(A) foxq2 expression at stages from 32-cell to hatched blastula, as well as fzl5/8 and six3 expression at mesenchyme blastula stage in control embryos (Aa–f), Fzl1/2/7 morpholino-injected embryos (Ah–m), and embryos treated with PKC inhibitor (Ao–t). (Ag, n, u) Neurons in control, Fzl1/2/7 morpholino-injected, and PKC inhibitor-treated 96-hpf pluteus larvae. White boxes outline the ANE. Serotonergic neurons (green), DAPI (nuclei, blue), synaptotagminB/1e11 (pan-neural, magenta). (B) Western blot showing that phosphorylation of PKC is blocked to similar extents with a PKC inhibitor and Fzl1/2/7 MO. (C, D) qPCR measurements from three different cultures of embryos showing that the early ANE regulatory genes are down-regulated in the absence of Fzl1/2/7 signaling at the 60-cell stage (C) and late blastula stages (D). The y-axis shows the fold change in gene expression level in Fzl1/2/7 morpholino-containing embryos relative to controls. The dotted line marks a 3-fold change in expression. nodal expression is used as an internal control.