduction Laboratories, Franklin Lakes, NJ; at a ratio of 1:100, 1 h at RT). The locations of these antigens were then revealed using secondary antibodies conjugated to either fluorescein isothiocyanate (FITC) or rhodamine (Jackson ImmunoResearch Laboratories, West Grove, PA; at a ratio of 1:50, 1 h at RT). All stains were mounted with the Vectashield mounting medium containing 4 6-diamino-2-phenylindole (DAPI; Vector Laboratories, Burlington, ON). Each staining experiment included a negative control with no primary antibody. All staining was visualized with a microscope (Leica, Deerfield, IL) equipped with a fluorescence attachment while images were captured with a high-resolution camera and associated software (Open-Lab; Improvision, Lexington, MA). Images were reproduced for publication with image-management software (Photoshop 7.0; Adobe Systems Inc., Mountain View, CA).


Expression of Pax6 and AP-2α in the developing mouse eye

To determine the timing and location in which Pax6 and AP-2α might mutually act during ocular development, colocalization studies were performed using immunofluorescence at a number of different developmental stages. At E9.5, Pax6 and AP-2α were found to be colocalized in cells of the lens placode (Figure 1C) whereas at this stage, only Pax6 was expressed in the optic vesicle (Figure 1A). At E10.5, Pax6 and AP-2α were expressed in cells lining the lens pit (Figure 1F). However, AP-2α expression appeared more intense at the distal ends of the lens pit as compared to the rest of the lens vesicle (Figure 1E). In E13.5, E16, and adult eye sections, Pax6 and AP-2α were colocalized in many cells of the corneal and lens epithelium (Figure 1I,L,O). In addition, strong AP-2α expression as well as a low level of Pax6 were also detected in the eyelid epidermis at E16 (Figure 1J,K). Expression was subsequently examined in the transitional zone of the lens at all stages and revealed that while Pax6 was expressed in the transitional zone of the lens both embryonically and post-natally, AP-2α was not and its expression terminated just prior to the transitional zone (Figure 2). Pax6 and AP-2α thus exhibited similar as well as distinct expression patterns within the lens placode derivatives.

Expression patterns of Pax6 and AP-2α were also examined in the developing optic cup. Sections of whole embryonic eyes revealed that while Pax6 was expressed in all of the retinal progenitor cells (RPC) in the optic cup at E13.5, AP-2α was only detected in a small subset of RPCs in the central cup region (Figure 3). In addition, colocalization studies revealed that at E13.5, E16, and E18.5 all of the RPCs that expressed AP-2α in the developing inner nuclear layer also expressed Pax6 (Figure 3C,F,I). In the adult retina, Pax6 and AP-2α were colocalized in amacrine cells of both the inner nuclear layer and the ganglion cell layer (Figure 3L). The expressing cells were known to be amacrine cells because in previous experiments, AP-2α colocalized with amacrine cell markers syntaxin-1, glycine transporter-1 (GlyT1), and γ-aminobutyric acid transporter 1 (GAT-1) [19]. In the same study, AP-2α-positive cells did not colocalize with cells positive for Brn3b and fluorogold, demonstrating that AP-2α-positive cells in the ganglion cell layer were in fact displaced amacrine cells and not ganglion cells [19]. Not all cells, however, appeared to be AP-2α -positive in these two layers; there appeared to be a population of cells that were only Pax6 positive. Nonetheless, Pax6 and AP-2α were observed to share similar expression patterns in the amacrine cells of the developing and adult retinas.

Pax6+/lacZ/AP-2α+/- double heterozygous mice exhibit a more severe lens phenotype than single heterozygous mice

In order to explicitly test the potential cooperation between Pax6 and Tcfap2a, Pax6+/lacZ heterozygous mice were crossed with AP-2α+/- heterozygous mice, producing Pax6+/lacZ/AP-2α+/- mice double heterozygous for both Pax6 and Tcfap2a. As previously reported, Pax6 heterozygous mice exhibit a small eye phenotype and an adhesion of the lens to the cornea [8,10]. In contrast, AP-2α+/- mice display no abnormal external phenotype or any obvious eye defect [17]. Our results corroborated the above findings wherein no aberrant ocular phenotype was observed in either embryonic (Figure 4B,F,J) or adult AP-2α+/- (Figure 5B) mouse eyes and both resembled wildtype eyes (Figure 4A,E,I and Figure 5A). Embryonic and adult Pax6+/lacZ mice demonstrated a lens separation defect and occasionally displayed the small eye phenotype. On the other hand, double heterozygous Pax6+/lacZ/AP-2α+/- mice exhibited more severe ocular defects than their single heterozygous littermates, both embryonically and post-natally. All results are summarized in Table 1.

