October 2016
Volume 57, Issue 13
Open Access
Retinal Cell Biology  |   October 2016
Loss of Axin2 Causes Ocular Defects During Mouse Eye Development
Author Affiliations & Notes
  • Ashley Alldredge
    Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Sabine Fuhrmann
    Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Correspondence: Sabine Fuhrmann, Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, Vanderbilt University Medical Center, 11425 Langford MRB IV, 2213 Garland Avenue, Nashville, TN 37232, USA; sabine.fuhrmann@vanderbilt.edu
Investigative Ophthalmology & Visual Science October 2016, Vol.57, 5253-5262. doi:10.1167/iovs.15-18599
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      Ashley Alldredge, Sabine Fuhrmann; Loss of Axin2 Causes Ocular Defects During Mouse Eye Development. Invest. Ophthalmol. Vis. Sci. 2016;57(13):5253-5262. doi: 10.1167/iovs.15-18599.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose: The scaffold protein Axin2 is an antagonist and universal target of the Wnt/β-catenin pathway. Disruption of Axin2 may lead to developmental eye defects; however, this has not been examined. The purpose of this study was to investigate the role of Axin2 during ocular and extraocular development in mouse.

Methods: Animals heterozygous and homozygous for a Axin2lacZ knock-in allele were analyzed at different developmental stages for reporter expression, morphology as well as for the presence of ocular and extraocular markers using histologic and immunohistochemical techniques.

Results: During early eye development, the Axin2lacZ reporter was expressed in the periocular mesenchyme, RPE, and optic stalk. In the developing retina, Axin2lacZ reporter expression was initiated in ganglion cells at late embryonic stages and robustly expressed in subpopulations of amacrine and horizontal cells postnatally. Activation of the Axin2lacZ reporter overlapped with labeling of POU4F1, PAX6, and Calbindin. Germline deletion of Axin2 led to variable ocular phenotypes ranging from normal to severely defective eyes exhibiting microphthalmia, coloboma, lens defects, and expanded ciliary margin. These defects were correlated with abnormal tissue patterning in individual affected tissues, such as the optic fissure margins in the ventral optic cup and in the expanded ciliary margin.

Conclusions: Our results reveal a critical role for Axin2 during ocular development, likely by restricting the activity of the Wnt/β-catenin pathway.

Signaling by Wnt/β-catenin controls several critical processes in the developing and adult vertebrate eye; it regulates proliferation, differentiation, dorsoventral patterning, differentiation of the RPE, ciliary body, and iris, and modulates lens development and retinal angiogenesis.121 In addition, the pathway is fine-tuned by antagonists that are critical for normal eye development,2231 and can be activated by Wnt-unrelated agonists (e.g., Norrin).32 In mice and humans, 19 Wnt ligands are identified that bind to several surface receptors, including the Frizzled family of transmembrane proteins that activate different pathways.33 The canonical Wnt/β-catenin pathway is the best characterized and functions through stabilization of β-catenin, its translocation into the nucleus, and activation of T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors. Axis inhibition proteins (AXIN1, AXIN2) act as scaffold proteins and associate with the Wnt signaling components Dishevelled, the serine/threonine kinase GSK3b, APC, and CKI to control β-catenin degradation3439 (for reviews, see Refs. 4042). In the absence of a Wnt signal, this “destruction” complex phosphorylates β-catenin, thereby targeting it for subsequent degradation through the ubiquitin-proteasome pathway. Activation of Frizzled receptors and LRP5/6 coreceptors perturbs formation of the destruction complex, allowing stable β-catenin to accumulate in the cytoplasm. Axis inhibition 1 (Axin1) is ubiquitously expressed and loss of function in mouse results in early embryonic lethality; Axin2 (conductin, axil) expression is more restrictive and is transcriptionally activated by Wnt/β-catenin signaling.43,44 Thereby, AXIN2 acts as a negative feedback regulator, and its activity is modulated by tankyrase and CDC20.43,4547 Consistent with being a Wnt target, lineage tracing in mouse revealed that Axin2 is expressed in cell populations responsive to Wnt/β-catenin signaling, often in cells with stem cell capacity.4851 In humans, Axin2 mutations are associated with colorectal cancer and oligodontia.52,53 Mice with an inactivated Axin2 gene survive, with defects in skull formation (premature fusion of the posterior-frontal suture, reminiscent of craniosynostosis in humans) and bone remodeling.5457 
Transgenic mouse reporter lines have been used to determine Wnt/β-catenin pathway activity during mouse eye development. Possibly due to variegation effects, expression in the developing and adult retina varies among the different reporter lines.5861 Importantly, we demonstrated in the TOPgal line that this TCF/LEF reporter is activated in embryonic retinal progenitor cells in the absence of β-catenin expression.62 Thus, TCF/LEF reporter lines can be expressed independently of Wnt/β-catenin signaling, which confounds a faithful analysis of Wnt/β-catenin activation. 
To obtain a more accurate and comprehensive picture of Wnt/β-catenin activation during embryonic and postnatal eye development, we analyzed expression of the Axin2lacZ reporter, which drives expression of LacZ from the endogenous Axin2 locus.63 It was generated by inserting lacZ into the endogenous start codon, thereby replacing most of exon 2 and inactivating the Axin2 gene.63 Our results show that Axin2lacZ activation starts during late embryogenesis in ganglion cells, and is postnatally upregulated in horizontal cells and amacrine cells, and occasionally in photoreceptors. Furthermore, it was recently noted that Axin2lacZ/lacZ mice display ocular abnormalities, but a detailed investigation is lacking.55,57 Here, we demonstrate that disruption of Axin2 results in severe ocular defects during optic cup morphogenesis, such as abnormal development of the anterior segment and a defect in closure of the optic fissure. 
Materials and Methods
Mice
Axin2lacZ mice were obtained from Jackson Laboratory (City, State, Country) and maintained on a C57BL/6 genetic background (Charles River, Hollister, CA, USA).63 Animals heterozygous and homozygous for the Axin2lacZ allele are here referred to as Axin2lacZ and Axin2lacZ/lacZ mice, respectively. Noon on the day of detection of the vaginal plug is counted embryonic day 0.5 (E0.5). Animals were genotyped by PCR using the following primer combinations: Cs: 5′-AAG CTG CGT CGG ATA CTT GAG A-3′, Cwt: 5′-AGT CCA TCT TCA TTC CGC CTA GC-3′, and ClacZ: 5′-TGG TAA TGC TGC AGT GGC TTG-3′. These primers produce the Axin wt (493 bp) and Axin lacZ amplicons (400 bp).57 In the course of this study, we found that the Axin2lacZ mouse line contained the Rd8 mutation, which is caused by a mutation in Crb1.64,65 Rd8 can lead to postnatal retinal disorganization, obvious at postnatal day 14 (P14), and subsequent photoreceptor degeneration66; however, we did not observe abnormal lamination of the adult retina. We also did not detect differences in Axin2lacZ reporter expression in adult eyes between Rd8 heterozygous and Rd8 homozygous mutants. The reporter expression did vary in the photoreceptor layer of adult animals that did not correlate with Rd8 heterozygosity or homozygosity. Animal experiments were performed according to the guidelines of the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University of Utah Institutional Animal Care and Use Committee. 
X-gal Labeling
Embryos were fixed with 4% paraformaldehyde for 10 to 15 minutes (E11 embryos) or 20 minutes (postnatal eyes) at room temperature. Standard X-gal labeling was performed on whole embryos (E11) or directly on cryostat sections (adult eyes) for 12 to 24 hours at 30°C and postfixed with paraformaldehyde. 
Immunohistochemistry
Heads and eyes were fixed in 4% paraformaldehyde, cryoembedded, and sectioned, usually at 12 μm. If necessary, cryostat sections were treated for antigen retrieval with hot citrate buffer (pH 6) or with 1% Triton X-100. The following primary antibodies or markers were used: BRN3A/POU4F1 (1:50, #AB1585; EMD Millipore, Billerica, MA, USA), BRN3 (1:50, sc-6026; Santa Cruz Biotechnology, Dallas, TX, USA), CALB1 (1:500, #AB1778; EMD Millipore), F-actin/Phalloidin (1:500, #A12379; Life Technologies, Carlsbad, CA, USA), β-galactosidase (1:5000, #55976; Cappel/MP Biomedicals, Aurora, OH, USA), β-galactosidase (1:750, generous gift from Nadean Brown, University of California, Davis, CA, USA), Laminin (1:2000, #ab30320; Abcam, Cambridge, MA, USA), LEF1 (1:100, #C12A5; Cell Signaling, Danvers, MA, USA), MITF (1:400, #X1405M; Exalpha, Exalpha Biologicals, Shirley, MA, USA), OTX1/2 (1:1500, #NG1734326; EMD Millipore), PAX2 (1:100, #PRB-276P; Covance/BioLegend, Dedham, MA, USA), PAX6 (1:300, #AB2237; EMD Millipore), PITX2 (1:1000, #PA1020-100; Capra Science, Angelholm, Sweden), PROX1 (1:1000, #11-002; AngioBio Co., San Diego, CA, USA), SOX9 (1:50, #AB5535; EMD Millipore), TCF4 (1:100, #2569; Cell Signaling Technology), and VSX2 (1:300, #X1180P; Exalpha Biologicals). These antibodies were used in combination with the following secondary antibodies: Alexa 488/568/647–conjugated secondary antibodies (1:1000; Life Technologies), donkey-anti-goat TRITC (1:500, #705-025-147; Jackson ImmunoResearch, West Grove, PA, USA), and donkey-anti-sheep TRITC (1:500, #713-165-003; Jackson ImmmunoResearch). We analyzed 5 and 4 Axin2lacZ/lacZ embryos with uni- or bilateral ocular defects at E12.5 and E15.5, respectively (see also Results for further details). Each marker was analyzed in two to four tissue sections in central regions of each eye, and several markers were analyzed repeatedly. 
Imaging
Epifluorescent images were taken with an Olympus XM10 camera (Olympus, Tokyo, Japan) on an upright Olympus BX51 microscope and were processed in Adobe Photoshop (CS3) (Adobe Systems, Inc., San Jose, CA, USA). Confocal images were taken with an Olympus FV1000 and processed using ImageJ (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) and Adobe Photoshop (CS3). All other images were taken with an Olympus U-CMAD3/Microfire camera mounted on the aforementioned microscope or on an Olympus SZX12 stereomicroscope. 
Results
Reporter activation of Axin2lacZ was detectable in the dorsal and ventral RPE of the optic cup at E11 (Figs. 1A, 1B), consistent with previous results in transgenic TCF/LEF reporters.2,62,6769 However, in contrast to the TOPgal and TCF/LEF reporter lines, Axin2lacZ was not activated in the embryonic retina up to E14.5 (Fig. 1C).62,68,69 At this age, few individual cells in the ganglion cell layer (GCL) started to show expression, and the number of cells in the GCL increased subsequently (Fig. 1D). At early postnatal ages, cells in the inner nuclear layer (INL) began to express Axin2lacZ (Fig. 1E). At approximately P15, reporter activation was detectable in the GCL, and in diverse populations in the INL (Fig. 1F), and this pattern did not change significantly in the GCL and INL (Figs. 1G, 1H); however, occasionally robust labeling of the photoreceptor layer was observed in adult eyes (Fig. 1H). This variability in reporter activation did not correlate with the Rd8 mutation; we observed either presence or absence of this LacZ expression pattern in the photoreceptor layer in animals heterozygous as well as homozygous for Rd8. Thus, the reason for the variable Axin2lacZ reporter activation in adult photoreceptors is unclear. Interestingly, Liu et al.50 observed an increase of X-gal labeling in the outer nuclear layer (ONL) when retinal explants of Axin2lacZ reporter mice are stimulated with exogenous R-spondin that can activate Wnt/β-catenin signaling in the presence of Wnts. This suggests that the Axin2lacZ reporter expression pattern in photoreceptors in our study may be activated in response to endogenous levels of Wnt signaling. Thus, endogenous activation of Wnt signaling may vary between individual animals, causing variable labeling of the ONL as observed in Figures 1G and 1H. 
Figure 1
 
