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).

WT Halocynthia roretzi fertilized egg probed for Fgf9/16/20 expression.
Fgf9/16/20 is not yet expressed.

WT Halocynthia roretzi 4-cell stage embryo probed for Fgf9/16/20 expression.
Fgf9/16/20 is not yet expressed.

Lateral view, anterior to the left and animal to the top (D) and anterior view with animal pole up (E) of a WT Halocynthia roretzi 8-cell stage embryo probed for Fgf9/16/20 expression.
Fgf9/16/20 expression starts at the 8-cell stage in the anterior vegetal blastomeres, A4.1 (arrows).
Diagram: 8-cell-stage embryo illustrating in orange the expression of Hr-FGF9/16/20.

Vegetal view of a WT Halocynthia roretzi 16-cell stage embryo probed for Fgf9/16/20 expression.
Fgf9/16/20 is expressed in A5.1, A5.2 and B5.1 blastomeres (arrows).
Diagram: 16-cell-stage embryo illustrating in orange the expression of Hr-FGF9/16/20.

Vegetal views with anterior up of WT Halocynthia roretzi early 32-cell stage embryos probed for Fgf9/16/20 expression.
Fgf9/16/20 is expressed in A6.1, A6.2, A6.3, A6.4 and B6.1 blastomere pairs.

Vegetal view of a Halocynthia roretzi 32-cell stages embryo probed for Fgf9/16/20 expression and injected with a control MO.
Fgf9/16/20 is expressed in A6.2, A6.1, A6.3, A6.4 and B6.1 blastomer pairs.

Vegetal view of a WT Halocynthia roretzi 32-cell stage embryo probed for Fgf9/16/20 expression.
Fgf9/16/20 is expressed in A6.1, A6.2, A6.3, A6.4 and B6.1 blastomer pairs.

Vegetal view of a WT Halocynthia roretzi 32-cell stage embryo probed for Fgf9/16/20 expression.
Fgf9/16/20 is expressed in A6.1, A6.2, A6.4, A6.3 and B6.1 blastomers.
Diagram: 32-cell-stage embryo illustrating in orange the expression of Hr-FGF9/16/20.

Vegetal view of a 64-cell stage Halocynthia roretzi embryo probed for Fgf9/16/20 expression.
The embryo showed Harore-Fgf9/16/20 expression in A7.4, A7.8, A7.3, A7.6, A7.7, B7.3, B7.4, B7.5 and B7.8 cells of the vegetal hemisphere.
Scale bar: 100 µm

Vegetal view of a WT Halocynthia roretzi 64-cell stage embryo probed for Fgf9/16/20 expression.
Fgf9/16/20 is expressed in A7.3, A7.4, A7.7, A7.8 and B7.4 blastomeres (arrows).
Original comment: At the 64-cell stage, the expression in the endoderm lineage disappears, while it persists in the presumptive nerve cord and notochord cells in the A-line (the A7.3, A7.4, A7.7 and A7.8 blastomeres) and becomes evident in part of the muscle precursors, the B7.4 blastomeres.
Diagram: 64-cell-stage embryo illustrating in orange the expression of Hr-FGF9/16/20.

Vegetal view of a 110-cell stage Halocynthia roretzi embryo probed for Fgf9/16/20 expression.
The embryo showed Harore-Fgf9/16/20 expression in the nerve cord precursors (A8.7, A8.8, A8.15, A8.16) and in B7.5, B8.7, B8.8 and B8.15 vegetal blastomers.
Scale bar: 100 µm

Vegetal view of a WT Halocynthia roretzi 110-cell stage embryo probed for Fgf9/16/20 expression.
Fgf9/16/20 is expressed in A8.7, A8.8, A8.15 and A8.16 blastomeres (arrows).
Original comment:By the 110-cell stage, the expression is observed only in the nerve cord lineage.
Diagram: 110-cell-stage embryo illustrating in orange the expression of Hr-FGF9/16/20.

Lateral view, anterior to the left and dorsal at the top (J) and dorsal view with anterior to the left (K) of a WT Halocynthia roretzi neurula stage embryo probed for Fgf9/16/20 expression.
Original comment:At the neurula stage, Hr-FGF9/16/20 is strongly expressed rather broadly in the dorsal region lateral to the closing neuropore (arrowheads).

WT Halocynthia roretzi mid tailbud stage embryos probed for Fgf9/16/20 expression.
Original comment: By the mid-tailbud stage Fgf9/16/20 becomes restricted to dotted cells that run bilaterally along the midline in the head region (arrows) and appear to be peripheral neuron precursors.
The embryo at the front is viewed from the cranial region with the dorsal side up and the one behind is viewed laterally with anterior to the left and dorsal to the top.