Fig. 4. Compensatory role of NKX6.2 for NKX6.1 function in - and ß-cell development. Confocal images showing immunofluorescence detection of glucagon (Glc) and insulin (Ins) in pancreas from wild-type, Nkx6.2^-/-, Nkx6.1^-/- and Nkx6.1^-/-;Nkx6.2^-/- embryos at E18.5 (A-D) and E14.5 (G-J). Nkx6.2^-/- (B,H) and wild-type (A,G) embryos show similar numbers and organization of insulin⁺ and glucagon⁺ cells in the pancreas. In Nkx6.1^-/- (I) and Nkx6.1^-/-;Nkx6.2^-/- (J) embryos, glucagon⁺ cells are normal, but insulin⁺ cells are reduced in number at E14.5. At E18.5, Nkx6.1^-/-;Nkx6.2^-/- embryos (D) show a further reduction in insulin⁺ cells as well as a reduction in glucagon⁺ cells compared with Nkx6.1^-/- embryos (C). (E) Quantification of hormone⁺ cells in wild type and Nkx6 compound mutants at E18.5. The average number of glucagon⁺ and insulin⁺ cells per section was determined by counting immunofluorescence-labeled cells. Five independent pancreata were evaluated for each genotype and the average number of cells in wild type arbitrarily set to 100. (F,K) Quantitative RT-PCR analysis of insulin and glucagon in pancreata from Nkx6 compound mutants at E18.5 (F) and E15.5 (K). Hormone expression levels are shown relative to the levels for -actin mRNA from two independent measurements of four pancreata for each genotype. For comparison, values are shown as a % of wild type, which was arbitrarily set to 100%. At E18.5, insulin mRNA levels were 6.2±0.78% of wild type in Nkx6.1^-/- and 1.7±0.30% of wild type in Nkx6.1^-/-;Nkx6.2^-/- embryos (F), compared with 2.2±0.16% of wild type in Nkx6.1^-/- and 2.0±0.84% of wild type in Nkx6.1^-/-;Nkx6.2^-/- embryos at E15.5 (K). The values shown represent mean±s.e.m. ^*P<0.01, as determined by Student's t-test. Scale bar: 50 µm.