Journal:

Article Title: Sexual Dimorphism in the Effect of GDF6 Deficiency on Murine Tendon

doi: 10.1002/jor.20916

Figure Lengend Snippet: Gene expression for Gdf5 and Gdf7 in GDF6 deficient (ko, shown in white) and wild type (wt, shown in grey) [A] male tail tendons; [B] male Achilles tendons; [C] female tail tendons; and [D] female Achilles tendons. Relative expression was calculated using the 2-ΔΔCt method, with the first Δ relative to internal 18s rRNA, and the second Δ relative to wild type in each age, sex, and site group. n = 4-8 per group. No two-fold or greater difference in expression was seen between GDF6 deficient and wild type tendons. Error bars calculated as described in reference 27.

Article Snippet: TaqMan Gene Expression Assays were ordered from ABI (Applied Biosystems) with the following catalog numbers (GDF5 inventoried #Mm00433564_m1; GDF7 inventoried #Mm00807130_m1).

Techniques: Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 alleviates LPS-induced ALI and pulmonary dysfunction in mice. (a) Mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI, and serum GDF7 levels were detected using an ELISA kit after 12 h ( n = 6). (b) The levels of Gdf7 mRNA in lung tissues with or without LPS instillation were detected using quantitative real-time PCR ( n = 6). (c) The levels of GDF7 protein in lung tissues with or without LPS instillation were detected using western blot ( n = 6). (d-e) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and serum and lung GDF7 levels in mice were detected using an ELISA kit after 24 h ( n = 6). (f) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, fresh lungs were harvested for the analysis of LDH activity using a commercial kit ( n = 6). (g) Lung wet to dry ratio ( n = 6). (h) Total proteins in BALF ( n = 6). (i) Arterial blood gas analysis results ( n = 6). (j) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). (k) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with a lethal dose of LPS (25 mg/kg). The survival rate was monitored every 12 h post-LPS treatment ( n = 20). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: Injection, Saline, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 inhibits inflammation and oxidative stress in LPS-treated mice. (a) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, BALF was collected for the analysis of total cells, neutrophils, and macrophages ( n = 6). (b) MPO activity in lung tissues ( n = 6). (c) IL-6 and TNF- α levels in lung tissues ( n = 6). (d) ROS content in lung tissues ( n = 6). (e) MDA and 4-HNE levels in lung tissues ( n = 6). (f-g) Total SOD activity and GSH content in lung tissues ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: Injection, Saline, Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 reduces LPS-stimulated inflammation and oxidative stress in primary macrophages. (a) Primary peritoneal macrophages were pretreated with rmGDF7 (10 nmol/L) for 24 h and then stimulated with LPS (100 ng/mL) for another 6 h. Cell medium was collected for the analysis of IL-6 and TNF- α ( n = 6). (b) ROS content in macrophages ( n = 6). (c) MDA and 4-HNE levels in macrophages ( n = 6). (d-e) Total SOD activity and GSH content in macrophages ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 attenuates LPS-induced ALI through activating AMPK in vivo. (a) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, lung tissues were harvested for the analysis of the AMPK pathway using western blot ( n = 6). (b) To inhibit AMPK, ALI mice were intraperitoneally injected with CpC (20 mg/kg) at 2 h pre- and 2 h post-rmGDF7 injection, and then IL-6 and TNF- α levels in lung tissues were detected ( n = 6). (c) ROS content in lung tissues ( n = 6). (d) MDA and 4-HNE levels in lung tissues ( n = 6). (e) LDH activity in lung tissues ( n = 6). (f) Lung wet to dry ratio ( n = 6). (g) Arterial blood gas analysis results ( n = 6). (h) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: In Vivo, Injection, Saline, Western Blot, Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 attenuates LPS-induced ALI through activating AMPK in vitro. (a) Primary peritoneal macrophages were pretreated with rmGDF7 (10 nmol/L) for 24 h and then stimulated with LPS (100 ng/mL) for another 6 h. To inhibit AMPK, macrophages were pretreated with CpC (10 μ mol/L) for 12 h before LPS stimulation. Cell medium was collected for the analysis of IL-6 and TNF- α ( n = 6). (b) ROS content in macrophages ( n = 6). (c) MDA and 4-HNE levels in macrophages ( n = 6). (d-e) Total SOD activity and GSH content in macrophages ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: In Vitro, Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 activates AMPK through downregulating STING in vivo. (a) Mice were subcutaneously injected with rmGDF7 (25 μ g per mouse), and 24 h later, ALI mice were intratracheally injected with LPS (5 mg/kg in 50 μ L saline) to generate sepsis-induced ALI. After 12 h, lung tissues were harvested for the analysis of STING using western blot ( n = 6). (b) To investigate the involvement of STING, STING KO mice were used, and lung tissues were harvested for the analysis of the AMPK pathway using western blot ( n = 6). (c) IL-6 and TNF- α levels in the lung tissues ( n = 6). (d) ROS content in lung tissues ( n = 6). (e) LDH activity in lung tissues ( n = 6). (f) Lung wet to dry ratio ( n = 6). (g) Arterial blood gas analysis results ( n = 6). (h) Respiratory function was detected by Buxco, including pulmonary ventilation, lung compliance, and airway resistance ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant. NS indicated no statistical significance.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: In Vivo, Injection, Saline, Western Blot, Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: GDF7 activates AMPK through downregulating STING in vitro. (a) Primary peritoneal macrophages were pretreated with rmGDF7 (10 nmol/L) for 24 h and then stimulated with LPS (100 ng/mL) for another 6 h. To investigate the involvement of STING, STING KO macrophages were used. Cells were harvested for the analysis of the AMPK pathway using western blot ( n = 6). (b) Cell medium was collected for the analysis of IL-6 and TNF- α ( n = 6). (c) ROS content in macrophages ( n = 6). (d) MDA and 4-HNE levels in macrophages ( n = 6). (e-f) Total SOD activity and GSH content in macrophages ( n = 6). All data were presented as mean ± SD, and ∗ P < 0.05 was regarded to be statistically significant. NS indicated no statistical significance.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques: In Vitro, Western Blot, Activity Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Growth Differentiation Factor 7 Prevents Sepsis-Induced Acute Lung Injury in Mice

