Vegetal views of Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions.
Original comment: We did not detect mis-expression of MA4 in B7.6 germline cells at the 110-cell stage after GSK126 treatment.
White or yellow arrows indicate the absence of ectopic gene expression in germline cells.
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with a control MO and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions.
Original comment: We did not detect mis-expression of MA4 in B7.6 germline cells at the 110-cell stage after GSK126 treatment.
White arrows indicate the absence of ectopic gene expression in germline cells.
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3. Nuclei were stained with SYTOX (A').
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions.
Original comment: Normally, B7.7 mesenchyme cells (purple arrows) are located vegetally to the B8.15 muscle cells (yellow arrows).
White arrows in A indicate the absence of ectopic gene expression in germline cells. White arrows in A’ show the positions of germline cells identified by nuclear staining and their smallest cell size.

Vegetal views of Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions.
Original comment: We did not detect mis-expression of MA4 in B7.6 germline cells at the 110-cell stage after GSK126 treatment.
White or yellow arrows indicate the absence of ectopic gene expression in germline cells.
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with a control MO and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions.
Original comment: We did not detect mis-expression of MA4 in B7.6 germline cells at the 110-cell stage after GSK126 treatment.
White arrows indicate the absence of ectopic gene expression in germline cells.
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3. Nuclei were stained with SYTOX (A').
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions.
Original comment: Normally, B7.7 mesenchyme cells (purple arrows) are located vegetally to the B8.15 muscle cells (yellow arrows).
White arrows in A indicate the absence of ectopic gene expression in germline cells. White arrows in A’ show the positions of germline cells identified by nuclear staining and their smallest cell size.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with control DMSO.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and weakly in B7.6.
Original comment: Ectopic MA4 expression in germline cells was observed after Pem MO injection (red arrow).
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal views of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control). Nuclei were stained with SYTOX (B').
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and ectopically in B7.6 (red arrow), B7.5 and B8.5.
Original comment: Normally, B7.7 mesenchyme cells (purple arrows) are located vegetally to the B8.15 muscle cells (yellow arrows). However, after Pem knockdown, the mesenchyme cells sometimes stuck out and pushed the B8.15 cells toward the midline of the embryo (yellow arrows); consequently, germline cells were no longer the posterior-most cells.
White arrows in B indicate the absence of ectopic gene expression in germline cells. White arrows in B’ show the positions of germline cells identified by nuclear staining and their smallest cell size

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control). Nuclei were stained with SYTOX (insert).
MA4 is expressed in B8.16, B8.7 and B8.8 blastomer pairs.
Original comment: Injection of Pem MO sometimes resulted in the loss of endogenous MA4 expression (light blue arrows).
White arrows indicate the absence of MA4 expression in germline cells; white arrows in inset the positions of germline cells identified by nuclear staining and their smallest cell size. Light blue arrows the loss of endogenous MA4 expression in B7.5 cells.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control).
MA4 expression is lost.
Original comment: Injection of Pem MO sometimes resulted in the loss of endogenous MA4 expression.

Vegetal views of a Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and weakly in B7.6.
Original comment: Significantly more embryos showed ectopic MA4 expression in the germline (red arrow) when Pem and H3K27me3 were simultaneously depleted than when Pem alone was knocked down.
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 expression in the muscle precursors (B7.5, B8.15, B8.16, B8.7 and B8.8) is strongly reduced, and ectopic expression is observed in B7.6.
Original comment: Significantly more embryos showed ectopic MA4 expression in the germline (red arrow) when Pem and H3K27me3 were simultaneously depleted than when Pem alone was knocked down.
The numbers represent the proportion of embryos showing ectopic gene expression in the germline.

Vegetal views of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3. Nuclei were stained with SYTOX (E').
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and ectopically in B7.6.
Original comment: White arrows in E’ show the positions of germline cells identified by nuclear staining and their smallest cell size, light blue arrow shows the loss of endogenous MA4 expression in B7.5 cells.

Vegetal views of a Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomers and in B7.6 (red arrow).
The numbers represent the proportion of embryos showing ectopic gene expression in the germline.

Vegetal views of a Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and weakly in B7.6.
Original comment: Significantly more embryos showed ectopic MA4 expression in the germline (red arrow) when Pem and H3K27me3 were simultaneously depleted than when Pem alone was knocked down.
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 expression in the muscle precursors (B7.5, B8.15, B8.16, B8.7 and B8.8) is strongly reduced, and ectopic expression is observed in B7.6.
Original comment: Significantly more embryos showed ectopic MA4 expression in the germline (red arrow) when Pem and H3K27me3 were simultaneously depleted than when Pem alone was knocked down.
The numbers represent the proportion of embryos showing ectopic gene expression in the germline.

