sox2 and sox3 cooperate to regulate otic/epibranchial placode induction in zebrafish.

Expression of sox3 is one of the earliest markers of Fgf-dependent otic/epibranchial placode induction. We report here that sox2 is also expressed in the early otic/epibranchial placode in zebrafish. To address functions of sox2 and sox3, we generated knockouts and heat shock-inducible transgenes. Mutant analysis, and low-level misexpression, showed that sox2 and sox3 act redundantly to establish a full complement of otic/epibranchial cells. Disruption of pax8, another early regulator, caused similar placodal deficiencies to sox3 mutants or pax8-sox3 double mutants, suggesting that sox3 and pax8 operate in the same pathway. High-level misexpression of sox2 or sox3 during early stages cell-autonomously blocked placode induction, whereas misexpression several hours later could not reverse placodal differentiation. In an assay for ectopic placode-induction, we previously showed that misexpression of fgf8 induces a high level of ectopic sox3, but not pax8. Partial knockdown of sox3 significantly enhanced ectopic induction of pax8, whereas full knockdown of sox3 inhibited this process. Together these findings show that sox2 and sox3 are together required for proper otic induction, but the level of expression must be tightly regulated to avoid suppression of differentiation and maintenance of pluripotency.