Figure 5

Localization of the HB-EGF reporter, β-gal, in the brain.

(a) The RMS of mice with a HB-EGF heterozygous allele visualized with DCX immunofluorescence at P21. Arrows in the rectangular inset is magnified to the right, demonstrating the beginning trajectory of the RMS (arrow and arrowhead). (a’) Coronal section of mice carrying HB-EGF homozygous alleles at P21 displaying localization of β-gal from the cortical layer V–VI to the pia mater: within the SVZ and adjacent layer (I-bar), β-gal localization was rarely detected. Str denotes striatum. (b) Sagittal views displaying localization of the β-gal reporting human HB-EGF protein in the SVZ at P21. Note that the β-gal is specifically localized in the region where the RMS is observed (longer arrowhead) in the HB-EGF heterozygous brain (left), whereas a widespread distribution of the human HB-EGF (shorter arrowheads) is detected in the homozygous mutant brain (right). Longer arrowheads indicate a longitudinal orientation of transgene distribution tangential to the ventricular surface (left: longitudinal; right: longitudinal and radial). Note also that β-gal is distant from the ventricular surface more than 100 μm in mice expressing human HB-EGF (dashed I-bars). (b’) Adjacent sagittal sections stained with GFAP. Note that GFAP+ cells in the SVZ are specifically organized in an orientation longitudinally towards the OB of the HB-EGF heterozygous mutant (left). In the HB-EGF homozygous mutant, GFAP+ cells are widely distributed radially in the layer V–VI next to the SVZ (right). (b”) A bar graph displaying the regional distribution of HB-EGF reporter, β-gal, of the HB-EGF heterozygous and homozygous mutant mice at P21: epend denotes ependyma. N = 3 in each group for all the experiments in this figure (a,b”). Single (*) and double asterisks (**) represent p < 0.05 and p < 0.01, as compared to the heterozygous control, respectively, by unpaired t test (two-tailed). AU denotes arbitrary unit. Scale bars, 50 μm (a,b’).