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First published online 30 August 2006
doi: 10.1242/dev.02579

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Tsukushi cooperates with VG1 to induce primitive streak and Hensen's node formation in the chick embryo

Kunimasa Ohta^1,2,*, Sei Kuriyama^1,3,, Tatsuya Okafuji^1,, Ryu Gejima^1,3, Shin-ichi Ohnuma⁴ and Hideaki Tanaka^1,3

¹ Department of Developmental Neurobiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
² PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama, Japan.
³ 21st Century COE, Kumamoto University, Kumamoto 860-8556, Japan.
⁴ Department of Oncology, The Hutchison/MRC Research Centre, University of Cambridge, Hills Road, Cambridge CB2 2XZ, UK.

View larger version (37K): [in a new window]   Fig. 1. Comparison of the primary structure between TSKA and TSKB. (A) Schematic drawing of the primary structure of TSKA and TSKB. The leucine-rich repeats (LRRs) are indicated as circles. Cysteine residues are indicated by red bars. (B) Alignment of C-terminal Tsukushi cDNA and amino acid sequences. The 990-1309 (red) nucleotide sequence is deleted by degenerate 5' splicing, resulting in the production of TSKA from TSKB. (C) Schematic drawing for the generation of TSKA and TSKB isoforms by alternative 5' splicing of chick Tsukushi gene. The numbers on the top correspond to the nucleotide sequence in B.

View larger version (55K): [in a new window]   Fig. 2. Expression of TSKA and TSKB in early chick embryo. (A-C) In situ hybridization and RT-PCR analyses of chick embryos at stages 2-4. The right panels show the expression patterns of Tsukushi mRNA. (A) Chick embryo at stage 2. (B) Chick embryos at stage 3 were categorized into anterior site (PS-A) and posterior site (PS-P). (C) Chick embryos at stage 4 were categorized into Hensen's node (HN), anterior site (PS-A), middle site (PS-M) and posterior site (PS-P). a, anterior; p, posterior.

View larger version (46K): [in a new window]   Fig. 3. The different BMP inhibitory activities between TSKA and TSKB. TSKA (A) or TSKB (B) mRNA was injected into a ventral vegetal blastomere at the eight-cell stage and the embryos were developed until stage 31. Arrow indicates a secondary axis. (C) Western blot analysis of TSKA-Myc-His, TSKB-Myc-His and BMP4-Flag proteins used. (D) Co-immunoprecipitation of TSKA-Myc-His or TSKB-Myc-His and BMP4-Flag. After immunoprecipitation with nickel-chelating resins, the complexes were washed under low or high stringency conditions. (E) Co-immunoprecipitation of TSKA-Myc-His or TSKB-Myc-His and BMP4-Flag with different amounts of BMP4. The numbers above the column show the volume (µl) of COS-7 cell supernatant, including BMP4 in the total volume (1 ml). (F) Co-immunoprecipitation of TSKB-Myc-His and BMP7-Flag. (G,J) Transplantation schemes. (H,I) TSKA- or TSKB-producing cells (orange in G) were grafted together with the middle primitive streak (MPS; green in G) on the right-hand side. The mock-transfected COS-7 cells (white in G) were grafted with MPS on the left-hand side. (K,L) TSKA or TSKB-producing cells (orange in J) were grafted together with cells expressing VG1 (purple in J) and WNT1 (blue in J) on the right-hand side. The mock-transfected COS-7 cells (white) were grafted with cells expressing VG1 and WNT1 on the left-hand side.

