Confocal Z-stack fluorescence images showing the localization of PEM protein (magenta) in a Halocynthia roretzi 1-cell stage embryo injected with PEM MO.
Blue arrowheads indicate the absence of nuclearPEM signals in PEM MO-injected embryos.

1st image : PEM localization
2nd image : Nuclei counterstained green by SYTOX dye (green).
3rd image : Merge

Confocal Z-stack fluorescence images showing the localization of PEM protein (magenta) in a Halocynthia roretzi 2-cell stage embryo injected with PEM MO.
Blue arrowheads indicate the absence of nuclearPEM signals in PEM MO-injected embryos.

1st image : PEM localization
2nd image : Nuclei counterstained green by SYTOX dye (green).
3rd image : Merge

Vegetal view with anterior to the left of a Halocynthia roretzi 4-cell stage embryo probed for Not (brown) and PEM (red) expressions, injected with morpholinos against PEM.
Not expression is observed in all the cells.
Original annotation:Ectopic zygotic transcription in PEM knockdown embryos is marked with yellow arrowheads.

Vegetal view with anterior left of a Halocynthia roretzi 4-cell stage embryo probed for Not (brown) and PEM (red) expressions, and injected with PEM MO.
PEM is expressed in the B-line
Original annotation: PEM mRNA is stained red for identification of germline cells.

Vegetal view with anterior to the left of a Halocynthia roretzi 4-cell stage embryo probed for Not (brown) and PEM (red) expressions, and injected with PEM mRNA.
Original annotation: Somatic expression disappears in PEM mRNA injected embryos.

Vegetal view with anterior to the left of a Halocynthia roretzi 4-cell stage embryo probed for Not (brown) and PEM (red) expressions, and injected with PEM mRNA.
No expression can be observed.

Confocal Z-stack fluorescence images showing the localization of PEM protein (magenta) in a Halocynthia roretzi 4-cell stage embryo injected with PEM MO.
Blue arrowheads indicate the absence of nuclear PEM signals in PEM MO-injected embryos.

1st image : PEM localization
2nd image : Nuclei counterstained green by SYTOX dye (green).
3rd image : Merge

Lateral view with anterior to the left of a Halocynthia roretzi 8-cell stage embryo probed for Not (dark blue) and PEM (red) expressions, and injected with morpholinos against PEM.
Not expression is ubiquitously observed.
Original annotation: Not ectopic zygotic transcription in PEM knockdown embryos is marked with yellow arrowheads.

Lateral view with anterior to the left of a Halocynthia roretzi 8-cell stage embryo probed for Not (dark blue) and PEM (red) expressions, and injected with morpholinos against PEM.
PEM is expressed in the B line (germline).

Lateral view with anterior to the left of a Halocynthia roretzi 8-cell stage embryo probed for Not (dark blue) and PEM (red) expressions, and injected with PEM mRNA.
Not expression is lost.
Original annotation: Somatic expression disappears in PEM mRNA injected embryos.

Lateral view with anterior to the left of a Halocynthia roretzi 8-cell stage embryo probed for Not (dark blue) and PEM (red) expressions, and injected with PEM mRNA.
PEM expression is lost.

Confocal Z-stack fluorescence images showing the localization of PEM protein (magenta) in a Halocynthia roretzi 8-cell stage embryo injected with PEM MO. Lateral view, anterior to the left.
Blue arrowheads indicate the absence of nuclear PEM signals in PEM MO-injected embryos.

1st image : PEM localization
2nd image : Nuclei counterstained green by SYTOX dye (green).
3rd image : Merge

Lateral view with anterior to the left (C) and posterior view with animal pole up (D) of a Halocynthia roretzi 8-cell stage embryo probed for PEM expression and injected with a Hr-macho-1 and Hr-β-catenin MOs.
PEM is expressed in the B4.1 blastomeres.
Original comment: In the β-catenin and macho-1 double-knockdown embryos, PEM RNA was detected in spots at the posterior ends of the B4.1 blastomeres, it was always shifted more vegetally with respect to the third cleavage plane.

Posterior view with animal pole up of a Halocynthia roretzi 8-cell stage embryo probed for PEM expression and injected with morpholino oligos against Hr-β-catenin.
PEM is expressed in the B4.1 blastomeres.
Original comment: Reduction in size of the area of RNA localization was also evident in single-knockdown embryos that had been treated with β-catenin MO.