doi: 10.1155/2022/3676444

Figure Lengend Snippet: Schematic diagram of the molecular mechanisms underlying GDF7-regulatedsepsis-induced ALI.

Article Snippet: Recombinant Mouse GDF7 Protein (rmGDF7, #779-G7), Mouse IL-6 Quantikine ELISA Kit (#M6000B), and Mouse TNF- α Quantikine ELISA Kit (#MTA00B) were purchased from R&D Systems, Inc. (Minneapolis, MN, Canada).

Techniques:

Journal: Journal of Tissue Engineering

Article Title: Therapeutic potential and mechanisms of umbilical cord mesenchymal stem cells differentiating into tendon cells and promotion of rotator cuff tendon-bone healing

doi: 10.1177/20417314251315185

Figure Lengend Snippet: Analysis of the induction effect of BMP-12, GDF-7, and CTGF on the differentiation of UCMSCs: (a) morphological changes of UCMSCs after induction treatment, (b) mRNA expression of MKX, SCX, and TNC detected by qPCR, (c) bands and protein expression of MKX, SCX, and TNC detected by WB, and (d) COL-I and COL-III expression detected by immunofluorescence (400×). ** p < 0.01 and *** p < 0.001 compared with the control group.

Article Snippet: Rat umbilical cord mesenchymal stem cells (UCMSCs, CP-R302, Pricella, China), 293T cells (Pricella, China), DMEM medium (L110KJ, Yuanpei, China), Fetal bovine serum(AC03L055, life-ilab, China), GDF-6 (HY-P79333, MedChemexpress, USA), GDF-7 (92004ES10, Yisheng, China), CTGF (HY-P72154, MedChemexpress Biotechnology, USA), GoldenstarTM RT6 cDNA Synthesis Kit Ver.2 (TSK302M, Tsingke, China), qPCR Mix (SYBR Green I) kit and PCR primer (TSE002, Tsingke, China), RIPA lysis buffer (Beyotime, China), TNC (A1927, abclonal, China), MKX (ab236400, abcam, UK), SCX (ab58655, abcam, UK), GAPDH (A19056, abcam, UK), Hes1 (ab71559, abcam, UK), Hras (ab32417, abcam, UK), GAPDH Rabbit mAb (A19056, abclonal, China), Lentiviral Packaging Kit (41102ES10, Yisheng, China), Fetal bovine serum (26050070, Thermo, USA), trypsin (LP0042, Thermo, USA), PBS (10010001, Thermo, USA).