View larger version (58K): [in a new window]   Fig. 4. Tsukushi interacts with VG1. (A-C) Expression of TSKB (A), VG1 (B) and WNT8C (C) in stage XII chick embryos. PMZ (arrows); Koller's sickle (arrowhead). (D-F) Expression of TSKB (D), VG1 (E) and WNT8C (F) in stage 4 chick embryos. Hensen's node (arrowheads); the middle primitive streak (arrows) (G) Co-immunoprecipitation of TSKA-Myc-His or TSKB-Myc-His and VG1-Myc. After immunoprecipitation with nickel-chelating resins, bound VG1 was detected by immunoblotting with anti-Myc antibody. (H) Co-immunoprecipitation of TSKB-Myc-His and VG1-Myc in the presence of BMP4. (I,J) VG1 mRNA (100 pg) or VG1 mRNA (100 pg) + TSKB mRNA (400 pg) was injected into ventral vegetal blastomeres at the eight-cell stage, and embryos were developed until stage 31 and in situ hybridized with a PAX2 probe. pn, pronephore; ov, optic vesicle. (K) TSKA- or TSKB-producing cells (orange) were grafted into the lateral marginal zone (a), the area opaca (b) or the area pellucida (c) with cells expressing VG1 (purple). (L) Induction of ectopic brachyury (arrow) was observed when TSKB-producing cells were grafted together into the area pellucida (`c' in K) with VG1-expressing cells.

View larger version (72K): [in a new window]   Fig. 5. Gene silencing of TSK results in the inhibition of organizer formation. (A) Transplantation scheme of siRNA experiments. Two chick embryos were electroporated with siRNA at stage 3+ and the excised MPSs were implanted into the lateral Hensen's node of the host embryo. (B) The embryo at stage 3+ was electroporated with control1, TSKA or TSKs siRNA. Hensen's node and the anterior fourth of the primitive streak were surgically removed. After 6 hours incubation, the level of VG1 or TSKB mRNA in the MPS was determined by RT-PCR. (C-J) After carrying out experiments according to the diagram in A, the ectopic induction of chordin (C-F) or goosecoid (G-J) was examined by in situ hybridization. (D,E) In the case of control1 and TSKA siRNAs, the MPS induced ectopic expression of chordin in 17/44 (39%) and 12/32 (38%), respectively, which are similar to embryos without electroporation (C) [20/50 (40%)]. (F) In the case of TSKs siRNA, the MPS induced ectopic expression of chordin in 8/40 (20%) embryos. (H,I) In the case of control1 and TSKA siRNAs, the MPS induced ectopic expression of goosecoid in 12/42 (29%) and 12/44 (27%), respectively, which are similar to embryos without electroporation (G) [12/40 (30%)]. (J) In the case of TSKs siRNA, the MPS induced ectopic expression of chordin in 7/53 (13%) embryos.

View larger version (42K): [in a new window]   Fig. 6. VG1+TSK can bypass the inhibition by the primitive streak. (A) Grafting a pellet expressing VG1 into the lateral margin of pre-streak stage embryos. Six hours later: (top) a second pellet expressing VG1, TSKA, TSKB, chordin, Nodal or FGF8B; (middle) a combination; (bottom) VG1+chordin + TSKA or TSKB were implanted into the opposite side. (B-D) A pellet of VG1 (B, purple), TSKA (C, pink) or TSKB (D, orange) was implanted into the opposite site. (E-H) A second pellet of VG1 (E-H, purple) and a pellet of TSKA (E, pink), TSKB (F, orange), Nodal (G, blue) or chordin (H, green) were implanted together into the opposite side. (I,J) When VG1 (purple) is implanted laterally, followed 6 hours later by a pellet of cells expressing VG1 (I,J; purple), chordin (I,J, green) and TSKA (I, pink) or TSKB (J, orange) on the opposite side, the combination of VG1+chordin and TSKA or TSKB can induce an ectopic primitive streak. VG1 was used as a probe for in situ hybridization to indicate the cell aggregates expressing VG1 (arrows) in I,J.

View larger version (10K): [in a new window]   Fig. 7. Model of molecular interactions during primitive streak formation. The successive stages in the development of the chick embryo are shown on the left; the sequential molecular signaling steps are shown on the right. (A) Just prior to the gastrulation (stages XI-XIII), WNT8C and TSK are expressed all around the marginal zone, and VG1 in PMZ. TSKA collaborates with chordin to decrease BMP activity in the PMZ. (B) At stages 2-3, the primitive streak starts to form under the influence of VG1+WNT8C and VG1+TSKB. VG1+WNT8C and VG1+TSKB signaling cascade synergize to induce the organizer in the central area where the TSKA+chordin serves to keep BMP expression clear of the center. TSKA also inhibits the activity of BMP7 expressed in the posterior primitive streak.