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The upstream ectoderm enhancer in Pax6 has an important role in lens induction

Patricia V. Dimanlig¹, Sonya C. Faber¹, Woytek Auerbach^1,2, Helen P. Makarenkova¹ and Richard A. Lang^1,*,

¹ Developmental Genetics Program, Skirball Institute for Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA
² Howard Hughes Medical Institute, Cell Biology Department, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA
^* Present address: Children’s Hospital Research Foundation, Developmental Biology Division, Department of Ophthalmology, 3333 Burnet Avenue, Cincinnati, OH45229-3039, USA

View larger version (23K): [in a new window]   Fig. 1. Generation of the Pax6 ectoderm enhancer null allele. (A) Schematic representation of the Pax6 ectoderm enhancer targeted deletion strategy. The purple box represents the 341 bp Pax6 ectoderm enhancer. The regions of sequence used as probes to assess the targeting procedure are pictured as white boxes. The 2.7 kb 5' targeting arm is represented by the yellow box, and the 3.6 kb 3' targeting arm is represented by the orange box. The loxP site-specific recombination sequences for cre recombinase are indicated by white triangles. The sizes of the restriction fragments detected by the probes are indicated by lines located above the corresponding map. Small arrows indicate the location of primer pairs (primers P1, P2 and P3) used for PCR genotyping. The sizes of the PCR products are indicated above the primer pairs. (B) Southern blotting to identify wild-type (+/+) and targeted (where +/– designates +/neoEE) ES cell-line genomic DNA for EcoRI and SphI restriction digests probed with the 5' and 3' probes, respectively. The fragment sizes are labeled next to the appropriate bands. (C) PCR genotyping of genomic DNA. The sizes of PCR products are indicated to the left and right of the gel panel. R, EcoRI; N, NcoI; Sa, SacII; Sp, SphI; A, AatII; K, KpnI; neo, neomycin phosphotransferase gene; tk, thymidine kinase gene; pA, polyadenylation signal; Pr, promoter.

View larger version (129K): [in a new window]   Fig. 2. Histological analysis of Pax6EE/EE mice. All panels show Hematoxylin and Eosin stained 4 µm paraffin sections. E9.5 eyes from WT (A), Pax6+/EE (B) and Pax6EE/EE (C) embryos. This shows the optic vesicle (ov) in close contact with the surface ectoderm (se) that in the wild-type is thickened in the region of the lens placode (pl). In Pax6EE/EE embryos, the nasal aspect of the placodal ectoderm is abnormally thin (arrow). E10.5 eyes from wild type (D), Pax6+/EE (E) and Pax6EE/EE (F) embryos. The optic cup (oc) and lens pit (lp) have formed through coordinated invagination of optic vesicle and lens placode. In Pax6EE/EE embryos, the lens pit and optic cup are small. Pax6+/EE embryos show an intermediate phenotype. E11.5 eyes from wild type (G), Pax6+/EE (H) and Pax6EE/EE (I) embryos. In the wild type, the lens vesicle (lv) has separated form the surface ectoderm (se). In the homozygous mutant, the lens vesicle is small and remains attached (red arrowhead). Heterozygotes have an intermediate phenotype (red arrowhead). E12.5 eyes from wild type (J), Pax6+/EE (K) and Pax6EE/EE (L) embryos. At this stage, primary fiber cells (pfc) have extended from the posterior lens vesicle towards the lens epithelium (le) in both wild type and mutant embryos, but the homozygous mutant lens is small with a persistent lens stalk (red arrowhead). The optic cup in Pax6EE/EE embryos is marginally smaller than in wild type. Again, heterozygotes have an intermediate phenotype. E17.5 eyes from wild type (M), Pax6+/EE (N) and Pax6EE/EE (O) embryos. In eyes of this stage, both primary and secondary fiber cells have differentiated in all genotypes. A smaller lens is apparent in homozygotes and a local invagination of the corneal epithelium (red arrowhead) indicates a persistent lens stalk. Morphologically, pseudostratification in the retina appears unaffected.

View larger version (62K): [in a new window]   Fig. 3. Assessment of placodal thickness and proliferation in Pax6EE/EE mice. Wild-type (A) and Pax6EE/EE (B) eyes at E9.5 in paraffin section. The numbered vertical lines indicate the points in the surface ectoderm at which the thickness of the lens placode was measured. Position 1 is on the nasal side and position 5 temporal. This was performed using digitized images and an arbitrary unit system. (C) Schematic of an E9.5 eye showing the section plane (purple shading) used for placodal thickness measurements. D, V, N and T indicate the dorsal, ventral, nasal and temporal aspects, respectively. (D) Graph showing, in arbitrary units, a comparison of placodal thickness in wild type (blue) Pax6+/EE (green) and Pax6EE/EE (red) embryos. This indicates that in approximately the nasal half of the lens placode, the Pax6EE/EE placode is thinner than in wild type. The difference is greatest in the central placode and minimal in the temporal domain. Pax6+/EE embryos have an intermediate phenotype. Vertical bars represent standard errors. Wild-type (E) and Pax6EE/EE eyes (F) at E9.5 in paraffin section stained with Hoechst 33258 to indicate nuclei (green labeling) and with anti-BrdU detection reagents (red labeling). Only the green component of the blue Hoechst signal has been included in these images for ease of visualization. The percentage of BrdU-positive cells within the lens placode (white lines) was determined and presented in a histogram (G) comparing wild-type and Pax6EE/EE embryos. This indicated that the level of proliferation was reduced in the Pax6EE/EE lens placode. Vertical bars represent standard errors.