Immunostaining of PEM protein in a Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin MO. Posterior view with animal pole up (B) and lateral view with anterior and animal pole to the left and top, respectively (F).
A signal is observed in the B4.1 at the CAB (centrosome-attracting body, white arrowheads).
Original comment: Injection of β-catenin MO alone did not affect normal PEM localization.

Immunostaining of PEM protein in a Halocynthia roretzi 8-cell stage embryo, injected with Hr-macho-1 MO. Posterior view with animal pole up (C) and lateral view with anterior and animal pole to the left and top, respectively (G).
Original comment: Injection of macho-1 MO alone resulted in elimination of the PEM signal from the CAB [100%, n= 10].

Immunostaining of PEM protein in a Halocynthia roretzi 8-cell stage embryo, injected with Hr-macho-1 MO. Posterior view with animal pole up (J) and lateral view with anterior and animal pole to the left and top, respectively (M).
A weak signal is observed in the B4.1 at the CAB (centrosome-attracting body, white arrowheads).
Original comment: Injection of macho-1 MO alone resulted in elimination of the PEM signal from the CAB [100%, n= 10].
This embryo was extracted, ie. treated with a detergent so that the sustained CAB structure is more clearly visible.

Immunostaining of PEM protein in a Halocynthia roretzi 8-cell stage embryo, injected with Hr-macho-1 MO. Posterior view with animal pole up (C) and lateral view with anterior and animal pole to the left and top, respectively (G).
Original comment: Injection of macho-1 MO alone resulted in elimination of the PEM signal from the CAB [100%, n= 10].

Immunostaining of PEM protein in a Halocynthia roretzi 8-cell stage embryo, injected with Hr-macho-1 MO. Posterior view with animal pole up (J) and lateral view with anterior and animal pole to the left and top, respectively (M).
A weak signal is observed in the B4.1 at the CAB (centrosome-attracting body, white arrowheads).
Original comment: Injection of macho-1 MO alone resulted in elimination of the PEM signal from the CAB [100%, n= 10].
This embryo was extracted, ie. treated with a detergent so that the sustained CAB structure is more clearly visible.

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs. Posterior view with animal pole up (D) and lateral view with anterior and animal pole to the left and top, respectively (H).
Original comment: When macho-1 and β-catenin were knocked down simultaneously, PEM protein was not detected at the CAB in any of the cases examined.

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs. Lateral view with anterior and animal pole to the left and top, respectively.
Original comment: When macho-1 and β-catenin were knocked down simultaneously, PEM protein was not detected at the CAB in any of the cases examined.

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs. Posterior view with animal pole up (K) and lateral view with anterior and animal pole to the left and top, respectively (N).
A very weak signal is observed in the B4.1 blastomeres at the CAB (cellular cortical structure known as centrosome-attracting body).

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs. Posterior view with animal pole up (D) and lateral view with anterior and animal pole to the left and top, respectively (H).
Original comment: When macho-1 and β-catenin were knocked down simultaneously, PEM protein was not detected at the CAB in any of the cases examined.

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs. Lateral view with anterior and animal pole to the left and top, respectively.
Original comment: When macho-1 and β-catenin were knocked down simultaneously, PEM protein was not detected at the CAB in any of the cases examined.

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs. Posterior view with animal pole up (K) and lateral view with anterior and animal pole to the left and top, respectively (N).
A very weak signal is observed in the B4.1 blastomeres at the CAB (cellular cortical structure known as centrosome-attracting body).

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs. Posterior view with animal pole up (D) and lateral view with anterior and animal pole to the left and top, respectively (H).
Original comment: When macho-1 and β-catenin were knocked down simultaneously, PEM protein was not detected at the CAB in any of the cases examined.

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs. Lateral view with anterior and animal pole to the left and top, respectively.
Original comment: When macho-1 and β-catenin were knocked down simultaneously, PEM protein was not detected at the CAB in any of the cases examined.

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs. Posterior view with animal pole up (K) and lateral view with anterior and animal pole to the left and top, respectively (N).
A very weak signal is observed in the B4.1 blastomeres at the CAB (cellular cortical structure known as centrosome-attracting body).

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs, and PEM mRNA. Posterior view with animal pole up (D) and lateral view with anterior and animal pole to the left and top, respectively (H).
Signal at the CAB of B4.1 blastomeres is lost.
Original comment: In addition to the elimination of the abrogation of unequal cleavages at the division to the 16-cell stage, the B4.1 blastomeres also protruded posteriorly at the 8-cell stage when PEM RNA was co-injected.

