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Figure 3. FGF signaling is required for lung and liver specification at the expense of pancreas. (A) In situ hybridization with the indicated probes in control, FGF-inhibited or FGF-activated embryos. Embryos were cultured in DMSO or PD173074 (FGFRi) from NF18 to NF35 to inhibit FGF signaling. Control uninjected or embryos injected with RNA encoding an inducible FGF receptor (caFGFR; 20 pg) into vegetal blastomeres at the 4/8-cell stage, were treated with the homodimerizing drug from NF18 to NF35 to activate FGF signaling. Yellow arrows indicate normal expression, red arrows absent expression, blue arrows ectopic expression; yellow dash outlines expression boundaries. p; pancreas, lv; liver, lu; lung, th; thyroid, h; heart. (B) Western blot analysis of pErk, total Erk, pAkt and total Akt in embryos cultured in either PD173074 (FGFRi) or DMSO from stage NF18 to NF35 (in triplicate). Western blot analysis of embryos injected with caFGFR(+) and treated from NF18 to NF35 with B/B Homodimerizer show increased FGF/pErk signaling compared to uninjected controls at Stage NF 23 (lanes1 + 2) and Stage NF 35 (lanes3 + 4). (C) Summary of gene expression in FGF-inhibited (FGFRi or dnFGFR; 3 ng) and FGF-activated (caFGFR; 20 pg) embryos. Controls include DMSO treated, β-gal injected and uninjected B/B dimerizing drug treated, none of which had an obvious impact on gene expression. (D) Quantification of average hhex and pdx1 expression areas in control and FGF-manipulated embryos. ImageJ software was used to measure the expression area with the average expression area of controls set to 1. Averages are based on n > 13 embryos for each condition and marker from at least two independent experiments, standard deviation and significance based on t-test (**p < 0.004). |
