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First published online 24 September 2003
doi: 10.1242/dev.00756

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The isthmic organizer links anteroposterior and dorsoventral patterning in the mid/hindbrain by generating roof plate structures

Régionalisation Nerveuse CNRS/ENS UMR 8542, Département de Biologie Ecole normale supérieure, 46 rue d'Ulm, 75005 Paris, France

View larger version (55K): [in a new window]   Fig. 1. An immobile pivotal domain in the caudal midbrain. (A,B) Posterior, (C) posterolateral, and (D) lateral views of the dissected (A-C) or whole-mount (D) neural tubes of HH19-21 chick embryos that received a graft or DiI crystals as schematized on the left. Small lateral grafts (A) and lateral DiI crystals (B) all converged towards the midline. They avoided a small medial circular area in the caudal midbrain (arrowheads in A,B). Small grafts (C) or DiI crystals (D) inserted into the center of the caudal mesencephalic vesicle apparently remained still (arrowheads in C and D) and ended in a protrusion or `beak' that marks the caudal midline of the mesencephalon. This is best seen on lateral views of the dissected neural tube (E,F arrowheads).

View larger version (62K): [in a new window]   Fig. 3. Expression of midbrain RP markers depends on IO-derived signals. (A-E) Posterior views of the MHB of HH19-21 embryos that were operated on at the 10-15ss, as schematized on the left, and treated for the detection of Gdf7 transcripts (purple). Corresponding transverse cryostat sections counterstained with nuclear Fast Red are illustrated in A'-C'. (A) In control HH19-21 chick (or quail) embryos Gdf7 expression marks the RP. (B) Gdf7 expression is completely (left, 4/6) or partially (right, 2/6) regenerated after ablations of the isthmic dorsal midline. (C-E) Homotopic quail to chick IO node grafts performed at different stages interfere with midbrain RP formation. Graft insertion into the IO node resulted in RP duplication in 14-15ss embryos (C, double arrowhead in C'; 5/5) and in the formation of a gap in Gdf7 expression in younger embryos (between arrowheads in D; 2/3). A medial bulge that expresses Gdf7 is observed in 10-12ss embryos (arrowhead in E; 3/4). (D') Transverse section of an E16 embryo that received a slightly larger graft centered on the IO node at the 12ss. The section was immunostained with QCPN to detect the grafted quail cells and counterstained with Cresyl Violet. Quail cells are detected at more caudal levels but the illustrated section is anterior to the graft limit. Note the absence of RP structure on the midline (arrowhead). Very few quail cells were detected in embryos that received transplants limited to the presumptive IO node (C-E) either because the low proliferation rate interfered with graft integration, or because of the high level of cell death detected by Nile blue sulfate staining on the midline of the isthmic region between the 14ss and 22ss (F-I) (see also Lumsden et al., 1991).

View larger version (48K): [in a new window]   Fig. 2. Isthmic origin of MHB roof plate structures. (A,D) Posterior views of the dissected neural tubes of HH19-21 embryos in which DiI crystals were inserted at the 10-13ss. The site of insertion is schematized on the left. Midline DiI crystals inserted at the level of the isthmic constriction (A) label the entire anterior hindbrain roof plate (RP). Paramedial DiI crystals inserted into the caudal midbrain vesicle (D) label the midbrain RP. (B,C,E,F) Midsagittal cryostat sections of two embryos (shown in the insets) similar to A and D. The DiI-labeled hindbrain (hb) RP ends beneath the beak (B,C). When it extends to the isthmic region (E,F), DiI labeling in the midbrain (mb) RP respects a similar sharp limit beneath the beak. (G) Fate map of the dorsal isthmic region focused on the central IO node. The cells that contribute to the roof plates of the midbrain (in red) and anterior hindbrain (purple) are organized around a central domain (gray) whose relative size decreases between the 10-13ss and HH19-21 stages. They migrate extensively along the midline but respect a common limit in the caudal midbrain. The 10-13ss and HH19-21 stage embryos are not drawn at the same scale.

View larger version (66K): [in a new window]   Fig. 4. Homeogenetic signaling induce the expression of RP markers. (A-D) Dorsal view of the midbrain region of HH19-21 embryos that received a small midbrain midline ablation at the 10-12ss as schematized on the left. Corresponding transverse cryostat sections counterstained with Nuclear Fast Red are illustrated in B',C'. The embryos were treated for the detection of Gdf7 (A-C) or Wnt1 (D) transcripts. The ablation resulted in complete regeneration (A, 1/6), or in the formation of partial (B, 4/6) or complete (C, 1/6) gaps in the midbrain Gdf7 expression domain. Scattered cells in the partial gaps expressed Gdf7 (B) or Wnt1 (D). (E,F) Lateral views (anterior is to the left) of Gdf7 midline expression in control (E) and ablated (F) embryos. Gdf7 expression is perturbed (*) on both sides of the ablation (delimited by the arrowheads). (G) Posterior view of a HH19 embryo illustrating the lack of Gdf7 expression (between arrowheads) on the midline of the mesencephalic vesicle after inversion of its anteroposterior axis as schematized in g. (H) Dorsal view of the midbrain of a 4-day-old chimera. An anteroposterior strip of quail midbrain neuroepithelium was transplanted at HH10 perpendicular to the host midline as schematized in h. H1-H3 illustrate the same chimera. The host roof plate (RP), labeled with chWnt1 (Fast Red), is seen in red using fluorescent optics (H1); the induced RP (purple arrowheads in H2 and H3), labeled with QWnt1 (NBT/BCIP), appears purple under bright-field optics (H2). After dissection of the dorsal midbrain, a faint QCPN labeling delineates the quail transplant (H3). Anterior is to the right.

View larger version (56K): [in a new window]   Fig. 5. Cell migration from the FGF8-induced IO marks the position of the ectopic roof plate (RP). Lateral views of three HH19-21 undissected embryos, 2 days after implantation in the lateral midbrain of DiI (A,A'), or FGF8/DiI (B,B',C,C') -soaked beads as schematized on the left. The same embryos photographed under rhodamine fluorescence optics in (A-C) were processed for the detection of Gdf7 (A',B') or Wnt1 (C') transcripts (purple). (A,A') Control embryo; a patch of fluorescent cells surrounds the DiI-labeled bead fragment (A). Midline Gdf7 (or Wnt1, not shown) expression is not perturbed (A'). In embryos that have received a FGF8/DiI bead fragment, a row of DiI-labeled cells (B,C) migrated from the FGF8-induced cerebellum in the direction of the host midline. These cells marked the position of ectopic RP structures labeled with Gdf7 (B') or Wnt1 (C'). The ectopic RP (arrowheads) either remained confined to the vicinity of the bead (B') or reached the endogenous midline (C'). The position of the bead is indicated with a red circle.

View larger version (36K): [in a new window]   Fig. 6. The immobile IO node and the regressing Hensen's node. Schematic drawing comparing the organizing properties of the IO node (left, same colour code as in Fig. 2G) and the regressing Hensen's node [right, the dotted area represents the neural plate; modified from Charrier et al. (Charrier et al., 1999)]. The IO and Hensen's node organize similar convergent extension cell movements and produce cells destined for axial structures. In both cases, the differentiation of the definitive midline neural structures depends on homeogenetic mechanisms (curved arrows). The stability of the IO node contrasts with the active caudalward movement of the regressing Hensen's node that leads to extension of the neural tube. The opposing centrifugal migrations arising from the IO node may explain why it remains still. Black arrows point to the direction of cell movements.