At E11.5, lens vesicle separation appeared to be delayed in the Pax6+/lacZ and Pax6+/lacZ/AP-2α+/- eyes as compared to the wildtype and AP-2α+/- eyes. The lens separation defect also appeared similar in both Pax6+/lacZ and Pax6+/lacZ/AP-2α+/- eyes (Figure 4C,D). However, by E15.5, a more notable difference in lens phenotype was observed between the single Pax6+/lacZ and double Pax6+/lacZ/AP-2α+/- heterozygous mouse eyes. At E15.5, the Pax6+/lacZ eyes demonstrated what appeared to be remnants of a lens stalk beneath the central corneal epithelium; the lens stalk did not protrude into the cornea, nor was there any direct attachment between the lens and cornea (Figure 4G). However, at E15.5, Pax6+/lacZ/AP-2α+/- eyes exhibited a lens that adhered to and protruded into the cornea (Figure 4H). This was considered a more severe phenotype than that observed in the Pax6+/lacZ mice at the same embryonic stage. At E18.5, the Pax6+/lacZ eyes also exhibited remnants of a lens stalk; the central corneal epithelial cells appeared to extend into the corneal stroma, yet no direct attachment was observed between the lens and cornea (Figure 4K). In contrast, the E18.5 Pax6+/lacZ/AP-2α+/- eyes demonstrated abnormal lens-corneal adhesion and this protruded directly into the cornea, forming a lens stalk. (Figure 4L). These data suggest that while single heterozygous Pax6+/lacZ and double heterozygous Pax6+/lacZ/AP-2α+/- embryonic eyes both demonstrated a lens separation defect, the Pax6+/lacZ/AP-2α+/- eyes consistently exhibited a more persistent lens stalk or corneal-lenticular adhesion phenotype by E15.5 and at later timepoints.

Histological examination of the adult Pax6+/lacZ and Pax6+/lacZ/AP-2α+/- mouse eyes with hematoxylin and eosin (H & E) staining revealed that similar to the embryonic stages, Pax6+/lacZ adult eyes exhibited what appeared to be remnants of a lens stalk (Figure 5C). Corneal epithelial cells appeared to extend into the corneal stroma, however, the lens was not directly adhered to the cornea. Extensive plaque formation was also detected in the area beneath the central lens epithelium in these mice (Figure 5C). In comparison to the adult Pax6+/lacZ mouse eyes, the adult Pax6+/lacZ/AP-2α+/- eyes displayed a more severe ocular defect. Similar to embryonic stages, the adult Pax6+/lacZ/AP-2α+/- eyes exhibited a centrally formed lens stalk or lens protrusion into the cornea (Figure 5D). This lens stalk phenotype, although observed in both pre-natal and post-natal stages, appeared thicker and more pronounced in the adult Pax6+/lacZ/AP-2α+/- eyes. These results were consistent in all phenotypic eyes sectioned, which comprised 76% of all double heterozygous adult eyes. Plaque formation was also observed beneath the lens stalks of the adult Pax6+/lacZ/AP-2α+/- eyes (Figure 5D). The remaining 24% of the double heterozygous Pax6+/lacZ/AP-2α+/- eyes only demonstrated plaque formation or corneal-lenticular adhesion and did not demonstrate a full lens stalk phenotype.

The adult eye sections were also subjected to periodic acid schiff (PAS) staining to observe aberrant matrix deposition in their plaques as well as for glycogen and carbohydrate content. The lens plaques of the Pax6+/lacZ adult eyes demonstrated substantial PAS staining (Figure 5G), confirming matrix deposition in these plaques. Lens capsule also stained positive for PAS due to its glycogen and carbohydrate content. In the Pax6+/lacZ/AP-2α+/- adult eyes, the lens stalks appeared to be surrounded by lens capsule as the positive stain surrounding the stalk was continuous with the lens capsule (Figure 5H). In addition, matrix deposition was observed in the plaques beneath the lens stalk (Figure 5H).

