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First published online 14 September 2005
doi: 10.1242/dev.02022

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Regulation of ocular lens development by Smad-interacting protein 1 involving Foxe3 activation

Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

View larger version (94K): [in a new window]   Fig. 1. Sip1 expression in early lens development. Distribution of Sip1 transcript detected by in situ hybridization of frontal sections through eye. Dorsal side is towards the right. (A) At E9.0 before lens induction, surface ectoderm does not express a significant level of Sip1. (B) At E9.5 soon after lens induction, Sip1 expression initiates in the lens placode. (C) At E10.5 in ectoderm derivatives, Sip1 expression is maintained only in invaginating lens cells but not in the adjacent surface ectoderm (future cornea). (D) At E11.5, all cells in the lens vesicle show Sip1 expression. (E) At E13.5, once mature lens fibers differentiate, they loose Sip1 expression, while immature lens fibers in the bow region and the lens epithelium show strong Sip1 expression. (F) Section of E10.5 embryo hybridized with sense control probe. se, surface ectoderm; ov, optic vesicle; lp, lens placode; lv, lens vesicle; le, lens epithelium; lf, mature lens fibers. Bow regions are encircled by broken lines. Scale bars: 100 µm.

View larger version (36K): [in a new window]   Fig. 2. Lens lineage-specific Cre recombinase system in ablation of Sip1 gene. (A) Scheme of Sip1 protein organization (top) compared with that of floxed allele of Sip1 (middle). NZF, N-terminal zinc fingers; CZF, C-terminal zinc fingers; SBD, Smad-binding domain; HD, homeodomain; CtBP, CtBP-binding sites. LoxP recombination sites flank major exon 7, and by the action of Cre recombinase, not only the deletion of this exon sequence occurs accompanying stop codon-generating splicing of exons 6 and 8, but detectable Sip1 protein synthesis is lost (Higashi et al., 2002). (B) Organization of two Pax6-Cre transgenes used in this study. Pax6(Lens)-Cre carries only 340 bp lens/cornea enhancer, while Pax6(LP)-Cre carries the entire 6.5 kb upstream sequence, including the pancreas enhancer in addition to the lens/cornea enhancer, both using the Pax6 P0 promoter. (C) Assessment of Cre recombinase activity by lacZ expression with R26R background. (a,c) Activity of Pax6(LP)-Cre. At E10.5, recombinase activity includes presumptive lens ectoderm forming a pit and neighboring region (mainly future cornea), but extends anteriorly to include the olfactory epithelium area (arrowhead in c). In addition, activity was detectable in the pancreas primordium (arrowhead in a), as reported previously (Kammandel et al., 1999; Williams et al., 1998). (b,d) Activity of Pax6(Lens)-Cre. Using Pax6(Lens)-Cre, the Cre activity domain is narrower and grossly confined to the future lens and cornea, without extending to the olfactory epithelium area (arrowhead in d). (e,f) Sections of the same embryos through plane indicated by arrows in c,d. Cre recombinase activity is evident in the lens pit and extends to surrounding ectoderm area, but the extension is limited in f. (g,h) Sections through eye at E11.5, demonstrating Cre activity in the lens and overlying cornea, and showing limited extraocular lacZ staining using Pax6(Lens)-Cre. Scale bars: 1 mm for a,b; 100 µm for e-h.

View larger version (63K): [in a new window]   Fig. 3. Defect in lens development caused by lens lineage-specific ablation of Sip1 gene. (A-F) Comparison of normal (A-C) and Sip1-defective (D-F) lenses. (B,E) After E11.5 Sip1-defective lens remains attached to surface ectoderm through persistent stalk (arrowhead). (C,F) In situ hybridization of E11.5 lens specimens analogous to (B,E) for Sip1 transcripts. Normal lens shows Sip1 expression throughout the lens vesicle (C), while the Sip1-defective vesicle shows no Sip1 transcripts in the stalk and anterior half of the vesicle, and residual low-level Sip1 transcripts in the posterior half (F). (G,I) Hematoxylin and Eosin (H-E) staining of E14.5 lenses. Normal lens shows full development of mature lens fiber cells (G), while Sip1-defective lens is still attached to the cornea through the stalk (arrowhead), and shows no development of mature lens fiber cells (I). (H,J) H-E staining of P0 lenses. Sip1-defective lens still attached to the cornea (arrowhead) as a small cell mass. (K) Homozygous dyl (Foxe3-defective) mouse lens at P0, showing morphological resemblance to Sip1-defective lens in J. (L) Distribution of apoptotic cells in the normal and Sip1-defective lens vesicles at E10.5, E11.5 and E12.5, where TUNEL-positive nuclei (yellow) among DAPI-stained nuclei (blue) are shown. Scale bars: 100 µm. (M) Statistics of apoptosis measured in meridian lens sections in their anterior and posterior halves. The fraction of TUNEL-positive nuclei in DAPI-stained nuclei is shown using data of two (E12.5) to six (other stages) lens specimens. Net increment of TUNEL+ apoptotic cell population under the Sip1-defective condition is indicated by hatched graph bars. le, lens epithelium; lf, mature lens fibers; cor, cornea; el, eyelid. Scale bars: 100 µm.

