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Three classes of signaling molecule, VG1, WNT and BMP, play crucial roles in axis formation in the chick embryo. Although VG1 and WNT signals have a pivotal function in inducing the primitive streak and Hensen's node in the embryo midline, their action is complemented by that of BMP antagonists that protect the prospective axial tissue from the inhibitory influence of BMPs secreted from the periphery. We have previously reported that a secreted factor, chick Tsukushi (TSK), is expressed in the primitive streak and Hensen's node, where it works as a BMP antagonist. Here, we describe a new crucial function for TSK in promoting formation of the primitive streak and Hensen's node by positively regulating VG1 activity. We provide evidence that TSK directly binds VG1 in vitro, and that TSK and VG1 functionally interact in axis formation, as shown by biological assays performed in chick and Xenopus embryos. Furthermore, we show that alternative splicing of TSK RNA leads to the formation of two isoforms (TSKA, originally designated as TSK, and TSKB) that differ in their C-terminal region. Biochemical and biological assays indicate that TSKB is a much weaker BMP antagonist than TSKA, although both isoforms efficiently interact with VG1. Remarkably, although both TSKA and TSKB are expressed throughout the early extending primitive streak, their expression patterns diverge during gastrulation. TSKA expression concentrates in Hensen's node, a well-known source of anti-BMP signals, whereas TSKB accumulates in the middle primitive streak (MPS), a region known to work as a node-inducing center where VG1 expression is also specifically localized. Loss-of-function experiments demonstrate that TSKB, but not TSKA, function is required in the MPS for induction of Hensen's node. Taken together, these results indicate that TSK isoforms play a crucial role in chick axis formation by locally modulating VG1 and BMP activities during gastrulation.
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 ( l) of COS-7 cell supernatant, including
BMP4 in the total volume (1 ml). (F) Co-immunoprecipitation of TSKBMyc-
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 lefthand
side.
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 TSKAMyc-
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 TSKBproducing
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.