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs, and PEM mRNA. Posterior view with animal pole up (D) and lateral view with anterior and animal pole to the left and top, respectively (H).
A very weak signal is observed in the B5.2 blastomeres at the CAB (cellular cortical structure known as centrosome-attracting body, white arrowheads).
This embryo was extracted, ie. treated with a detergent so that the sustained CAB structure is more clearly visible.

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs, and PEM mRNA. Posterior view with animal pole up (D) and lateral view with anterior and animal pole to the left and top, respectively (H).
Signal at the CAB of B4.1 blastomeres is lost.
Original comment: In addition to the elimination of the abrogation of unequal cleavages at the division to the 16-cell stage, the B4.1 blastomeres also protruded posteriorly at the 8-cell stage when PEM RNA was co-injected.

Immunostaining of PEM protein in a WT Halocynthia roretzi 8-cell stage embryo, injected with Hr-β-catenin and Hr-macho-1 MOs, and PEM mRNA. Posterior view with animal pole up (D) and lateral view with anterior and animal pole to the left and top, respectively (H).
A very weak signal is observed in the B5.2 blastomeres at the CAB (cellular cortical structure known as centrosome-attracting body, white arrowheads).
This embryo was extracted, ie. treated with a detergent so that the sustained CAB structure is more clearly visible.

Vegetal view with anterior to the top of a Halocynthia roretzi 16-cell stage embryo probed for Not (dark blue) and PEM (red) expressions, and injected with PEM MO.
Not is ubiquitously expressed.
Original annotation: Not ectopic zygotic transcription in PEM knockdown embryos is marked with yellow arrowheads.

Vegetal view with anterior to the top of a Halocynthia roretzi 16-cell stage embryo probed for Not (dark blue) and PEM (red) expressions, and injected with PEM MO.
PEM is expressed in B5.2 cell pair (germline cells).

Vegetal view with anterior to the top of a Halocynthia roretzi 16-cell stage embryo probed for Not (dark blue) and PEM (red) expressions, and injected with PEM mRNA.
Not somatic expression is lost.

Vegetal view with anterior to the top of a Halocynthia roretzi 16-cell stage embryo probed for Not (dark blue) and PEM (red) expressions, and injected with PEM mRNA.
PEM expression is lost.

Confocal Z-stack fluorescence images showing the localization of PEM protein (magenta) in a Halocynthia roretzi 16-cell stage embryo injected with PEM MO. Vegetal view, anterior to the top.
Blue arrowheads indicate the absence of nuclear PEM signals in PEM MO-injected embryos.

1st image : PEM localization
2nd image : Nuclei counterstained green by SYTOX dye (green).
3rd image : Merge

Vegetal view with anterior up of the B5.2 blastomeres of a Halocynthia roretzi 16-cell stage embryo probed for PEM protein localization and injected with 100pg of PEM MO.
PEM proteine is still localized in the B5.2 blastomere pair.
Original annotation: Arrowheads represent signals detected at the CAB (a posterior-most cortical structure known as the centrosome-attracting body . A slight amount of PEM protein remained (100% (n= 21)) when a decreased amount of PEM MO was injected.

Vegetal view of a Halocynthia roretzi 16-cell stage embryo stained with anti-Pem antibody and injected with morpholino oligos against Popk-1.
Signal is observed in B5.2 blastomere pair, white arrows indicate the faint nuclear signals, and white arrowheads the CAB signals.
Original comment: Popk-1 knockdown resulted in a reduction of the Pem protein level in the germline nuclei.
The digits indicate proportions of embryos with nuclear Pem signals.
Smaller panels in the top left corners show entire views of the embryo. G’ is the DIC image of G fluorescent image. G and G’ are on the same focal planes, indicating that faint signals in the nuclei in G are not due to being out of focus for the nuclear level.

Vegetal view of a WT Halocynthia roretzi 64-cell stage embryo stained with anti-Pem antibody and injected with morpholinos against Zf-1.
Signal is observed in B7.6 blastomers.
Original comment: Pem protein signal was observed at a higher level (than WT) in the nuclei (arrows) in the Zf-1 knockdown embryos.
The digits show the proportion of embryos with high expression level of nuclear Pem signals.
In K', the nuclei are showed using SYTOX green.
These panels are single focal planes, where the Pem protein signals were strongest in the Z-series through the nuclei. K and K’ are on the same focal planes. Smaller panels in the top left corners show entire embryos.