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First published online 11 April 2007
doi: 10.1242/dev.001495

Development 134, 1873-1885 (2007)
Published by The Company of Biologists 2007
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pygopus 2 has a crucial, Wnt pathway-independent function in lens induction

Ni Song1,2,3,4, Kristopher R. Schwab5, Larry T. Patterson6, Terry Yamaguchi7, Xinhua Lin2,4, Steven S. Potter2,4 and Richard A. Lang1,2,3,4,*

1 Divisions of Pediatric Ophthalmology, Children's Hospital Research Foundation, Cincinnati, OH45229, USA.
2 Divisions of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, OH45229, USA.
3 Department of Ophthalmology, College of Medicine, University of Cincinnati, Cincinnati, OH45229, USA.
4 Graduate Program of Molecular and Developmental Biology, College of Medicine, University of Cincinnati, Cincinnati, OH45229, USA.
5 Department of Pathology, University of Michigan, 109 Zina Pitcher Drive, Ann Arbor, MI48109, USA.
6 Division of Nephrology, Children's Hospital Research Foundation, Cincinnati, OH45229, USA.
7 Cancer and Developmental Biology Laboratory, Cell Signaling in Vertebrate Development Section, National Cancer Institute, Frederick, MD 21701-1201, USA.


Figure 1
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  		Fig. 1. Pygo1 and Pygo2 mutant alleles and expression of Pygo2 in the early mouse eye. (A,C) Gene targeting for Pygo1 and Pygo2 alleles. Green and gray boxes represent coding and non-coding exons, respectively. Light orange box indicates positive selection marker Pgk-Neo. Dark orange bars are frt sites. Blue arrowheads denote loxP sites. The left and right targeting arms (LTA and RTA), N box, PHD domain and nuclear localization signal (NLS) are indicated. Exons are numbered. Black arrows indicate the location of amplification primers used in B,D. (B,D) PCR amplification of genomic DNA or cDNA (the lower panel in D) for Pygo1 (B) or Pygo2 (D) allelic series. (E-J) Immunofluorescence for Pygo2 (E,F,H,I) or Pax6 (G,J) in eye region cryosections from the embryonic stages and genotypes indicated. A gray line between panels indicates that different channels of the same image are displayed. ple, presumptive lens ectoderm; op, optic pit; lp, lens placode; ov, optic vesicle.

 

Figure 2
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  		Fig. 2. Pygo2 is required for lens development in the mouse. (A-C) Hematoxylin and Eosin-stained sections of eyes in wild-type (WT) and Pygo2-/- mice show mildly (B, small lens) and severely (C, no lens) affected mutants. (D-F) GFP fluorescence in whole-mount embryos again showing small (E) or absent (F) lenses in the mutant. (G-I) Eye region cryosections labeled for the lens marker ß-crystallin (green) and for nuclei with Hoechst 33258 (blue). A small group of ß-crystallin-positive cells is indicated by the arrow in I. (J,L) Whole-mount embryos showing the WT and gross Pygo2-/- phenotype. The arrow in L indicates pigmented tissue within the eye cup. (K) Box plot of the lens:optic cup diameter ratio in allelic series of Pygo1 and Pygo2 mutants. Each bar represents measurements from ten eyes in five embryos. One-way ANOVA indicated statistically significant changes as compared with the wild-type control (**, P≤0.01≥0). ret, retina; mes, mesenchyme; pce, presumptive conjunctival epithelium; pr, presumptive retina.

 

Figure 3
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  		Fig. 3. Pygo2 regulates Pax6 expression in the mouse lens placode. (A-P) Eye region cryosections labeled for nuclei with Hoechst 33258 (A-N, blue), Pax6 (A-F, red), GFP (G,H, green), Sox2 (I,J, red), Pygo2 (K-N, red) or unlabeled differential interference contrast (DIC) images (O,P). The arrowhead in N (lower panel) indicates a small group of Pygo2-labeled cells in the presumptive lens. ple, presumptive lens ectoderm; ov, optic vesicle; lp, lens placode; pi, lens pit; pr, presumptive retina. A gray line between panels indicates that different channels of the same image are displayed. (Q) Box plot showing the ratio of lens to optic cup diameter in E12.5 embryos of the indicated genotypes. Mutant values (green and red bars) were significantly different (**, P≤0.01) from the wild-type and Le-cre controls (yellow bars). In addition, the Le-cre conditional mutant (green bar) is significantly different (**, P≤0.01) from the germline null (red bar). The wild-type control value incorporates the ten samples from Fig. 2K and a new set of 12 control values from this experiment which yielded the Le-cre conditional mutants. Germline null values are reproduced from Fig. 2K for the purposes of comparison.

