Journal: Developmental biology
Article Title: Myo/Nog Cell Regulation of Bone Morphogenetic Protein Signaling in the Blastocyst is Essential for Normal Morphogenesis and Striated Muscle Lineage Specification
Figure Lengend Snippet: Effects of Myo/Nog cell ablation on the expression of regulators of BMP signaling in the stage 2 embryo Untreated embryos and G8 or D4/complement treated stage X–XII embryos were incubated to stage 2 and double labeled with antibodies. The colors of the fluorescent tags are indicated in each panel. The photograph of the stage 2 embryo (A) illustrates the regions shown in the fluorescence micrographs. Overlap of red and green appears yellow/white in merged images. After ablating G8+ cells in the stage X–XII epiblast, neither G8 (B, C, E–I, K–N), noggin (NogP) (B) nor follistatin (Fol) (C) was detected in the stage 2 embryo. Ablation with the D4 MAb did not affect follistatin expression (D). Staining for chordin (Chor) was reduced along Koller’s sickle (ks) in G8 ablated embryos (E). In untreated stage 2 embryos, BMP4+ cells surrounded Koller’s sickle (F) and BMP2 was present below Koller’s sickle (bks) (G). P-Smad1/5/8 was detected only below Koller’s sickle (H) and did not overlap with G8+ cells in the posterior/medial epiblast (pme) (I). Cytoplasmic staining for Smad4 was present in the pme (J). In G8 ablated embryos, staining for BMP4 was increased in the pme (K), BMP2 remained below Koller’s sickle (L) and p-Smad1/5/8 was expanded into the pme (M) and central epiblast (ce) (N). P-Smad1/5/8 was not present above Koller’s sickle in embryos ablated with the D4 MAb (O). Bar, 1,250 µm in A; 9 µm in B–T.
Article Snippet: Double labeling was also carried out with IgG MAbs to Pax3 , sarcomeric myosin heavy chain (MF20 MAb) , neurofilament associated antigen (3A10 MAb) , the notochord marker NOT1, cardiac troponin I (TI-1 MAb) , Shh (5E1 MAb) ( ) (all obtained from the Developmental Studies Hybridoma Bank, Iowa City, IA), MyoD1 (NCL-MyoD1 MAb from Vector Laboratories, Burlingame, CA), BMP2 (Sigma-Aldrich, St. Louis, MO) and cardiac troponin T, isoform Ab-1 (MS-295-PO, Thermo Scientific, Fremont, CA), and the following polyclonal antisera: goat anti-mouse noggin (AF719, R & D Systems, Minneapolis, MN), rabbit anti-mouse chordin (ab24562, Abcam, Cambridge, MA), goat anti-human follistatin (sc-23553, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human BMP4 (5674R-100, Biovision, Mountain View, CA), rabbit anti-human p-Smad1/5/8 (9511, Cell Signaling Technology, Danvers, MA), rabbit anti-human Wnt3a (ab28472, Abcam), rabbit anti-human GATA4 (ab25992, Abcam) and rabbit anti-human Smad4 (sc-7154, Santa Cruz Biotechnology).
Techniques: Expressing, Incubation, Labeling, Fluorescence, Staining