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Anti-apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis

Institut d’Embryologie Cellulaire et Moléculaire, CNRS FRE2160, 49bis Avenue de la Belle Gabrielle, 94736 Nogent-sur-Marne Cedex, France

View larger version (30K): [in a new window]   Fig. 1. (A) Dorsal and lateral views of the excision of the axial-paraxial hinge (APH) in chick or quail embryos, at E1.5 (V, ventral; D, dorsal). The APH is the region encompassing the caudal Hensen’s node (HN) and the rostral primitive streak (PS). (B) One day after the operation (i.e. 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. (C) In experiment A, operated embryos are kept in ovo, (1) without a graft, as control; (2) grafted with a fragment of notochord or floor plate; (3) grafted with SHH-producing cells. (D) In experiment B, the neural tube (NT) deprived of midline cells is enzymatically isolated and grafted into a stage-matched chick embryo in place of a segment of its own neural tube-notochord complex, (1) alone; (2) above a notochord or a floor plate fragment; (3) above a layer of SHH-producing cells. (E) In experiment C, the neural plate (NP) and paraxial mesoderm (PM) are separated from the notochord (No) and floor plate (FP) by a slit penetrating the 3 germ layers, from the level of the last formed somites (S) to Hensen’s node (HN) level, in chick embryos at the 10- to 12-somite stage. (F) In experiment D, the neural tube (NT) is separated from the paraxial mesoderm or somites (So) by a slit extending from the level of the 2 or 3 last formed somites (S) to the non-segmented region in chick embryos at the 12- to 15-somite stage. Ao, aorta.

View larger version (173K): [in a new window]   Fig. 2. Experiment A (see Fig. 1C). One day after APH excision (E2.5), (A) a fragment of notochord or (B) SHH-producing cells were grafted between the neural tube deprived of midline cells and the paraxial mesoderm over a length of several somites. (F,J) Dorsal views, at E3.5, of embryos grafted respectively with a notochord or SHH-producing cells. Consecutive sections, at the levels indicated, either in the notochord-grafted region (C-E), or in the non-grafted region of the same embryo (G-I), or in the region grafted with SHH-producing cells (K-M), were hybridized with Shh (C,G,K) or Pax3 (D,H,L) probes or examined for apoptosis using TUNEL (arrowheads) (E,I,M). En, endoderm; No, notochord; NT, neural tube; Shh, SHH-producing cells; So, somite.

View larger version (114K): [in a new window]   Fig. 3. Experiment B (see Fig. 1D). (A,E,I) Dorsal views of E3 chick embryos grafted at E2 with a fragment of neural tube deprived of midline cells (A) alone (experiment B1); (E) with a notochord (experiment B2); (I) on a layer of SHH-producing cells (experiment B3). (B-D, F-H and J-L) Consecutive sections of each embryo at the level of the graft, hybridized with Shh (B,F,J) and Pax3 probes (C,G,K) or examined for cell death using the TUNEL assay (arrowheads) (D,H,L). En: endoderm; No, notochord; NT, neural tube; Shh, SHH-producing cells; So, somite.

View larger version (127K): [in a new window]   Fig. 4. Results at E4 of experiment B1 (see Fig. 1D1 and Fig. 3A-D). (A) Lateral view of a host chick embryo 2 days after the operation. The position of the graft is apparent from a notch at the cervical level. (B-E) Consecutive cross sections at the level of the graft, hybridized with Shh (B), Pax3 (D) or BMP4 (E) probes or treated with the QCPN antibody for detection of quail cells (C). In C, the neural tube (NT) and dorsal root ganglia (DRG) are indicated. Ao, aorta.

View larger version (141K): [in a new window]   Fig. 5. Expression patterns of Shh (A-D) and Ptc (E-H) on serial sections at E3. (A,E) control level of expression; (B,F) midline cell-deprived neural tube level; (C,G) SHH-producing cell grafted level (experiment A3); (D,H) midline cell-deprived neural tube grafted on a layer of SHH-producing cells in a host (experiment B3). Ptc is upregulated (arrowheads) in the presence of SHH; Shh, SHH-producing cells.

View larger version (96K): [in a new window]   Fig. 6. Results of experiment C (see Fig. 1E). (A-C) Dorsal views of embryos hybridized in whole mount with Pax1 (A), Pax3 (B) and MyoD (C) probes, one day after the surgery (E2.5). (D) 2.5 days later (i.e. E5), the side deprived of midline cells is very short compared to the contralateral side, which developed normally and became U-shaped. (E-G) Serial sections at E2.5 hybridized with Shh (E), Pax1/Pax6 (F) probes or examined for apoptosis using TUNEL (arrowheads) (G). DM, dermomyotome; En, endoderm; FP, floor plate; NT, neural tube; No, notochord; Scl, sclerotome; *, ink.

View larger version (136K): [in a new window]   Fig. 7. Results of experiment D (see Fig. 1F). Cross sections 1 day after operation (E2.5) hybridized with Shh (A), Pax3 (B), Pax1/Pax6 (C) probes. (D) TUNEL assay for detection of apoptosis (arrowheads). Dm, dermomyotome; En, endoderm, Scl, sclerotome.

View larger version (127K): [in a new window]   Fig. 8. Results of experiment D (see Fig. 1F) 3 days after operation (E5.5). (A) Dorsal view of an embryo. (B-D) Serial cross sections hybridized with Pax1/Pax6 (B), HNF3ß/MyoD (C) and Pax3 probes (D). DRG, dorsal root ganglion; FP, floor plate; M, dorsal muscle; No, notochord; SC, spinal cord; V, vertebra; *, haemorrhagic area.

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