Journal: bioRxiv

Article Title: Time, Space and Single-Cell Resolved Molecular Trajectory of Cell Populations and the Laterality of the Body Plan at Gastrulation

doi: 10.1101/2023.04.19.537134

Figure Lengend Snippet: a. Experimental strategy of ex vivo culture and analysis of embryos following chemical inhibition of BMP activity for 3, 6, 9, 12 and 15 hours beginning on E7.0. b. Whole-mount RNAscope analysis of embryos after 30 hours of ex vivo culture, showing the expression of Nodal in the lateral mesoderm and the node (ventral view). The frequency of left-sided expression of Nodal is shown for Group A (untreated control) and Group B (3 hours treatment), and the frequency of abnormal (bilateral) expression of Nodal is shown for Groups C (6 hours treatment), D (9 hours treatment), E (12 hours treatment) and F (15 hours treatment). c. Table showing the pattern of Nodal expression in the cultured embryos collected at 30 hours in vitro (equivalent to E8.25). d. Whole-mount in situ hybridization of Dand5 / Cerl2 in cultured embryos from indicated experimental groups. e. Experimental strategy of ex vivo culture and analysis of embryos following chemical inhibition of BMP activity for 6 hours beginning at E7.25 (Group G and H), E7.5 (Group I and J), and E7.75 (Group K and L) stages. f. Whole-mount in situ hybridization of Lefty2 after ex vivo culture (equivalent to E8.25), showing the expression of Lefty2 in the lateral mesoderm (ventral view). The frequency of annotated pattern of Lefty2 is also shown for each group. g. Table showing the pattern of Lefty2 expression in the cultured embryos collected at equivalent E8.25 stages. h. Whole-mount in situ hybridization of Dand5 / Cerl2 in cultured embryos from indicated experimental groups.

Article Snippet: 1 μM chemical inhibitor of BMP signaling (LDN193189, Selleck, S2618-2mg) was added to the culture medium in the respective experimental groups.

Techniques: Ex Vivo, Inhibition, Activity Assay, RNAscope, Expressing, Control, Cell Culture, In Vitro, In Situ Hybridization

Journal: bioRxiv

Article Title: Time, Space and Single-Cell Resolved Molecular Trajectory of Cell Populations and the Laterality of the Body Plan at Gastrulation

doi: 10.1101/2023.04.19.537134

Figure Lengend Snippet: a. Schematic showing the workflow of GEO-seq for ex vivo cultured embryos. b. The BMP signaling activity in the right-side lateral mesoderm region (MR) was curtailed by LDN193189 treatment. c. Heatmap showing the expression pattern of right-side specific genes of proximal lateral mesoderm (n = 801) in Control (Ctrl) and 9 hours BMP Inhibition embryos. A few right-side specific genes of proximal lateral mesoderm were not expressed in cultured embryos, these genes were then marked with white lines. d. Violin plot showing the expression level of right-side specific genes of proximal lateral mesoderm in control (Ctrl) and 9 hours BMP Inhibition embryos. The left-right differences were abrogated in the inhibition group. Rank sum test, ***, p < 0.001; N.S., no significant difference. e. Region-specific (the right-side proximal lateral mesoderm) perturbation of BMP signaling by multiple siRNA knockdowns. Top panel: The experiment snapshot of siRNAs microinjection. Bottom panel: Region-specific transfection of corresponding siRNAs can be observed at embryos of equivalent E7.5 stage. f. Heatmap showing the expression pattern of right-side specific genes of proximal lateral mesoderm (n = 801) in Control (Ctrl) and siRNA knockdown embryos. A few right-side specific genes of proximal lateral mesoderm were not expressed in cultured embryos, these genes were then marked with white lines. g. Violin plot showing the expression level of right-side specific genes of proximal lateral mesoderm in control (Ctrl) and siRNA knockdown embryos. The left-right differences were abrogated in the Bmp siRNA microinjection group. Rank sum test, ***, p < 0.001; N.S., no significant difference. h. Experimental strategy of siRNA knockdown in the left-side mesoderm and ex vivo culture. i. Whole-mount in situ hybridization of Lefty2 of cultured embryo at equivalent E8.25 stage, showing Lefty2 expression in the lateral mesoderm. j. Pattern of Lefty2 expression in the cultured embryos at equivalent E8.25 stages.

Article Snippet: 1 μM chemical inhibitor of BMP signaling (LDN193189, Selleck, S2618-2mg) was added to the culture medium in the respective experimental groups.

Techniques: Ex Vivo, Cell Culture, Activity Assay, Expressing, Control, Inhibition, Microinjection, Transfection, Knockdown, In Situ Hybridization

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Optimization of Differentiation of Nonhuman Primate Pluripotent Cells Using a Combinatorial Approach

doi: 10.1007/978-1-4939-9007-8_14

Figure Lengend Snippet: Analysis of the effects of three small-molecule factors on genes that respond to activation of three different intracellular pathways in marmoset iPS cells. In each case “0” = mRNA level before treatment and “-” = level following 24 hours in basal medium only. DM = dorsomorphin at a range of concentrations from 0 to 2 μM; B = 40 ng/ml BMP4. BMP4 treatment induced a statistically significant upregulation of ID1 and ID3 mRNA; co-treatment with dorsomorphin resulted in lowered ID1, but not a statistically significant lowering of ID3. SB = SB4321542 at a range of concentrations from 0 to 3 μM; T = 10 ng/ml TGF-β1. Treatment with TGF-β1 increased expression of SERPINE and HES1, while SB431542 coadministration resulted in dosage-dependent reduction in expression of both genes. B = BIO at a range of concentrations from 0 to 2 μM. BIO increased the expression of Wnt/β-catenin targets SP5 and AXIN2. See Note 10.

Article Snippet: Small molecule differentiation factors Y-27632 Dorsomorphin (Fisher Scientific); a selective small molecule inhibitor of BMP signaling SB431542 (Selleckchem); inhibitor of the TGF-β/Activin/NODAL pathway that inhibits ALK5, ALK4 and ALK7 PD325901 (Biotang); inhibitor of MEK 1 and 2 BIO (Enzo); GSK-3 inhibitor IWR-1 (Sigma); Wnt inhibitor, promotes β-catenin destruction DAPT (Tocris); Notch inhibitor all -trans retinoic acid (Sigma) BMP4 ligand (Peprotech) TGF-β1 (R&D Systems) FGF2 (basic FGF) (Stemgent) 2.4.

Techniques: Activation Assay, Expressing

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Optimization of Differentiation of Nonhuman Primate Pluripotent Cells Using a Combinatorial Approach

doi: 10.1007/978-1-4939-9007-8_14

Figure Lengend Snippet: Concentrations of factors used. For details see .

Article Snippet: Small molecule differentiation factors Y-27632 Dorsomorphin (Fisher Scientific); a selective small molecule inhibitor of BMP signaling SB431542 (Selleckchem); inhibitor of the TGF-β/Activin/NODAL pathway that inhibits ALK5, ALK4 and ALK7 PD325901 (Biotang); inhibitor of MEK 1 and 2 BIO (Enzo); GSK-3 inhibitor IWR-1 (Sigma); Wnt inhibitor, promotes β-catenin destruction DAPT (Tocris); Notch inhibitor all -trans retinoic acid (Sigma) BMP4 ligand (Peprotech) TGF-β1 (R&D Systems) FGF2 (basic FGF) (Stemgent) 2.4.

Techniques: