Figure 1
Apc/ Apc expression in the cerebral cortex during normal development and following Cre-mediated deletion of Apc. (A, D, F, I) Immunohistochemistry showing Apc expression in the developing forebrain of control embryos at embryonic day (E)11.5 (A), E12.5 (D), E13.5 (F), and E14.5 (I). (B, E, G, J) Higher power magnification of the control cerebral cortex. (B) At E11.5, Apc is expressed throughout the cerebral cortex (cc) with prominent expression at its apical (a) surface compared to the basal (b) surface. (E) At E12.5, Apc is expressed throughout the cerebral cortex in the ventricular zone (vz) and the newly formed cortical plate (cp) where its expression levels are highest. (G, J) At E13.5 (G) and E14.5 (J), Apc is expressed throughout the cerebral cortex c. Apc expression extends from the apical surface of the ventricular zone to the pial edge with the highest levels of expression in the cortical plate and intermediate zone (iz). (C, H, K) Apc expression is reduced in the cerebral cortex of mutant embryos compared to controls at E11.5 (C), E13.5 (H) and E14.5 (K). The white dotted lines demarcate the mutant cerebral cortex. (L, M) Apc immunocytochemistry of dissociated E13.5 control (L) and mutant (M) cells showing that Apc expression is reduced in the mutant. Nuclei stained red with Propidium (PI). (N, O) Controls showing that the secondary antibody used to detect Apc does not stain neural cells at E12.5 (N) and E13.5 (O), although blood vessels (arrows) are strongly fluorescent. (P) Apc transcript levels increase during normal corticogenesis from E12.5 to E13.5 and E13.5 to E15.5. Histogram shows levels of Apc relative to GAPDH in mRNA extracted from cerebral cortex at E12.5, E13.5 and E15.5. Black bars are control and white bars are mutant. Student's t-test p-values are indicated above the bars (comparison between all groups ANOVA p = 0.002). (Q, R) LacZ staining (blue) of E12.5 (Q) and E16.5 (R) Emx1Cre/+; R26R forebrain, confirming the ability of Cre recombinase to recombine a floxed R26R reporter allele and turn on LacZ expression in the cerebral cortex. (S) Mutant cerebral cortex has significantly reduced levels of wild-type Apc transcripts compared to controls. Histogram shows the ratio of transcripts containing exon 14 (deleted following Cre-mediated recombination of Apc580Sallele) to exon 4 at E12.5, E13.5, and E15.5. Student's t-test p-values are indicated above the bars (comparison between all groups ANOVA p = 0.001). There is no significant difference in the ratio obtained from control embryos at the ages examined (ANOVA p = 0.390). (D, E, I, J, K, O) Vibratome sections; (A-C, F-H, N) wax sections; (L, M) dissociated cells embedded in collagen. Additional abbreviations: h, hippocampus; t, thalamus; vtel, ventral telencephalon. All sections are coronal and dorsal is up. Scale bars: (A, D, F, I) 200 μm; (B, C, E, G, H, J, K, N, O) 50 μm; (L, M) 25 μm. Error bars in (P, S) are standard error of the mean.