Oocytes of a single individual of Halocynthia roretzi (Aomori, Japan) were fertilized by sperms of an another single individual and development was followed microscopically. Embryos were snapfrozen in liquid nitrogen at the appropriate stage and kept at -80°C. Total RNA was prepared with the AGPC method and the quality was checked with the Bioanalyzer, most samples had a RIN value of higher than 9. Strand specific libraries were constructed and the sequencing (50PE) was performed with Illumina HiSeq 2000 technology (BGI, Hong Kong, China).

Confocal Z-stack fluorescence images showing the localization of PEM protein (magenta) in a WT Halocynthia roretzi 1-cell stage embryo.
White arrowheads indicate PEM signals in the nucleus.
Nuclear PEM signals are visible in the germline throughout early embryogenesis.

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

The expression pattern of mRNA encoding CDK9 in Halocynthia egg. CDK9 and PEM mRNA are stained purple and red, respectively. PEM mRNA is shown to indicate the germline cells. Anterior is to the left. Lateral view.

The expression pattern of mRNA encoding PEM in Halocynthia fertilized egg. CDK9 and PEM mRNA are stained purple and red, respectively. PEM mRNA is shown to indicate the germline cells. Anterior is to the left. Lateral view.

Confocal Z-stack fluorescence images showing the localization of PEM protein (magenta) in a WT Halocynthia roretzi 2-cell stage embryo.
White arrowheads indicate PEM signals in the nucleus.
Nuclear PEM signals are visible in the germline throughout early embryogenesis.

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 WT Halocynthia roretzi 4-cell stage embryo.
White arrowheads indicate PEM signals in the nuclei of B3 cell pair.
Nuclear PEM signals are visible in the germline throughout early embryogenesis.

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

Vegetal view, anterior to the left of a 4-cell stage Halocynthia roretzi embryo probed for Not (brown) and PEM (red) expressions
Not is expressed in the A-line
Original annotation:Zygotic transcription is detected in the somatic, but not in the germline lineage. PEM mRNA is stained red for identification of germline cells.

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

The expression pattern of mRNA encoding CDK9 in 4-cell stage Halocynthia embryo. CDK9 and PEM mRNA are stained purple and red, respectively.
Halocynthia CDK9 mRNA was detected in the entire embryo.
PEM mRNA is shown to indicate the germline cells. Anterior is to the left.

The expression pattern of mRNA encoding PEM in 4-cell stage Halocynthia embryo. CDK9 and PEM mRNA are stained purple and red, respectively. PEM mRNA is shown to indicate the germline cells, in the B-line. Anterior is to the left.

Confocal Z-stack fluorescence images showing the localization of PEM protein (magenta) in a WT Halocynthia roretzi 8-cell stage embryo. Lateral view, anterior to the left.
White arrowheads indicate PEM signals in the nuclei of b4.2 and B4.1.
Nuclear PEM signals are visible in the germline throughout early embryogenesis.

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 WT Halocynthia roretzi 8-cell stage embryo probed for Not (dark blue) and PEM (red) expressions.
Not is expressed in a, A and b lines.
Original annotation: Not zygotic transcription is detected in the somatic, but not in the germline lineage.

Lateral view with anterior to the left of a WT Halocynthia roretzi 8-cell stage embryo probed for Not (dark blue) and PEM (red) expressions.
PEM is expressed in the B4.1 cell pair.
Original annotation: PEM mRNA is stained red for identification of germline cells.

The expression pattern of mRNA encoding CDK9 in 8-cell stage Halocynthia embryo. CDK9 and PEM mRNA are stained purple and red, respectively.
Halocynthia CDK9 mRNA was detected in the entire embryo.
PEM mRNA is shown to indicate the germline cells. Anterior is to the left. Lateral view.

The expression pattern of mRNA encoding PEM in 8-cell stage Halocynthia embryo. CDK9 and PEM mRNA are stained purple and red, respectively. PEM mRNA is shown to indicate the B-line (germline cells). Anterior is to the left. Lateral view.

Lateral view with anterior to the left (A) and posterior view with animal pole up (B) of a Halocynthia roretzi 8-cell stage embryo probed for PEM expression and injected with a control MO.
PEM is expressed in the B4.1 blastomeres.
Original comment: c-MO-injected control embryos showed a moustache-shaped area of RNA localization that constituted the CAB (centrosome-attracting body) .
Scale bar: 100 μm.