Opposing expression patterns of six3 and sox4. Whole-mount in situ hybridization (WISH) analysis of the developmental expression pattern of the transcription factors six3 and sox4. (a to e) six3; (f to j) sox4. The animal side is located to the top in all images. Six3 expression starts as early as 8 hours of development (8 hours embryo in panel a and 10 hours in panel b) at the animal side of the embryo. At the mesenchyme blastula stage (20 hours in panel c and flattened embryo in panel d), the animal expression clears from the central apical plate (apical organ) and at the same time forms a ring-like expression around the vegetal pole as well. In the pluteus (panel e) expression is detectable in a part of one coelomic pouch and at the forgut-midgut constriction. In contrast, sox4 is initially expressed on the vegetal side (14 hours embryo in panel f). Starting from 18 hours (panel g, and 20 hours in panel h) of development, expression also starts in the apical plate. At gastrula stage (panel i) expression is detected at the archenteron tip, and in the pluteus (panel j) expression can be detected in various secondary mesoderm cell derivatives, including some coelomic pouch cells.