Vegetal view of a Halocynthia roretzi 8-cell stage embryo probed for m.actin expression and injected with Hr-macho-A-EnR mRNA.
m.actin expression is lost.
Original comment: Injection of RNA that encodes a dominant negative version of macho-1, in which the full length of macho-1 is fused at its C-terminus with the Drosophila engrailed repressor domain (known as EnR), completely eliminated muscle actin expression [100%, n= 7].

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with Tbx6 MO.
Muscle actin expression is strongly reduced in muscle lineage (only 3 over 21 embryos showed expression in muscle cells at a similar level to the control).

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with Tbx6 MO and Tbx6 mRNA.
Muscle actin is expressed in muscle cells (B8.15, B8.8 and B8.7).
Original comment: The injection of Tbx6 RNA was able to rescue the MO-induced knockdown of Muscle actin expression.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 32-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, and ectopically in B8.5 and B7.7.
Original comment: Embryos treated with MEK inhibitor from the 32-cell stage showed ectopic Muscle actin expression in nearly all cells of both the anterior (B8.5, 97.2% [n = 36]) and posterior (B7.7, 100% [n = 36]) mesenchyme lineages at the 110-cell stage.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 44-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, and ectopically in B8.5 and B7.7.
Original comment: Treatment starting from the 44-cell stage resulted in ectopic expression in all posterior-lineage blastomeres (B7.7, 100% [n = 38]), but in only approximately 60% of those of the anterior lineage (B8.5, 60.5% [n = 38]).

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 44-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, and ectopically in B8.5 and B7.7.
Original comment: Treatment starting from the 44-cell stage resulted in ectopic expression in all posterior-lineage blastomeres (B7.7, 100% [n = 38]), but in only approximately 60% of those of the anterior lineage (B8.5, 60.5% [n = 38]).

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 44-cell stage.
Muscle actin is expressed in B8.15, B8.8 and B8.7, and ectopically in B7.7.
Original comment: Treatment starting from the 44-cell stage resulted in ectopic expression in all posterior-lineage blastomeres (B7.7, 100% [n = 38]), but in only approximately 60% of those of the anterior lineage (B8.5, 60.5% [n = 38]).

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 44-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, and ectopically in B8.5 and B7.7.
Original comment: Treatment starting from the 44-cell stage resulted in ectopic expression in all posterior-lineage blastomeres (B7.7, 100% [n = 38]), but in only approximately 60% of those of the anterior lineage (B8.5, 60.5% [n = 38]).

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 44-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, and ectopically in B8.5 and B7.7.
Original comment: Treatment starting from the 44-cell stage resulted in ectopic expression in all posterior-lineage blastomeres (B7.7, 100% [n = 38]), but in only approximately 60% of those of the anterior lineage (B8.5, 60.5% [n = 38]).

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 44-cell stage.
Muscle actin is expressed in B8.15, B8.8 and B8.7, and ectopically in B7.7.
Original comment: Treatment starting from the 44-cell stage resulted in ectopic expression in all posterior-lineage blastomeres (B7.7, 100% [n = 38]), but in only approximately 60% of those of the anterior lineage (B8.5, 60.5% [n = 38]).

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 44-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, and ectopically in B8.5 and B7.7.
Original comment: Treatment starting from the 44-cell stage resulted in ectopic expression in all posterior-lineage blastomeres (B7.7, 100% [n = 38]), but in only approximately 60% of those of the anterior lineage (B8.5, 60.5% [n = 38]).

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 44-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, and ectopically in B8.5 and B7.7.
Original comment: Treatment starting from the 44-cell stage resulted in ectopic expression in all posterior-lineage blastomeres (B7.7, 100% [n = 38]), but in only approximately 60% of those of the anterior lineage (B8.5, 60.5% [n = 38]).

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 44-cell stage.
Muscle actin is expressed in B8.15, B8.8 and B8.7, and ectopically in B7.7.
Original comment: Treatment starting from the 44-cell stage resulted in ectopic expression in all posterior-lineage blastomeres (B7.7, 100% [n = 38]), but in only approximately 60% of those of the anterior lineage (B8.5, 60.5% [n = 38]).

