Figure 5

Secreted NRGs have a repellent effect on cells migrating from MGE explants in collagen co-cultures. (A-D) MGE explants collected at E14.5 from eGFP-expressing mice were co-cultured with aggregates of 293 cells transiently transfected with a control expression construct (A), or a construct expressing the functional Nrg1α EGF-like domain (B), Nrg1β EGF-like domain (C) or Nrg3 EGF-like domain (D). In the control, cells migrate symmetrically from the MGE explant (A). The pattern of cell migration from the MGE explants is asymmetric in the presence of the 293 cell aggregates transfected with the functional NRG EGF-like domains (B-D), with a significant preference for cells to migrate away from the transfected cell aggregates. (E, F) Quantification of eGFP fluorescence done blind to the transfection type confirmed the statistical significance of the symmetric versus asymmetric migration. The dashed set of perpendicular lines shown in (A) divide MGE explants into four quadrants. The numbers of cells within the quadrants on the sides of the MGE explants proximal (P) and distal (D) to the transfected cell aggregates were estimated by automated analysis of eGFP fluorescence measuring total pixel number (E) or total pixel intensity (F) as described in Pak et al. [48]. Compared to the symmetric pixel measurements in the controls, cells migrating from MGE explants in the presence of the 293 cell aggregates transfected with the functional NRG EGF-like domains exhibit asymmetric pixel measurements, with more pixels in the distal (D) than proximal (P) quadrants (*P < 0.1; **P < 0.01; unpaired Student's t-test). Scale bar: 200 μm.