Dynamic activation of Axin2lacZ in the developing and mature mouse eye. Whole embryos, and coronal and transversal cryostat ocular sections were stained with X-gal substrate to detect β-galactosidase activity or labeled with a β-galactosidase antibody. (A) At E11.5, the Axin2lacZ reporter is activated in many regions of the mouse embryo, including the eye, limbs, facial primordia, and the dorsal brain. (B) In the E11.5 optic cup, Axin2lacZ is expressed in the dorsal and ventral RPE (arrows), extraocular and corneal mesenchyme (arrowheads), and in the optic stalk (asterisks). (C) At E14.5, Axin2lacZ expression is robust in the extraocular and eyelid mesenchyme (arrowheads). (D) At E17.5, expression is detectable cells in the differentiated/ presumptive GCL (arrows) and in the ciliary margin (arrowhead). (E) Shortly after birth (postnatal day 5: P5), cells in the GCL and several cells in the presumptive INL show reporter activation (arrowheads). (F) At P15, the Axin2lacZ reporter is robustly expressed in several cell populations in the INL and in cells in the GCL. (G, H) Axin2lacZ reporter activation in the adult retina of Rd8 homozygous mutant mice. (G) The Axin2lacZ expression pattern is largely maintained in the adult retina. (H) In addition, we observed several animals exhibiting variable Axin2lacZ reporter activation in the photoreceptor layer, shown here in most if not all photoreceptor cells (arrow). We found the presence or absence of this LacZ expression pattern in the photoreceptor layer in animals heterozygous and homozygous for Rd8; thus, we conclude that it did not correlate with Rd8. Scale bars: (A) 1 mm, in (B, C, D) 100 μm, in (E) 50 μm, in (G) 20 μm.
Figure 1
 
Dynamic activation of Axin2lacZ in the developing and mature mouse eye. Whole embryos, and coronal and transversal cryostat ocular sections were stained with X-gal substrate to detect β-galactosidase activity or labeled with a β-galactosidase antibody. (A) At E11.5, the Axin2lacZ reporter is activated in many regions of the mouse embryo, including the eye, limbs, facial primordia, and the dorsal brain. (B) In the E11.5 optic cup, Axin2lacZ is expressed in the dorsal and ventral RPE (arrows), extraocular and corneal mesenchyme (arrowheads), and in the optic stalk (asterisks). (C) At E14.5, Axin2lacZ expression is robust in the extraocular and eyelid mesenchyme (arrowheads). (D) At E17.5, expression is detectable cells in the differentiated/ presumptive GCL (arrows) and in the ciliary margin (arrowhead). (E) Shortly after birth (postnatal day 5: P5), cells in the GCL and several cells in the presumptive INL show reporter activation (arrowheads). (F) At P15, the Axin2lacZ reporter is robustly expressed in several cell populations in the INL and in cells in the GCL. (G, H) Axin2lacZ reporter activation in the adult retina of Rd8 homozygous mutant mice. (G) The Axin2lacZ expression pattern is largely maintained in the adult retina. (H) In addition, we observed several animals exhibiting variable Axin2lacZ reporter activation in the photoreceptor layer, shown here in most if not all photoreceptor cells (arrow). We found the presence or absence of this LacZ expression pattern in the photoreceptor layer in animals heterozygous and homozygous for Rd8; thus, we conclude that it did not correlate with Rd8. Scale bars: (A) 1 mm, in (B, C, D) 100 μm, in (E) 50 μm, in (G) 20 μm.
To identify retinal cell types expressing the Axin2lacZ reporter, we performed colabeling with several antibodies recognizing differentiated retinal cell populations in P15 to P21 eyes. The calcium-binding protein Calbindin (CALB1) is present in horizontal cells and amacrine cells in the INL and in the GCL (Fig. 2A).70 Activation of Axin2lacZ overlapped with CALB1-positive horizontal cells in the INL and few CALB1-positive amacrine cells in the GCL (Figs. 2B, 2C); Axin2lacZ was also expressed in PAX6-positive amacrine cells in the INL and GCL (Figs. 2D–F). In addition, Axin2lacZ reporter expression was observed in many ganglion cells that are labeled for the transcription factor POU4F1 (Figs. 2G–I). Furthermore, we investigated whether Müller glia labeled for SOX9 show Axin2lacZ expression. As previously reported,50 we observed little overlap (Figs. 2K, 2L). Finally, transcriptional activation downstream of Wnt/β-catenin signaling is mediated by TCF/LEF transcription factors. With the exception of a few cells, Axin2lacZ reporter-expressing cells did not show TCF4 colabeling (Figs. 2N–P). Because LEF1 is not detectable in the adult central retina (Supplementary Fig. S1), this suggests that other downstream effectors may be used in Axin2lacZ-expressing cells, such as TCF1; however, we were not able to find a satisfying TCF1 antibody. Overall, our results showed that the Axin2lacZ reporter is activated in ganglion and horizontal cells, as well as in amacrine cell populations. Subsequently, we examined the role of Axin2 in development of these cell populations by analyzing mice homozygous for the Axin2lacZ reporter (Axin2lacZ/lacZ), which results in global inactivation of the Axin2 gene. Interestingly, although we detected severe defects in early ocular development in some Axin2lacZ/lacZ mice (see below), no changes in differentiation of retinal cell populations labeled for PAX6, CALB1, POU4F1, TCF4, and SOX9 protein were observed in mutant eyes without obvious abnormalities (Supplementary Fig. S2). 
Figure 2
 
The Axin2lacZ reporter is present in specific cell populations in the postnatal retina. Transversal sections were double-labeled with antibodies detecting cell type–specific markers (red; A, D, G, K, N) and β-gal protein (green; B, E, H, L, O), merge shown in (C, F, I, M, P). (A–C) At P15, Axin2lacZ reporter-positive horizontal cells and cells in the GCL colabel for CALB1 (arrows and open arrowheads, respectively). (D–F) PAX6-positive amacrine cells (arrows) and cells in the GCL (arrowheads) show Axin2lacZ reporter activation at P15. (G–I) Reporter activation in P15 ganglion cells is confirmed by POU4F1 colabeling (arrowheads). (K–M) Very rarely, labeling of the Axin2lacZ reporter overlapped with the Müller Glia marker SOX9, shown here at P21 (arrows). (N–P) Few Axin2lacZ reporter-expressing cells colabel for TCF4 protein (P, arrows). Scale bars: (C) 30 μm, in (F, M) 20 μm, in (P) 10 μm.
Figure 2
 
