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Fig. S7. BMP/pSMAD1 signaling is upregulated in Osr1/2-depleted embryos and temporal Smad1 overexpression can inhibit lung specification. (A) Immunofluorescence analysis of phospho-SMAD1/5/8 (green) and fibronectin (red) at stage35/36 in Osr1/2-MO bilaterally injected embryos. Section 1 through the foregut is anterior to section 2. Yellow arrows indicate ectopic pSMAD1/5/8 in dorsal foregut endoderm, and yellow asterisks indicate ectopic pSMAD1/5/8 and reduced fibronectin at the level of the nascent lung buds. (B) Embryos were injected with RNA encoding a hormone-inducible GR-Smad1 (800 pg) at the four/eight-cell stage into 1 side of the embryo. Between stages 25 and 35, embryos were treated with or without 1 µM dexamethasone (dex) to activate the construct, thus inducing active BMP signaling. In situ hybridization at stage 35 shows that activated GR-Smad1 is inhibitory to wnt2b, nkx2.1 and sox2 expression. (C) Overexpression of GR-SMAD1 does not cause cell death. Embryos were injected and treated as in B, and analyzed at stage 35/36 by immunofluorescence for Sox2 (red) and activated caspase 3 (green). No cell death was detectable in either condition. (D) LDN treatment rescues the elevated pSMAD1/5/8 signaling observed in Osr1/2-MO embryos. Embryos were injected as in A and treated from stages 25-35 with DMSO vehicle or 10 µM LDN193189 and assayed by immunofluorescence for phospho-SMAD1/5/8 (green). Image published in: Rankin SA et al. (2012) Copyright © 2012. Image reproduced with permission of the Publisher. Experiment + Assay Source Phenotypes and Disease Xtr Wt + osr1 MO + osr2 MO + NF35/36 (immunohistochemistry) fig.S7.a Anatomical Phenotype abnormal development of foregut abnormal development of lung Expression Phenotype decreased amount fn1 expression in lung bud increased amount smad1 expression in dorsal foregut increased amount smad1 expression in lung bud Xtr Wt + smad1-Rno.GR + DEX + NF35/36 (in situ hybridization) fig.S7.b Expression Phenotype decreased amount nkx2-1 expression in lung bud decreased amount wnt2b expression in lateral plate mesoderm Xtr Wt + smad1-Rno.GR + DEX + NF35/36 (immunohistochemistry) fig.S7.c Expression Phenotype decreased amount sox2 expression in foregut Xtr Wt + osr1 MO + osr2 MO + NF35/36 (immunohistochemistry) fig.S7.d Expression Phenotype increased amount smad1 expression in foregut Larger Image Printer Friendly View

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