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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. 3. Rfx6 is required for pancreas differentiation. Whole mount in situ hybridizations of NF42 whole guts from morphant (MO1) and mis-match (control) tadpoles. (A–D) Expression of pancreatic transcription factors pdx1 (n = 18) and ptf1a (n = 18) is almost completely abolished in morphant tissue. (E, F) Expression of acinar marker elastase is reduced (n = 16). (G, H) Schematic illustrating organs in the whole gut. Pink, liver; red, gall bladder; blue, pancreas; yellow, stomach, duodenum and intestine. (I, J) β cell marker insulin is reduced (72/75 reduced). (K–N) Glucagon and somatostatin, both markers of endocrine cells in the pancreas and stomach are reduced (glucagon n = 18, somatostatin 15/16 reduced). (O, P) NeuroD, developmental endocrine cell marker, is reduced (n = 17). |
