Transplantation experiments. (A-D) Paramedial (type 3) grafts. (A-C) Grafts placed close to the midline (type 3a and 3b) are found elongated bordering on the midline. When placed at the isthmus (type 3a), they end up either in the midbrain (A) or straddling the MHB (B), while when placed rostral to the isthmus (type 3b), they become elongated in the caudal midbrain (C). (D) Grafts placed rostral to the isthmus and at a distance from the midline (type 3c) move rostromedially in the midbrain but are no longer elongated and have increased in size relatively to the grafts placed closer to the midline. (E-H) Bilateral dorsal strip (type 4) grafts of the caudal midbrain move rostromedially but also caudally in the hindbrain where they contribute to thin medial streams of cells that avoid the roof plate. (I,J) Small midline (type 5) grafts. Small grafts centered on the isthmus contribute modestly to the midline either exclusively in the hindbrain (I; flat-mount view of the grafted embryo) or straddling the MHB (J; ibid.).

(A-H) DiI labeling of cells at the isthmus. (A-D) Examples of embryos labeled with DiI at the 10ss and fixed 1 hour after labeling. (A) Dorsomedial, (B) small midline, (C) rostral to constriction, and (D) paramedial DiI application sites. (E,F) Examples of embryos fixed at E4; the MHB region is flat-mounted. Dorsomedially located cells participate in the cerebellar midline (E), or, in addition, to the midline of the caudal midbrain (F). Cells located medially but anterior to the isthmus, are confined to the caudal midbrain (G). Cells at paramedial isthmic locations are found on both sides of the MHB (H). Arrows indicate DiI application sites. (I-L) Cell movements in the isthmus. (I) Summary of medial and paramedial grafts at E2 (HH10). (J) At E4, medially located isthmic cells (in green) have preferentially moved in a caudal direction, while cells rostral (in blue) or caudal (in bright pink) to them, have moved, respectively, to the caudal midbrain or the edge of the cerebellar plate. Paramedially located cells (in purple and pink) move rostromedially towards the midline. (K) Summary of lateral grafts at E2 (HH10). (L) At E4, laterally located isthmic cells (in red) have moved rostromedially or caudomedially. Cells located rostrolaterally to the constriction (in blue and orange), have moved rostromedially. In J and L, compare the relative size of the grafts at E4, which depends on the initial transplantation site.

The strip grafts (type 4) contribute to the cerebellar midline but produce none of the main cerebellar cell types. Sagittal sections through the MHB region of E16 (A-D) and E18 (F,G,I,J) chick-quail chimeras that received type 4 transplants. (A,B) The grafts contribute to the velum medullaris (arrowheads) and to the medial cerebellum (arrows). (C,D) Higher magnifications illustrating the graft-derived cell types. None of the grafts produced granule cells or deep nuclear neurons. Half of the grafts produced typical cerebellar cell types i.e. Purkinje and Golgi cells (arrowheads in C and F). The other half produced exclusively glial cells and interneurons of the molecular layer (D) suggesting that they arose from the frontier of the cerebellar anlage. (E,H) The number of molecular layer interneurons that express GAD67 increases significantly between E16 and E18 in the normal chick embryo. (F,G,I,J). The grafts produced only scarce Purkinje cells (F) or molecular layer interneurons (G), both detected by parvalbumin immunohistochemistry, a few QH1-positive vascular elements in the molecular and granular layers (I), and some astrocytes (J).

Cerebellar midline fusion and variations in the expression of roof plate markers at the MHB junction. (A) Sagittal section of the cerebellum of a newborn mouse. PCs and GCPs are labeled by in situ hybridization for the detection of Rorα (red) and Math1 (blue) transcripts, respectively. (B,C) Math1 is expressed in the hindbrain rhombic lip including the cerebellar midline (arrowhead in B) until E13.5. Math1 expression is downregulated on the cerebellar midline at E14.5 (arrowhead in C) when the GCPs begin to form the external granular layer. (D,E) The proliferating GCPs, labeled by incorporation of BrdU, extend rostrally between E14.5 and E15.5, in particular in the midline region (arrowheads). (F-I) PCs are detected at successive developmental stages (top left of each picture) in whole-mount cerebellum (F) or transverse vibratome sections (G-I) by Rorα in situ hybridization (F-H) or CaBP (calbindin, I) immunocytochemistry. PCs are at first excluded from the midline region (arrowhead in F,G), which becomes secondarily colonized by E15.5 (arrowhead in H,I). (J) In mouse the cerebellar midline expresses high levels of GAD67 transcripts suggesting its specialization in the production of GABAergic interneurons. (K-N) Expression of dorsal midline markers is distinctly modulated in the MHB region (arrowheads) of chick (K,L) and mouse (M,N) embryos.

Development of the velum medullaris in the mouse. (A,B,D-G) Sagittal and (C) horizontal sections through the MHB junction of mice (A-D,G) or rat (E,F). (A-G) The developmental age (top left) and marker used (bottom) are indicated on each image. (A) In late embryos, the Pax6-labeled EGL reaches the midbrain boundary; the velum medullaris (arrowhead) is very short at this stage. It develops during the following days and expresses high levels of En1, detected in B and C by X-gal staining of En1^Lki/+ mice Asterisk in B and D indicate cerebellar midline ependyme. The plane of section in C is indicated by the dashed line in A. (D) The high number of cells that incorporate BrdU in the velum compared to the ependymal surface of the cerebellum indicates that velum growth depends on active proliferation. (E,F) A specific set of radial glia cells labeled for vimentin (vim) link the velum (arrowhead in E) and the cerebellum ependyma. (F) Higher magnification of E. Black dashed line indicates the direction of cerebellar cortex retraction; white dashed line outlines the future extent of the velum medullaris. (G) These glia are outlined by the diffusible fluorescent β-galactosidase substrate FDG (arrow) in En1^Lki/+ pups indicating that they express high levels of En1. The arrowheads point to the velum medullaris. ic: inferior colliculus, sc: superior colliculus.

Morphogenetic movements in the dorsal neural tube at the MHB junction, based on observations in chick and mouse. Midbrain-derived cells, blue; isthmus-derived, pink; cerebellum, green (darker for the vermis). Double-headed arrows indicate AP orientation. (Top) The two drawings on the left depict the formation of the cerebellum (purple) and caudal midbrain (gray) roof plate structures derived from a restricted midline region in chick HH10. The cerebellum then fuses (third drawing, chick HH24 and mouse E15) over an isthmus-derived territory. The fourth drawing illustrates the medial retraction of the cerebellar cortex in perinatal mice and the growth of the velum medullaris. Pink circles represent isthmic nuclei. The white area derives from the hindbrain. (Bottom) Seesaw motion of the cerebellar cortex over the isthmic-derived territory. Schematic sagittal sections through the isthmo-cerebellar region close to the midline. The choroid plexus (black coil) marks the posterior pole of the region. Red arrows indicate the direction of cerebellar cortex displacement. Global tissue displacements are illustrated in the upper panels 1 and 2, and in 3. The orientation of radial glia (thin pink and green lines) in the same sections is illustrated in the lower panels 1 and 2. In 2, the radial glia become bent by the sliding cerebellum; pink dots in 2 and 3 represent isthmus-derived interneurons.