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).

Frontal view (dorsal to the top) of a WT Halocynthia roretzi mid neurula stage embryo probed for Pitx expression.
Original comment: Hr-Pitx was expressed in eight cells of the anterior trunk epidermis, which were aligned in an arc shape.

Frontal view (dorsal to the top) of a WT Halocynthia roretzi late neurula stage embryo probed for Pitx expression.
Original comment: In the late neurula stage, the number of anterior trunk epidermis cells expressing Hr-Pitx increased from 8 to 16.

Frontal (c, dorsal to the top) and dorsal (e,anterior to the left) views of a WT Halocynthia roretzi initial-to-early tailbud stage embryo probed for Pitx expression.
Original comment: In the early tailbud stage, Hr-Pitx expression in the anterior trunk was downregulated except for three spots where papillae would form in the larva. Hr-Pitx was expressed on the left-side of the epidermis.

Frontal (d, dorsal to the top) and dorsal (f,anterior to the left) views of a WT Halocynthia roretzi early tailbud stage embryo probed for Pitx expression.
Original comment: Expression of Hr-Pitx in the papilla-forming region was maintained up to the mid tailbud stage. Hr-Pitx was expressed on the left-side of the epidermis.
Scale bar: 100 μm.

Anterior (c) and dorsal (d) views of a WT Halocynthia roretzi mid tailbud stage embryo probed for Pitx expression.
Hr-Pitx was detected in the left epidermis and in the papilla-forming region.

Lateral view of a WT mid-to-late tailbud embryo probed for Pitx expressionstages.
Original comment: Open arrowhead indicates the expression of Hr-Pitx in photoreceptor cell lineages.
Expression of Hr-Pitx in the papilla-forming region disappeared by the mid-to-late tailbud stage, while it is still expressed on the left-side of the epidermis.

Dorsal (h) and lateral (a) view of a WT Halocynthia roretzi swimming larva probed for Pitx expression.
Original comment: Pitx expression is detected in photoreceptor cells (white arrowhead), which are located on the right-ventral side of the ocellus pigment cell. It is also expressed in the stomodeum region (arrow) and on the left side of the anterior sensory vesicle of the larvae (black arrowhead).
Scale bar indicate 100 µm.

Lateral view (anterior to the right) of a WT Halocynthia roretzi swimming larva probed for Pitx expression.
Original comment: Pitx expression is detected in photoreceptor cells (white arrowhead), which are located on the right-ventral side of the ocellus pigment cell. It is also expressed in the stomodeum region (arrow).

Lateral view (anterior to the right) a WT Halocynthia roretzi swimming larva probed for Pitx expression.
Original comment: Pitx expression is detected in the stomodeum region (arrow) and on the left side of the anterior sensory vesicle of the larvae (black arrowhead).