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Dual origin of the floor plate in the avian embryo

Institut d’Embryologie Cellulaire et Moléculaire, CNRS and Collège de France, UMR 7128, 49bis Avenue de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex, France

View larger version (34K): [in a new window]   Fig. 1. (A) Dorsal and lateral views of the sinus rhomboidalis of an E1.5 quail embryo after the excision of the axial-paraxial hinge (APH) (V, ventral; D, dorsal). APH is the region encompassing caudal Hensen’s node (HN) and the rostral primitive streak (PS). (B) One day after the operation (at E2.5) the neural tube caudal to somite 20 (S20) is deprived of midline structures (floor plate and notochord) and is smaller in diameter than normal as seen in cross section (C). (D) In a second experiment, the quail neural tube (NT) deprived of midline cells is enzymatically isolated and grafted in a stage-matched chick embryo in the place of a segment of its own neural tube-notochord complex: (a) above a notochord; (b) above a floor-plate fragment; and (c) above a layer of SHH-producing cells.

View larger version (116K): [in a new window]   Fig. 2. (A) Whole-mount in situ hybridisation of a 15 ss chick embryo with the CSox1 probe. (B,C) Cross-sections of this embryo at the neural tube level (B,C) as indicated in A. CSox1 transcripts are present only in the neuroepithelium and not in the floor plate (FP) or the chordo-neural hinge (CNH), which is present at the posterior neuropore level at this stage. (D) Cross-section of a 15 ss quail-chick chimera (quail Hensen’s node graft as shown in E) at the same level as C, stained with the quail specific mAb QCPN. The CNH is made up of quail cells. (E) Schematic representation of quail-chick grafts of Hensen’s node (in red) and posterior neural plate (in blue) at the 5-6 ss. (F-H) Serial cross-sections of a chimera grafted with a quail Hensen’s node (red in E), 2 days after the operation (E3.5). The expression pattern of HNF3ß (F) and Shh (G) genes is wider than the node-derived region revealed by the QCPN mAb (H). (I-M) Serial sections of another Hensen’s node chimera fixed 5.5 days after the graft (E7). CSox1 (I) is not expressed in the quail QCPN+ node-derived region (shown in L), which is where expression of HNF3ß is now restricted (J). This region also constitutes the medial floor plate (MFP). Shh (K) is expressed both in the node-derived region and in a neural plate-derived area where Nkx2.2 transcripts (M) are also present. The latter constitutes the lateral floor plate (LFP). (N-P) Serial sections of a quail-chick chimera grafted with a posterior neural plate (blue in E), 5.5 days after the operation (E7). As in Hensen’s node chimeras at the same stage, HNF3ß transcripts (N) are localised in the node-derived (host) region as seen in P, while Shh transcripts are distributed over a larger area covering both node-derived (MFP) and QCPN+ neural plate-derived tissues, including the LFP (O). Arrowheads, MFP limits; arrows, LFP lateral limit.

View larger version (16K): [in a new window]   Fig. 3. The distribution of complete floor plate (with medial and lateral components) (red) or lateral floor-plate cells only (blue) induced in the lateral neural tube of E5 to E7 chick embryos mapped as a function of the somitic stage (ss) of the chick hosts (x-axis) and of the quail donors (y-axis) of inducer tissues [notochord (No; closed circles) or floor plate (FP; open circles)] at the time of the graft. For SHH-producing cell grafts (squares) only chick host age is considered. The number of cases for each experimental series is indicated. It appears that MFP induction can occur only if the chick host is younger than 15 ss when the stage of the quail donor is between 10 and 25 ss.

View larger version (99K): [in a new window]   Fig. 4. (A-D) Graft of a 9 ss quail notochord (No') lateral to the caudal neural tube of a 10 ss chick embryo. Serial sections performed 2 days after the graft (E4) show that the neural epithelium has increased in size on the side of the graft when compared with the contralateral side. Shh (A) and HNF3ß (B) are expressed ectopically in the region facing the graft. Pax6 (C) is not found in this region. (D) QCPN labelling of the graft. (E-L) Serial sections 4 (E-H) and 5 (I-L) days after the same experiment (E6 and E7) show that the molecular characteristics of a complete floor plate with its medial and lateral components are progressively acquired in the region close to the graft: wide expression of Shh (E, I) and Netrin1 (K); presence of HNF3ß transcripts in a restricted medial region (F,J) where Shh (E,I) and Netrin1 (K) are upregulated and CSox1 is downregulated (L); expression of Nkx2.2 (G) and Sim1 (H) lateral to the HNF3ß+ region. Arrowheads, MFP limits; arrows, LFP lateral limit.