Vegetal views of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3. Nuclei were stained with SYTOX (E').
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and ectopically in B7.6.
Original comment: White arrows in E’ show the positions of germline cells identified by nuclear staining and their smallest cell size, light blue arrow shows the loss of endogenous MA4 expression in B7.5 cells.

Vegetal views of a Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomers and in B7.6 (red arrow).
The numbers represent the proportion of embryos showing ectopic gene expression in the germline.

Vegetal views of Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions.
Original comment: We did not detect mis-expression of MA4 in B7.6 germline cells at the 110-cell stage after GSK126 treatment.
White or yellow arrows indicate the absence of ectopic gene expression in germline cells.
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with a control MO and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions.
Original comment: We did not detect mis-expression of MA4 in B7.6 germline cells at the 110-cell stage after GSK126 treatment.
White arrows indicate the absence of ectopic gene expression in germline cells.
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3. Nuclei were stained with SYTOX (A').
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions.
Original comment: Normally, B7.7 mesenchyme cells (purple arrows) are located vegetally to the B8.15 muscle cells (yellow arrows).
White arrows in A indicate the absence of ectopic gene expression in germline cells. White arrows in A’ show the positions of germline cells identified by nuclear staining and their smallest cell size.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with control DMSO.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and weakly in B7.6.
Original comment: Ectopic MA4 expression in germline cells was observed after Pem MO injection (red arrow).
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal views of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control). Nuclei were stained with SYTOX (B').
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and ectopically in B7.6 (red arrow), B7.5 and B8.5.
Original comment: Normally, B7.7 mesenchyme cells (purple arrows) are located vegetally to the B8.15 muscle cells (yellow arrows). However, after Pem knockdown, the mesenchyme cells sometimes stuck out and pushed the B8.15 cells toward the midline of the embryo (yellow arrows); consequently, germline cells were no longer the posterior-most cells.
White arrows in B indicate the absence of ectopic gene expression in germline cells. White arrows in B’ show the positions of germline cells identified by nuclear staining and their smallest cell size

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control). Nuclei were stained with SYTOX (insert).
MA4 is expressed in B8.16, B8.7 and B8.8 blastomer pairs.
Original comment: Injection of Pem MO sometimes resulted in the loss of endogenous MA4 expression (light blue arrows).
White arrows indicate the absence of MA4 expression in germline cells; white arrows in inset the positions of germline cells identified by nuclear staining and their smallest cell size. Light blue arrows the loss of endogenous MA4 expression in B7.5 cells.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control).
MA4 expression is lost.
Original comment: Injection of Pem MO sometimes resulted in the loss of endogenous MA4 expression.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with control DMSO.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and weakly in B7.6.
Original comment: Ectopic MA4 expression in germline cells was observed after Pem MO injection (red arrow).
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal views of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control). Nuclei were stained with SYTOX (B').
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and ectopically in B7.6 (red arrow), B7.5 and B8.5.
Original comment: Normally, B7.7 mesenchyme cells (purple arrows) are located vegetally to the B8.15 muscle cells (yellow arrows). However, after Pem knockdown, the mesenchyme cells sometimes stuck out and pushed the B8.15 cells toward the midline of the embryo (yellow arrows); consequently, germline cells were no longer the posterior-most cells.
White arrows in B indicate the absence of ectopic gene expression in germline cells. White arrows in B’ show the positions of germline cells identified by nuclear staining and their smallest cell size

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control). Nuclei were stained with SYTOX (insert).
MA4 is expressed in B8.16, B8.7 and B8.8 blastomer pairs.
Original comment: Injection of Pem MO sometimes resulted in the loss of endogenous MA4 expression (light blue arrows).
White arrows indicate the absence of MA4 expression in germline cells; white arrows in inset the positions of germline cells identified by nuclear staining and their smallest cell size. Light blue arrows the loss of endogenous MA4 expression in B7.5 cells.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control).
MA4 expression is lost.
Original comment: Injection of Pem MO sometimes resulted in the loss of endogenous MA4 expression.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with control DMSO.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and weakly in B7.6.
Original comment: Ectopic MA4 expression in germline cells was observed after Pem MO injection (red arrow).
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal views of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control). Nuclei were stained with SYTOX (B').
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and ectopically in B7.6 (red arrow), B7.5 and B8.5.
Original comment: Normally, B7.7 mesenchyme cells (purple arrows) are located vegetally to the B8.15 muscle cells (yellow arrows). However, after Pem knockdown, the mesenchyme cells sometimes stuck out and pushed the B8.15 cells toward the midline of the embryo (yellow arrows); consequently, germline cells were no longer the posterior-most cells.
White arrows in B indicate the absence of ectopic gene expression in germline cells. White arrows in B’ show the positions of germline cells identified by nuclear staining and their smallest cell size

