Shifley ET , Kenny AP , Rankin SA , Zorn AM .

K08 HL105661 NHLBI NIH HHS , R01 DK078392 NIDDK NIH HHS , T32HD07463 NICHD NIH HHS , R01 DK070858 NIDDK NIH HHS

Xla Wt + {dn}fgfr1 (fig.3.c)
Xla Wt + {dn}fgfr1 (fig.4.a)
Xla Wt + {dn}fgfr1 (Fig S2 A row4)
Xla Wt + endoderm explant (Fig 1B)
Xla Wt + endoderm explant (Fig 1B)
Xla Wt + endoderm explant (Fig 1B)
Xla Wt + endoderm explant (Fig 1B)
Xla Wt + endoderm explant (Fig 1B)
Xla Wt + endoderm explant (Fig 1B)
Xla Wt + endoderm explant (Fig 1C column1)
Xla Wt + endoderm explant + mesoderm explant (Fig 1C column1)
Xla Wt + fgfr1 (Fig 4A)
Xla Wt + fgfr1 (Fig 4B)
Xla Wt + fgfr1 (Fig 4B)
Xla Wt + LY294002 (fig.6.a)
Xla Wt + LY294002 (fig.6.b,c)
Xla Wt + LY294002 (fig.6.b,c)
Xla Wt + LY294002 (fig.S3.b)
Xla Wt + LY294002 (Fig S4 A, B)
Xla Wt + LY294002 (fig.S4.a,b)
Xla Wt + LY294002 (Fig S4 C, D)
Xla Wt + LY294002 (Fig S4 C, D)
Xla Wt + LY294002 (fig.S4.c,d)
Xla Wt + LY294002 (fig.S4.c,d)
Xla Wt + LY294002 + U0126 (fig.6.b,c)
Xla Wt + LY294002 + U0126 (fig.6.b,c)
Xla Wt + Mmu.fgfr1{del}-Mmu.fkbp1a{del} + B/B Homodimerizer (Fig 3)
Xla Wt + Mmu.fgfr1{del}-Mmu.fkbp1a{del} + B/B Homodimerizer (fig.3.a)
Xla Wt + Mmu.fgfr1{del}-Mmu.fkbp1a{del} + B/B Homodimerizer (fig.3.a,c)
Xla Wt + Mmu.fgfr1{del}-Mmu.fkbp1a{del} + B/B Homodimerizer (fig.3.a,c,d)
Xla Wt + Mmu.fgfr1{del}-Mmu.fkbp1a{del} + B/B Homodimerizer (fig.3.b)
Xla Wt + PD173074 (Fig 3)
Xla Wt + PD173074 (fig.3.a,c)
Xla Wt + PD173074 (fig.3.a,c,d)
Xla Wt + PD173074 (fig.3.a,d)
Xla Wt + PD173074 (fig.3.b)
Xla Wt + PD173074 (Fig 5B,C)
Xla Wt + PD173074 (Fig 5B,C)
Xla Wt + PD173074 (Fig 5B,C)
Xla Wt + PD173074 (Fig 5B,C)
Xla Wt + PD173074 (fig.5.b;fig.S3.a)
Xla Wt + PD173074 (fig.5.b;fig.S3.a)
Xla Wt + PD173074 (fig.5.b;fig.S3.a)
Xla Wt + PD173074 (fig.6.b,c)
Xla Wt + PD173074 (fig.6.b,c)
Xla Wt + PD173074 (fig.S2.a)
Xla Wt + PD173074 (Fig S2 A row2)
Xla Wt + PD173074 (Fig S2 B)
Xla Wt + PD173074 (Fig S2 B)
Xla Wt + PD173074 (fig.S2.b)
Xla Wt + PD173074 (fig.S2.b)
Xla Wt + PD173074 (fig.S3.b)
Xla Wt + PD173074 (Fig S4 A, B)
Xla Wt + PD173074 (fig.S4.a,b)
Xla Wt + PD173074 (Fig S4 C, D)
Xla Wt + PD173074 (Fig S4 C, D)
Xla Wt + su5402 (fig.6.b,c)
Xla Wt + U0126 (fig.6.a)
Xla Wt + U0126 (fig.6.b,c)
Xla Wt + U0126 (fig.6.b,c)
Xla Wt + U0126 (Fig S4 A, B)

	Figure 1. Foregut organ specification requires prolonged contact with the cardiac-lateral plate mesoderm. (A) Diagram of the experimental design. Ventral explants were cultured with (+Meso) or without mesoderm (−Meso) during the indicated time points and assayed by in situ hybridization at stage NF37. (B) Summary of experimental results showing the percentage of explants with mesoderm removed at different times expressing liver (nr1h5), lung/thyroid (nkx2.1) and pancreas (pdx1 or ptf1a) markers at stage NF37, n > 20 explants for each condition and each probe. (C) Representative explants cultured from stage NF16 to NF37 with or without mesoderm and corresponding whole embryo controls assayed by in situ hybridization with the indicated markers. Endodermal (gjb1) or mesodermal (foxf1 and tnni3) specific markers demonstrated clean separation of the tissues.
