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Fig. 2. Rfx6 LOF leads to decreased expression of foregut and pancreas markers early in pancreas development. (A) Western blot with anti-flag antibody of in vitro TnT assay of translation. This demonstrates that MO1 prevents translation of rfx6 mRNA, while the mis-match morpholino (MM) does not. Rfx6 mRNA, flag-tagged rfx6 mRNA plasmid; MM, mis-match morpholino. (B) Diagramatic representation of RT-PCR test of MO2 efficiency. (C) RT-PCR performed on wild-type (WT) and MO2-injected tadpoles. WT mRNA is processed leaving the reverse primer unable to bind resulting in no amplification in the Rfx6 panel. MO2 blocks mRNA processing at the intron 2 donor site (5' end of the intron) as demonstrated by the presence of PCR product in MO2 lane. Ef1α is a housekeeping gene used as a loading control. (D, E) Hnf6 expression is reduced in the morphant foregut endoderm at NF25 (24/28 reduced). (F, G) FoxA2 expression is reduced in the morphant foregut endoderm at NF25 (17/28). (H, I) Hnf6 expression is reduced in the morphant foregut at NF35 (10/13 reduced). (J, K) Pdx1 expression is reduced in the morphant (17/19 reduced). (L, M) Ptf1a expression is reduced in both the dorsal (arrow) and ventral (arrowhead) pancreatic buds of the morphant (n = 18). (N, O) Early insulin expression in the dorsal pancreatic bud (arrow) is lost in morphant tissue (56/62 reduced). Control tadpoles were injected with 25 ng mis-match morpholino. |
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Fig. 7. In situ hybridization confirms differential expression of organ marker genes for stomach, lung, heart and kidney in LOF microarray. (A, B) Isolated whole gut NF42 showing expression of stomach marker agr2 almost entirely abolished in morphant tissue (n = 23). (C, D) Expression of stomach marker frp5 was reduced (n = 18). (E–J) Whole tadpoles at NF42 with ventral skin removed for in situ hybridization. (E, F) Lung marker surfactant C (SurfC) is decreased in morphant tissue, closed arrows (10/14 reduced). (G, H) Heart marker nkx2.5 is decreased in morphant tissue, open arrow (8/14 reduced). (I, J) Kidney marker kcnJ1 is increased in morphant tissue, arrowhead (8/11 increased). (K,L) Expression of liver marker hex is unaffected by rfx6 knockdown (n = 32). (M, N) Expression of gall bladder and duodenum marker hnf6 is reduced in duodenum but not gall bladder (17/18). (O, P) Gall bladder marker sox17α was not reduced (18/20 showed normal expression). |