The Axin2lacZ reporter is present in specific cell populations in the postnatal retina. Transversal sections were double-labeled with antibodies detecting cell type–specific markers (red; A, D, G, K, N) and β-gal protein (green; B, E, H, L, O), merge shown in (C, F, I, M, P). (A–C) At P15, Axin2lacZ reporter-positive horizontal cells and cells in the GCL colabel for CALB1 (arrows and open arrowheads, respectively). (D–F) PAX6-positive amacrine cells (arrows) and cells in the GCL (arrowheads) show Axin2lacZ reporter activation at P15. (G–I) Reporter activation in P15 ganglion cells is confirmed by POU4F1 colabeling (arrowheads). (K–M) Very rarely, labeling of the Axin2lacZ reporter overlapped with the Müller Glia marker SOX9, shown here at P21 (arrows). (N–P) Few Axin2lacZ reporter-expressing cells colabel for TCF4 protein (P, arrows). Scale bars: (C) 30 μm, in (F, M) 20 μm, in (P) 10 μm.
Homozygous Axin2 inactivation resulted in variable ocular defects in 36% of the Axin2lacZ/lacZ embryos and 25% of Axin2lacZ/lacZ eyes between E12.5 and E15.5 (Fig. 3; n = 44 embryos, 17 litters). We analyzed five Axin2lacZ/lacZ embryos with ocular defects in eight eyes at E12.5 and four Axin2lacZ/lacZ embryos with four abnormal eyes at E15.5. We observed coloboma, anterior segment with variable lens abnormalities and ventral optic cup defects in Axin2lacZ/lacZ embryos at E12.5 (Figs. 3A–F; see below). At later stages, eyelid closure defects were occasionally detectable and affected eyes can be severely microphthalmic (Figs. 3G–K). Axin2lacZ/lacZ mice showed cranial defects as previously described (craniosynostosis; Figs. 3I, 3K, arrowheads). 
Figure 3
 
Eye development defects in Axin2lacZ/lacZ embryos, in particular abnormal pigment and lens formation, coloboma and microphthalmia. (A) Lateral view of control eye at E12.5. (B–F) Range of severities of ocular phenotypes found in E12.5 Axin2lacZ/lacZ embryos, for example, hyperpigmentation (B, arrow) and coloboma (B, arrowhead) or, in rare cases, largely absent pigment and severe microphthalmia (C). (D) Control eye at higher magnification. (E) Mild ocular defect with ventral coloboma (arrowhead). (F) Severe hyperpigmentation dorsally, possibly ventral rotation of the eye resulting in severely reduced ventral optic cup (arrow). (G) Lateral view of control embryo at E17.5 showing closed eyelid. (H) Axin2lacZ/lacZ embryos at E17.5 with eyelid closure defect and hyperpigmentation. (I) Extreme microphthalmia in Axin2lacZ/lacZ embryos at E17.5. (J) Adult control animal at P23. (K) Adult Axin2lacZ/lacZ embryos with microphthalmia (arrow). White arrowheads in I, K point to cranial defects (craniosynostosis).
Figure 3
 
Eye development defects in Axin2lacZ/lacZ embryos, in particular abnormal pigment and lens formation, coloboma and microphthalmia. (A) Lateral view of control eye at E12.5. (B–F) Range of severities of ocular phenotypes found in E12.5 Axin2lacZ/lacZ embryos, for example, hyperpigmentation (B, arrow) and coloboma (B, arrowhead) or, in rare cases, largely absent pigment and severe microphthalmia (C). (D) Control eye at higher magnification. (E) Mild ocular defect with ventral coloboma (arrowhead). (F) Severe hyperpigmentation dorsally, possibly ventral rotation of the eye resulting in severely reduced ventral optic cup (arrow). (G) Lateral view of control embryo at E17.5 showing closed eyelid. (H) Axin2lacZ/lacZ embryos at E17.5 with eyelid closure defect and hyperpigmentation. (I) Extreme microphthalmia in Axin2lacZ/lacZ embryos at E17.5. (J) Adult control animal at P23. (K) Adult Axin2lacZ/lacZ embryos with microphthalmia (arrow). White arrowheads in I, K point to cranial defects (craniosynostosis).
Histologic and immunohistochemical analysis of the ocular defects in Axin2lacZ/lacZ embryos revealed that colobomatous eyes in Axin2lacZ/lacZ embryos exhibited a defect in closure of the optic fissure, confirmed by persistent presence of the basement membrane marker Laminin (Figs. 4A–D; six of six colobomatous eyes; n = 4 embryos). The transcription factor PAX2 is required for optic fissure closure during optic cup morphogenesis and Pax2 mutations in humans cause coloboma, among other developmental abnormalities. (for review, see Ref. 71) We observed that PAX2 labeling showed a largely normal pattern in both margins of the optic fissure of Axin2lacZ/lacZ optic cups (Fig. 4F; four of five colobomatous eyes; n = 3 embryos). In addition, apicobasal polarity appeared undisturbed, because F-actin showed normal apical distribution in the fissure margins (Fig. 4H; four of five colobomatous eyes; n = 3 embryos). The transcription factor Pitx2 is expressed in the periocular mesenchyme and positively regulated by Wnt/β-catenin signaling.72 Mutations in Pitx2 cause Axenfeld-Rieger syndrome resulting from abnormal anterior segment development, which can lead to coloboma. However, we observed no obvious changes in mesenchymal PITX2 labeling in Axin2lacZ/lacZ embryos (Fig. 4J; five of five colobomatous eyes; n = 3 embryos). 
Figure 4
 
Defects in closure of the optic fissure in Axin2lacZ/lacZ embryos. Sagittal views of E12.5 optic cups. (A, B) Hematoxylin and eosin–stained control (A) and Axin2lacZ/lacZ optic cup (B). Arrow points to the abnormally open optic fissure in the ventral optic cup of mutant eyes. (C) Laminin labeling in the basement membrane surrounds ocular tissues in control eyes and is not detectable in the optic fissure (asterisk). (D) Persistent Laminin labeling in the Axin2lacZ/lacZ ventral optic cup (arrow). Asterisk marks the optic fissure. (E, F) Pax2 is present in the ventral optic cup of controls (E) and Axin2lacZ/lacZ eyes (F, arrows). (G) F-Actin localization marks the apical border of retina and RPE in controls. (H) Apical distribution of F-Actin is maintained in Axin2lacZ/lacZ optic cups (arrow). (I) The transcription factor PITX2 is present in the extraocular mesenchyme surrounding the optic cup. (J) In Axin2lacZ/lacZ embryos, extraocular mesenchyme labeling of PITX2 appears normal. Scale bar: 100 μm.
Figure 4
 
Defects in closure of the optic fissure in Axin2lacZ/lacZ embryos. Sagittal views of E12.5 optic cups. (A, B) Hematoxylin and eosin–stained control (A) and Axin2lacZ/lacZ optic cup (B). Arrow points to the abnormally open optic fissure in the ventral optic cup of mutant eyes. (C) Laminin labeling in the basement membrane surrounds ocular tissues in control eyes and is not detectable in the optic fissure (asterisk). (D) Persistent Laminin labeling in the Axin2lacZ/lacZ ventral optic cup (arrow). Asterisk marks the optic fissure. (E, F) Pax2 is present in the ventral optic cup of controls (E) and Axin2lacZ/lacZ eyes (F, arrows). (G) F-Actin localization marks the apical border of retina and RPE in controls. (H) Apical distribution of F-Actin is maintained in Axin2lacZ/lacZ optic cups (arrow). (I) The transcription factor PITX2 is present in the extraocular mesenchyme surrounding the optic cup. (J) In Axin2lacZ/lacZ embryos, extraocular mesenchyme labeling of PITX2 appears normal. Scale bar: 100 μm.
Although general patterning of the optic cup neuroepithelium into retina and RPE appeared normal in Axin2lacZ/lacZ optic cups (see Fig. 6), we observed changes of gene expression particularly in cells lining the margins of the optic fissure (Fig. 5). Visual system homeobox 2 (VSX2) protein is normally found in retinal progenitors throughout the optic cup at E12.5 (Fig. 5A). Several affected Axin2lacZ/lacZ optic cups showed absent VSX2 labeling in cells comprising the margins of the open optic fissure (Fig. 5B; arrows; three of five colobomatous eyes, n = 4 embryos). Retinal pigment epithelium markers such as OTX and MITF are normally not present in the region of the optic fissure after closure is completed, except in the RPE in the ventral optic cup (Figs. 5C, 5E, 5G). Interestingly, in Axin2lacZ/lacZ optic cups, OTX1/2 and MITF were extended into the optic fissure margins that exhibited a defect in closure, suggesting that RPE tissue persists in the optic fissure (Figs. 5D–H) (MITF: eight of eight colobomatous eyes; n = 5 embryos; OTX2: five of eight colobomatous eyes; n = 5 embryos). Immunolabeling for LacZ did not reveal a consistent, robust increase in activity of the Axin2lacZ reporter, occasionally the reporter appeared weakly upregulated in the margins of Axin2lacZ/lacZ eyes (Fig. 5J, arrow; four of eight colobomatous eyes, n = 5 embryos). Normally LEF1 labeling is not robustly detectable in the ventral RPE in the optic fissure (Supplementary Fig. S3A), and we did not observe an increase of LEF1 in Axin2lacZ/lacZ eyes (Supplementary Fig. S3B). Thus, Wnt/β-catenin activation in the domain of persistent RPE in the optic fissure margins was not clearly detectable. 
Figure 5
 
Persistence of RPE tissue in the optic fissure margins in Axin2lacZ/lacZ ventral optic cups. Sagittal views of E12.5–E13.0 ventral optic cups. (A) Control embryo showing continuous VSX2 labeling in the ventral retina (arrows), also in the region of the fused optic fissure (asterisks). (B) In some of the affected Axin2lacZ/lacZ ventral optic cups, VSX2 labeling is discontinuous in the region of the optic fissure (arrows). (C) In controls, OTX1/2 labeling in the ventral optic cup is confined to the RPE and absent in the optic fissure (arrows). (D) The Axin2lacZ/lacZ ventral optic cup shows persistent OTX1/2 labeling in the margins of the optic fissure that is continuous with the RPE in the ventral optic cup (arrows). (E, F) Merge of VSX2 and OTX1/2 staining in control (E) and Axin2lacZ/lacZ optic cups (F). (G) Similar to OTX1/2, MITF is restricted to the ventral RPE and excluded from the optic fissure region in control embryos (arrows). (H) In the margins of the defective optic fissure of Axin2lacZ/lacZ embryos, MITF is persistent (arrows). (I, J) In Axin2lacZ/lacZ optic cups, Axin2lacZ reporter expression does not show robust upregulation in the optic fissure margins and in the RPE, as shown by β–GAL immunolabeling (J, arrows). Scale bar: 30 μm.
Figure 5
 
Persistence of RPE tissue in the optic fissure margins in Axin2lacZ/lacZ ventral optic cups. Sagittal views of E12.5–E13.0 ventral optic cups. (A) Control embryo showing continuous VSX2 labeling in the ventral retina (arrows), also in the region of the fused optic fissure (asterisks). (B) In some of the affected Axin2lacZ/lacZ ventral optic cups, VSX2 labeling is discontinuous in the region of the optic fissure (arrows). (C) In controls, OTX1/2 labeling in the ventral optic cup is confined to the RPE and absent in the optic fissure (arrows). (D) The Axin2lacZ/lacZ ventral optic cup shows persistent OTX1/2 labeling in the margins of the optic fissure that is continuous with the RPE in the ventral optic cup (arrows). (E, F) Merge of VSX2 and OTX1/2 staining in control (E) and Axin2lacZ/lacZ optic cups (F). (G) Similar to OTX1/2, MITF is restricted to the ventral RPE and excluded from the optic fissure region in control embryos (arrows). (H) In the margins of the defective optic fissure of Axin2lacZ/lacZ embryos, MITF is persistent (arrows). (I, J) In Axin2lacZ/lacZ optic cups, Axin2lacZ reporter expression does not show robust upregulation in the optic fissure margins and in the RPE, as shown by β–GAL immunolabeling (J, arrows). Scale bar: 30 μm.
Figure 6
 
Axin2lacZ/lacZ embryonic eyes show ectopic expansion of the peripheral optic cup. Coronal views of E15.5 optic cups. (A) Protein labeling for the homeobox transcription factor VSX2 in retinal progenitor cells in controls. (B) VSX2 labeling pattern in the central retina of Axin2lacZ/lacZ optic cups appears normal. Arrow points to the expanded area in the dorsal ciliary margin that exhibits reduced VSX2 labeling (dotted lines). (C, D) The paired homeobox transcription factor Pax6 is present in many ocular and extraocular tissues. (D) Pax6 labeling is found in the ectopic tissue in Axin2lacZ/lacZ dorsal optic cups (arrow). Arrowheads label the eyelid primordia. (E, F) Otx1/2 protein is normally found in the RPE, surface ectoderm, retinal progenitors, and ciliary margin. (F) In Axin2lacZ/lacZ embryos, the abnormally expanded region of the dorsal optic cup shows a OTX1/2 labeling pattern typical for the ciliary margin. (G, H) PROX1 labeling in the embryonic lens is not significantly altered in Axin2lacZ/lacZ embryos. (I, J) The HMG transcription factor LEF1 is present in the eyelid and corneal mesenchyme, and in the ciliary margin (arrowheads). (J) The abnormal expansion of the dorsal optic cup in Axin2lacZ/lacZ mutants shows increased LEF1 labeling (arrow). (K, L) PITX2 labeling in the extraocular mesenchyme is not altered in Axin2lacZ/lacZ embryos (L). Arrows in (B, D, F, H, J, L) point to abnormal, expanded area in the dorsal optic cup of Axin2lacZ/lacZ embryos. Scale bar: 300 μm.
Figure 6
 
