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Fig. 8 Specific regulation of Wnt signaling mimics the GF and ZM conditions. Representative images of whole mount in situ hybridization on 2 dpf conventional embryos, germ-free embryos, germ-free embryos treated with known Wnt activator BIO, and conventional embryos treated with known Wnt inhibitor XAV939 for genes ascl1a (A–D) and axin2 (E–H). All embryos were treated in parallel and stained for the same period of time. Black bar identifies the hindbrain region where there is less staining in both the GF and conventional treated with Wnt inhibitor than the CV or GF treated with Wnt activator. Hollow white arrows represent the posterior recess of the hypothalamus [41, 42]. Experiment was conducted using 8–10 embryos per group. Scale bars represent 100um