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 64-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, and ectopically in B7.7.
Original comment: When MEK inhibitor treatment was initiated at the 64-cell stage, no anterior- (B8.5, 0% [n = 40]) and 57.5% (n = 40) of posterior (B7.7) -lineage blastomeres exhibited ectopic expression.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and treated with the MEK inhibitor U0126 from the 76-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, as in WT conditions.
Original comment: Almost no cells in the mesenchyme lineage exhibited Muscle actin expression when the MEK signal was blocked from the 76-cell stage 0% (n = 40) in B8.5 and 7.5% (n = 40) in B7.7.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with FGF9/16/20 MO in the B6.1 blastomer at the 32-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, and ectopically in the posterior mesenchyme cell B7.7.
Original comment: The result showed that the FGF signal from B6.1 is required for suppression of the muscle fate in B7.7
The numbers in the corner indicate embryos showing ectopic expression in B7.7 blastomeres.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with FGF9/16/20 MO in the B6.2 blastomer at the 32-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, and ectopically in the posterior mesenchyme cell B7.7.
Original comment: The result showed that the FGF signal from B6.2 is required for suppression of the muscle fate in B7.7
The numbers in the corner indicate embryos showing ectopic expression in B7.7 blastomeres.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with FGF9/16/20 MO in the A5.2 blastomer at the 16-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, as in WT conditions.
The numbers in the corner indicate embryos showing ectopic expression in B7.7 blastomeres.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with FGF9/16/20 MO in the B5.1 blastomer at the 16-cell stage.
Muscle actin is expressed in B7.5, B8.15, B8.8 and B8.7, and ectopically in the posterior mesenchyme cell B7.7.
Original comment: MO injection into B5.1, the mother cell of B6.1 and B6.2 at one-cell cycle earlier, still resulted in 14.3% (n = 7) of B8.5 blastomeres showing ectopic Muscle actin expression, while it resulted in 100% (n = 7) of B7.7.
The numbers in the corner indicate embryos showing ectopic expression in B7.7 blastomeres.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with WT Tbx6 mRNA.
Muscle actin is expressed in B7.5, B8.15, B8.7 and B8.8, and ectopically in nerve chord precursors : A8.7, A8.8, A8.15 and A8.16.
The numbers in the lower right-hand corner indicate embryos with ectopic expression in nerve cord cells, whereas the number in the upper right-hand represents the average number of nerve cord cells per embryo with ectopic expression.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with WT-Tbx6 and FGF9/16/20 mRNAs.
Muscle actin is expressed in B7.5, B8.15, B8.7 and B8.8, and ectopically in the nerve chord precursor A8.7.
Original comment: Co-injection of embryos with FGF9/16/20 and Tbx6 mRNAs significantly reduced both the percentage of embryos (39.1%, n = 23; P < 0.05, κ2-test) and average number of cells (1.06, P < 0.01, κ2-test) expressing Muscle actin.
The numbers in the lower right-hand corner indicate embryos with ectopic expression in nerve cord cells, whereas the number in the upper right-hand represents the average number of nerve cord cells per embryo with ectopic expression.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with Tbx6(PPAP) mRNA. This mutant carries a C-terminal mutation (PPTP>PPAP).
Muscle actin is expressed in B7.5, B8.15, B8.7 and B8.8, and ectopically in nerve chord precursors (A8.15 and A8.16). Signal was also detected in mesenchyme precursors B8.5 and B7.7, but only in a small percentage (19.0%).
The numbers in the lower right-hand corner indicate embryos with ectopic expression in nerve cord cells, whereas the number in the upper right-hand represents the average number of nerve cord cells per embryo with ectopic expression.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with FGF9/16/20 and Tbx6(PPAP) mRNAs.
Muscle actin is expressed in B7.5, B8.15, B8.7 and B8.8, and ectopically in nerve chord precursors (A8.15 and A8.16 in one side).
Original comment: The C-terminal mutation (PPTP>PPAP) did not significantly change the degree of ectopic expression of Muscle actin in the presence and absence of FGF, indicating that the mutant no longer responded to FGF.
The numbers in the lower right-hand corner indicate embryos with ectopic expression in nerve cord cells, whereas the number in the upper right-hand represents the average number of nerve cord cells per embryo with ectopic expression.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with Tbx6(PNAP) mRNA. This RNA carries a mutant T-box domain.
Muscle actin is expressed in B7.5, B8.15, B8.7 and B8.8, and ectopically in nerve chord precursors (A8.15 and A8.16 in one side).
Original comment: The mutant mRNA reduced the transcriptional activity, as observed by the reduced percentage of embryos with ectopic expression (69.2–24.5%) and number of such cells (3.46–0.74).
The numbers in the lower right-hand corner indicate embryos with ectopic expression in nerve cord cells, whereas the number in the upper right-hand represents the average number of nerve cord cells per embryo with ectopic expression.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for Muscle actin expression and injected with FGF9/16/20 and Tbx6(PNAP) mRNAs. This RNA carries a mutant T-box domain.
Muscle actin is only expressed in muscle precursors (B8.15, B8.7 and B8.8).
Original comment: The transcriptional activity of the T-box domain mutant was significantly affected by the FGF signal, indicating that the mutant still responded to FGF.
The numbers in the lower right-hand corner indicate embryos with ectopic expression in nerve cord cells, whereas the number in the upper right-hand represents the average number of nerve cord cells per embryo with ectopic expression.