recombinant frem1 protein  (R&D Systems)

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Frem1 activity is regulated by Sonic hedgehog signaling in the cranial neural crest mesenchyme during midfacial morphogenesis.

doi: 10.1002/dvdy.555

Figure Lengend Snippet: FIGURE 1 Frem1 is expressed in the cNCC mesenchyme during midfacial morphogenesis. The bilaterally paired facial growth centers that form the midface are shown in an intact GD11 embryo (A), after microdissection (B), and after enzymatic digestion to separate the ectodermal epithelium and cNCC-derived mesenchyme (C–F). Gene expression in epithelial and mesenchymal compartments of GD11 MNP and MxP tissue was determined by qPCR (G,H). Individual values are plotted along with the mean ± SEM of four independently collected and pooled tissue samples. Whole-mount tissue (I) and sections through the facial growth centers (J–L) were stained by ISH to visualize expression of Frem1 (I–J), Frem2 (K), and Fras1 (L). At GD11, the facial growth centers fuse to form a lambdoidal junction as shown in schematic (M). MNP, medial nasal process; MxP, maxillary process; LNP, lateral nasal process; MdP, mandibular process. Scale bars: 0.25 mm

Article Snippet: O9-1 cells were plated at 5 104 cells/mL (0.4 mL per well in a 24-well plate) in Matrigel-coated wells and allowed to attach in complete O9-1 media for 16 h. Recombinant FREM1 protein (R&D Systems, Minneapolis, MN) dissolved in DPBS was diluted to final concentrations of 7.5, 2.5, and 0.83 μg/mL in DMEM containing 1% FBS, and 400 μL treatment or DPBS vehicle media were added to each well.

Techniques: Laser Capture Microdissection, Derivative Assay, Gene Expression, Staining, Expressing

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Frem1 activity is regulated by Sonic hedgehog signaling in the cranial neural crest mesenchyme during midfacial morphogenesis.

doi: 10.1002/dvdy.555

Figure Lengend Snippet: FIGURE 2 Frem1 expression overlaps with Shh pathway target Gli1 during midfacial morphogenesis. The spatial expression of Frem1 and Gli1 was assessed by ISH at key stages of midfacial morphogenesis. Embryonic stages and planes of section are depicted by schematics in the left column. GD10.25 embryos were sectioned to visualize nascent MNP, LNP (A–C, plane of section 1), or MxP tissues (D–F, plane of section 2). GD11 embryos were sectioned to visualize MNP and LNP tissues along their proximal to distal axes (G–I, plane of section 3). GD13 embryos were sectioned to visualize the MxP-derived palatal shelves situated vertically along the sides of the tongue (J–L, plane of section 4). Staining is shown on adjacent sections for each stage/plane of section. Areas of apparent overlap in Gli1 and Frem1 expression are shown in schematics (C,F,I,L). MNP, medial nasal process; MxP, maxillary process; LNP, lateral nasal process; MdP, mandibular process; T, tongue; PS, palatal shelf. Scale bar: 0.25 mm

Article Snippet: O9-1 cells were plated at 5 104 cells/mL (0.4 mL per well in a 24-well plate) in Matrigel-coated wells and allowed to attach in complete O9-1 media for 16 h. Recombinant FREM1 protein (R&D Systems, Minneapolis, MN) dissolved in DPBS was diluted to final concentrations of 7.5, 2.5, and 0.83 μg/mL in DMEM containing 1% FBS, and 400 μL treatment or DPBS vehicle media were added to each well.

Techniques: Expressing, Derivative Assay, Staining

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Frem1 activity is regulated by Sonic hedgehog signaling in the cranial neural crest mesenchyme during midfacial morphogenesis.

doi: 10.1002/dvdy.555

Figure Lengend Snippet: FIGURE 3 Frem1 is regulated by the Shh pathway in cNCCs. O9-1 cNCCs were cultured with or without SHH ligand (0.4 μg/mL) and with or without the Smoothened antagonist vismodegib (Vismo, 100 nM) (A,B). SHH ligand caused an increase in Gli1 and Frem1 expression, which was blocked by the addition of vismodegib. Expression of Gli1 and Frem1 are increased in cNCCs expressing a constitutively active form of human Smoothened (SMOM2) relative to a GFP expressing line (C,D). cNCCs cultured with the Smoothened agonist SAG (50 nM) similarly demonstrated increased expression of Gli1 and Frem1 compared to vehicle alone (E–F). Values represent the mean ± SEM of N = 5 biological replicates for each condition. *P < 0.05, ** P < 0.01 (ANOVA with Tukey's post hoc test or two-tailed t-test)

Article Snippet: O9-1 cells were plated at 5 104 cells/mL (0.4 mL per well in a 24-well plate) in Matrigel-coated wells and allowed to attach in complete O9-1 media for 16 h. Recombinant FREM1 protein (R&D Systems, Minneapolis, MN) dissolved in DPBS was diluted to final concentrations of 7.5, 2.5, and 0.83 μg/mL in DMEM containing 1% FBS, and 400 μL treatment or DPBS vehicle media were added to each well.