Previous data has demonstrated that when both Tcfap2a alleles are conditionally deleted from lens placode derivatives, E-cadherin expression in the lens is reduced [20]. Furthermore, Sey+/- mutant mice demonstrate an abnormal corneal epithelial cell adhesion, which suggests a role for Pax6 in proper cell-cell adhesion [16]. To determine if reduced dosage of both Pax6 and AP-2α affected E-cadherin expression in the lenses of our Pax6+/lacZ and Pax6+/lacZ/AP-2α+/- adult eyes, immunohistochemistry was performed. In wildtype eyes, E-cadherin was expressed at the cell-cell junctions of corneal and lens epithelial cells (Figure 6A). E-cadherin expression was also detected in the lens epithelium and in the lens plaques of the Pax6+/lacZ eyes (Figure 6B). In the Pax6+/lacZ/AP-2α+/- eyes, both the lens stalks and the plaques beneath the lens stalks stained positive for E-cadherin (Figure 6C). Protein expression appeared normal in the remaining areas of the adult lens epithelium. Thus, E-cadherin expression remained normal in the lens epithelium of both Pax6+/lacZ and Pax6+/lacZ/AP-2α+/- adult eyes but was expressed throughout the plaques of the single Pax6+/lacZ heterozygote and lens stalks of double heterozygous Pax6+/lacZ/AP-2α+/- eyes.

Apoptosis is known to play an important role during lens development and as reported earlier, is essential for lens vesicle separation [21]. We thereby examined cell death using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis in embryonic sections of both Pax6+/lacZ and Pax6+/lacZ/AP-2α+/- eyes. TUNEL staining did not reveal a significant difference between single and double heterozygous mouse eyes, indicating no change in levels of programmed cell death between these groups (data not shown). To determine if the abnormal phenotypes observed in the Pax6+/lacZ and Pax6+/lacZ/AP-2α+/- eyes were due to a change in the levels of cellular proliferation, embryonic sections were stained with Ki67, a marker for cell proliferation. However, similar to the TUNEL analysis, no notable difference in levels of cellular proliferation was observed between the single and double heterozygous mouse eyes (data not shown).


Ocular morphogenesis is controlled by the intricate interaction of multiple transcription factors. Pax6 and AP-2α have been previously identified as important regulators of eye development [5,6,12]. Pax6-/- mutant mice die embryonically and heterozygous Pax6 small eye mice exhibit severe ocular defects including microphthalmia, corneal-lenticular adhesion, and cataracts [8,22]. AP-2α-/- mutant mice demonstrate similar developmental abnormalities as well as early death [17]. Moreover, Le-AP-2α mutant mice, in which Tcfap2a is conditionally deleted from the lens placode and its derivatives, display lens and corneal defects similar to Pax6+/- mice [14,15]. Previous studies have also shown that Pax6 and AP-2α protein are colocalized in ocular epithelial cells [3] and specifically, Pax6 and AP-2α were shown to cooperate in regulating the gelatinase B (gelB) promoter [3]. Based on these findings and the comparable ocular phenotypes displayed by the mutant mice, we hypothesized that Pax6 and AP-2α may share similar signaling pathways during lens development and work together to coregulate genes involved in cascades governing lens morphogenesis. The findings in our current study support this hypothesis in that Pax6 and AP-2α were found to be colocalized in cells of the central lens epithelium. Moreover, Pax6+/lacZ/AP-2α+/- double heterozygous mice exhibited a more severe lens phenotype than single heterozygote littermates.

