Journal: Developmental biology

Article Title: Deletion of the Dishevelled family of genes disrupts anterior-posterior axis specification and selectively prevents mesoderm differentiation.

doi: 10.1016/j.ydbio.2020.05.010

Figure Lengend Snippet: Fig. 6. Effects of Inhibiting BMP, Canonical Wnt, and Nodal signaling on Germ Lineage Differentiation. A-B) Representative images of Brachyury (green), Otx2 (red), and DAPI (blue) immunostaining of WT and Dvl TKO EBs on day 7 of differentiation after inhibition with BMP (LDN-193189), canonical and non-canonical Wnt (IWP L6) and Nodal (SB-431542) drug treatments. Scale bar ¼ 100 μm. C-D) Quantification of Brachyury and Otx2-positive cells in WT and Dvl EBs differentiated for 5–7 days (n ¼ 20/group). Brachyury-positive cells are not detectable in Dvl TKO EBs, and there are no significant differences in Otx2-positive cells. E-F) Quantitative RT-PCR analysis of Brachyury (mesoderm) and GATA4 (endoderm) mRNA expression to determine differences in differentiation potential in WT and Dvl TKO EBs over a 6-day time course (n ¼ 3). Expression is normalized to the untreated controls in Fig. 5G (Brachyury) and 5H (GATA4). E) Brachyury expression in WT EBs is attenuated with inhibition of BMP, Wnt, and Nodal signaling, similar to Dvl TKO EBs. F) Inhibition of BMP and Wnt signaling significantly enhances endoderm differentiation in WT and Dvl TKO EBs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The following drug treatments were added at the time of plating and removed after 48 h: 100 ng/ml Activin A (R&D Systems), 50 ng/ml BMP4 (R&D Systems), 3uM CHIR-99021 (Tocris), 100 ng/ml Wnt3a (R&D Systems), 10 μM SB-431542 (EMD Millipore), 200 nM LDN-193189 (Stemgent), 0.5 μM IWP L6 (Tocris).

Techniques: Immunostaining, Inhibition, Quantitative RT-PCR, Expressing

Journal: Developmental biology

Article Title: Deletion of the Dishevelled family of genes disrupts anterior-posterior axis specification and selectively prevents mesoderm differentiation.

doi: 10.1016/j.ydbio.2020.05.010

Figure Lengend Snippet: Fig. 7. Effects of Developmental Pathway Modulation on Germ Lineage Differentiation. Activation of BMP (BMP4), canonical Wnt (Wnt3a and CHIR-99021) and Nodal (Activin A) pathways in WT EBs enhances mesoderm (Bra) differentiation, while endoderm (GATA4) differentiation remains low, and ectoderm (Otx2) dif- ferentiation diminishes over time. Treatment with these activators in Dvl TKO EBs does not rescue mesoderm induction. The relative level of endoderm marker expression is higher in Dvl TKO EBs compared to WT EBs treated with Wnt activators, Wnt3a and CHIR, but not BMP4 or Activin A. Dvl loss also delays the decrease in ectoderm lineage expression. Inhibition of BMP (LDN-198189), canonical and non-canonical Wnt (IWP L6) and Nodal (SB431542) pathways in WT EBs results in the suppression of mesoderm differentiation, recapitulating the Dvl TKO phenotype. Pathway inhibitor treatment in WT EBs, leads to similar trends of differentiation as Dvl TKO EBs. Endoderm differentiation is greatly enhanced in Dvl TKO EBs with LDN-193189 and IWP L6 treatment.

Article Snippet: The following drug treatments were added at the time of plating and removed after 48 h: 100 ng/ml Activin A (R&D Systems), 50 ng/ml BMP4 (R&D Systems), 3uM CHIR-99021 (Tocris), 100 ng/ml Wnt3a (R&D Systems), 10 μM SB-431542 (EMD Millipore), 200 nM LDN-193189 (Stemgent), 0.5 μM IWP L6 (Tocris).

Techniques: Activation Assay, Marker, Expressing, Inhibition