Axin2lacZ/lacZ embryonic eyes show ectopic expansion of the peripheral optic cup. Coronal views of E15.5 optic cups. (A) Protein labeling for the homeobox transcription factor VSX2 in retinal progenitor cells in controls. (B) VSX2 labeling pattern in the central retina of Axin2lacZ/lacZ optic cups appears normal. Arrow points to the expanded area in the dorsal ciliary margin that exhibits reduced VSX2 labeling (dotted lines). (C, D) The paired homeobox transcription factor Pax6 is present in many ocular and extraocular tissues. (D) Pax6 labeling is found in the ectopic tissue in Axin2lacZ/lacZ dorsal optic cups (arrow). Arrowheads label the eyelid primordia. (E, F) Otx1/2 protein is normally found in the RPE, surface ectoderm, retinal progenitors, and ciliary margin. (F) In Axin2lacZ/lacZ embryos, the abnormally expanded region of the dorsal optic cup shows a OTX1/2 labeling pattern typical for the ciliary margin. (G, H) PROX1 labeling in the embryonic lens is not significantly altered in Axin2lacZ/lacZ embryos. (I, J) The HMG transcription factor LEF1 is present in the eyelid and corneal mesenchyme, and in the ciliary margin (arrowheads). (J) The abnormal expansion of the dorsal optic cup in Axin2lacZ/lacZ mutants shows increased LEF1 labeling (arrow). (K, L) PITX2 labeling in the extraocular mesenchyme is not altered in Axin2lacZ/lacZ embryos (L). Arrows in (B, D, F, H, J, L) point to abnormal, expanded area in the dorsal optic cup of Axin2lacZ/lacZ embryos. Scale bar: 300 μm.
At later embryonic ages, ectopic expansion of the optic cup periphery became more evident (Figs. 3H, 6). At E15.5, the ectopic tissue in the Axin2lacZ/lacZ optic cup margin did not show labeling for the retinal marker VSX2 (Fig. 6B; arrow; three of four affected eyes; n = 4 embryos). Paired box 6 (Pax6) protein was present in the ciliary margin of controls (Fig. 6C) and in the ectopic tissue in the margin of Axin2lacZ/lacZ optic cups (Fig. 6D; three of four affected eyes; n = 4 embryos). In control eyes, OTX1/2 protein is normally found in the RPE, retinal progenitors, and lower levels are present diffusely in the presumptive ciliary body and iris (Fig. 6E; arrow). In Axin2lacZ/lacZ eyes, the ectopic tissue in the dorsal optic cup showed a widespread OTX1/2 labeling pattern, reminiscent of the ciliary margin in controls (Fig. 6F; arrow; four of four affected eyes; n = 4 embryos). Compared with control eyes, lens vesicles appeared smaller in three of four affected Axin2lacZ/lacZ eyes and labeled for Pax6 and Prox1 protein (Figs. 6D, 6H), suggesting that some aspects of lens differentiation can be maintained. Elevated expression of the Wnt target LEF1 confirmed that the ectopic tissue in Axin2lacZ/lacZ eyes acquired a peripheral retina fate (Fig. 6J; arrow; three of four affected eyes; n = 4 embryos). Similar to E12.5, PITX2 showed largely normal expression in Axin2lacZ/lacZ mesenchyme (Fig. 6L; three of three affected eyes; n = 3 embryos). Collectively, our data suggest that the hyperplastic tissue in Axin2lacZ/lacZ eyes shows features of presumptive ciliary body and iris. 
Discussion
The purpose of our study was to characterize activation of the universal target and negative regulator of the Wnt/β-catenin pathway Axin2 in the developing mouse eye using the knock-in LacZ reporter Axin2lacZ.63 In addition, we investigated the ocular phenotype in Axin2lacZ/lacZ eyes. Our results demonstrate that Axin2lacZ is dynamically expressed; it becomes activated in subpopulations of diverse retinal cell types mostly during postnatal differentiation. During optic cup morphogenesis, Axin2lacZ is activated in the presumptive RPE, ciliary body, and iris, and in the extraocular mesenchyme. Furthermore, we show that Axin2 exerts distinct roles during ocular development; on disruption of the Axin2 gene, microphthalmia, coloboma, and an expanded ciliary margin are observed. These phenotypes occur with varying severity and incomplete penetrance, suggesting that other factors may interact with Axin2 to regulate ocular development.73 In humans, ocular defects have not been associated with Axin2 mutations, thus, it is possible that Axin2 has distinct, species-specific functions in the developing eye of mouse and humans. 
Optic Cup Morphogenesis Requires Proper Levels of Wnt Signaling
Axin2 acts as an inhibitor of the Wnt/β-catenin pathway; therefore, homozygous disruption in Axin2lacZ/lacZ mice is likely to result in increased nuclear localization of β-catenin and possibly in ectopic Wnt pathway activation.56,57,69 However, Axin2 is considered a relatively weak inhibitor,46 which could explain the variable penetrance and range of early ocular defects in Axin2lacZ/lacZ mutants. In Axin2lacZ/lacZ optic cups that display a closure defect of the optic fissure, we observed that labeling for the RPE markers OTX1/2 and MITF are persistent in the fissure margins. The Wnt/β-catenin pathway transcriptionally regulates expression of MITF and OTX2.1,2,4 Thus, we expected that loss of Axin2 leads directly to elevated Wnt activity resulting in abnormal maintenance or upregulation of RPE gene expression in the optic fissure margins; however, our results do not indicate a robust increase of Axin2 activation in the optic fissure. It is possible that the reporter is not sensitive enough to detect potentially small changes of pathway activation in the optic fissure margins. Alternatively, the ubiquitous Axin1 may compensate for loss of Axin2 and prevent significant upregulation of the pathway. Finally, we cannot exclude that Axin2 is regulated independent of Wnt/β-catenin signaling. 
Interestingly, disruption of FGF signaling, which is required for retinal patterning in the optic cup, shows a very similar effect of persistent RPE labeling in the optic fissure margins, associated with colobomata.74,75 Collectively, these and our present study show that proper acquisition of cell organization in the marginal cells of the optic fissure is crucial for the progression to closure and fusion. It will be interesting to further investigate the nature of the downstream molecular events that ensure subsequent attachment and fusion of the fissure margins. 
In addition, our results demonstrate that the expanded ciliary margin shows elevated LEF1 labeling, consistent with an enlarged domain of Wnt/β-catenin pathway activation in this area (Fig. 6J). The observed defects in the anterior segment in Axin2lacZ/lacZ eyes are in agreement with previous studies demonstrating that balanced levels of Wnt/β-catenin pathway activation are required for proper eye development. A secreted antagonist, Dkk1, binds to the coreceptor LRP5/6, resulting normally in downregulation of the pathway.22 Haploinsufficiency of Dkk1 leads to defects in optic cup morphogenesis similar to Axin2lacZ/lacZ mutants, such as microphthalmia, coloboma, lens abnormalities, and anterior segment defects.26 Similar abnormalities are observed on ablation of the forkhead transcription factor Foxg1.7678 The abnormal expansion of ciliary margin tissue in Foxg1 mutants could be explained by ectopic Wnt pathway activation. However, shh expression is decreased early in Foxg1 mutants, which may contribute to the ocular defects.76,77 In apc mutant zebrafish, an expanded ciliary margin zone and coloboma were observed.27,29 Consistent with a role in cell fate determination, ectopic Wnt/β-catenin pathway activation in the retina by constitutively active β-catenin leads to transdifferentiation into peripheral fate.5,79 Finally, several of the above-mentioned mouse models exhibit microphthalmia and coloboma to some degree, which may be due to ectopic Wnt/β-catenin activity, as discussed previously. Together with mouse models displaying reduced or absent Wnt/β-catenin pathway activity that are associated with coloboma and peripheral eye defects, our data are in agreement with the notion that Wnt/β-catenin activity needs to be tightly regulated during normal optic cup morphogenesis.12,21 
Comparison Between Axin2lacZ and Transgenic Wnt/β-Catenin Reporter Lines
Our study shows Axin2lacZ activation in the dorsal optic cup, extraocular mesenchyme, differentiating ciliary body and iris, and optic stalk in the embryonic eye, which is consistent with other transgenic Wnt/β-catenin reporter lines. In addition, we observed Axin2lacZ reporter expression in the differentiating cells of the GCL and inner INL in a pattern similar to the transgenic TCF/LEF line.69 Our results demonstrate that Axin2lacZ is activated in the adult retina in horizontal and occasionally in photoreceptor cells (Fig. 1), both of which are novel findings. However, a TCF/LEF-independent regulation of the Axin2 gene cannot be excluded, which could be responsible for some of the reporter activation in retinal cell populations.80,81 
In agreement with others, we observed very little overlap of Axin2lacZ with markers for Müller glia (this study).50 Under certain conditions, however, Müller glia show stem cell potential (for review, see Ref. 82) and exhibit Axin2lacZ reporter expression in retinal explants after exogenous Wnt pathway activation.50 Interestingly, ectopic Wnt pathway activation, due to complete inactivation of Axin2, causes Müller glia to proliferate and produce retinal progenitors.50 
Recently, it was shown that retinal amacrine cells expressing the G-protein–coupled receptor Lgr5 display regenerative potential.83 Lgr5 is a Wnt pathway target and bound by secreted R-spondin proteins to modulate Wnt pathway activation. It is possible that Axin2lacZ overlaps with expression of Lgr5 in amacrine cells, because both populations express Pax6 in a similar pattern (this study: Fig. 2F; Ref. 83: Supplementary Fig. S2G). Both Axin2 and Lgr5 are stem cell markers in other systems; thus, some of the adult expression pattern of Axin2lacZ may reflect a regenerative potential of retinal cells. Further studies are required to address the precise role of Axin2 in the diverse cell types in the adult retina. 
Acknowledgments
The authors thank Ed Levine for critical reading of the manuscript, Nadean Brown for providing antibodies, and members of the Fuhrmann and Ed Levine laboratories for technical support and helpful comments. 
Supported by National Institutes of Health Grants EY014954 and EY024373 (SF), Core Grants EY014800; and in part by Unrestricted Grants from Research to Prevent Blindness, Inc., New York, NY, to the Departments of Ophthalmology and Visual Sciences, University of Utah and Vanderbilt University. 
Disclosure: A. Alldredge, None; S. Fuhrmann None 
References
Westenskow PD, McKean JB, Kubo F, Nakagawa S, Fuhrmann S. Ectopic Mitf in the embryonic chick retina by co-transfection of beta-catenin and Otx2. Invest Ophthalmol Vis Sci. 2010; 51: 5328–5335.
Westenskow P, Piccolo S, Fuhrmann S. Beta-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression. Development. 2009; 136: 2505–2510.
Agathocleous M, Iordanova I, Willardsen MI, et al. A directional Wnt/beta-catenin-Sox2-proneural pathway regulates the transition from proliferation to differentiation in the Xenopus retina. Development. 2009; 136: 3289–3299.
Fujimura N, Taketo MM, Mori M, Korinek V, Kozmik Z. Spatial and temporal regulation of Wnt/beta-catenin signaling is essential for development of the retinal pigment epithelium. Dev Biol. 2009; 334: 31–45.
Cho SH, Cepko CL. Wnt2b/beta-catenin-mediated canonical Wnt signaling determines the peripheral fates of the chick eye. Development. 2006; 133: 3167–3177.
Kubo F, Takeichi M, Nakagawa S. Wnt2b controls retinal cell differentiation at the ciliary marginal zone. Development. 2003; 130: 587–598.
Hagglund AC, Berghard A, Carlsson L. Canonical Wnt/beta-catenin signalling is essential for optic cup formation. PLoS One. 2013; 8: e81158.
Liu H, Xu S, Wang Y, et al. Ciliary margin transdifferentiation from neural retina is controlled by canonical Wnt signaling. Dev Biol. 2007; 308: 54–67.
Ye X, Wang Y, Cahill H, et al. Norrin, frizzled-4 and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization. Cell. 2009; 139: 285–298.
Das AV, Bhattacharya S, Zhao X, et al. The canonical Wnt pathway regulates retinal stem cells/progenitors in concert with Notch signaling. Dev Neurosci. 2008; 30: 389–409.
Antosova B, Smolikova J, Borkovcova R, et al. Ectopic activation of Wnt/beta-catenin signaling in lens fiber cells results in cataract formation and aberrant fiber cell differentiation. PLoS One. 2013; 8: e78279.
Bankhead EJ, Colasanto MP, Dyorich KM, Jamrich M, Murtaugh LC, Fuhrmann S. Multiple requirements of the focal dermal hypoplasia gene porcupine during ocular morphogenesis. Am J Pathol. 2015; 185: 197–213.
Carpenter AC, Smith AN, Wagner H, et al. Wnt ligands from the embryonic surface ectoderm regulate ‘bimetallic strip' optic cup morphogenesis in mouse. Development. 2015; 142: 972–982.
Chen Y, Stump RJ, Lovicu FJ, Shimono A, McAvoy JW. Wnt signaling is required for organization of the lens fiber cell cytoskeleton and development of lens three-dimensional architecture. Dev Biol. 2008; 324: 161–176.
Grocott T, Johnson S, Bailey AP, Streit A. Neural crest cells organize the eye via TGF-beta and canonical Wnt signalling. Nat Commun. 2011; 2: 265.
Ouchi Y, Baba Y, Koso H, et al. beta-Catenin signaling regulates the timing of cell differentiation in mouse retinal progenitor cells. Mol Cell Neurosci. 2011; 46: 770–780.
Robitaille J, MacDonald ML, Kaykas A, et al. Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy. Nat Genet. 2002; 32: 326–330.
Smith AN, Miller LA, Song N, Taketo MM, Lang RA. The duality of beta-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm. Dev Biol. 2005; 285: 477–489.
Veien ES, Rosenthal JS, Kruse-Bend RC, Chien CB, Dorsky RI. Canonical Wnt signaling is required for the maintenance of dorsal retinal identity. Development. 2008; 135: 4101–4111.
Zhang J, Fuhrmann S, Vetter ML. A nonautonomous role for retinal frizzled-5 in regulating hyaloid vitreous vasculature development. Invest Ophthalmol Vis Sci. 2008; 49: 5561–5567.
Zhou CJ, Molotkov A, Song L, et al. Ocular coloboma and dorsoventral neuroretinal patterning defects in Lrp6 mutant eyes. Dev Dyn. 2008; 237: 3681–3689.
Glinka A, Wu W, Delius H, Monaghan AP, Blumenstock C, Niehrs C. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature. 1998; 391: 357–362.
Heisenberg CP, Houart C, Take-Uchi M, et al. A mutation in the Gsk3-binding domain of zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon and eyes to diencephalon. Genes Dev. 2001; 15: 1427–1434.
Hunter DD, Zhang M, Ferguson JW, Koch M, Brunken WJ. The extracellular matrix component WIF-1 is expressed during, and can modulate, retinal development. Mol Cell Neurosci. 2004; 27: 477–488.
Kim CH, Oda T, Itoh M, et al. Repressor activity of Headless/Tcf3 is essential for vertebrate head formation. Nature. 2000; 407: 913–916.
Lieven O, Ruther U. The Dkk1 dose is critical for eye development. Dev Biol. 2011; 355: 124–137.
Nadauld LD, Chidester S, Shelton DN, et al. Dual roles for adenomatous polyposis coli in regulating retinoic acid biosynthesis and Wnt during ocular development. Proc Natl Acad Sci U S A. 2006; 103: 13409–13414.
Ruiz JM, Rodriguez J, Bovolenta P. Growth and differentiation of the retina and the optic tectum in the medaka fish requires olSfrp5. Dev Neurobiol. 2009; 69: 617–632.
Stephens WZ, Senecal M, Nguyen M, Piotrowski T. Loss of adenomatous polyposis coli (apc) results in an expanded ciliary marginal zone in the zebrafish eye. Dev Dyn. 2010; 239: 2066–2077.
Vacik T, Stubbs JL, Lemke G. A novel mechanism for the transcriptional regulation of Wnt signaling in development. Genes Dev. 2011; 25: 1783–1795.
van de Water S, van de Wetering M, Joore J, et al. Ectopic Wnt signal determines the eyeless phenotype of zebrafish masterblind mutant. Development. 2001; 128: 3877–3888.
Xu Q, Wang Y, Dabdoub A, et al. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell. 2004; 116: 883–895.
Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell. 2012; 149: 1192–1205.
Behrens J, Jerchow BA, Wurtele M, et al. Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science. 1998; 280: 596–599.
Ikeda S, Kishida S, Yamamoto H, Murai H, Koyama S, Kikuchi A. Axin a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin. EMBO J. 1998; 17: 1371–1384.
Kishida S, Yamamoto H, Ikeda S, et al. Axin, a negative regulator of the wnt signaling pathway directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin. J Biol Chem. 1998; 273: 10823–10826.
Yamamoto H, Kishida S, Uochi T, et al. Axil, a member of the Axin family, interacts with both glycogen synthase kinase 3beta and beta-catenin and inhibits axis formation of Xenopus embryos. Mol Cell Biol. 1998; 18: 2867–2875.
Zeng L, Fagotto F, Zhang T, et al. The mouse Fused locus encodes Axin an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell. 1997; 90: 181–192.
Kim SE, Huang H, Zhao M, et al. Wnt stabilization of beta-catenin reveals principles for morphogen receptor-scaffold assemblies. Science. 2013; 340: 867–870.
MacDonald BT, He X. A finger on the pulse of Wnt receptor signaling. Cell Res. 2012; 22: 1410–1412.
Song X, Wang S, Li L. New insights into the regulation of Axin function in canonical Wnt signaling pathway. Protein Cell. 2014; 5: 186–193.
Tacchelly-Benites O, Wang Z, Yang E, Lee E, Ahmed Y. Toggling a conformational switch in Wnt/beta-catenin signaling: regulation of Axin phosphorylation. The phosphorylation state of Axin controls its scaffold function in two Wnt pathway protein complexes. Bioessays. 2013; 35: 1063–1070.
Jho EH, Zhang T, Domon C, Joo CK, Freund JN, Costantini F. Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2 a negative regulator of the signaling pathway. Mol Cell Biol. 2002; 22: 1172–1183.
Leung JY, Kolligs FT, Wu R, et al. Activation of AXIN2 expression by beta-catenin-T cell factor. A feedback repressor pathway regulating Wnt signaling. J Biol Chem. 2002; 277: 21657–21665.
Bernkopf DB, Hadjihannas MV, Behrens J. Negative-feedback regulation of the Wnt pathway by conductin/axin2 involves insensitivity to upstream signalling. J Cell Sci. 2015; 128: 33–39.
Hadjihannas MV, Bernkopf DB, Bruckner M, Behrens J. Cell cycle control of Wnt/beta-catenin signalling by conductin/axin2 through CDC20. EMBO Rep. 2012; 13: 347–354.
Huang SM, Mishina YM, Liu S, et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature. 2009; 461: 614–620.
Bowman AN, van Amerongen R, Palmer TD, Nusse R. Lineage tracing with Axin2 reveals distinct developmental and adult populations of Wnt/beta-catenin-responsive neural stem cells. Proc Natl Acad Sci U S A. 2013; 110: 7324–7329.
Lim X, Tan SH, Koh WL, et al. Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling. Science. 2013; 342: 1226–1230.
Liu B, Hunter DJ, Rooker S, et al. Wnt signaling promotes Müller cell proliferation and survival after injury. Invest Ophthalmol Vis Sci. 2013; 54: 444–453.
Zeng YA, Nusse R. Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture. Cell Stem Cell. 2010; 6: 568–577.
Lammi L, Arte S, Somer M, et al. Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer. Am J Hum Genet. 2004; 74: 1043–1050.
Marvin ML, Mazzoni SM, Herron CM, Edwards S, Gruber SB, Petty EM. AXIN2-associated autosomal dominant ectodermal dysplasia and neoplastic syndrome. Am J Med Genet A. 2011; 155A: 898–902.
Behr B, Longaker MT, Quarto N. Absence of endochondral ossification and craniosynostosis in posterior frontal cranial sutures of Axin2(-/-) mice. PLoS One. 2013; 8: e70240.
Dao DY, Yang X, Flick LM, Chen D, Hilton MJ, O'Keefe RJ. Axin2 regulates chondrocyte maturation and axial skeletal development. J Orthop Res. 2010; 28: 89–95.
Yan Y, Tang D, Chen M, et al. Axin2 controls bone remodeling through the beta-catenin-BMP signaling pathway in adult mice. J Cell Sci. 2009; 122: 3566–3578.
Yu HM, Jerchow B, Sheu TJ, et al. The role of Axin2 in calvarial morphogenesis and craniosynostosis. Development. 2005; 132: 1995–2005.
Ahrens MJ, Romereim S, Dudley AT. A re-evaluation of two key reagents for in vivo studies of Wnt signaling. Dev Dyn. 2011; 240: 2060–2068.
Al Alam D, Green M, Tabatabai Irani R, et al. Contrasting expression of canonical Wnt signaling reporters TOPGAL, BATGAL and Axin2(LacZ) during murine lung development and repair. PLoS One. 2011; 6: e23139.
Barolo S. Transgenic Wnt/TCF pathway reporters: all you need is Lef? Oncogene. 2006; 25: 7505–7511.
Fuhrmann S. Wnt signaling in eye organogenesis. Organogenesis. 2008; 4: 60–67.
Fuhrmann S, Riesenberg AN, Mathiesen AM, Brown EC, Vetter ML, Brown NL. Characterization of a transient TCF/LEF-responsive progenitor population in the embryonic mouse retina. Invest Ophthalmol Vis Sci. 2009; 50: 432–440.
Lustig B, Jerchow B, Sachs M, et al. Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol Cell Biol. 2002; 22: 1184–1193.
Mattapallil MJ, Wawrousek EF, Chan CC, et al. The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells and confounds ocular induced mutant phenotypes. Invest Ophthalmol Vis Sci. 2012; 53: 2921–2927.
Chang B, Hawes NL, Hurd RE, Davisson MT, Nusinowitz S, Heckenlively JR. Retinal degeneration mutants in the mouse. Vision Res. 2002; 42: 517–525.
Mehalow AK, Kameya S, Smith RS, et al. CRB1 is essential for external limiting membrane integrity and photoreceptor morphogenesis in the mammalian retina. Hum Mol Genet. 2003; 12: 2179–2189.
Burns CJ, Zhang J, Brown EC, et al. Investigation of Frizzled-5 during embryonic neural development in mouse. Dev Dyn. 2008; 237: 1614–1626.
Liu H, Mohamed O, Dufort D, Wallace VA. Characterization of Wnt signaling components and activation of the Wnt canonical pathway in the murine retina. Dev Dyn. 2003; 227: 323–334.
Liu H, Thurig S, Mohamed O, Dufort D, Wallace VA. Mapping canonical Wnt signaling in the developing and adult retina. Invest Ophthalmol Vis Sci. 2006; 47: 5088–5097.
Uesugi R, Yamada M, Mizuguchi M, Baimbridge KG, Kim SU. Calbindin D-28k and parvalbumin immunohistochemistry in developing rat retina. Exp Eye Res. 1992; 54: 491–499.
Gregory-Evans CY, Williams MJ, Halford S, Gregory-Evans K. Ocular coloboma: a reassessment in the age of molecular neuroscience. J Med Genet. 2004; 41: 881–891.
Zacharias AL, Gage PJ. Canonical Wnt/beta-catenin signaling is required for maintenance but not activation of Pitx2 expression in neural crest during eye development. Dev Dyn. 2010; 239: 3215–3225.
Attisano L, Wrana JL. Signal integration in TGF-beta, WNT and Hippo pathways. F1000Prime Rep. 2013; 5: 17.
Cai Z, Tao C, Li H, et al. Deficient FGF signaling causes optic nerve dysgenesis and ocular coloboma. Development. 2013; 140: 2711–2723.
Chen S, Li H, Gaudenz K, et al. Defective FGF signaling causes coloboma formation and disrupts retinal neurogenesis. Cell Res. 2013; 23: 254–273.
Fotaki V, Smith R, Pratt T, Price DJ. Foxg1 is required to limit the formation of ciliary margin tissue and Wnt/beta-catenin signalling in the developing nasal retina of the mouse. Dev Biol. 2013; 380: 299–313.
Huh S, Hatini V, Marcus RC, Li SC, Lai E. Dorsal-ventral patterning defects in the eye of BF-1-deficient mice associated with a restricted loss of shh expression. Dev Biol. 1999; 211: 53–63.
Xuan S, Baptista CA, Balas G, Tao W, Soares VC, Lai E. Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres. Neuron. 1995; 14: 1141–1152.
Liu B, Yu HM, Hsu W. Craniosynostosis caused by Axin2 deficiency is mediated through distinct functions of beta-catenin in proliferation and differentiation. Dev Biol. 2007; 301: 298–308.
Evans AL, Faial T, Gilchrist MJ, et al. Genomic targets of Brachyury (T) in differentiating mouse embryonic stem cells. PLoS One. 2012; 7: e33346.
Olsen AK, Coskun M, Bzorek M, et al. Regulation of APC and AXIN2 expression by intestinal tumor suppressor CDX2 in colon cancer cells. Carcinogenesis. 2013; 34: 1361–1369.
Gallina D, Todd L, Fischer AJ. A comparative analysis of Müller glia-mediated regeneration in the vertebrate retina. Exp Eye Res. 2014; 123: 121–130.
Chen M, Tian S, Glasgow NG, et al. Lgr5(+) amacrine cells possess regenerative potential in the retina of adult mice. Aging Cell. 2015; 14: 635–643.
Figure 1
 
