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Experiment details for hhex

Timing is everything: Reiterative Wnt, BMP and RA signaling regulate developmental competence during endoderm organogenesis.

Timing is everything: Reiterative Wnt, BMP and RA signaling regulate developmental competence during endoderm organogenesis.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 10.25 endoderm , anterior

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  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).

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 10.25 endoderm , anterior

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  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.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 10.5 endoderm , anterior
hhex.L laevis NF stage 11.5 to NF stage 12 endoderm , anterior

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  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).