	Figure 2. FGF signaling is active in the foregut endoderm during organ induction. Confocal immunostaining of bisected Xenopus embryos show active FGF-MEK signaling with di-phospho ERK1/2 (pErk) (white) in the developing foregut tissue at the indicated stages (anterior left, ventral down). Images are 10X magnification with independent scans of the boxed regions at 20X magnification. (A) Stage NF15 embryos show a low level of FGF signaling in the migrating anterior mesendoderm (ame). (B) Stage NF19, (C) NF23 and (D) NF28 show pErk staining in the ventral foregut endoderm (fg) as well as the underlying cardiac mesoderm (cm). The dashed yellow line indicates the boundary between the endoderm and mesoderm. (E) Mid-sagittal and (F) transverse optical sections of stage NF35 embryos show pErk staining in the liver epithelium (lv), heart (ht) and nascent lung buds (lu) and lateral plate mesoderm (lpm). (G and H) Stage NF42 gut tubes show pErk in liver bud (lv), gal bladder (gb), dorsal (dp) and ventral pancreatic buds (vp), the stomach (st) and the distal tips of the lung buds (lu). (I) Stage NF23 control embryos with no primary antibody.
	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).
	Figure 4. Inhibition of FGF signaling in Xenopus embryos. (A) Confocal immunostaining of pErk (red) and a GFP lineage tracer (green) in the foregut region of bisected stage NF23 Xenopus embryos that were injected into the presumptive foregut endoderm cells at the 16-cell stage with RNA encoding either β-gal (3 ng) and GFP or dnFGFR (3 ng) and GFP. The β-gal/GFP injected embryos show pErk in GPF + cells whereas in dnFGFR/GFP injected embryos pErk is undetectable in GFP + foregut cells. (B) A representative stage NF42 gut tube from βgal/GFP and dnFGFR/GFP injected embryos assayed by in situ hybridization for gfp RNA to show mosaic contribution of labeled cells to different organ buds. p; pancreas, lv; liver, s; stomach, i; intestine. (C) Summary showing percentage of organ buds containing labeled cells indicates a decreased contribution to the lung and liver buds in dnFGFR injected embryos (n = 120) compared to βgal controls (n = 70).
	Figure 5. Prolonged FGF signaling is required for lung and liver induction. (A) Experimental design showing the time-course of FGFRi treatment. (B) Embryos treated with DMSO or FGFRi at the indicated stages were assayed at stage NF35 by in situ hybridization for markers of liver (nr1h5), lung (nkx2.1), and heart (tnni3). Representative examples are shown and the graph summarizes the % of embryos with normal, weak, or severely reduced/absent expression.
