Zebrafish pou2, which encodes a class V POU transcription factor considered to be an orthologue of mouse Oct-3/4, has been implicated by mutant analysis in dorsoventral (DV) patterning, gastrulation, and endoderm formation in early embryos and later in the regionalization of the neural plate. A series of gain-of-function experiments were conducted in the present study to directly reveal the roles pou2 plays in embryogenesis. We first revealed that injecting low-dose wild-type pou2 mRNA ventralizes embryos. Similar overexpression of activated (vp-) pou2 resulted in the same effects, whereas repressive (en-) pou2 caused dorsalization, supporting the previously proposed idea that pou2 is involved in DV patterning and that pou2 is a transcriptional activator. In contrast, high-dose mRNA for pou2 and its modified genes affected convergent extension (CE) movement. We observed similar activities for mouse Oct-3/4, suggesting conservation of the roles of this POU family in vertebrate development. To determine the critical stage for the functions of pou2 in embryos, we established a transgenic (Tg) fish line harboring en-pou2 under regulation of a heat-shock promoter (HEP) and found that the exposure of HEP Tg embryos to heat shock at the midblastula (sphere) stage dorsalized embryos, whereas induction of HEP at the late blastula stage (30-50% epiboly) affected CE movement. The defects due to HEP induction were rescued by introducing wild-type pou2 mRNA before the heat treatments. Collectively, these data demonstrated that pou2 regulates DV patterning and CE movement in zebrafish embryos at the midblastula and late blastula stages, respectively.