The colocalization experiments carried out in this study revealed an overlap in Pax6 and AP-2α expression in multiple ocular tissues including the corneal epithelium, the lens epithelium, and the inner nuclear layer of the retina. We were particularly interested in the overlap observed in the lens. Colocalization was detected in the central lens epithelium which, importantly, coincided with the region in which the severe lens defect was observed in the Pax6+/lacZ/AP-2α+/- double heterozygous mouse eyes. Contrasting protein expression patterns were also observed in that Pax6 expression continued into the transitional zone of the lens whereas AP-2α expression terminated just before. Studies from this laboratory have previously shown that AP-2α is important in regulating the lens epithelial cell phenotype by possibly acting as a negative regulator of fiber cell differentiation [23]. Additional studies have described AP-2α as a suppressor of terminal differentiation during embryonic development [24]. Thus, the expression pattern of AP-2α in the lens epithelium in which it ceases before the transitional zone is consistent with these roles. Pax6 expression is also maintained in the lens epithelium and while we observed that it continues somewhat into the transitional zone, like AP-2α, the decrease in Pax6 expression in fiber cells is required for normal lens development [25]. Thus, our Pax6 and AP-2α colocalization studies, described herein, highlight the potential overlapping roles for these transcription factors in lens epithelial cell morphogenesis. However, further studies are required to understand if there is any significance to the contrasting expression patterns of Pax6 and AP-2α in the transitional zone of the lens.

In order to further investigate the potential collaborative roles of Pax6 and AP-2α in the lens at the genetic level, we created a double heterozygous mouse model in which Pax6+/lacZ heterozygous mice were crossed with AP-2α+/- heterozygous mice to produce double heterozygous Pax6+/lacZ/AP-2α+/- mice. The rationale behind this particular genetic cross was that if Pax6 and AP-2α cooperate during lens development, then Pax6+/lacZ/AP-2α+/- mouse eyes will demonstrate a more severe lens phenotype than single Pax6+/lacZ heterozygous mice. As expected, single heterozygous AP-2α+/- mouse eyes resembled wildtype eyes [17] while single heterozygous Pax6+/lacZ mouse eyes exhibited previously reported phenotypes including microphthalmia, corneal-lenticular adhesion, and lens plaques [8,22]. The double heterozygous Pax6+/lacZ/AP-2α+/- eyes, however, demonstrated a more severe lens phenotype than the single heterozygote Pax6+/lacZ eyes, both embryonically and post-natally. These Pax6+/lacZ/AP-2α+/- eyes displayed a persistent, thickened lens stalk that protruded into the body of the corneal stroma, creating a direct corneal-lenticular attachment. This lens protrusion, or lens stalk phenotype, was observed in 19 of the 25 adult eyes and was only seen in the Pax6+/lacZ/AP-2α+/- mice. Interestingly, the AP-2α+/- mice did not demonstrate a lens defect or any other aberrant external ocular phenotypes as previously reported [17]. Thus, the severity of the lens phenotype observed in the double heterozygotes cannot be simply explained by an 'additive effect' of individual ocular phenotypes.

The fact that lens defects were observed in the Pax6+/lacZ but not in the AP-2α+/- heterozygote mice suggests that lens development is more sensitive to Pax6 dosage than it is to the dosage of AP-2α. Since the Pax6 heterozygote phenotype can be made more severe with the loss of one allele of Tcfap2a, it can be proposed that the normal function of Pax6 is dependent on normal dosage of AP-2α. Indeed, the ability of Pax6 to bind to gene promoters has been shown to be dependent on its interaction with AP-2α in corneal epithelial cells [3]. Pax6 and AP-2α likely coregulate additional genes in the lens. For example, Pax6 and AP-2αalong with PROX1, have been shown to activate the Sox2 promoter [26]. In addition, microarrays performed in our lab on lens tissue from the Le-AP-2α mutant mice revealed an alteration in a number of downstream genes involved in lens development and differentiation including L1cam, Bfsp1, Etv6, Olig2, Sema3b, and Sema4a (unpublished). Interestingly, these genes have been previously identified as downstream targets of Pax6 [27-29], implying that Pax6 and AP-2α may coregulate other genes in the lens development pathway.

A recent study, carried out by Zaki et al. [30], utilized a comparable approach to examine the cooperative role of Pax6 and Gli3, the latter of which is a transcription factor essential for brain, limb, and eye development. It was observed that mice heterozygous for both Gli3 and Pax6 demonstrated a greater range of ocular defects than single Gli3 or Pax6 heterozygous mice. Based on these findings, it was surmised that the requirement for normal Gli3 dosage in eye development is more important in the absence of normal Pax6 dosage, demonstrating their cooperative roles during ocular development [30]. The Pax6+/-;Gli3+/- mice also exhibited abnormal contact between the iris and the lens as well as between the lens and cornea [30] which suggests that the Pax6/Gli3 interaction may have affected the same processes as that of the Pax6/AP-2α interaction in our study. Creation and subsequent examination of a Tcfap2a/Gli3 mutant may reveal cooperation between these two transcription factors indicating that Pax6, AP-2α, and Gli3 may all be linked during lens development.