 

Figure 4
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  		Fig. 4. Mesenchymal Pygo2 participates in lens development. (A,C,G,I) Whole-mount mouse embryos visualized for GFP (A,C) or in bright-field (G,I). (B,D-F,H,J-N) Unlabeled, DIC-illuminated cryosections (H,J), and cryosections labeled for nuclei with Hoechst 33258 (B,D-F, blue), Ap2{alpha} (B,D,K-N, red), GFP (B,D, green), Pygo2 (E,F, red) or Pax6 (K-N, green). A gray line between panels indicates that different channels of the same image are displayed. In D, the red channel within the dashed box has been enhanced to show weak Ap2{alpha} immunoreactivity in the ocular mesenchyme, the asterisks indicate GFP-negative blood vessels, and the arrow indicates remaining mesenchymal cells between presumptive lens and retinal epithelia. In E,F, the dashed line encloses approximately equivalent regions of the OM. In F, arrows point to Pygo2-positive blood vessels. In M,N are shown the green (Pax6) and red (Ap2{alpha}) channel intensities for a line interval passing through the nuclei of the lens placode. The bracket indicates the regions in the lens placode with a reduced Pax6:Ap2{alpha} ratio in the Pygo2 mutant. ple, presumptive lens ectoderm; ov, optic vesicle; om, ocular mesenchyme; lp, lens placode; pr, presumptive retina.

 

Figure 5
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  		Fig. 5. Mesenchymal and ectodermal Pygo2 cooperate in mouse lens development. (A,C,G,I) Whole-mount embryos unstained (G,I) or visualized for GFP (A,C). (B,D-F,H,J,K-N) Unlabeled, DIC-illuminated cryosections (H,J) and cryosections labeled for nuclei (B,E,F, blue), GFP (B,D, green), F-actin (D, red), Pygo2 (E,F, red), Pax6 (K-N, green) and Ap2{alpha} (K-N, red). In E,F, the dashed line encloses approximately equivalent regions of mesenchyme and surface ectoderm. In M,N are shown the green (Pax6) and red (Ap2{alpha}) channel intensities for a line interval passing through the nuclei of the lens placode. ov, optic vesicle; 1ba, first branchial arch; otv, otic vesicle; ple, presumptive lens ectoderm; om, ocular mesenchyme; lp, lens placode; pr, presumptive retina. A gray line between panels indicates that different channels of the same image are displayed. (O) Box plot showing the ratio of lens to optic cup diameter in E12.5 embryos of the indicated genotypes. Mutant values (green and red bars) were significantly different from the wild-type control (yellow bar); * within the box, P≤0.05>0.01 (Wnt1-cre); ** within the box, P≤0.01≥0 (Le-cre, Le-cre; Wnt1-cre double, Ap2{alpha}-cre, germline null). Conditional mutant values (green bars) were significantly different (**, P≤0.01≥0) from the germline null (red bar). Wnt1-cre and Le-cre conditional mutants were also significantly different (**, P≤0.01≥0) from the Ap2{alpha}-cre conditional mutant. (P) Box plot showing the ratio of average Pax6:Ap2{alpha} labeling intensity across the lens placode (as in Fig. 4M,N and Fig. 5M,N) for the genotypes indicated. Mutant values (green and red bars) were significantly different from the wild-type control (yellow bar); ** within the box, P≤0.001≥0). Conditional mutant values (green bars) were significantly different (** within the box, P≤0.001≥0) from the germline null (red bar). Wnt1-cre conditional mutants were also significantly different (** within the box, P≤0.001≥0) from the Ap2{alpha}-cre conditional mutants.

 

Figure 6
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  		Fig. 6. ß-catenin/Wnt signaling in the OM is not required for lens development. (A-F) Whole-mount mouse embryo heads and eye region sections stained for ß-gal activity. The section shown in E defines the regions where quantification was performed for Fig. 7A. Dashed green line, dorsal neural tube (DNT); dashed purple line, optic vesicle (OV); dashed red line, ocular mesenchyme (OM). (G,H) Cryosections labeled for ß-catenin (red), Pax6 (green) and nuclei (blue). The dashed lines enclose regions of OM negative for ß-catenin immunoreactivity. Asterisks indicate blood vessels that remain immunoreactive to ß-catenin. (I-L) Whole-mount (I,K) and DIC-illuminated eye region sections (J,L). ov, optic vesicle; 1ba, first branchial arch; ple, presumptive lens ectoderm; om, ocular mesenchyme; lp, lens placode; pr, presumptive retina. A gray line between panels indicates that different channels of the same image are displayed.

 

Figure 7
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  		Fig. 7. Wnt-independent function of mesenchymal Pygo2 in lens development. (A) Quantification of lacZ-positive cells or total cells relative to control in the ocular mesenchyme of E8.5 mouse embryos of the indicated genotypes. The regions quantified are shown in Fig. 6E. *, P≤0.05>0.01. (B) Box plot showing the ratio of lens to optic cup diameter in E12.5 embryos of the indicated genotypes. **, P≤0.01≥0. The wild-type control value incorporates the ten samples from Fig. 2K, 12 samples from the Fig. 3Q experiment that yielded the Wnt1-cre conditional mutants and a new set of four control values from this experiment that yielded the Wnt1-cre; ß-catenin conditional mutants. The germline null and Wnt1-cre conditional null values are reproduced from Fig. 2K and Fig. 5O, respectively, for the purposes of comparison. (C) Summary of cre expression patterns. Schematic of the right-hand side of E8.5 (top row) and E9.5 (bottom row) mouse embryos anterior to the midpoint of the optic vesicle. The green regions indicate the tissue domains in which the listed Cre drivers can perform allele conversions. om, ocular mesenchyme; op, optic pit; ov, optic vesicle; hse, head surface ectoderm. (D) Model for the involvement of Pygo2 in lens development (see text for details). Pax6pp represents Pax6pre-placode.

 




© The Company of Biologists Ltd 2007