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Fig. 3. Upregulation of NKX6.2 expression in Nkx6.1 mutant embryos. Immunofluorescence detection of NKX6.1 with NGN3 (A), insulin (Ins) (B), glucagon (Glc) (C) or amylase (Amy) (D) in pancreas from E14.5 (A) and E15.5 (B-D) embryos. NKX6.1 is expressed in a subset of NGN3⁺ cells (A), in all insulin⁺ cells (B), and in very few glucagon⁺ cells (arrowhead in C). No co-expression of NKX6.1 and amylase is observed (D). (E-P) Confocal images showing immunofluorescence staining for NKX6.2 together with various pancreatic markers in wild-type and Nkx6.1^-/- embryos. At E12.5, the number of NKX6.2⁺ cells is increased in the pancreatic epithelium of Nkx6.1 mutants (E,F). Upregulation of NKX6.2 in Nkx6.1 mutants is not seen in insulin⁺ (Ins) (arrowheads in G,H indicate insulin⁺/NKX6.2^- cells) or glucagon⁺ (Glc) cells (arrowheads in K,L indicate glucagon⁺/NKX6.2^- cells). NGN3⁺ cells are NKX6.2^- in wild type and Nkx6.1 mutants (I,J). The ectopic Nkx6.2⁺ cells in Nkx6.1 mutants do not produce amylase (Amy) (M,N). At E15.5, the majority of ectopic NKX6.2-expressing cells co-express PDX1 (O,P). (Q) Quantification of NKX6.2⁺ cells in wild-type and Nkx6.1^-/- embryos. The average number of NKX6.2⁺ cells per section was determined by counting immunofluorescence-labeled cells at E10.5, E12.5 and E15.5. Four independent pancreata were evaluated for each genotype. The average number of cells are shown as a % of wild type (arbitrarily set to 100%) ±s.e.m. Scale bar: 50 µm.

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