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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 on Xenbase with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
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