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Fig. 8. Expression of Nkx6.1 or Nkx6.2 in the Pdx1⁺ domain restores beta-cell development and maturation in Nkx6.1 mutant embryos. Immunofluorescence detection of Glut2 (A-D, red), MafA (E-H, red), Nkx6.1 (I-K, red) or ß-galactosidase (ß-gal; L, red) with insulin (Ins, green) in pancreata from wild-type (A,E,I), Nkx6.1^-/- (B,F,J), Nkx6.1^-/-;Pdx1-Nkx6.1 (C,G,K) and Nkx6.1^-/-;Pdx1-Nkx6.2 (D,H,L) embryos at E18.5. (A,B) Whereas Glut2 expression is confined to insulin⁺ cells in wild-type embryos (A), insulin⁺ cells are Glut2-negative in Nkx6.1^-/- mutants (B). (B) Notably, Nkx6.1^-/- embryos display clusters of Glut2⁺, but insulin-negative, cells adjacent to the insulin⁺ domain. (C,D) The Pdx1-Nkx6.1 and Pdx1-Nkx6.2 transgenes restore the formation of insulin/Glut2-co-expressing cells in the Nkx6.1-deficient background. Some insulin⁺/Glut2-negative as well as Glut2⁺/insulin-negative cells remain in transgene-expressing embryos. (F) In Nkx6.1^-/- embryos, insulin⁺ cells do not express MafA. (G,H) MafA expression is restored in the majority of, but not in all, insulin⁺ cells by the Pdx1-Nkx6.1 and Pdx1-Nkx6.2 transgenes (arrows point to MafA-negative/insulin⁺ cells). (K,L) Sustained expression of the Pdx1-Nkx6.1 and Pdx1-Nkx6.2 transgenes in rescued insulin⁺ cells is confirmed by the detection of Nkx6.1 (K) or ß-gal (L), respectively. Scale bar: 50 µm.

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