Techniques: Expressing, Immunofluorescence, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 2. Characterization of recombinant GDF6, GDF7, and BMP10. A, immuno- blotting analyses of recombinant GDF6 and GDF7. Recombinant proteins were analyzed under reduced or nonreduced conditions following blotting with specific antibodies. B, biochemical properties of purified recombinant human BMP10. BMP10 with an N-termi- nal His6 tag was purified from conditioned media of 293T cells using metal chelate chro- matography. BMP10 was detected by Coomassie Blue staining (lanes 1–3) and immuno- blotting using antibodies against polyhistidine His6 (lanes 4 and 5) under nonreduced or reduced conditions. Some samples were treated with peptide N-glycosidase F (PNGase) to remove N-linked carbohydrate side chains.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Recombinant, Purification, Staining

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 3. Treatment with GDF6, GDF7, and BMP10 stimulated the Smad1/5/8 pathway but not the Smad2/3 pathway in mouse MC3T3 cells. Cells were transiently transfected with the BMP-responsive reporter BRE (A) or the TGF/ac- tivin-responsive CAGA reporter (B). At 48 h after transfection, cells were incubated for 20 h in the absence(Ct)orpresenceofGDF6(10nM),GDF7(10 nM), or BMP10 (3 nM). Cells treated with BMP2 (0.1 nM), BMP7 (3 nM), and TGF (0.1 nM) served as pos- itive controls. C, cells were transfected with the BRE reporter and treated with increasing doses of GDF6, GDF7, or BMP10. The relative luciferase activity was normalized based on the -galacto- sidase activity to correct for variations in transfec- tion efficiency. Results are presented as the mean S.E. D, immunoblotting analysis of Smad1 and Smad2 phosphorylation was performed using cell extracts following treatment of MC3T3 cells with BMP2 (0.1 nM), TGF (0.1 nM), GDF6 (10 nM), GDF7 (10 nM), or BMP10 (3 nM). Treatment with GDF6, GDF7, or BMP10 induced the phosphoryla- tion of Smad1 (top) but not Smad2 (Bottom). BMP2 and TGF served as positive controls. Arrows indi- cate the immunoreactive bands of phosphoryl- ated Smad proteins. Ct, control.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Incubation, Luciferase, Activity Assay, Western Blot, Phospho-proteomics, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 4. Antagonistic effects of inhibitory Smad proteins on the stimulation of the BRE promoter by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of plasmids encoding the inhibitory Smad6 or Smad7. After transfection, cells were incubated for 20 h with or without GDF6 (10 nM), GDF7 (10 nM), BMP10 (3 nM), or BMP2 (0.1 nM), before determina- tion of luciferase activity. The relative luciferase activity was normalized based on the -galactosidase activity to correct for variations in transfection efficiency. Results are presented as the mean S.E.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 5. Overexpression of ALK3 or ALK6 confers ligand signaling by GDF6, GDF7, or BMP10. A, COS7 cells were transfected with 500 ng of the BRE reporter construct together with 30 ng of different ALK plasmids or increasing amounts of the ALK3 or ALK6 plasmid. After 5 h of incubation, cells were treated with GDF6 (1 nM), GDF7 (1 nM), or BMP10 (3 nM) for 24 h. The relative luciferase activity was normalized based on the -galactosidase activity. Results are presented as the mean S.E. B, confirmation of the overexpression of different human ALK receptor transcripts in transfected cells using semi-quantitative PCR. , transfection with the empty vector; , transfection with individual ALK plasmid. In select cases, the endogenous monkey transcripts were amplified in COS7 cells because of sequence conservation.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Over Expression, Transfection, Construct, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Amplification, Sequencing