View larger version (139K): [in a new window]   Fig. 5. (A-D) Graft of a 15 ss quail floor plate (FP') lateral to the caudal neural tube of a 14 ss chick embryo. Serial sections collected 2 days after the operation (E4) show that the size of the neural tube has expanded on the side of the graft. At this stage, Shh (A), HNF3ß (B) and Nkx2.2 (C) are co-expressed in a region that is still CSox1+ (D), close to the graft. (E-H) Graft of a 12 ss quail FP' lateral to the caudal neural tube of a 13 ss chick embryo. Serial sections performed 5 days after the operation (E7) hybridised with Shh (E), HNF3ß (F), Nkx2.2 (G) and CSox1 (H) probes show that a complete floor plate has differentiated with its medial (Shh+, HNF3ß+, Nkx2.2–, CSox1–) and lateral (Shh+, HNF3ß–, Nkx2.2+, CSox1+) components. Grafted MFP plus LFP (FP') generally become circular. A new MFP is induced in the host neural tube close to the MFP part of the graft (HNF3ß+). Arrowheads, MFP limits; arrows, LFP lateral limit.

View larger version (145K): [in a new window]   Fig. 6. (A-K) Graft of SHH-producing cells between the caudal neural tube and the presomitic mesoderm of 10-15 ss chick embryos. (A) Immediately after the graft, clumps of cells are aligned to the left of the enlarged neural tube (between arrowheads). (B) Whole-mount Shh in situ hybridisation 1 day after the graft, at 25 ss (E3). The grafted cells are localised lateral to the neural tube (arrowheads). (C) A cross-section of the whole mount in B shows that the cells are still grouped (arrow) and that the neuroepithelium has expanded dorsoventrally on the side of the graft. (D,E) Serial cross-sections 3 days after the graft (E5) hybridised with Shh (D) and HNF3ß (E) probes. SHH-producing cells (arrow) are now dispersed and the Shh+, HNF3ß– lateral floor plate has widened. (F-K) Serial cross-sections 5 days after the graft (E7) show that the ventral and lateral neural tube is enlarged and perturbed on the side of the graft. Shh (F), HNF3ß (G), Netrin1 (H), Nkx2.2 (I), Sim1 (J) and CSox1 (K) expression patterns in the region facing the SHH-producing cells are characteristic of the lateral floor plate. RP, roof plate. Arrowheads, MFP limits; arrows, LFP lateral limit.

View larger version (155K): [in a new window]   Fig. 7. (A-L) Back grafts of quail neural tubes deprived of midline cells by APH excision into chick embryos from which a fragment of their own neural tube and notochord had been previously excised (see Fig. 1). Fixation was 5 days after the operation (E7). (A-D) Grafted over a notochord (No') the quail neural tube, labelled with the quail specific mAb QCPN (A), develops a typical floor plate with its medial and lateral components distinguished by the characteristic distribution of the Shh (B), HNF3ß (C) and (D) NKx2.2 transcripts. (E-H) Grafted over a floor plate (FP'), the QCPN+ quail neural tube (E) weakly expresses Shh (F) and very little HNF3ß (G) but strongly expresses Nkx2.2 (H) in the region close to FP'. This pattern recalls that of the lateral floor plate (LFP) as defined in Fig. 2. (I-L) In contact with SHH-producing cells (arrow in J), the quail neural tube deprived of midline cells develops a large LFP-like structure that expresses CSox1 (I), Shh (J), little HNF3ß (K) and Nkx2.2 (L). Arrowheads, MFP limits; arrows, LFP lateral limit.

View larger version (28K): [in a new window]   Fig. 8. Model of avian floor-plate development from E1.5 to E7 at the thoracic level. Hensen’s node is schematically represented at 5 ss (E1.5) when it becomes inserted into the neural plate which expresses the gene Sox1 (Sox1+). One day later (E2.5) at the same level, Hensen’s node has split into floor plate and notochord. The node-derived floor plate (ND FP, in red), like the node itself, expresses HNF3ß and Shh. ND FP and perhaps also the notochord, induce adjacent neural plate cells (arrows) to express HNF3ß and Shh, thus forming at E3.5 a lateral floor plate (LFP, in blue), which retains Shh expression while loosing HNF3ß expression (E7). LFP expresses continuously neural genes (Sox1, Nkx2.2 and Sim1) that are never expressed in the node-derived medial floor plate (MFP). Netrin1 is expressed both in MFP and LFP cells.