Fig. 2. Retinal vascularization correlates with differentiation of retinal astrocytes. Retinal wholemounts from P5 mice were analyzed for proliferation (A-G) and stained for PDGFRa (A-C,E), VEGF (G,H) or GFAP (F,I) mRNA. Retinal astrocytes were visualized by PDGFRa in situ hybridization (dark signal in A-C,E) and cell proliferation was assessed by BrdU incorporation (red nuclei in A-C,E-G). Blood vessels were stained with anti-collagen type IV antibody (green in A,C, white in H). Proliferation of retinal astrocytes is high in peripheral, avascular areas (B) but low in central, vascularized areas (C). This was quantified by counting cells in the avascular (B in panel D) or vascularized (C in panel D) retina in whole-mount preparations double labeled for PDGFRa mRNA and BrdU incorporation (data points represent the mean±s.d. from five different animals). High magnification reveals that BrdU incorporation in the peripheral retina is limited to retinal astrocytes identified by PDGFRa mRNA (E), GFAP mRNA (F) and VEGF mRNA (G) expression. VEGF mRNA (dark signal in H) is strongly expressed in avascular areas, whereas GFAP mRNA (black in I) is expressed strongly in the presence of blood vessels. Upper box in A refers to B, lower box to C. Scale bars: 50 µm.