Examination of retinas in the single and double heterozygous mouse eyes demonstrated no gross morphological differences corresponding to what was observed in the lens. However, there may be subtle defects in vision that we are not able to detect due to the inherent lens defects present in these mice. Results obtained from this study indicate that lens morphogenesis may be more sensitive to Pax6 and AP-2α dosage than that of retinal development. This is supported by the fact that mice carrying a conditional deletion of Tcfap2a in the developing retina do not demonstrate phenotypic abnormalities [19] whereas Le-AP-2α mice, in which Tcfap2a is conditionally deleted from lens placode derivatives, exhibit both lens and corneal defects [14,15]. If in fact dosage effects are involved in retinal development, perhaps the loss of one copy of the Pax6 gene combined with a conditional knock out of Tcfap2a in the retina will help uncover this.

It has recently been shown that conditional deletion of Tcfap2a in the lens placode leads to a reduction of E-cadherin expression in the placode and its derivatives [14,15]. Furthermore, Pax6 is known to play a role in corneal epithelial cell adhesion [16]. Cell-cell adhesion molecules have also been implicated in regulating lens vesicle separation [31]. Thus, we investigated whether the hemizygous deletion of Tcfap2a and Pax6 would effect E-cadherin expression in the lens. E-cadherin immunostaining of the mutant lenses revealed positive staining in the lens epithelium and in the cells of the plaques in both the Pax6+/lacZ and Pax6+/lacZ/AP-2α+/- mice with no obvious difference in expression levels between the single and double heterozygotes. These findings indicate that deletion of both Tcfap2a alleles is needed to produce a reduction in E-cadherin expression in the ocular epithelium and further suggest that the E-cadherin gene is not likely to be cooperatively regulated by AP-2α and Pax6. Interestingly, recent studies involving conditional deletion of the E-cadherin gene in the lens have shown that this does not result in a lens stalk phenotype [32]. Thus, additional genes, perhaps those coregulated by Pax6 and AP-2α, are involved in regulating normal separation of the lens vesicle during development.

Apoptosis has been known to play a role during lens vesicle separation [21]. We, therefore, examined cell death using TUNEL staining in embryonic sections of Pax6+/lacZ and Pax6+/lacZ/AP-2α+/- eyes. No significant difference was observed between the single and double heterozygotes. Investigation of the levels of cellular proliferation in embryonic Pax6+/lacZ and Pax6+/lacZ/AP-2α+/- eye sections using Ki67 staining also showed no visible difference between the single and double heterozygotes. Together, these results indicate that cellular death and proliferation did not contribute to the more severe lens phenotype observed in double heterozygous mice.

Additional studies are required for determining the interaction between the Pax6 and AP-2α pathways, however, this study has provided initial evidence to support their cooperative roles during lens development. It is well known that Pax6 lies near the top of the genetic hierarchy in controlling lens and eye development [6,7]. More recently, Pax6 has also been associated with certain forms of cancer [33]. Similarly, while AP-2α has been shown to be important in ocular morphogenesis, it has been investigated in various forms of cancer [34,35]. In fact, the levels of both Pax6 and AP-2α have been used as prognostic indicators of glioblastoma, a malignant form of brain cancer [33,35]. Further elucidation of the interaction between Pax6 and AP-2α may therefore help lead to further advances in multiple fields including those for eye development and oncology.


This work was supported by National Institutes of Health Grants EY11910 (J.W.M.) and DE-12728 (T.W.) and Research to Prevent Blindness (J.W.M.). We thank Yu Ji and Morgan Singleton for assistance with mouse husbandry and genotyping as well as Dr. Peter Gruss and Dr. Martyn Goulding for providing us with the Pax6+/lacZ mice. We also thank Dr. Dhruva Dwivedi and Giuseppe Pontoriero for their helpful advice and guidance throughout this project.


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