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 7. Overexpression of the ALK3 shRNA suppressed the stimulation of BRE promoter activities by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of the ALK3 shRNA or control shRNA. Two days after transfection, cells were incubated for 20 h with or without GDF6 (10 nM), GDF7 (10 nM), or BMP10 (3 nM). As a negative control, cells were transfected with the ALK3 shRNA before treatment with TGF (0.1 nM). The relative lucif- erase activity was normalized based on the -galactosidase activity to correct for varia- tions in transfection efficiency. Results are presented as the mean S.E.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Over Expression, shRNA, Transfection, Plasmid Preparation, Control, Incubation, Negative Control, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Receptors and Signaling Pathways for Orphan Bone Morphogenetic Protein/Growth Differentiation Factor Ligands Based on Genomic Analyses

doi: 10.1074/jbc.m504629200

Figure Lengend Snippet: FIGURE 8. Overexpression of BMPRII shRNA or ActRIIA shRNA blocked the stimula- tion of the BRE promoter activity by GDF6, GDF7, and BMP10. MC3T3 cells were transfected with 150 ng of the BRE reporter plasmid with or without increasing amounts of the BMPRII, ActRIIA, or control shRNA. As a negative control, cells were transfected with the BMPRII shRNA or ActRIIA shRNA before treatment with TGF. The relative lucif- erase activity was normalized based on the -galactosidase activity to correct for varia- tions in transfection efficiency. Results are presented as the mean S.E.

Article Snippet: RecombinantmouseGDF6 and GDF7 as well as human BMP2, BMP7, and TGF- 1 were from R & D Systems (Minneapolis, MN).

Techniques: Over Expression, shRNA, Activity Assay, Transfection, Plasmid Preparation, Control, Negative Control

Journal: PLoS Biology

Article Title: Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck Feathering

doi: 10.1371/journal.pbio.1001028

Figure Lengend Snippet: (A) Adult Na / Na . Feathers are absent on the neck and head, excepting the crown. (B) E8.5 embryos hybridized with a β - catenin probe to mark the patterning field and feather primordia. Punctate expression of β - catenin in feather placodes is seen on the body but not the neck of the mutant. WT, wild type; Na/Na , Naked neck. (C) E12.5 embryos showing limited lateral tract expansion (arrows) in Na / Na , reducing body feather coverage. (D) Quantitative RT-PCR determination of BMP12 expression in body and neck skin of E7.5 and E8.5 wild type and Na / Na embryos. (E,F) In situ hybridization detecting BMP12 in wild type and Na/Na embryos at (E) E7.5 and (F) E8.5. Wild type and mutant embryos were hybridized and photographed together. Na/Na embryos have elevated and diffuse expression of BMP12 in the skin. (G) Sequence traces of PCR products from E8.5 Na/+ . Genomic DNA PCR products display double peaks following a TA indel polymorphism in the BMP12 3′UTR. RT-PCR products from neck and body skin show a single trace throughout, indicating predominant expression of the Naked neck BMP12 allele, while both alleles are detected in RT-PCR products from other tissues. (H) Schematic showing insertion of chromosome 1 sequences into chromosome 3 at the Naked neck locus. Chromosome coordinates, the Naked neck identical by descent segment, gene names, exons, untranslated regions, and non-coding elements conserved between chicken and human genomes, based on the ENSEMBL genome viewer, are indicated.

Article Snippet: Recombinant human BMP4 and mouse BMP12 (R&D Systems) were used.

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, In Situ Hybridization, Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck Feathering

doi: 10.1371/journal.pbio.1001028

Figure Lengend Snippet: (A) Application of recombinant BMP12 to cultured skin for 15 h leads to elevation of SOSTDC1 expression, determined by quantitative RT-PCR. (B–E) Detection of SOSTDC1 expression by in situ hybridization. (B) At E7.5 wild type embryos have two rows of feather placodes running up the neck. SOSTDC1 is expressed at the periphery of the placodes and is not detected in the medial region between the lateral rows of placodes. (C) By E8.5 the medial region of the neck is populated by feather placodes. (D) E7.5 Na/Na embryos have placodes on the dorsum, but widespread SOSTDC1 expression on the neck, including the medial region. (E) At E8.5 the Naked neck skin maintains a high level of widespread SOSTDC1 expression, with peri-placode expression visible on the body. (F) Ex vivo rescue of the Naked neck phenotype by suppression of BMP signaling. E7.0 Na/Na skin was cultured in the presence of dorsomorphin (DM, used at 8 µM) and SB203580 (SB, used at 5 µM), pharmacological inhibitors of BMP signal transduction, for 48 h. This permitted feather development across most of the mutant neck skin.