View larger version (145K): [in a new window]   Fig. 4. Expression and distribution of differentiation markers in Pax6EE/EE mice. All panels show fluorescently labeled 4 µm paraffin sections counterstained with Hoechst 33258 to indicate nuclei (blue labeling). E11.5 eyes from wild-type (A), Pax6+/EE (B) and Pax6EE/EE (C) embryos labeled with anti--crystallin antibodies. Pax6EE/EE embryos show reduced per cell labeling and a smaller number of positive cells in the posterior lens vesicle. -crystallin labeling at E12.5 of wild type (D), Pax6+/EE (E) and Pax6EE/EE (F) embryos emphasizes that there are fewer positive cells in the lens of the homozygous mutant. E11.5 eyes from wild-type (G), Pax6+/EE (H) and Pax6EE/EE (I) embryos labeled with anti-ß-crystallin antibodies. Pax6EE/EE embryos show a reduced level of labeling on a per cell basis and fewer positive cells in the posterior lens vesicle. This indicates a suppression of fiber cell differentiation consistent with morphological findings. This observation is emphasized in comparing wild type (J), Pax6+/EE (K) and Pax6EE/EE (L) E12.5 embryos where primary fiber cell extension is minimal in the homozygous mutant.

View larger version (88K): [in a new window]   Fig. 5. Pax6 and Foxe3 expression in Pax6EE/EE mice. (A-C) P6 5.0-lacz reporter animals stained with X-gal. (A) At E8.75 X-gal staining appears in a teardrop-shaped region of surface ectoderm that overlies the optic vesicle (broken line) and extends temporally (arrow). (B) By E9.5 expression is most intense in a crescent-shaped region that corresponds to the nasoventral lens placode. The broken line indicates the section plane used for C-E and the arrowhead the intense X-gal staining on the nasal side of the lens placode. (C) Frozen section from an X-gal stained E9.5 P6 5.0-lacz reporter animal showing stronger staining in the nasal region (black arrowheads) of the surface ectoderm overlying the optic vesicle. (D,E) Pax6 immunofluorescence in cryosections of wild-type (D) and Pax6EE/EE (E) eye primordia at E9.5. The broken white line indicates the border between surface ectoderm and optic vesicle (ov). This analysis indicates that the level of Pax6 immunoreactivity is greatly diminished in Pax6EE/EE embryos in the nasal ectoderm of the lens placode (compare arrowed region in D with the equivalent region in E). The zone of diminished Pax6 immunoreactivity in Pax6EE/EE embryos corresponds to the region where X-gal staining is strongest in the P6 5.0-lacz reporter (arrowed region in C). (F,G) Pax6 immunofluorescence in cryosections of wild-type (F) and Pax6EE/EE (G) eyes at E13.0. Wild-type (H) and Pax6EE/EE (I) E9.5 embryos subject to whole-mount in situ hybridization with an antisense Foxe3 probe. This indicates that in wild-type embryos, Foxe3 expression is found in the ectoderm of the lens placode as expected. In homozygous mutant embryos, Foxe3 expression is lost from the lens placode, but not from the midbrain region (red arrowheads). The optic cup that surrounds the lens pit is marked by a broken line. ov, optic vesicle; le, lens epithelium; pfc, primary fiber cells; pr, presumptive retina.

View larger version (21K): [in a new window]   Fig. 6. Proposed models for Pax6 ectoderm enhancer involvement in lens induction and development. (A) Pathway assembled from the data reported here, and (B) with data incorporated from previous analyses. The black arrows indicate demonstrated genetic interactions, the gray arrows interactions that are implied. The highest component of the proposed pathway is the first phase of Pax6 expression in the pre-placodal ectoderm (defined as Pax6pre-placode). Previous work has shown that Pax6pre-placode is required for the placodal phase of Pax6 expression (defined as Pax6placode). The reduced, but still present, Pax6 expression observed in Pax6EE/EE embryos argues for the presence of multiple enhancer elements (denoted as ectoderm enhancer and enhancer 2) that together confer complete placodal Pax6 expression. Significantly, reduction of Pax6 protein in the lens placode results in loss of Foxe3 expression, showing that a threshold level of Pax6 is required for its expression. This indicates that Foxe3, a forkhead transcription factor necessary for vesicle closure, separation and proliferation, is genetically downstream of Pax6placode. Recent work has shown that Fgf receptor activity and Bmp7 cooperate in maintaining Pax6placode, and that Pax6placode and Bmp4 within the optic vesicle are required for Sox2 expression in the lens placode. The genetic relationship between Foxe3 and Sox2 remains to be determined.