Fig. 4. Expression of Nkx6.1 in the Pdx1⁺ domain can fully restore insulin expression in Nkx6.1 mutant embryos. (A-F) Immunofluorescence detection of insulin (Ins, green) and glucagon (Glc, red). (B,E) Nkx6.1^-/- embryos have reduced insulin but normal glucagon cell numbers at E14.5 (B) and E18.5 (E) compared to wild type (A,D). (C,F) Expression of the Pdx1-Nkx6.1 transgene in Nkx6.1^-/- embryos restores the formation of insulin⁺ cells at E14.5 (C), and restores normal islet morphology and size at E18.5 (F). (G) Morphometric quantification of the insulin⁺ area over total pancreatic area at E14.5 reveals an almost complete absence of insulin⁺ cells in Nkx6.1^-/- embryos and a significant rescue of insulin⁺ cells in a subset of Nkx6.1^-/- embryos carrying the Pdx1-Nkx6.1 transgene (n=4). (H) At E18.5, the insulin⁺ area is reduced by a threefold average in Nkx6.1^-/- embryos (n=5) compared to wild-type mice (n=5). The Pdx1-Nkx6.1 transgene restores the insulin⁺ area in Nkx6.1^-/- mutants to wild-type values in two embryos, whereas five embryos showed partial or no rescue (n=7). Scale bar: 50 µm.