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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.





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