wnt5b and no tail expression prior to formation of midline bifurcations. (A–X) wnt5b (A–L) and ntl (M-X) expression during gastrulation of standard and heat-treated WT and MZsqt embryos. Embryos were cultured at 28 °C or heated (34 °C) starting at the 64-cell stage and then fixed at different stages for whole-mount in situ analysis. No difference in wnt5b expression was seen between control and heated WT embryos at all tested stages. However, heated MZsqt embryos showed reduced wnt5b expression in the dorsal region at all stages tested. Similarly, no differences in ntl expression was seen between control and heated WT embryos at all stages. However, reduced dorsal expression of ntl is obvious in control MZsqt embryos. Heated MZsqt embryos display further reduced dorsal expression of ntl at the 40% epiboly stage (P) and the 75% epiboly stage (T), and bifurcated ntl expression is often seen at the bud stage (X). Embryo orientations: 40% epiboly, animal pole view, dorsal on the right; 75% epiboly, dorsal view, animal pole on the top; bud stage, vegetal pole view, dorsal on the right for wnt5b embryos and dorsal view, animal pole on the top for ntl embryos. (Y) Up-regulated Wnt/β-catenin signaling in heated MZsqt embryos. WT and MZsqt embryos injected with 23 pg of reporter plasmids were reared at 28 °C or heated (34 °C) starting at the 64-cell stage and lysed at the bud stage for the TCF/LEF reporter assay. Average values and standard deviations from triplicate experiments are shown.
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