Prolonged FGF signaling is necessary for lung and liver induction in Xenopus

Abstract

Background: FGF signaling plays numerous roles during organogenesis of the embryonic gut tube. Mouse explant studies suggest that different thresholds of FGF signaling from the cardiogenic mesoderm induce lung, liver, and pancreas lineages from the ventral foregut progenitor cells. The mechanisms that regulate FGF dose in vivo are unknown. Here we use Xenopus embryos to examine the hypothesis that a prolonged duration of FGF signaling from the mesoderm is required to induce foregut organs.

Results: We show that both mesoderm and FGF signaling are required for liver and lung development in Xenopus; formally demonstrating that this important step in organ induction is conserved with other vertebrate species. Prolonged contact with the mesoderm and persistent FGF signaling through both MEK and PI3K over an extended period of time are required for liver and lung specification. Inhibition of FGF signaling results in reduced liver and lung development, with a modest expansion of the pancreas/duodenum progenitor domain. Hyper-activation of FGF signaling has the opposite effect expanding liver and lung gene expression and repressing pancreatic markers. We show that FGF signaling is cell autonomously required in the endoderm and that a dominant negative FGF receptor decreases the ability of ventral foregut progenitor cells to contribute to the lung and liver buds.

Conclusions: These results suggest that the liver and lungs are specified at progressively later times in development requiring mesoderm contact for different lengths of time. Our data suggest that this is achieved at least in part through prolonged FGF signaling. In addition to providing a foundation for further mechanistic studies on foregut organogenesis using the experimental advantages of the Xenopus system, these data have implications for the directed differentiation of stem cells into foregut lineages.

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.