Oocytes of a single individual of Ciona intestinalis (Roscoff, France) were fertilized by a mixture of sperm of two individuals and development was followed microscopically. Embryos were snapfrozen in liquid nitrogen at the appropriate stage and kept at -80°C. Samples at stage 0, 8, 12, 15, 21, 26 were collected from the same fertilzation, while sample from stage 11 was independently collected from a different individual. Total RNA was prepared with the RNA II kit of Magerey Nagel and the quality was checked with the Bioanalyzer, all 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). Reads were aligned onto C.robusta genome and considered to reflect the expression of the C.robusta orthologous genes.

Lateral view of an unfertilized egg. Distribution of maternal and zygotic mRNAs of Ci-macho1, as revealed by whole-mount in situ hybridization.

Original annotation: in unfertilized eggs, the mRNA was detected in the peripheral cytoplasm, except for the animal pole region.

Conversion to Aniseed format of data from Yutaka Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :macho-1.
Expression pattern : Pem-like localized signal was detected.
Comment : The expression in the posterior end/endodermal strand is maternal.

Lateral view of a fertilized egg. Distribution of maternal and zygotic mRNAs of Ci-macho1, as revealed by whole-mount in situ hybridization. The animal pole is up and the vegetal pole is down.

Original annotation: after the first phase of ooplasmic segregation, transcripts of Ci-macho1 became concentrated near the vegetal pole (arrow, maternal signal).

Lateral view of an eight-cell stage embryo. Distribution of maternal and zygotic mRNAs of Ci-macho1, as revealed by whole-mount in situ hybridization. The animal pole is up and anterior is left.

Original annotation: in the 8-cell stage embryo, the distribution of the transcripts of Ci-macho1 is restricted to the posterior region of B4.1 cells (arrow, maternal signal).

"Zic-r.a was observed in all nuclei of the PVD and in the most posterior region where Zic-r.a mRNA is localized."

Conversion to Aniseed format of data from Yutaka Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :macho-1.
Expression pattern : No distinct zygotic signal was detected.
Comment : The expression in the posterior end/a pair of cells in the endodermal strand is maternal.

Conversion to Aniseed format of data from Yutaka Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :macho-1.
Expression pattern : No distinct zygotic signal was detected.
Comment : The expression in the posterior end/a pair of cells in the endodermal strand is maternal.

Conversion to Aniseed format of data from Yutaka Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :macho-1.
Expression pattern : No distinct zygotic signal was detected.
Comment : The expression in the posterior end/a pair of cells in the endodermal strand is maternal.

112-cell stage wild type embryos probed for Ci-macho1 expression.

Original Annotation : Control embryos expressing zygotic Ci-macho1 in germ-line precursors (yellow arrowhead).

Conversion to Aniseed format of data from Yutaka Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :macho-1.
Expression pattern : No distinct zygotic signal was detected.
Comment : The expression in the posterior end/a pair of cells in the endodermal strand is maternal.

Vegatal view of a 110-cell stage embryo. Distribution of maternal and zygotic mRNAs of Ci-macho1, as revealed by whole-mount in situ hybridization. Posterior is down (arrow, maternal signal).

Conversion to Aniseed format of data from Yutaka Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :macho-1.
Expression pattern : No distinct zygotic signal was detected.
Comment : The expression in the posterior end/a pair of cells in the endodermal strand is maternal.

Conversion to Aniseed format of data from Yutaka Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :macho-1.
Expression pattern : nervous system.
Comment : The expression in the posterior end/a pair of cells in the endodermal strand is maternal.

Dorsal and lateral views of a late neurula stage embryo. Distribution of maternal and zygotic mRNAs of Ci-macho1, as revealed by whole-mount in situ hybridization. The anterior is left.

Original annotation: a strong signal was first seen in posterior cells of the nerve cord, and a weak signal in the anterior sensory vesicle (arrow, maternal signal; arrowhead, zygotic signal).

Conversion to Aniseed format of data from Yutaka Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :macho-1.
Expression pattern : nervous system, nerve cord.
Comment : The expression in the posterior end/a pair of cells in the endodermal strand is maternal.

Lateral ans dorsal views of a early tailbud stage embryo. Distribution of maternal and zygotic mRNAs of Ci-macho1, as revealed by whole-mount in situ hybridization. The anterior is left.

Original annotation: after the early tailbud-stage, a signal was found in the anterior sensory vesicle and nerve cord (arrow, maternal signal; arrowhead, zygotic signal).

Lateral view of a wild type early tailbud embryo probed for Macho-1 expression.

Original Annotation :
Macho-1 was expressed in the dorsal nerve cord (b10.75, b10.76, b10.73 and b10.74 cell pairs) and the ventral nerve cord (A10.28, A10.27, A10.26 and A10.30, A10.25 and A10.29 cell pairs).

Aniseed Annotation :
According to the picture, there is also a staining in the endodermal strand and in anterior sensory vesicle.

Mid tailbud embryos probed for Ci-macho1 expression.
First picture is low-magnification microphotographs of embryos. Individual embryos are oriented with anterior to the left, dorsal up in the second picture.

Original Annotation : Control embryos expressing zygotic Ci-macho1 in germ-line precursors (yellow arrowhead), sensory vesicle and nerve cord (blue arrowheads).

Conversion to Aniseed format of data from Yutaka Satous gene expression profiles of transcription factors and signalling molecule ISH screen (Development. 2004 131:4047-58).

Original annotation :
Gene name :macho-1.
Expression pattern : nervous system, nerve cord.
Comment : The expression in the posterior end/a pair of cells in the endodermal strand is maternal.

Immunostaining of (C) Zic-r.a with specific antibodies. Images are Z-projected image stacks overlaid in pseudocolor. (C) Zic-r.a mRNA is expressed widely in the nervous system [8, 15], and Zic-r.a protein was detected in nuclei of cells in the nervous system.
Coding sequences of Gata.a, Tcf7, and Zic-r.a were cloned into pET16b (Novagen), and Histagged proteins were produced in E. coli. After purification with NiNTA resin (Qiagen), recombinant proteins were used for immunizing rabbits and polyclonal antibodies were obtained. To detect protein localization, embryos were fixed with 3.7% formaldehyde in PBS, treated with 3% H2O2 for 30 minutes, and then incubated with the antibodies in Can-Get-Signal- Immunostain Solution B (Toyobo). The signal was visualized with a TSA kit (Invitrogen) using horseradish peroxidase-conjugated goat anti-rabbit IgG and Alexa Fluor 488 tyramide. Control embryos incubated without primary antibodies yielded no signal.