Dynamic activation of Axin2lacZ in the developing and mature mouse eye. Whole embryos, and coronal and transversal cryostat ocular sections were stained with X-gal substrate to detect β-galactosidase activity or labeled with a β-galactosidase antibody. (A) At E11.5, the Axin2lacZ reporter is activated in many regions of the mouse embryo, including the eye, limbs, facial primordia, and the dorsal brain. (B) In the E11.5 optic cup, Axin2lacZ is expressed in the dorsal and ventral RPE (arrows), extraocular and corneal mesenchyme (arrowheads), and in the optic stalk (asterisks). (C) At E14.5, Axin2lacZ expression is robust in the extraocular and eyelid mesenchyme (arrowheads). (D) At E17.5, expression is detectable cells in the differentiated/ presumptive GCL (arrows) and in the ciliary margin (arrowhead). (E) Shortly after birth (postnatal day 5: P5), cells in the GCL and several cells in the presumptive INL show reporter activation (arrowheads). (F) At P15, the Axin2lacZ reporter is robustly expressed in several cell populations in the INL and in cells in the GCL. (G, H) Axin2lacZ reporter activation in the adult retina of Rd8 homozygous mutant mice. (G) The Axin2lacZ expression pattern is largely maintained in the adult retina. (H) In addition, we observed several animals exhibiting variable Axin2lacZ reporter activation in the photoreceptor layer, shown here in most if not all photoreceptor cells (arrow). We found the presence or absence of this LacZ expression pattern in the photoreceptor layer in animals heterozygous and homozygous for Rd8; thus, we conclude that it did not correlate with Rd8. Scale bars: (A) 1 mm, in (B, C, D) 100 μm, in (E) 50 μm, in (G) 20 μm.
Figure 1
 
