Rankin SA , McCracken KW , Luedeke DM , Han L , Wells JM , Shannon JM , Zorn AM .

R01 HL098319 NHLBI NIH HHS , R01 HL114898 NHLBI NIH HHS , R01 DK070858 NIDDK NIH HHS , U01 DK103117 NIDDK NIH HHS , R01 DK092456 NIDDK NIH HHS , U19 AI116491 NIAID NIH HHS , R01 HD073179 NICHD NIH HHS , P30 DK078392 NIDDK NIH HHS , P01 HD093363 NICHD NIH HHS , U01 DK085532 NIDDK NIH HHS

             BMP signaling pathway
             regulation of endodermal cell fate specification
             retinoic acid receptor signaling pathway

	Fig. 1. Respiratory competence ofXenopusendoderm is restricted by the early gastrula stage. (A) Experimental diagram of the Xenopus respiratory competence assay. Bisected early gastrula stage (NF10.25) Xenopus embryos assayed by in-situ hybridization show the expression of sox17α throughout the definitive endoderm (DE), hhex in anterior endoderm (AE) and ventx2 in posterior endoderm (PE). DE, AE, or PE explants were dissected at NF10.25, cultured in isolation until NF14, treated +/− 50 nM RA from NF14-25, and then in 3.5 μM Bio + 50 ng/mL BMP4 from NF25-38, at which time they were fixed and analyzed by in-situ hybridization for the respiratory endoderm markers nkx2-1(B-G) and sftpc(H-M).
	Fig. 4. Wnt/BMP-mediated A-P patterning impacts developmental competence. (A,B) Experimental diagram. Xenopus embryos at the 16-cell stage with clear pigment differences (animal pole, dorsal-anterior, and ventral-posterior views of such embryos are shown) were injected with 25 pg of Noggin or 100 pg of Dkk1 RNAs (to inhibit BMP and Wnt/βcatenin signaling, respectively) into each ventral-vegetal V2.1 blastomere along with 25 pg eGFP RNA as a lineage tracer, which targets the ventx1/2-expressing posterior mesendoderm. Hhex+ AE tissue was targeted by injection of each dorsal-vegetal D1.1 blastomere with 25 pg eGFP RNA. At gastrula stage NF10.25, GFP fluorescence was monitored and used to dissect AE, PE, and BMP- or Wnt-inhibited PE explants, which were then treated as indicated in panel B (50 nM RA from NF14-25 followed by 3.5 μM Bio + 50 ng/mL BMP4 from NF25-38). (C)In-situ hybridization analysis of hhex and ventx1+2 (both probes mixed together) in bisected gastrula NF10.25 embryos confirms effective inhibition of the posteriorizing Wnt and BMP pathways by dkk1 and noggin RNA injection. Numbers in the lower left corner indicate numbers of embryos assayed with the gene expression pattern shown. (D-M) Relative gene expression analysis (RT-qPCR) of different anterior-posterior endoderm lineages assayed in explants as prepared/treated in panels A,B. Graphs display the average 2−δδCt value +/- SEM of 3 biological replicates. (N-W)In-situ hybridization of control embryos showing the endogenous spatial domains of expression along the anterior-posterior axis.
	Fig. 6. Model depicting the reiterative use of Wnt, BMP, and RA signals during endoderm development. (A) In situ hybridization showing expression of the RA-synthesizing enzyme raldh2 (aldh1a2) during early Xenopus development. Abbreviations: AE, anterior endoderm; PE, posterior endoderm; lpm, lateral plate mesoderm; FG, foregut; MG, midgut; HG, hindgut; sm, somitic mesoderm; ant, anterior; post, posterior. (B) Schematic depicting the reiterative roles for Wnt, BMP and RA signals during endoderm vertebrate development.
	Supplemental Figure S1. (A) Xenopus early somite stage NF14 and NF20 foregut endoderm explants contain a restricted domain of respiratory-competent cells that can respond to RA followed by Wnt/BMP pathway stimulation to activate respiratory fate. NF14 endoderm requires exogenous RA (50nM) prior to Bio (3.5uM) +BMP4 (50ng/mL) in order to express the respiratory markers nkx2-1 and sftpc in only a subset of each explant. NF20 endoderm is competent, and does not require exogenous RA in order to express respiratory markers in response to Bio+BMP4. This competence is RA-dependent, as culture of embryos in the Raldh inhibitor DEAB (10uM) prior to foregut dissection blocks the ability of Bio+BMP4 to induce the lung markers. (B-J) Gastrula anterior endoderm contributes to foregut organ lineages. Immunofluorescence analysis of optical sections through hhex:GFP transgenic X.laevis embryos. GFP expression driven by the Xenopus hhex promoter is localized to gastrula anterior endoderm (C; plane of section indicated in B) and acts as a short-term lineage label (Rankin et al., 2011). (E-I) Analysis of NF34 hhex:GFP embryos (planes of section indicated in D) reveal GFP expression is present in lateral and ventral Sox2+ pharyngeal endoderm (E,F,G), Nkx2-1+thyroid endoderm (E,E”), Sox2+ esophageal endoderm (G,H, J, J”), Nkx2-1+ respiratory endoderm (G-G”,H-H”), ventral pancreatic endoderm (I), and stomach endoderm (I, J, J”), confirming that gastrula stage AE contributes to foregut lineages. J and J” show staining of the lung/esophageal/stomach region of NF38 embryos, confirming that gastrula AE contributes to both lung bud, esophageal, and stomach endoderm. Scale bars 100uM. Abbreviations: DE, definitive endoderm; AE, anterior endoderm; PE, posterior endoderm; fb, forebrain; phe, pharyngeal endoderm; thy, thyroid; v fge, ventral foregut endoderm; d fge, dorsal foregut endoderm; sto, stomach; vp, ventral pancreas; tr, trachea; eso, esophagus; lb, lung bud; stom, stomach.
	Supplemental Figure S2 (A). Dynamic expression of the RA-synthesizing enzymes rdh10 and raldh2 and RA-degrading enzyme cyp26a1 during early Xenopus development. Panel A shows in situ hybridization analysis of rdh10, raldh2, cyp26a1, hhex, and ventx1 at the indicated stages of Xenopus development. Abbreviations: AE, anterior endoderm; PE, posterior endoderm; AM, anterior mesoderm; PM, posterior mesoderm; vlpm, ventral lateral plate mesoderm; fg, foregut; hg, hindgut; lpm, lateral plate mesoderm. Note the robust expression of rdh10 and raldh2 in the ventral lpm at NF20 but not NF14, which correlates with the endogenous timing of RA-dependent respiratory competence of the endoderm (Supplemental Fig.S1A). Boxed flow chart in (A) is a simple schematic of where the enzymes act in the metabolism and catabolism pathway of RA biogenesis. (B). Early RA treatment of Xenopus DE is inhibitory to lung induction and promotes pancreatic/stomach and intestinal fate. Experimental diagram to test two different RA treatment periods on Xenopus gastrula DE. RT-qPCR analysis of DE explants treated either from NF10.5 to NF25 or NF15-25 with 50nM RA and then from NF25-38 with 3.5M Bio + 50ng/mL BMP4. Gene expression in each condition was normalized to the housekeeping gene ODC and then log2 fold changes in experimental gene expression were determined using the 2−δδCt method relative to the experimental gene’s ODC-normalized expression in either AE or PE cultured in isolation as indicated. Graphs display the average 2−δδCt value +/- SEM of 3 biological replicates (each biological replicate contained n=4 explants).

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