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control). Nuclei were stained with SYTOX (insert).
MA4 is expressed in B8.16, B8.7 and B8.8 blastomer pairs.
Original comment: Injection of Pem MO sometimes resulted in the loss of endogenous MA4 expression (light blue arrows).
White arrows indicate the absence of MA4 expression in germline cells; white arrows in inset the positions of germline cells identified by nuclear staining and their smallest cell size. Light blue arrows the loss of endogenous MA4 expression in B7.5 cells.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with DMSO (control).
MA4 expression is lost.
Original comment: Injection of Pem MO sometimes resulted in the loss of endogenous MA4 expression.

Vegetal views of a Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and weakly in B7.6.
Original comment: Significantly more embryos showed ectopic MA4 expression in the germline (red arrow) when Pem and H3K27me3 were simultaneously depleted than when Pem alone was knocked down.
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 expression in the muscle precursors (B7.5, B8.15, B8.16, B8.7 and B8.8) is strongly reduced, and ectopic expression is observed in B7.6.
Original comment: Significantly more embryos showed ectopic MA4 expression in the germline (red arrow) when Pem and H3K27me3 were simultaneously depleted than when Pem alone was knocked down.
The numbers represent the proportion of embryos showing ectopic gene expression in the germline.

Vegetal views of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3. Nuclei were stained with SYTOX (E').
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and ectopically in B7.6.
Original comment: White arrows in E’ show the positions of germline cells identified by nuclear staining and their smallest cell size, light blue arrow shows the loss of endogenous MA4 expression in B7.5 cells.

Vegetal views of a Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomers and in B7.6 (red arrow).
The numbers represent the proportion of embryos showing ectopic gene expression in the germline.

Vegetal views of a Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and weakly in B7.6.
Original comment: Significantly more embryos showed ectopic MA4 expression in the germline (red arrow) when Pem and H3K27me3 were simultaneously depleted than when Pem alone was knocked down.
The numbers represent that of embryos showing ectopic gene expression in the germline.

Vegetal view of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 expression in the muscle precursors (B7.5, B8.15, B8.16, B8.7 and B8.8) is strongly reduced, and ectopic expression is observed in B7.6.
Original comment: Significantly more embryos showed ectopic MA4 expression in the germline (red arrow) when Pem and H3K27me3 were simultaneously depleted than when Pem alone was knocked down.
The numbers represent the proportion of embryos showing ectopic gene expression in the germline.

Vegetal views of a Halocynthia roretzi 110-cell stage embryo probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3. Nuclei were stained with SYTOX (E').
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomer pairs, as in WT conditions, and ectopically in B7.6.
Original comment: White arrows in E’ show the positions of germline cells identified by nuclear staining and their smallest cell size, light blue arrow shows the loss of endogenous MA4 expression in B7.5 cells.

Vegetal views of a Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression, injected with Pem MOs and treated with GSK126, a selective inhibitor of EZH2 used to inhibit H3K27me3.
MA4 is expressed in B7.5, B8.15, B8.16, B8.7 and B8.8 blastomers and in B7.6 (red arrow).
The numbers represent the proportion of embryos showing ectopic gene expression in the germline.

Vegetal views of a Halocynthia roretzi 110-cell stage embryos probed for MA4 (Muscle actin) expression, injected with Pem MOs and EnR-Macho-1 mRNA, and treated with GSK126 (a selective inhibitor of EZH2 used to inhibit H3K27me3). EnR-Macho-1 mRNA is used to inhibit the function of Macho-1.
MA4 expression is lost

Original annotation: Inhibition of Macho-1 function eliminated ectopic MA4 expression caused by downregulation of Pem and H3K27me3 (33%, n ​= ​12) in almost all cases (4.8% expression, n ​= ​21) as well as endogenous expression.
The numbers represent the proportion of embryos showing ectopic gene expression in the germline.