The Engrailed homeobox genes determine the different foliation patterns in the vermis and hemispheres of the mammalian cerebellum

Abstract

Little is known about the genetic pathways and cellular processes responsible for regional differences in cerebellum foliation, which interestingly are accompanied by regionally distinct afferent circuitry. We have identified the Engrailed (En) homeobox genes as being crucial to producing the distinct medial vermis and lateral hemisphere foliation patterns in mammalian cerebella. By producing a series of temporal conditional mutants in En1 and/or En2, we demonstrate that both En genes are required to ensure that folia exclusive to the vermis or hemispheres form in the appropriate mediolateral position. Furthermore, En1/En2 continue to regulate foliation after embryonic day 14, at which time Fgf8 isthmic organizer activity is complete and the major output cells of the cerebellar cortex have been specified. Changes in spatially restricted gene expression occur prior to foliation in mutants, and foliation is altered from the onset and is accompanied by changes in the thickness of the layer of proliferating granule cell precursors. In addition, the positioning and timing of fissure formation are altered. Thus, the En genes represent a new class of genes that are fundamental to patterning cerebellum foliation throughout the mediolateral axis and that act late in development.

Fig. 1.

The Rosa26^CreERT2 allele produces efficient recombination in the embryonic brain until late gestation. (A-L) X-galactosidase staining of Cb sagittal sections of Rosa26^CreERT2/R embryos (A-J) or adults (K,L) treated with tamoxifen (TM) at the times indicated. Panels on right are higher powerered images of the areas outlined in the left panels. Anterior is to the left. Scale bars: 100 μm in A,C,E,G,I; 25 μm in B,D,F,H; 400 μm in K; 50 in μm L.

Fig. 2.

En2 is required after ~E12 to regulate the Cb foliation pattern. (A,B) Schematics (not drawn to scale) of conditional En2^flox (A) or En2^floxlZ conditional (B) alleles and the corresponding experimental time-course used to ablate or activate En2, respectively. Green rectangles and red lines represent the periods when En2 was expressed or ablated, respectively; arrows indicate the time of TM administration. Hematoxylin and Eosin (H&E)-stained sagittal sections are shown of the vermis (C,F,I,L,O) and hemispheres (D,G,J,M,P) of the mutants indicated. Inactivation of En2 up to ~E12 produces an En2 null mutant phenotype, whereas activation at ~E12 rescues the foliation defects. (E,H,K,N,Q) Images of immunostaining for En2 protein in the boxed vermis regions. Defective folia are outlined by dotted lines. S, Simplex; CI, CrusI; CII, CrusII; Pm, Paramedian. Scale bar in E: 200 μm for C,D,F,G,I,J,L,M,O,P; 25 μm for E,H,K,N,Q.

Fig. 3.

Inactivation of both En1 and En2 preferentially affects the lobules specific to the vermis and the hemispheres. (A-J) H&E-stained sagittal sections of adult Cb from the mutants indicated. (K) Schematics of idealized surface renderings of foliation of a flattened Cb based on sections and micro-MRI of all mutants. (L) A series of medial to lateral sagittal sections of adult wild-type (WT) Cb are shown with shading of lobules I-V (yellow), VIa-b (green), VI-IX (red) and X (blue). The earlier En1/En2 expression is removed, lobule I-V and VIII in the vermis are reduced in size, remnants of lobules I-V and VIII/IX are extended more laterally than normal, lobules VI/VII in the vermis are relatively larger than normal, and CII and Pm in the hemispheres are fused. Scale bar: 200 μm.

Fig. 4.

Inactivation of both En1 and En2 produced a more homogeneous foliation pattern along the ML axis. Volumetric micro-MRI data was used to generate dorsal and oblique ventral (3D) views of surface rendered images, as well as coronal sections (2D) of the cerebellum at P14 (A) and P21 (B). Coloring is as in Fig. 3. Arrowheads indicate approximate morphological borders between vermis and hemispheres (dorsal views); asterisks indicate lateral extension of posterior lobules in mutants (coronal sections). All mice analyzed with micro-MRI were treated with TM at E10.5.

Fig. 5.

En1/En2 are required for robust expression of Fgf8 in the isthmic organizer after ~E11. (A-D) Medial (A,B) and slightly lateral (C,D) sagittal sections showing that Fgf8 RNA expression is reduced in the dorsal medial mid/hindbrain junction at E12.5 in En1/2-E10.5 CKOs compared with in controls. (E,F) Expression of Otx2 protein does not extend into the hindbrain in mutants. Cb primordium is outlined with a white dotted line. Scale bar: 25 μm.

Fig. 6.

En1/En2 determine the timing of fissure formation. (A-I) H&E staining of vermis and hemisphere sagittal sections of the developing Cb in the mutants indicated compared with controls. (J,K) Outlines of a medial-to-lateral (left to right) series of sagittal sections of P1 wild type (J) and P3 En1/2-E10.5 CKOs (K) illustrate foliation pattern. Regions highlighted as in Fig. 3L. Scale bar in A: 100 μm for A-F,H,I; 25 μm for G,G′.

Fig. 7.

Regional markers reveal that AP patterning of the Cb is altered at ~E18.5, prior to fissure formation in En1/2 CKOs. (A-P) H&E staining (A-D) and RNA in situ hybridization of sagittal sections through the vermis of controls (R26^CreER/+; En1^+/lox; En2^+/lox) and En1/2-E10.5 CKOs showing Otx2 (E-H), Rnx (I-L) and Gli1 (M-P) expression. (Q-T) Outlines of sagittal sections of P2 or adult brains shaded as in Fig. 3L to illustrate the relationship between lobules at two stages. (U) Graph comparing lengths of surface of the Cb at P2 and volumes at P14 of different lobules. *P'0.0001. (V) Percentage of BrdU- and Pax6-positive cells in the EGL (Pax6⁺) is shown graphically for the top of lobule VI-V compared with the top of VI at E18.5 and P2. Scale bar: 200 μm.

Fig. 8.

Summary of the altered timing of vermis fissure formation in En1/2-E10.5 CKOs. The order of fissure formation is color coded and indicated numerically above the arrowheads, and the times when the fissures form in the wild type (WT) and in En1/2-E9.5 CKOs are indicated in brackets.