Kurmann AA , Serra M , Hawkins F , Rankin SA , Mori M , Astapova I , Ullas S , Lin S , Bilodeau M , Rossant J , Jean JC , Ikonomou L , Deterding RR , Shannon JM , Zorn AM , Hollenberg AN , Kotton DN .

R01 HL114898 NHLBI NIH HHS , R01 HL095993 NHLBI NIH HHS , R01 HL108678 NHLBI NIH HHS , R01 HL111574 NHLBI NIH HHS , R01 HL122442 NHLBI NIH HHS , T32 DK007516 NIDDK NIH HHS , U01 HL110967 NHLBI NIH HHS , U01HL110-967 NHLBI NIH HHS , Canadian Institutes of Health Research

Xla Wt + DMH-1 (Fig.S4.b)
Xla Wt + DMH-1 (Fig.S4.c)
Xla Wt + Hsa.BMP4 + Hsa.FGF2 + foregut endoderm explant (Fig.4.c)
Xla Wt + LY294002 (Fig.S4.b)
Xla Wt + LY294002 (Fig.S4.c)
Xla Wt + PD161570 (Fig.S4.b)
Xla Wt + PD161570 (Fig.S4.c)
Xla Wt + su5402 (Fig.4.b)
Xla Wt + su5402 (Fig.S4.b)
Xla Wt + su5402 (Fig.S4.c)
Xla Wt + {dn}bmpr1a (Fig.S4.b)
Xla Wt + {dn}bmpr1a (Fig.S4.c)
Xla Wt + {dn}fgfr1 (Fig.S4.b)
Xla Wt + {dn}fgfr1 (Fig.S4.c)

Figure 4. ESC Models Predict the Evolutionarily Conserved Pathways That Are Necessary and Sufficient for Thyroid Specification in Mouse and Xenopus Embryos (A) BMP signaling blockade abrogates thyroid bud formation in mouse: whole foreguts from E8.0 embryos (6–8 ss) were cultured for 2–3 days in the presence of the BMP antagonist DMH-1. Co-localization of Nkx2-1+ and Pax8+ shows a discrete thyroid bud formed in control cultures (upper left). No thyroid bud was apparent when BMP signaling was inhibited (lower left). Reduced pSmad1/5 content in the presence of DMH-1 (right; western blot). (B) In vivo pharmacological loss of function using antagonists of type I BMP receptor (DMH-1) or FGF receptor (SU5402) activity in Xenopus embryos. Whole embryos were cultured in the presence of the antagonists from stage NF13-20 and assayed for the indicated genes at stage NF35. The number of embryos with the displayed phenotype is indicated. (C) FGF and BMP signaling is sufficient to induce thyroid gene expression in dissected Xenopus foregut endoderm explants. Explants were dissected at stage NF15, cultured until NF35, and assayed for the indicated genes. (D) Schematic of inhibition of BMP4 or FGF2 signaling blocking thyroid specification.

Figure S4. Role of BMP and FGF signaling in Xenopus and mouse whole embryo thyroid specification, Related to Figure 4. (A) Analysis of phosphorylated SMAD1/5/8, ERK1/2 and AKT during Xenopus thyroid development. Confocal immunofluorescence analysis on sagittal sections of stage NF20 Xenopus embryos showing nuclear phospho-SMAD1/5/8 (A-A’), phospho-ERK1/2 (B-B’) and phospho-AKT (C-C’) in the ventral anterior foregut endoderm and mesoderm where the thyroid will form (anterior left). (D-G”) Confocal immunofluorescence analysis at stage NF33/34 on transverse sections through the ventral pharyngeal region show that the nascent Nkx2-1+ thyroid primordium budding from the Sox2+ pharyngeal floor (D-D”) stains positive for phospho-SMAD1/5/8 (E-E”), phospho-ERK1/2 (F-F”), and phospho-AKT ser473. Fibronectin staining outlines the mesoderm (G-G”). Scale bars 50 μm. (B) BMP and FGF, but not RA or Wnt/β-catenin, signals during stage NF13-20 are necessary for induction of thyroid nkx2-1 in Xenopus. Xenopus embryos were either: 1) injected with mRNA encoding dominant negative BMP receptors (dnBMPR) or dominant negative FGF receptors (dnFGFR), or 2) cultured in the indicated pathway antagonists from stage NF13 to NF20. In-situ hybridization analysis for nkx2-1 expression at stage NF35 was used as marker of thyroid specification. The number of embryos for each condition with the displayed phenotype is indicated. (C) Immunostaining of pAKT, pERK and pSmad1/5/8, confirmed the efficacy of BMP and FGF pathway inhibition in the indicated conditions. (D) In-situ hybridization analysis for nkx2-1 expression at stage NF35 of Xenopus embryos cultured in the indicated pathway antagonists from stage NF20 to NF35. The number of embryos for each condition with the displayed phenotype is indicated.

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