Dynamic activation of Axin2lacZ in the developing and mature mouse eye. Whole embryos, and coronal and transversal cryostat ocular sections were stained with X-gal substrate to detect β-galactosidase activity or labeled with a β-galactosidase antibody. (A) At E11.5, the Axin2lacZ reporter is activated in many regions of the mouse embryo, including the eye, limbs, facial primordia, and the dorsal brain. (B) In the E11.5 optic cup, Axin2lacZ is expressed in the dorsal and ventral RPE (arrows), extraocular and corneal mesenchyme (arrowheads), and in the optic stalk (asterisks). (C) At E14.5, Axin2lacZ expression is robust in the extraocular and eyelid mesenchyme (arrowheads). (D) At E17.5, expression is detectable cells in the differentiated/ presumptive GCL (arrows) and in the ciliary margin (arrowhead). (E) Shortly after birth (postnatal day 5: P5), cells in the GCL and several cells in the presumptive INL show reporter activation (arrowheads). (F) At P15, the Axin2lacZ reporter is robustly expressed in several cell populations in the INL and in cells in the GCL. (G, H) Axin2lacZ reporter activation in the adult retina of Rd8 homozygous mutant mice. (G) The Axin2lacZ expression pattern is largely maintained in the adult retina. (H) In addition, we observed several animals exhibiting variable Axin2lacZ reporter activation in the photoreceptor layer, shown here in most if not all photoreceptor cells (arrow). We found the presence or absence of this LacZ expression pattern in the photoreceptor layer in animals heterozygous and homozygous for Rd8; thus, we conclude that it did not correlate with Rd8. Scale bars: (A) 1 mm, in (B, C, D) 100 μm, in (E) 50 μm, in (G) 20 μm.
Figure 2
 
The Axin2lacZ reporter is present in specific cell populations in the postnatal retina. Transversal sections were double-labeled with antibodies detecting cell type–specific markers (red; A, D, G, K, N) and β-gal protein (green; B, E, H, L, O), merge shown in (C, F, I, M, P). (A–C) At P15, Axin2lacZ reporter-positive horizontal cells and cells in the GCL colabel for CALB1 (arrows and open arrowheads, respectively). (D–F) PAX6-positive amacrine cells (arrows) and cells in the GCL (arrowheads) show Axin2lacZ reporter activation at P15. (G–I) Reporter activation in P15 ganglion cells is confirmed by POU4F1 colabeling (arrowheads). (K–M) Very rarely, labeling of the Axin2lacZ reporter overlapped with the Müller Glia marker SOX9, shown here at P21 (arrows). (N–P) Few Axin2lacZ reporter-expressing cells colabel for TCF4 protein (P, arrows). Scale bars: (C) 30 μm, in (F, M) 20 μm, in (P) 10 μm.
Figure 2
 
The Axin2lacZ reporter is present in specific cell populations in the postnatal retina. Transversal sections were double-labeled with antibodies detecting cell type–specific markers (red; A, D, G, K, N) and β-gal protein (green; B, E, H, L, O), merge shown in (C, F, I, M, P). (A–C) At P15, Axin2lacZ reporter-positive horizontal cells and cells in the GCL colabel for CALB1 (arrows and open arrowheads, respectively). (D–F) PAX6-positive amacrine cells (arrows) and cells in the GCL (arrowheads) show Axin2lacZ reporter activation at P15. (G–I) Reporter activation in P15 ganglion cells is confirmed by POU4F1 colabeling (arrowheads). (K–M) Very rarely, labeling of the Axin2lacZ reporter overlapped with the Müller Glia marker SOX9, shown here at P21 (arrows). (N–P) Few Axin2lacZ reporter-expressing cells colabel for TCF4 protein (P, arrows). Scale bars: (C) 30 μm, in (F, M) 20 μm, in (P) 10 μm.
Figure 3
 
Eye development defects in Axin2lacZ/lacZ embryos, in particular abnormal pigment and lens formation, coloboma and microphthalmia. (A) Lateral view of control eye at E12.5. (B–F) Range of severities of ocular phenotypes found in E12.5 Axin2lacZ/lacZ embryos, for example, hyperpigmentation (B, arrow) and coloboma (B, arrowhead) or, in rare cases, largely absent pigment and severe microphthalmia (C). (D) Control eye at higher magnification. (E) Mild ocular defect with ventral coloboma (arrowhead). (F) Severe hyperpigmentation dorsally, possibly ventral rotation of the eye resulting in severely reduced ventral optic cup (arrow). (G) Lateral view of control embryo at E17.5 showing closed eyelid. (H) Axin2lacZ/lacZ embryos at E17.5 with eyelid closure defect and hyperpigmentation. (I) Extreme microphthalmia in Axin2lacZ/lacZ embryos at E17.5. (J) Adult control animal at P23. (K) Adult Axin2lacZ/lacZ embryos with microphthalmia (arrow). White arrowheads in I, K point to cranial defects (craniosynostosis).
Figure 3
 
