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Display additional annotations [+]
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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.
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Display additional annotations [+]
Gene |
Clone |
Species |
Stages |
Anatomy |
foxe1
|
|
laevis
|
NF stage 37 and 38
|
head region
,
endoderm
,
thyroid primordium
,
pharyngeal region
|
cdx2
|
|
laevis
|
NF stage 37 and 38
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endoderm
,
midgut
,
hindgut
,
dorsal fin
|
pdx1
|
|
laevis
|
NF stage 37 and 38
|
endoderm
,
foregut
,
dorsal pancreatic bud
,
ventral pancreatic bud
|
nkx2-1
|
|
laevis
|
NF stage 37 and 38
|
brain
,
forebrain
,
head region
,
neuroectoderm
,
endoderm
,
[+]
|
hhex
|
|
laevis
|
NF stage 10.25
|
endoderm
,
anterior
|
foxa1
|
|
laevis
|
NF stage 37 and 38
|
epidermis
,
head region
,
notochord
,
endoderm
,
hindgut
,
[+]
|
gata4
|
|
laevis
|
NF stage 37 and 38
|
heart
,
endoderm
,
midgut
,
foregut
|
sftpc
|
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laevis
|
NF stage 37 and 38
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lung bud
|
ventx2.2
|
|
laevis
|
NF stage 10.25
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ventral
,
endomesoderm
,
posterior
|
ventx1.2
|
|
laevis
|
NF stage 10.25
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ventral
,
endomesoderm
,
posterior
|
sox9
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|
laevis
|
NF stage 37 and 38
|
otic vesicle
,
brain
,
notochord
,
endoderm
,
eye
,
[+]
|
satb2
|
|
laevis
|
NF stage 37 and 38
|
notochord
,
endoderm
,
hindgut
,
mouth primordium
,
mouth
,
[+]
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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.
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