Journal: Development (Cambridge, England)

Article Title: Requirements for endoderm and BMP signaling in sensory neurogenesis in zebrafish.

doi: 10.1242/dev.01936

Figure Lengend Snippet: Fig. 5. BMP gene expression in the pharyngeal pouches during epibranchial development, and defects in cas mutants. Lateral views, anterior to the left. Whole- mount in situ hybridization was performed in wild types at 22 hpf (top row), 30 hpf (second row) and 40 hpf (third row), and in cas mutants at 40 hpf (bottom row), for bmp2a (A,E,I,M), bmp2b (B,F,J,N), bmp4 (C,G,K,O) and bmp5 (D,H,L,P). Arrows indicate initial expression at the ventrolateral edges of each pouch. Arrow in H denotes bmp5 expression in the dorsal pouches. 1-5, pharyngeal pouches; ot, otic capsule.

Article Snippet: Human recombinant BMP4, BMP5, BMP7 and Noggin proteins (R&D Systems) were used for bead implantation experiments.

Techniques: Gene Expression, In Situ Hybridization, Expressing

Journal: Development (Cambridge, England)

Article Title: Requirements for endoderm and BMP signaling in sensory neurogenesis in zebrafish.

doi: 10.1242/dev.01936

Figure Lengend Snippet: Fig. 8. BMP4-coated beads induce, and NOG-coated beads inhibit, epibranchial formation. Lateral views, anterior to the left. In situ hybridization for phox2b mRNA to label epibranchials at 48 hpf. (A) Wild-type control embryo. (B-F) Embryos that received Affi-Gel Blue beads soaked in either PBS (B), BMP4 (10 μg/ml, C; 1 μg/ml, D) or NOG protein (500 pg/ml, E; 300 pg/ml, F). Arrows in C and D indicate ectopic phox2b+ cells adjacent to the bead; arrows in E and F denote missing ganglia adjacent to the NOG-coated bead. Asterisks indicate beads. gVII, facial; gIX, glossopharyngeal; gX, vagal; ot, otic vesicle. Scale bar: 50 μm.

Article Snippet: Human recombinant BMP4, BMP5, BMP7 and Noggin proteins (R&D Systems) were used for bead implantation experiments.

Techniques: In Situ Hybridization, Control

Journal: mAbs

Article Title: Discovery of ginisortamab, a potent and novel anti-gremlin-1 antibody in clinical development for the treatment of cancer

doi: 10.1080/19420862.2023.2289681

Figure Lengend Snippet: Ginisortamab IC50 for the inhibition of gremlin-1 antagonism of BMP-4/7 heterodimer activity, as measured in the BMP signaling reporter gene assay.

Article Snippet: Human gremlin-1 (R&D Systems, 5190-GR) or mouse gremlin-1 (R&D Systems, 956-GR) protein was added to the antibody titration and pre-incubated for 45 min at 37°C, 5% CO 2 , followed by a second pre-incubation with BMP-4/-7 heterodimer (R&D Systems, 3727-BP), BMP-4 (R&D Systems, 314-BP), or BMP-7 (R&D Systems, 354-BP) for 45 min at 37°C, 5% CO 2 , prior to addition to the cells.

Techniques: Inhibition, Activity Assay

Journal: Nature genetics

Article Title: Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation.

doi: 10.1038/ng1304

Figure Lengend Snippet: Figure 3 Hh signaling restricts Wnt targets BMP4 and Engrailed-1 to the crypt base in the adult rat colon in vivo. (a) Ptc is expressed by epithelial cells throughout the crypt and in several stromal cell types (arrows). (b) A gradient of expression of Engrailed-1 (En-1) in epithelial cells. (c) In colon of control rats, engrailed-1 was expressed at low levels in superficial enterocytes. (d) Engrailed-1 accumulated in the superficial enterocytes in cyclopamine-treated rats (arrow). (e–g) The gradient of BMP4 expression from the base of the crypts to the superficial enterocytes in control rats (e,g) was lost in cyclopamine-treated rats (f,h). (i) Western blots. The first seven lanes contained colonic homogenates of seven individual control rats; the last seven lanes contained samples from cyclopamine-treated rats. En-1, Engrailed-1. (j) Quantification of blots shown in i. Mean ± s.e.m. of the relative expression compared to the mean of the seven controls. P values (Student’s t-test): BMP2, P = 0.25; BMP4, P = 0.0001, Engrailed-1 (En-1), P = 0.002, Cyclin D1, P = 0.01. (k) The numbers of cells positive for 5-bromodeoxyuridine (BrdU) per crypt was counted (mean of ten crypts) in cyclopamine-treated rats (Cyclo) and controls (Co; P = 0.01).

