In En2 mutants, the timing of the formation of two anchoring centers is altered. (a) Midsagittal section of P7 wild-type (WT) Cb shows a normal foliation pattern. (b) The Cb of En2 mutants at P7 displays the final vermis foliation defect. Lobule VIII (red outline) is associated with the lobule IX rather than with lobules VI/VII, the secondary fissure (white arrow) is shallower than in the WT and the prepyramidal fissure (yellow arrow) is longer than in the WT. (c, d) Midsagittal section of an E17.5 WT and En2 mutant Cb shows that, only in the WT, the Pcl invaginates in the areas where three of the four future principal fissures will form (black asterisks). The orange bracket indicates the thicker EGL in the wild-type Cb. The black bracket demarcates the Pcl. (e) Sagittal section of a WT Cb at P0 shows the four principal fissures (black asterisks). (f) In a sagittal section at P0 of an En2 mutant Cb, only three shallow principal fissures (black asterisks) are apparent; the secondary (sec) fissure is absent. (g-l) Anti-Calbindin immunostaining (red) and DAPI staining (blue) of WT (g, i, m) and En2 (h, j, n) mutant Cb at P0 (g, h), P1 (i, j), and P2 (m, n) show a delay in formation of the secondary (sec) fissure and premature formation of the prepyramidal (ppd) fissure. Medial sagittal sections of P1 WT (k) and En2 mutant (l) embryos with anti-BLBP immunostaining show that Bg fibers point to an anchoring center in both secondary and prepyramidal fissure. Scale bars: (a, b,) 25 μm; (c-f) 100 μm; (g-n) 50 μm.