View larger version (63K): [in a new window]   Fig. 4. Crystallin expression and its regulation in Sip1-defective lenses. (A-E) Normal lenses and (F-J) Sip1-defective lenses, subjected to immunostaining (A-C,F-H) and in situ hybridization (D,E,I,J). (A,F) At E12.5, -crystallins are expressed throughout the lens under either normal (A) or Sip1-defective (F) conditions. ßA-crystallins are normally expressed widely in nonepithelial domains through immature to mature lens fibers (B), but in Sip1-defective lens, its expression was confined to the innermost domain (G). -Crystallin expression marking mature lens fiber cells in normal lens (C) is totally missing in Sip1-defective lens (H), clearly indicating a defect in lens fiber maturation. Broken lines indicate the boundaries of the bow region. However, two known key transcription factor genes for -crystallin regulation, Sox1 (Kamachi et al., 1995; Nishiguchi et al., 1998) and Maf (Kawauchi et al., 1999; Kim et al., 1999; Ring et al., 2000), are expressed in normal (D,E) and Sip1-defective (I,J) lenses. Scale bars: 100 µm.

View larger version (77K): [in a new window]   Fig. 5. Loss of Foxe3 expression in the epithelial compartment of Sip1-defective lenses. (A-D) Normal lenses, (E-G) Sip1-defective lenses and (H) dyl (Foxe3-defective) mutant lens. Foxe3 is expressed strongly in the anterior compartment (indicated by the broken line) of the lens vesicle that later contributes to lens epithelium (A). In Sip1-defective lens, however, Foxe3 expression in the corresponding anterior compartment is missing (E) (a section adjacent to Fig. 3F). Accordingly, Pdgfra downstream of Foxe3 (Blixt et al., 2000) (B) is attenuated in the corresponding compartment of the Sip1-defective lens (F). However, Pax6, which is known to be involved in activation of Foxe3 (Brownell et al., 2000), is expressed at similar levels in normal (C) and Sip1-defective lenses (G). Expression of Sip1 in normal lens (D) is maintained in dyl (Foxe3) homozygous lenses (H). Scale bars: 100 µm.

View larger version (27K): [in a new window]   Fig. 6. Activation of Foxe3 promoter by Sip1 and Smad8. (A) Scheme of full-length Sip1 (FL) and SBD-deleted Sip1 (SBD-) (left), and Foxe3 pro (-6.2k)-luciferase reporter construct used for transfection (right). (B) Exogenous Sip1, either full-length or SBD-deleted form, activates the promoter in a dose-dependent manner in transfected lens cells. Typically, 100 ng of Sip1 expression vector causes sevenfold activation and 500 ng of the vector elicits 20-fold activation. Using the EF1 expression vector, only a marginal activation effect was observed. (C) Effect of exogenous Smads on Sip1-dependent activation of Foxe3 promoter in transfected lens cells. Any of the Smads, alone, has no effect on promoter activity, as exemplified by Smad8. Of the eight Smads examined, only Smad8 augmented Sip1-dependent activation level. This effect was absent when Sip1 lacked SBD. (D) Activation of 3GC2-Luciferase reporter gene by expression vectors for Smad1, Smad5 and Smad8 in transfected lens cells. (E) Effect of inhibitory Smads on Smad8-mediated augmentation of Sip1-dependent promoter activation. Smad6 or Smad7 individually cancelled Smad8 effect. (F) Amount-dependent augmenting effect of exogenous Smad8 on Sip1-dependent activation of Foxe3 promoter. Regardless of the initial activation level by exogenous Sip1, the effect of Smad8 saturates at approximately threefold augmentation level. (G) Effect of length of Foxe3 promoter sequence on activation by Sip1 and augmentation by Smad8. In the scheme for the 6.2 kb promoter sequence, CACCT sequences are indicated by dots for comparison. A 1.26 kb promoter sequence was sufficient to show Sip1-dependent activation and Smad8-dependent augmentation. A 287 bp sequence still showed significant response to Sip1 and Smad8, but the shortening of the promoter to 127 bp resulted in loss of the responses. Data using 100 ng of Sip1 expression vector is shown, but basically the same promoter-length-dependent effect was observed using 500 ng of Sip1 expression vector as tabulated in the right panel.

View larger version (33K): [in a new window]   Fig. 7. Lens-specific regulation of Foxe3 promoter in transgenic mouse embryos. (A) The 6.2 kb promoter sequence upstream of the SmaI site (Brownell et al., 2000) was divided into four blocks, A-D, from the proximal side, and in combination ligated to a lacZ expression cassette, and primary transgenic embryos were produced. Panels in the middle show transgene expression in the head (lens and brain) under low magnification (left) and in the lens under high magnification (right). The numbers in the right panel indicate cases of transgene expression in transgene (PCR)-positive embryos. (B) Schematic presentation of Sip1/Smad8-responsive region, and major and minor lens elements in Foxe3 promoter.