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Fig. 1. Identification, characterization and regulation of Xenopus apoc1. (A-C) Bar charts showing the expression levels, displayed as fragments per kilobase of transcript per million fragments mapped (FPKM), for apoc1 (A), the Wnt regulated gene nodal3.2 (B), and the non-Wnt regulated gene odc1 (C) in Xenopus tropicalis embryos that were either uninjected (blue), injected with 105 pg axin1 mRNA (pink) or 24 pg ctnnb1 mRNA (green). (D) Schematic drawing of the Apoc1 secretion experiment. mRNA was injected at the two-cell stage. The blastopore fluid was collected at the gastrula stage and subjected to western blot (WB) analysis to determine whether flag-tagged Apoc1 protein is secreted from cells during embryonic development. (E) Apoc1, but not Ctnnb1, protein was detected by WB in the blastocoel fluid after injection of flag-tagged apoc1 (500 pg) and flag-tagged ctnnb1 (1 ng). The tubulin blot shows that there is no cellular contamination in the blastopore fluid. (F) Wnt regulation of apoc1 in Xenopus laevis embryos. (F) 300 pg of wnt3a or 200 pg of chrd and 300 pg wnt3a were injected into the animal side of two blastomeres at the two-cell stage. Animal cap explants were dissected from stage 9 embryos, cultured until stage 10, and processed for RT-PCR analysis. Expression of apoc1, and the known Wnt target gene nodal3.2, was induced by wnt3a alone, and chrd+wnt3a. Odc1 was used as loading control. (G) Apoc1, gbx2.2 and pax3 are immediate-early target gene of Wnt/Ctnnb1 signaling. 200 pg of GR-ctnnb1 and 100 pg lacZ mRNA were injected into one side of a dorsoanimal blastomere of 8-cell stage embryos and the embryos were treated with cycloheximide 30 min prior to DEX treatment at stage 11.5. The expression of apoc1, gbx2.2, pax3 and snail2 were examined by whole mount in situ hybridization. Orange arrows indicate ectopic localization of the tested genes.
