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The BMP signaling pathway is required together with the FGF pathway for notochord induction in the ascidian embryo

¹ Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
² Laboratoire de Génétique et Physiologie du Développement, Institut de Biologie du Développement de Marseille, CNRS-INSERM-Université de la Méditerranée-AP de Marseille, Campus de Luminy, Case 907, F-13288 Marseille Cedex 9, France

View larger version (21K): [in a new window]   Fig. 1. Schematic representation of the notochord lineage. Primary notochord precursors are indicated in dark pink and secondary notochord precursors in light pink. (A) Eight-cell stage embryo. Lateral view. Anterior is towards the left, animal is upwards. (B) 24-cell stage. (C) 44-cell stage. (D) 110-cell stage. Vegetal view, anterior is upwards in B-D. (E) Mid-tailbud. The 32 anterior cells constitute the primary notochord, while the eight posterior cells form the secondary notochord. (F) Lineage tree of the primary notochord starting from the eight-cell stage. (G) Lineage tree of the secondary notochord. Red rectangles correspond to blastomeres that develop into notochord after isolation.

View larger version (29K): [in a new window]   Fig. 2. chordin reduces notochord formation. (A-D) Immunostaining for Not-1, a notochord-specific antigen. (A) Control mid-tailbud. 40 cells are stained. (B) 17 cells are stained after injection of 200 pg of chordin mRNA. (C) When cleavage was arrested at the 110-cell stage, 10 notochord cells were eventually stained at the mid-tailbud stage. (D) Only four cells are positive after injection of 200 pg of chordin. Scale bar: 100 µm. (E) Number of Not-1-positive cells per embryo in cleavage arrested control embryos (blue, total number of embryos examined; n=31) and chordin-injected embryos (purple, n=28).

View larger version (52K): [in a new window]   Fig. 3. Expression pattern and its schematic representation of BMPb (A,C) and chordin (B,D) at the 64-cell stage. (E) When A6.2/A6.4 were isolated at the 24-cell stage, they did not express chordin at the 64-cell stage. (F) BMP-4 induced chordin expression in A6.2/A6.4 isolated blastomeres. Scale bar: 100 µm.

View larger version (36K): [in a new window]   Fig. 4. BMP-4 induced notochord in the primary lineage. Immunofluorescence for Not-1 antigen. (A) Notochord precursors isolated at the 24-cell stage did not form notochord. (B) Treatment with BMP-4 induced notochord formation. (C) U0126 blocked notochord induction by BMP-4. Scale bar: 50 µm.

View larger version (16K): [in a new window]   Fig. 5. Treatment of isolated primary notochord precursors with BMP, FGF, U0126 (MEK inhibitor) and SU5402 (FGFR1 inhibitor) starting from 24- and 44-cell stages. The formation of notochord evaluated by immunostaining for Not-1 antigen is indicated on the right-hand side.

View larger version (88K): [in a new window]   Fig. 6. B-line secondary notochord induction. (A-H) bFGF and BMP-4 cooperate to induce B-line notochord. (A-D) Bright field view. (E-H) Immunofluorescence for Not-1 antigen. (A,E) BSA-treated control B6.2 blastomere. Upon this treatment, all constituent cells develop into large muscle cells (Kim and Nishida, 1999). (B,F) BMP-4 protein treatment. (C,G) bFGF protein treatment. Upon this treatment, all constituent cells develop into small mesenchyme cells (Kim et al., 2000). (D,H) Simultaneous treatment with bFGF and BMP-4 proteins. Four notochord cells were induced in the partial embryo. (I-K) Unequal cleavage of B7.3. When B7.3 was isolated at the 44-cell stage, the cleavage was equal and produced daughter cells of similar size (I). Treatment with BMP-4 protein provoked a slightly unequal cleavage (J), similar to that observed when B7.3 was isolated at the late 64-cell stage (K). Scale bars: 50 µm.

View larger version (21K): [in a new window]   Fig. 7. Co-isolation experiments. Schematic representation of the B-line notochord precursor and its neighbors at the 24-cell stage (A) and at the 44-cell stage (B) with vegetal views of whole embryos at the top. The b5.4 presumptive epidermis blastomere indicated outside diagram of the top figure is a cell in the animal hemisphere. Each drawing represents a given type of co-isolation experiment. The occurrence of notochord formation is shown as percentage of resulting partial embryos positive for Not-1 antigen. The number of partial embryos is indicated in parentheses.

View larger version (23K): [in a new window]   Fig. 8. Model for notochord induction in ascidian embryos. At the 24-cell stage, a bFGF-like signal is secreted from all endodermal precursors. Then at the 44-cell stage, BMPb is expressed in the anterior endoderm and completes the induction in combination with bFGF-like signal.