	Figure 6. Both PI3K and MEK signaling contribute to lung and liver development. (A) Western blot analysis of embryos cultured in DMSO, PI3Ki (LY294002), or MEKi (U0126) from stages NF18 to NF35 in triplicate shows a dramatic decrease in pAkt and pErk levels with PI3Ki and MEKi treatment respectively. (B) Embryos cultured in DMSO or the indicated inhibitors from NF18 to NF35 were analyzed at stage NF35 for liver (nr1h5), lung (nkx2.1), pancreas (ptf1α), liver/thyroid (hhex), and pancreatic/duodenal (pdx1) expression. Embryos cultured in DMSO or the indicated inhibitors from NF18 to NF35 were analyzed at stage NF42 for organ bud appearance with hnf4α-stained gut tubes. Yellow arrows indicate normal expression, red arrows indicate reduced or absent expression. Foregut organ buds are outlined in dashed lines (lu; lung, p; pancreas, lv; liver, s; stomach, i; intestine, th; thyroid, p/d; pancreas/duodenum). (C) Summary of the percentage of inhibited embryos with expanded, normal, weak, or severely reduced/absent foregut marker expression compared to controls with the number of embryos analyzed listed for each condition. FGFRi data is repeated from Figure 3 for comparison.
	Confocal immunostaining of bisected Xenopus embryo, NF stage 42, shows active FGF-MEK signaling with di-phospho ERK1/2 (pErk) (white) in the developing foregut (g). G' illustrates position of gall bladder primordium (gb), liver primordium ( lv), and stomach (st), which is flanked by the ventral pancreatic bud (vp) and dorsal pancreatic bud (dp).
	Figure S1. Exogenous FGF is not sufficient to induce lung or liver lineages in explants. (A) Foregut explants with or without mesoderm were cultured from stage NF18 to NF35 in BSA or FGF2 and analyzed for expression of liver (nr1h5), lung (nkx2.1) and pancreas (pdx1) markers. (B) Western blot analysis of explants shows an increase in pErk levels upon FGF2 treatment. (
	Figure S2. FGF signaling is not required for maintaining foregut progenitors, but is required for lung and liver induction in a dose-dependent manner. (A) Embryos cultured in DMSO and FGFRi from stages NF15 to NF23 or injected with RNA encoding β-gal (3 ng) or dnFGFR (3 ng) were analyzed for expression of the foregut progenitor marker hhex and the cardiac progenitor marker nkx2.5. The graph summarizes the percentage of embryos with normal, weak or severely reduced/absent expression. (B) Embryos cultured from stages NF18 to NF35 in DMSO or FGFRi at the indicated concentrations and the percent of embryos with normal, weak, or severely reduced/absent nr1h5 and nkx2.1 expression was scored at stage NF35.
	Figure S3. Prolonged MEK and PI3K signaling are required for a full lung and liver induction. (A) Percentage of embryos treated with either; DMSO, FGFRi, PI3Ki or MEKi for the indicated stages that exhibit with normal expression of nr1h5, nkx2.1, ptf1α, or tnni3 (n > 14 embryos for each condition and probe). Inhibition of MEK or PI3K over various intermediate durations results in a reduction in specification markers, suggesting that prolonged signaling is required for full foregut organ gene expression. (B) Quantification of average hhex and pdx1 expression areas in control and inhibited 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 < .004) as indicated. FGFRi data is repeated from Figure 3 for comparison.
	Figure S4. Cell Proliferation and apoptosis in FGF-inhibited embryos. (A) Analysis of cell proliferation by phospho-histone H3 (pHH3) immunostaining in embryos treated with DMSO, FGFRi, PI3Ki or MEKi. At stages NF23 (mid-sagittal) and NF35 (transverse section) embryos were assayed by 80 μM confocal Z-stack of pHH3 immunostaining (white dots), whereas stage NF42 isolated gut tubes were assayed by pHH3 immunohistochemistry (blue dots). Yellow dashed lines outline the foregut region quantified. (B) Summary of mean number of pHH3+ cells in the foregut region +/− SD (n > 5 embryos for each condition and stage). (C) Analysis of apoptosis by activated Caspase-3 immunostaining in embryos treated with DMSO, FGFRi, PI3Ki or MEKi. At stages NF23 (mid-sagittal) and NF35 (transverse section) embryos were assayed by 80 μM confocal Z-stack immunostaining (white dots). Yellow dashed lines outline the foregut region quantified. (D) Summary of mean number of active caspase-3+ cells in the foregut region +/− SD (n > 5 embryos for each condition and stage)

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