Pair cell assay

CNgV were microdissected from entire E10.5 litters (5 independent experiments/5 litters, wild type; n=4 independent experiments/4 litters, LgDel) and dissociated as described previously (Lehtinen et al., 2011; Shen et al., 2002; Tucker et al., 2010). Dissociated cells were plated at clonal density (35 cells/µl; 14 µl total volume/well) on poly-D-lysine-coated Terasaki plates. Cultures were incubated for 21 h at 37°C with 5% CO₂, fixed and immunolabeled for Sox2 (progenitor marker) and βIII-tubulin (neuronal marker), as well as DAPI to identify nuclei. Pairs of cells were identified based upon DAPI labeling in individual wells based upon apposition of two cells isolated from any other cells. For each isolated DAPI-labeled pair, the expression of progenitor and neural markers was visualized and scored.