Article Snippet: Recombinant human BMP4 and mouse BMP12 (R&D Systems) were used.

Techniques: Recombinant, Cell Culture, Expressing, Quantitative RT-PCR, In Situ Hybridization, Ex Vivo, Transduction, Mutagenesis

Journal: PLoS Biology

Article Title: Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck Feathering

doi: 10.1371/journal.pbio.1001028

Figure Lengend Snippet: (A,B) β - catenin in situ hybridization revealing the effects of recombinant BMP12 application on feather periodicity and regional distribution in wild type skin after 48 h. (C,D) Dose effects of BMP12 on the number of feather placode rows on the spinal tract of the body. Feather primordia are visualized by β - catenin in situ hybridization. (E) SOSTDC1 expression on control and 80 ng/ml BMP12 treated skin explants. Feather placodes express SOSTDC1 at their periphery on both body and neck. Upon application of BMP12, the non-placode skin of the neck expresses a higher level of SOSTDC1 than does the body (compare signal intensity in the red boxed area to that of the blue boxed area). (F) Schematic of reaction-diffusion regulatory interactions. Adjacent numbering refers to mathematical terms in the supporting methods. C I represents the constitutive, ubiquitous production of the Inhibitor. (G) Quantification of periodicity of Activator foci in simulated neck and body with differential sensitivities to Inhibitor. C I increases along the x -axis. (H) Pattern outcomes from reaction-diffusion dynamics in a field with graded sensitivity to the Inhibitor. Abolition of Activator foci in the more sensitive part of the field is achieved with little effect on periodic spacing in the remainder of the field, producing a macropatttern that matches the effects of BMP12 treatment on cultured skin. Colors denote local Activator concentrations, with black representing the highest and white the lowest Activator levels. Areas with high Activator concentration represent placodes.

Article Snippet: Recombinant human BMP4 and mouse BMP12 (R&D Systems) were used.

Techniques: In Situ Hybridization, Recombinant, Expressing, Control, Diffusion-based Assay, Cell Culture, Concentration Assay

Journal: PLoS Biology

Article Title: Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck Feathering

doi: 10.1371/journal.pbio.1001028

Figure Lengend Snippet: (A) RA administration reduces the density of placodes, which are detected by β - catenin in situ hybridization, completely inhibiting placode formation at high doses. Suppression of BMP signaling with 4 µM dorsomorphin and 5 µM SB203580 rescues placode formation in the presence of RA. (B) Quantification of placode density on neck and body upon RA treatment. With increasing doses of RA the feather density on body and neck converges and ultimately all feather placode formation is suppressed. (C) RA sensitizes body skin to BMP-driven inhibition of feather development. The application of 0.1 µM RA has little effect on the placode pattern and application of 40 ng/ml BMP12 permits placode formation on the body. Co-treatment with RA and BMP12 has a synergistic effect, completely suppressing feather development on the body. Conversely, treatment of skin with the RA synthesis inhibitor Citral renders the neck resistant to suppression of placode formation by BMPs.

Article Snippet: Recombinant human BMP4 and mouse BMP12 (R&D Systems) were used.

Techniques: In Situ Hybridization, Inhibition

Journal: Reproductive toxicology (Elmsford, N.Y.)

Article Title: Determinants of Orofacial Clefting I: Effects of 5-Aza-2′-deoxycytidine on Cellular Processes and Gene Expression during Development of the First Branchial Arch

doi: 10.1016/j.reprotox.2016.11.016

Figure Lengend Snippet: Genes Differentially Expressed within 1 st Branchial Arches Following 12 hour in utero Exposure to 1 mg/kg AzaD on GD 9.5.

Article Snippet: Gdf7 , Growth differentiation factor 7 , Mm04213617_sH , 3.50 , 0.00.

Techniques: In Utero, TaqMan Assay, Binding Assay, Membrane, Histone Deacetylase Assay, Translocation Assay, Ubiquitin Proteomics