Eye development defects in Axin2lacZ/lacZ embryos, in particular abnormal pigment and lens formation, coloboma and microphthalmia. (A) Lateral view of control eye at E12.5. (B–F) Range of severities of ocular phenotypes found in E12.5 Axin2lacZ/lacZ embryos, for example, hyperpigmentation (B, arrow) and coloboma (B, arrowhead) or, in rare cases, largely absent pigment and severe microphthalmia (C). (D) Control eye at higher magnification. (E) Mild ocular defect with ventral coloboma (arrowhead). (F) Severe hyperpigmentation dorsally, possibly ventral rotation of the eye resulting in severely reduced ventral optic cup (arrow). (G) Lateral view of control embryo at E17.5 showing closed eyelid. (H) Axin2lacZ/lacZ embryos at E17.5 with eyelid closure defect and hyperpigmentation. (I) Extreme microphthalmia in Axin2lacZ/lacZ embryos at E17.5. (J) Adult control animal at P23. (K) Adult Axin2lacZ/lacZ embryos with microphthalmia (arrow). White arrowheads in I, K point to cranial defects (craniosynostosis).
Figure 4
 
Defects in closure of the optic fissure in Axin2lacZ/lacZ embryos. Sagittal views of E12.5 optic cups. (A, B) Hematoxylin and eosin–stained control (A) and Axin2lacZ/lacZ optic cup (B). Arrow points to the abnormally open optic fissure in the ventral optic cup of mutant eyes. (C) Laminin labeling in the basement membrane surrounds ocular tissues in control eyes and is not detectable in the optic fissure (asterisk). (D) Persistent Laminin labeling in the Axin2lacZ/lacZ ventral optic cup (arrow). Asterisk marks the optic fissure. (E, F) Pax2 is present in the ventral optic cup of controls (E) and Axin2lacZ/lacZ eyes (F, arrows). (G) F-Actin localization marks the apical border of retina and RPE in controls. (H) Apical distribution of F-Actin is maintained in Axin2lacZ/lacZ optic cups (arrow). (I) The transcription factor PITX2 is present in the extraocular mesenchyme surrounding the optic cup. (J) In Axin2lacZ/lacZ embryos, extraocular mesenchyme labeling of PITX2 appears normal. Scale bar: 100 μm.
Figure 4
 
Defects in closure of the optic fissure in Axin2lacZ/lacZ embryos. Sagittal views of E12.5 optic cups. (A, B) Hematoxylin and eosin–stained control (A) and Axin2lacZ/lacZ optic cup (B). Arrow points to the abnormally open optic fissure in the ventral optic cup of mutant eyes. (C) Laminin labeling in the basement membrane surrounds ocular tissues in control eyes and is not detectable in the optic fissure (asterisk). (D) Persistent Laminin labeling in the Axin2lacZ/lacZ ventral optic cup (arrow). Asterisk marks the optic fissure. (E, F) Pax2 is present in the ventral optic cup of controls (E) and Axin2lacZ/lacZ eyes (F, arrows). (G) F-Actin localization marks the apical border of retina and RPE in controls. (H) Apical distribution of F-Actin is maintained in Axin2lacZ/lacZ optic cups (arrow). (I) The transcription factor PITX2 is present in the extraocular mesenchyme surrounding the optic cup. (J) In Axin2lacZ/lacZ embryos, extraocular mesenchyme labeling of PITX2 appears normal. Scale bar: 100 μm.
Figure 5
 
Persistence of RPE tissue in the optic fissure margins in Axin2lacZ/lacZ ventral optic cups. Sagittal views of E12.5–E13.0 ventral optic cups. (A) Control embryo showing continuous VSX2 labeling in the ventral retina (arrows), also in the region of the fused optic fissure (asterisks). (B) In some of the affected Axin2lacZ/lacZ ventral optic cups, VSX2 labeling is discontinuous in the region of the optic fissure (arrows). (C) In controls, OTX1/2 labeling in the ventral optic cup is confined to the RPE and absent in the optic fissure (arrows). (D) The Axin2lacZ/lacZ ventral optic cup shows persistent OTX1/2 labeling in the margins of the optic fissure that is continuous with the RPE in the ventral optic cup (arrows). (E, F) Merge of VSX2 and OTX1/2 staining in control (E) and Axin2lacZ/lacZ optic cups (F). (G) Similar to OTX1/2, MITF is restricted to the ventral RPE and excluded from the optic fissure region in control embryos (arrows). (H) In the margins of the defective optic fissure of Axin2lacZ/lacZ embryos, MITF is persistent (arrows). (I, J) In Axin2lacZ/lacZ optic cups, Axin2lacZ reporter expression does not show robust upregulation in the optic fissure margins and in the RPE, as shown by β–GAL immunolabeling (J, arrows). Scale bar: 30 μm.
Figure 5
 
Persistence of RPE tissue in the optic fissure margins in Axin2lacZ/lacZ ventral optic cups. Sagittal views of E12.5–E13.0 ventral optic cups. (A) Control embryo showing continuous VSX2 labeling in the ventral retina (arrows), also in the region of the fused optic fissure (asterisks). (B) In some of the affected Axin2lacZ/lacZ ventral optic cups, VSX2 labeling is discontinuous in the region of the optic fissure (arrows). (C) In controls, OTX1/2 labeling in the ventral optic cup is confined to the RPE and absent in the optic fissure (arrows). (D) The Axin2lacZ/lacZ ventral optic cup shows persistent OTX1/2 labeling in the margins of the optic fissure that is continuous with the RPE in the ventral optic cup (arrows). (E, F) Merge of VSX2 and OTX1/2 staining in control (E) and Axin2lacZ/lacZ optic cups (F). (G) Similar to OTX1/2, MITF is restricted to the ventral RPE and excluded from the optic fissure region in control embryos (arrows). (H) In the margins of the defective optic fissure of Axin2lacZ/lacZ embryos, MITF is persistent (arrows). (I, J) In Axin2lacZ/lacZ optic cups, Axin2lacZ reporter expression does not show robust upregulation in the optic fissure margins and in the RPE, as shown by β–GAL immunolabeling (J, arrows). Scale bar: 30 μm.
Figure 6
 
Axin2lacZ/lacZ embryonic eyes show ectopic expansion of the peripheral optic cup. Coronal views of E15.5 optic cups. (A) Protein labeling for the homeobox transcription factor VSX2 in retinal progenitor cells in controls. (B) VSX2 labeling pattern in the central retina of Axin2lacZ/lacZ optic cups appears normal. Arrow points to the expanded area in the dorsal ciliary margin that exhibits reduced VSX2 labeling (dotted lines). (C, D) The paired homeobox transcription factor Pax6 is present in many ocular and extraocular tissues. (D) Pax6 labeling is found in the ectopic tissue in Axin2lacZ/lacZ dorsal optic cups (arrow). Arrowheads label the eyelid primordia. (E, F) Otx1/2 protein is normally found in the RPE, surface ectoderm, retinal progenitors, and ciliary margin. (F) In Axin2lacZ/lacZ embryos, the abnormally expanded region of the dorsal optic cup shows a OTX1/2 labeling pattern typical for the ciliary margin. (G, H) PROX1 labeling in the embryonic lens is not significantly altered in Axin2lacZ/lacZ embryos. (I, J) The HMG transcription factor LEF1 is present in the eyelid and corneal mesenchyme, and in the ciliary margin (arrowheads). (J) The abnormal expansion of the dorsal optic cup in Axin2lacZ/lacZ mutants shows increased LEF1 labeling (arrow). (K, L) PITX2 labeling in the extraocular mesenchyme is not altered in Axin2lacZ/lacZ embryos (L). Arrows in (B, D, F, H, J, L) point to abnormal, expanded area in the dorsal optic cup of Axin2lacZ/lacZ embryos. Scale bar: 300 μm.
Figure 6
 
Axin2lacZ/lacZ embryonic eyes show ectopic expansion of the peripheral optic cup. Coronal views of E15.5 optic cups. (A) Protein labeling for the homeobox transcription factor VSX2 in retinal progenitor cells in controls. (B) VSX2 labeling pattern in the central retina of Axin2lacZ/lacZ optic cups appears normal. Arrow points to the expanded area in the dorsal ciliary margin that exhibits reduced VSX2 labeling (dotted lines). (C, D) The paired homeobox transcription factor Pax6 is present in many ocular and extraocular tissues. (D) Pax6 labeling is found in the ectopic tissue in Axin2lacZ/lacZ dorsal optic cups (arrow). Arrowheads label the eyelid primordia. (E, F) Otx1/2 protein is normally found in the RPE, surface ectoderm, retinal progenitors, and ciliary margin. (F) In Axin2lacZ/lacZ embryos, the abnormally expanded region of the dorsal optic cup shows a OTX1/2 labeling pattern typical for the ciliary margin. (G, H) PROX1 labeling in the embryonic lens is not significantly altered in Axin2lacZ/lacZ embryos. (I, J) The HMG transcription factor LEF1 is present in the eyelid and corneal mesenchyme, and in the ciliary margin (arrowheads). (J) The abnormal expansion of the dorsal optic cup in Axin2lacZ/lacZ mutants shows increased LEF1 labeling (arrow). (K, L) PITX2 labeling in the extraocular mesenchyme is not altered in Axin2lacZ/lacZ embryos (L). Arrows in (B, D, F, H, J, L) point to abnormal, expanded area in the dorsal optic cup of Axin2lacZ/lacZ embryos. Scale bar: 300 μm.
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