XB-ART-53579Elife. September 25, 2017; 6
SMOC can act as both an antagonist and an expander of BMP signaling.
PubMed ID: 28323621
Article link: Elife.
Genes referenced: ag1 bmp2 dspp fst ggt1 nrp1 otx2 pnmt sdc2
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|Figure 1. Download figureOpen in new tab Figure 1. Xenopus SMOC-1 and Drosophila pent are orthologues that inhibit BMP signaling downstream of the BMP receptor. (A) Schematic representation of vertebrate SMOC and Drosophila Pent: SP- signal peptide, FS – Follistatin-like domain, Tg1 – Thyroglobulin type I-like domain, EC- Extracellular calcium binding domain (B) Dorsalized phenotypes of stage 35 Xenopus embryos following overexpression of mRNAs for XSMOC-1 or codon-optimized pent (co-pent): Xenopus embryos were injected bilaterally at the two-cell stage with 200 pg of mRNA for either GFP (control), XSMOC-1, or co-pent. The exaggerated dorsal/anterior structures and diminished posterior structures observed following XSMOC-1 or co-pent overexpression were observed in 95% of embryos in four independent experiments (n = 130). (C) RT-PCR analysis of animal cap (AC) explants removed from stage 8/9 embryos injected bilaterally at the two-cell stage with either 450 pg of GFP (Control), 150 pg of constitutively active BMP receptor IB (caBMPR-IB) plus GFP (300 pg), or 150 pg of caBMPR-1B plus XSMOC-1 or co-pent (300 pg) mRNAs. The AC explants were incubated until stage 20 before RNA extraction. Induction of the BMP signaling target gene, XVent, by caBMPR-1B was blocked by co-expression of either XSMOC-1 or co-pent. –RT control, without reverse transcriptase. DOI: http://dx.doi.org/10.7554/eLife.17935.003|
|Figure 1—figure supplement 2. Nucleotide alignment of pentagone and codon optimized pentagone (co-pentagone). Amino acid identity is 100%; nucleotide identity is 73%. DOI: http://dx.doi.org/10.7554/eLife.17935.005|
|Figure 2—figure supplement 1. Dimeric XSMOC-1 is not dissociated by chelation or reduction. (A) SEC profile showing that XSMOC-1, refolded in the presence of 2 mM CaCl2, continues to migrate as a dimer (↓) upon the subsequent chelation of calcium ions by dialysis in the presence of 10.5 mM EDTA. (B) SDS-PAGE analysis of XSMOC-1, XSMOC-1 ∆EC, and XSMOC-1 EC in the presence (+) and absence (−) of β-mercaptoethanol shows that the XSMOC-1, XSMOC-1 ∆EC dimers are not linked by disulfide bonds. DOI: http://dx.doi.org/10.7554/eLife.17935.007|
|Figure 5—figure supplement 1. The EC domains of SMOC and Pent are conserved and share structural homology to BM-40. (A) Predicted heparin binding sequence of hSMOC-1 aligned with the same region of XSMOC-1 and Pent. Conserved amino acids are shown in bold and basic amino acids are shown in red. (B) Unsupervised homology model for the XSMOC-1 EC domain, based on the structure of the EC domain of BM-40 (1SRA.pdb), constructed using the Protein Homology/analogy Recognition Engine (PHYRE, RRID:SCR_010270). The XSMOC-1 EC domain aligns in the exact orientation with the BM-40 model; alpha helices E and F are noted. (C) Electrostatic surface potential maps (based on solvent accessibility) of the EC domains of XSMOC-1 and BM-40 showing an area of positive charge (shown in blue) in XSMOC-1 that is absent in the equivalent region of BM-40. DOI: http://dx.doi.org/10.7554/eLife.17935.012|
|Figure 6. Immunofluorescence assay demonstrating that XSMOC-1 can promote BMP signaling at a distance from its source by competitive binding to HSPGs. BMP4-soaked beads (100 μm) were implanted into 0.5 μl drops of 0.7% low melting point (LMP) agarose (A, D), LMP agarose containing 10 μg/ml heparan sulfate (B, E), or LMP agarose containing heparan sulfate (10 μg/ml) and 100 μg/ml XSMOC-1EC (C, F) on 8-well chamber slides. C33A-2D2-09 cells, harboring luciferase under the control of a BMP response element (BRE) were seeded at 2 × 104 cells/well and incubated for 48 hr in serum-free medium. Luciferase immunofluorescence (green) indicates cells positive for BMP signaling. Cell nuclei (blue) were stained with DAPI. Dashed lines in A-C indicate the boundaries between C33A-2D2-09 cells and the agarose drops. Analysis of four fields of view in three separate experiments demonstrated the number of luciferase-positive cells to be lower (16% ± 8%) adjacent to matrices containing HS alone (B, E) compared to those containing both HS and XSMOC-1EC (69% ± 26%) (C, F). Representative fields are shown. DOI: http://dx.doi.org/10.7554/eLife.17935.013 Figure 6—source data 1. Percentage of luciferase positive cells per field of view. The number of Luciferase positve cells in five fields of view were calculated as a percentage of the total number of cells in each field. DOI: http://dx.doi.org/10.7554/eLife.17935.014 Download source data [figure-6—source-data-1.media-2.docx]|
|Figure 7. In vivo assay demonstrating that XSMOC-1 can expand the range of BMP2 signaling. (A) Schematic diagram of the host/donor animal cap (AC) transfer assay. (B–F) Donor AC grafts expressing mCherry (red) and host/donor immunofluorescent nuclear staining of pSmad 1/5/8 (green). (B) Control host + mCherry mRNA (200 pg)-injected donor AC (mCherry AC); endogenous pSmad is not detectable, (C) BMP2 mRNA (300 pg)-injected host + mCherry AC; pSmad is detected throughout the host tissue and donor AC, (D) BMP2 mRNA (30 pg)-injected host (BMP2-30 pg host) + mCherry AC; pSmad is detected in the host tissue and at the host tissue/AC boundary, (E) BMP2-30 pg host + mCherry/XSMOC-1 mRNA (10 pg)-injected AC; pSmad is detected in the host tissue and 4–5 cell diameters into the AC (F) BMP2-30pg host + mCherry/XSMOC-1 mRNA (300 pg)-injected AC; pSmad is not detected in the AC and is also absent at the host tissue/AC boundary. DOI: http://dx.doi.org/10.7554/eLife.17935.015|