Article Snippet: Antibodies to BMP2 (clone 355; 1:1,000; 1:2,000) and to BMP4 (clone 757; 1:500; 1:2,000) were from R&D Systems.

Techniques: In Vivo, Expressing, Control, Western Blot

Journal: Nature genetics

Article Title: Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation.

doi: 10.1038/ng1304

Figure Lengend Snippet: Figure 4 Ihh negatively regulates the Wnt pathway through downregulation of nuclear TCF4–β-catenin in vitro. (a) DLD-1 cells were transiently transfected with the TOPflash (filled bars) and FOPflash (open bars) reporter constructs in the presence or absence of an Ihh expression construct. A β-catenin expression construct was used as a positive control. Data are mean of three independent experiments (n = 12); P < 0.001 for TOP versus TOP+Ihh. (b) Western blots showing the expression of Wnt targets (BMP4, Cyclin D1 and c-Met), β-catenin and β-actin (as a loading control) in duplicate cultures of DLD-1 cells transfected with either pcDNA3.1(−) (Co) or an Ihh expression construct and harvested 24 h after transfection. Data are representative of three independent experiments with duplicate cultures. (c) There were no changes in nuclear levels of either β-catenin or TCF4 in DLD-1 cells transfected with control vector (+) relative to untransfected cells (−). (d) In nuclear extracts of cells transfected with Ihh (+), TCF4 disappeared almost completely and β- catenin was less abundant relative to untransfected cells (−). Data are representative of three independent experiments with duplicate cultures.

Article Snippet: Antibodies to BMP2 (clone 355; 1:1,000; 1:2,000) and to BMP4 (clone 757; 1:500; 1:2,000) were from R&D Systems.

Techniques: In Vitro, Transfection, Construct, Expressing, Positive Control, Western Blot, Control, Plasmid Preparation

Journal: Nature genetics

Article Title: Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation.

doi: 10.1038/ng1304

Figure Lengend Snippet: Figure 5 Loss of Ihh expression precedes the development of dysplasia in colon carcinogenesis. (a) An adenoma (asterisks) bordering histologically normal epithelium (arrows). (b) Histologically normal epithelial cells expressed Ihh. (c) Ihh was not expressed in the superficial dysplastic epithelial cells of the adenoma. (d–h) Immunohistochemical detection of Ihh and β-catenin in adjacent sections of tissue from an individual with FAP. (d,f) Ihh staining. Expression of Ihh was lost in a few morphologically normal crypts (asterisks), with a sharp transition to normal Ihh expression in the adjacent crypts on both sides (arrows). (e,g,h) β-catenin stain. The same crypts showed abnormal localization of β-catenin (f–h). (i) Western blots of DLD-1 cells carrying a doxycycline- inducible dnTCF4 construct. On treatment with doxycycline, FLAG epitope–tagged dnTCF4 was induced. An induction of Ihh expression was observed, whereas Wnt targets c-Met, BMP4 and Cyclin D1 (used as positive control) were downregulated. Data are representative of three independent experiments. (j) A model of the proposed Hh-Wnt axis in the colonic crypt. At the base of the crypt, Wnt factors are produced (probably by mesenchymal cell types1) that drive β-catenin–TCF signaling in epithelial precursor cells. Ihh expression is negatively regulated by β-catenin–TCF signaling and thus lost on mutation of APC during colorectal carcinogenesis. Ihh is expressed by mature colonocytes and regulates their differentiation. Hh signaling represses β-catenin–TCF signaling and restricts the expression of Wnt targets to the base of the colonic crypt.

Article Snippet: Antibodies to BMP2 (clone 355; 1:1,000; 1:2,000) and to BMP4 (clone 757; 1:500; 1:2,000) were from R&D Systems.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Construct, FLAG-tag, Positive Control, Produced, Mutagenesis