Journal: Stem Cell Reports

Article Title: Anteroposterior Wnt-RA Gradient Defines Adhesion and Migration Properties of Neural Progenitors in Developing Spinal Cord

doi: 10.1016/j.stemcr.2020.08.016

Figure Lengend Snippet: Wnt/β-catenin Signaling Determines AP Gradients of Cell-Cell Adhesions (A) Schematic diagram of E12 embryo, and diagram of cell-sorting assay where cells pre-treated with DMSO, Chiron, Dkk1, or RA were allowed to self-sort based on their adhesion affinity. The gradient of purple color indicates the activation rate of different signals upon chemical treatment. (B) Quantification of the percentage of sorting patterns. NPCs treated with chemicals were from E12 T or Br. Br versus T resulted in significantly (80%) higher enveloped phenotype than segregated 5% and checkerboard 15%. Br versus T-Dkk1 resulted in significantly (58%) higher checkerboard phenotype than segregated 14% and enveloped 28% phenotypes. Br versus T-RA resulted in significantly (70%) higher checkerboard than 12% segregated and 18% enveloped. Br-Chiron versus T resulted in significantly (47%) higher checkerboard phenotype than segregated 30% and segregated 23% phenotypes. Br-Chiron versus T-Chiron E12 resulted in significantly (52%) higher enveloped phenotype than segregated 3% and checkerboard 45%. Br versus Br-DMSO resulted in significantly (88%) higher checkerboard phenotype than enveloped 12% phenotype. Br versus Br-Chiron resulted in significantly (72%) higher enveloped phenotype than checkerboard 28%. Data are shown as percentage; number of independent experiments = 3; number of examined neurospheres = 1,638. (C) RT-PCR analyses present the mRNA fold change in β-catenin , Lef1 , Tcf4 , Axin2 , and Cyclin D1 expression levels upon activation of Wnt signaling with 2 μM Chiron. Data are shown as mean ± SD; number of independent experiments = 3. ∗ p < 0.001 via Student's t test. (D) qRT-PCR analysis shows the fold change in mRNA levels of Wnt -related genes pre-treated with chemicals. Data are shown as mean ± SD; number of independent experiments = 3; ∗ p < 0.001 via one-way ANOVA. (E) qRT-PCR of Wnt -related genes in tail NPCs pre-treated with DMSO or 1 μM RA. Data are shown as mean ± SD; number of independent experiments = 3; ∗ p < 0.001 via Student's t test. Orange shading indicates the downstream genes of RA signaling. (F and I) Box plots for the directionality of brachial (F) and tail (I) NPCs pre-treated with chemicals during the scratch assay. Data are shown as median ± SD; number of independent experiments = 4; number of examined cells = 401; ∗ p < 0.001 via one-way ANOVA on ranks. (G and J) Top shows cell trajectory of migrating brachial (G) and tail (J) NPCs, and the bottom shows the angle histograms (polar graph) representing the direction of migrating cells at the leading and following domains. Different lengths of the angle bar represent different grouped cells with a particular angle. Front (angle 0°–180°) and rear (angle 180°–360°) cells were grouped based on the position of the centrosome. Number of independent experiments = 3; number of examined cells = 395. (H and K) Measurement of front-to-rear ratio of brachial (H) and tail (K) NPCs that were determined in polar graphs following chemical treatment. Data are shown as mean ± SD; number of independent experiments = 3; number of examined cells = 395; ∗ p < 0.001 via one-way ANOVA.

Article Snippet: For pre-treatment, NPCs at passage 0 were pre-treated with DMSO, 3 μM Chiron, 200 ng/mL DKK-1, 1 μM XAC-939, 1 μM Wnt-C59, or 1 μM RA for 4 consecutive days.

Techniques: FACS, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Wound Healing Assay