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Fig. 1
Microbes are necessary for timely neural gene expression. A WMISH of target genes in conventionally raised (CV) and germ-free (GF) embryos at 2dpf. RNA expression of target genes notch1b (N = 6) ngn1 (N = 3), ascl1a (N = 2), fgf8 (N = 6), and phox2bb (N = 1) is reduced in the absence of microbes at 2dpf. Expression of isl1 (N = 3) shows no appreciable difference between groups. B WMISH of target genes in conventionally raised and germ-free embryos at 4dpf. RNA expression of target genes notch1b (N = 4), ngn1 (N = 2), and ascl1a (N = 1) shows an increase in expression in the GF group compared to their CV counterparts at 4dpf. Expression of fgf8 (N = 2) and phox2bb (N = 2) remains reduced in comparison to the CV group C, D Whole homogenized single CV (left) or GF (right) embryos plated on brain heart infusion media and left at C 28.5°C or D 37°C for 24 h. E RT-qPCR analysis of universal 16S rRNA gene in CV and GF embryos (* = p < 0.05 in a one-way ANOVA, based on delta, delta Ct), normalized to ef1α (error bars represent SEM). RNA was extracted from a pool of five embryos for each group and experiment was conducted in triplicate. F Live images of 2dpf zebrafish embryos for morphological comparison. N values represent the number independent biological replicates each of which contained approximately 8–10 embryos. Scale bars represent 100um