Formation of embryonic axis is an essential step for animal development but its mechanism is not well understood. For axial determination in the fish embryos, participation of the yolk syncytial layer (YSL; a unique multinucleated structure formed in the yolk cell) has been shown. To investigate relationship between the YSL and axial specification, we examined whether or not expressions of the mesodermal marker genes no tail (Brachyury) or goosecoid are dependent on the YSL in explants isolated from the medaka embryos. The results of whole-mount in situ hybridization showed that these genes were expressed in the explants irrespective of the YSL, indicating that activation of these genes is a separate process from YSL-dependent axis formation.