Techniques: Cell Culture, Expressing, Two Tailed Test

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Frem1 activity is regulated by Sonic hedgehog signaling in the cranial neural crest mesenchyme during midfacial morphogenesis.

doi: 10.1002/dvdy.555

Figure Lengend Snippet: FIGURE 5 FREM1 promotes concentration-dependent cNCC proliferation. Scratch assays were performed on O9-1 cNNCs ± recombinant FREM1, and migration rate was determined over the period of scratch closure for up to 8 h. FREM1 had no impact on the rate of O9-1 cNCC migration at any concentration tested (A). Representative images from vehicle- and 7.5 μg/mL FREM1-treated cells show scratch closure over time (B–G). EdU incorporation was used to assess cell proliferation in FREM1-treated O9-1 cNCCs. Addition of recombinant FREM1 protein to culture media increased proliferation in a concentration-dependent manner (H). Representative images of vehicle- and FREM1-treated O9-1 cNCCs stained with Hoechst (blue) and EdU (green) (I,J). Values represent the mean ± SEM of N = 3 biological replicates for each condition. *P < 0.05, **P < 0.01 (one-way ANOVA)

Article Snippet: O9-1 cells were plated at 5 104 cells/mL (0.4 mL per well in a 24-well plate) in Matrigel-coated wells and allowed to attach in complete O9-1 media for 16 h. Recombinant FREM1 protein (R&D Systems, Minneapolis, MN) dissolved in DPBS was diluted to final concentrations of 7.5, 2.5, and 0.83 μg/mL in DMEM containing 1% FBS, and 400 μL treatment or DPBS vehicle media were added to each well.

Techniques: Concentration Assay, Recombinant, Migration, Staining

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Frem1 activity is regulated by Sonic hedgehog signaling in the cranial neural crest mesenchyme during midfacial morphogenesis.

doi: 10.1002/dvdy.555

Figure Lengend Snippet: FIGURE 4 GLI binding to the Frem1 promoter during facial morphogenesis. GLI3 ChIP-seq data from GD11.5 whole face (MNP, MxP, LNP, MdP) were analyzed for GLI3 binding at Shh pathway target genes. GLI3 peaks (rectangles below ChIP-seq tracks) were present at both RefSeq56 annotated transcription start sites for Frem1 (A) but not Frem2 (B) or Frem3 (C). GLI3 ChIP-seq signal at the Frem1 promoters was comparable to GLI3 signal at the promoters of known Shh targets Gli1, Ccnd2, and Foxf2 (D–F).

Article Snippet: O9-1 cells were plated at 5 104 cells/mL (0.4 mL per well in a 24-well plate) in Matrigel-coated wells and allowed to attach in complete O9-1 media for 16 h. Recombinant FREM1 protein (R&D Systems, Minneapolis, MN) dissolved in DPBS was diluted to final concentrations of 7.5, 2.5, and 0.83 μg/mL in DMEM containing 1% FBS, and 400 μL treatment or DPBS vehicle media were added to each well.

Techniques: Binding Assay, ChIP-sequencing

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Frem1 activity is regulated by Sonic hedgehog signaling in the cranial neural crest mesenchyme during midfacial morphogenesis.

doi: 10.1002/dvdy.555

Figure Lengend Snippet: FIGURE 6 Frem1 expression is diminished by Shh pathway inhibition during pathogenesis of midfacial hypoplasia/cleft lip. In utero exposure to the Shh pathway antagonist cyclopamine from GD8.25 to 9.5 disrupts midfacial morphogenesis, resulting in either midfacial hypoplasia (B) or cleft lip (C) compared to control (A), as shown at GD17. Frontonasal prominence (FNP) tissue that gives rise to the MNPs and LNPs was microdissected from vehicle- and cyclopamine-exposed embryos at GD9.25, and gene expression was determined by qPCR (D). Expression data from pooled tissue of an entire litter are shown as individual points along with mean ± SEM. Expression of both Gli1 and Frem1 was significantly reduced in FNP tissue from embryos exposed to cyclopamine vs vehicle alone. * P < 0.05, ** P < 0.01 (two-tailed t-test). Sections through MNP and LNP tissue produced from GD10 vehicle- and cyclopamine-exposed embryos were stained by ISH to visualize expression of Gli1 (E,F) and Frem1 (G,H). Dashed outlines surround the mesenchyme of the MNP and LNP tissues. Cyclopamine exposure reduced expression of Gli1 and Frem1 in the MNP mesenchyme. MNP, medial nasal process; LNP, lateral nasal process; NE, neuroectoderm. Scale bar: 0.125 mm

Article Snippet: O9-1 cells were plated at 5 104 cells/mL (0.4 mL per well in a 24-well plate) in Matrigel-coated wells and allowed to attach in complete O9-1 media for 16 h. Recombinant FREM1 protein (R&D Systems, Minneapolis, MN) dissolved in DPBS was diluted to final concentrations of 7.5, 2.5, and 0.83 μg/mL in DMEM containing 1% FBS, and 400 μL treatment or DPBS vehicle media were added to each well.

Techniques: Expressing, Inhibition, In Utero, Control, Gene Expression, Two Tailed Test, Produced, Staining