Fig. 5. Reelin signaling affects Blbp content and morphology of radial glial cells in vitro. (A) Fluorescent micrographs of Rc2- (red) and Blbp- (blue) immunoreactive cells dissociated from E14 cortex and cultured for 24 hours in control (top) or reelin-conditioned (bottom) medium. Note the increase in the number of Blbp-immunoreactive cells cultured in reelin-conditioned medium (middle bottom panel). Arrows indicate double-labelled cells. (B-D) Histograms of the percent of Rc2-only (red bars) or Rc2- and Blbp-immunoreactive cells (yellow bars) dissociated from different brain regions of E14 embryos cultured for 2 or 24 hours in control or reelin-conditioned medium. Note that the proportion of Blbp-immunoreactive cells significantly increases (Student's t-test, ^***P<0.01) after a 24 hour exposure to reelin-conditioned medium. This increase also occurred in cells from reeler cortex to the same number of Blbp-positive cells as induced in wild-type cortex, suggesting that the decrease in Blbp-positive cells of the reeler cortex can be fully rescued by the addition of reelin. [Number of cells analysed: (B) 2 hours, n(Ctrl-med)=1652, n(Rln-med)=1523; (B) 24 hours n(Ctrl-med)=3478, n(Rln-med)=3457; (C) n(Ctrl-med)=1263; n(Rln-med)=1274; (D) n(GE)=973, n(SC)=886.] (E) Western blot of chemically defined culture medium conditioned for 48 hours by cells stably transfected with a control (CTRL) or reelin-expression plasmid (see D'Arcangelo et al., 1997; Beffert et al., 2002; Förster et al., 2002) showing a high amount of reelin in the reelin-conditioned, but not the control medium. (F,G) Morphological analysis of radial glial cells after exposure to reelin in vitro. (F) Two corresponding micrographs depicting examples of an Rc2-only positive precursor without processes (arrowheads) and two Rc2/Blbp-doubleimmunoreactive bipolar cells with processes longer than their cell soma after culturing in reelin-conditioned medium. The histograms in G depict the proportion of Rc2-positive cells from E14 wild-type (light gray) or reeler (dark gray) cortex with a bipolar morphology. Note that cells with bipolar morphology increase significantly (Student's t-test, ^***P<0.01) after exposure to reelin-conditioned medium for 24 hours. [Number of cells analysed: Ctrl-med, n(wild type)=429, n(Rln)=204; Rln-med, n(wild type)=396, n(Rln)=232.]