Fig. 3. Induction of otic placode markers occurs in the absence of neural or mesodermal markers. Presumptive trigeminal ectoderm was cultured in the presence of 50 ng/ml Fgf2 for between 3 and 24 hours, and examined for the expression of neural and mesodermal markers. Tailless marks the anterior CNS, Krox20 marks rhombomeres 3 and 5, Wnt8c marks rhombomere 4 and the caudal neural plate, while Sax1 exclusively marks the caudal neural plate. Sip1 and Zic2 are pan-neural markers, while brachyury marks early mesoderm. The panels show two controls: the normal expression pattern of each gene in vivo and tissue positive for each marker processed for in situ hybridization in collagen gels. These are compared with the expression seen in cultured epiblast +Fgf2 after 24 hours. Epiblast explants cultured in the presence of Fgf2 were completely negative for all markers at all time points examined (Table 3).