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First published online 24 September 2003
doi: 10.1242/dev.00736

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The isthmic neuroepithelium is essential for cerebellar midline fusion

Angeliki Louvi^1,*, Paula Alexandre¹, Christine Métin^1,, Wolfgang Wurst² and Marion Wassef^1,

¹ Régionalisation Nerveuse CNRS/ENS UMR 8542, Ecole normale supérieure, 46 rue d'Ulm, 75005 Paris, France
² GSF-Research Centre, Institute of Mammalian Genetics, Ingolsträdter Landstrasse 1, D-85764 Neuherberg and Max-Planck-Institute of Psychiatry, Kraepelinstrasse 2-10, D-80809 Munich, Germany

View larger version (101K): [in a new window]   Fig. 1. Transplantation experiments. In this and the next figure, schematic representations of isotopic and isochronic graft types are shown on the left, and the grafted quail cells (in brown) are visualized by anti-QCPN immunohistochemistry. (A-D) Dorsomedial (type 1) grafts. (A,B) Grafts centered on the isthmus (type 1a) move caudally and are detected in the hindbrain (A; 78% of the cases) or straddling the MHB (mid/hindbrain) (B; 11%). (C,D) When grafts are placed caudal (type 1b; C) or rostral (type 1c; D) to the isthmus, they are detected, respectively, in the hindbrain or the midbrain. (E-G) Lateral (type 2) grafts. (E) Example of an embryo with a type 2a graft, fixed 2 hours post-transplantation. (F) Grafts positioned laterally (example of a graft to the left side) at the level of the isthmus (type 2a) move rostromedially and are detected significantly enlarged in the midbrain. (G) Grafts positioned just rostral to the isthmus (type 2c) move rostromedially.

View larger version (85K): [in a new window]   Fig. 2. 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.).

View larger version (89K): [in a new window]   Fig. 3. (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.

View larger version (98K): [in a new window]   Fig. 4. 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).

View larger version (92K): [in a new window]   Fig. 5. 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 Ror (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 Ror 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.

View larger version (70K): [in a new window]   Fig. 6. 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 En1Lki/+ 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 ß-galactosidase substrate FDG (arrow) in En1Lki/+ pups indicating that they express high levels of En1. The arrowheads point to the velum medullaris. ic: inferior colliculus, sc: superior colliculus.

View larger version (41K): [in a new window]   Fig. 7. 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.

View larger version (69K): [in a new window]   Fig. 8. Mid/hindbrain mutations interfere with cerebellar midline fusion. Whole-mount view (A,C) and midsagittal sections (B, D) through the cerebellum of adult wild-type (A,B) and En1hd/hd survivor mutants (C,D). Compared to wild type, the velum medullaris is shortened in adult En1hd/hd mutant survivors (arrowhead in D), and the ependymal surface of the cerebellum is widened (asterisks in B and D) indicating that En1 function is required for the partitioning of the ependymal surface between the velum and the vermis. (E-G) The postnatal cerebellum of Wnt1sw/sw at P20 (E) and En1+/Otx2lacZ at P4 (F) has a distinct gap on the midline that is reduced to a fissure in Wnt1sw/sw adults (G). (H,J) In adult En1+/Otx2lacZ mutants, the two cerebellar halves are separated by a wide sheet of cells that loops above the surface of the cerebellum (arrowheads). (I) In Wnt1sw/sw, the anterior cerebellum is fused to the inferior colliculus (arrowhead). (K) Posterior views of E11.5 embryos where the choroid plexus has been labeled by Ttr in situ hybridization. Compared to wild type (wt) and En1+/Otx2lacZ (En1Otx2) mutant embryos, the choroid plexus in Wnt1sw/sw (sw, arrowhead) extends anteriorly separating the cerebellar halves. (L-N) Modification of the cerebellar midline domain in En1+/Otx2lacZ mutants. (L) Math1 expression is interrupted on the midline in E11.5 embryos. (M,N) Wnt1 expression on the roof plate is prolonged in the hindbrain at E9.5 (compare with wild type; arrowhead, M). This expression becomes wider and fuzzy at later stages (E12.5, N). (O) In Wnt1sw/sw (sw) mutants at E10.5, Wnt1 expression (purple) is not interrupted in the cerebellar plate as observed in wild type. Axons are labeled in brown by neurofilament immunocytochemistry. (P) Schematic interpretation of the midline phenotypes of Wnt1sw/sw and En1+/Otx2lacZ mutants compared to wild type. Same color code as in Fig. 7.