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Fig. 5. Induction of mesoderm is disrupted by XPACE4 depletion. (A) A schematic presentation of the Nieuwkoop Assay details the Materials and methods. The marker expression of animal cap explants (ac) co-cultured with control (Un vm) and XPACE4-depleted vegetal masses [P(-) vm] is analyzed by real-time RT-PCR. Animal caps incubated alone (ac alone) do not express significant levels of mes-endodermal genes. The induction of organizer genes chordin, goosecoid, mesodermal genes Fgf8 and Xbra, and endodermal gene XSox17{alpha} are all reduced in caps cultured with XPACE4-depleted vegetal masses [ac P(-) vm] compared with controls (ac Un vm). Uninjected whole embryo (Uninj WE) is used for the dilution series and the quantification. (B) XPACE4-depleted embryos [P(-)+ARE] have reduced ARE-luciferase reporter activity compared with controls (Un+ARE). (C) Western blot analysis of control (UN) and XPACE4-depleted [P(-)] gastrulae show phospho-Smad2 levels are reduced by XPACE4 depletion. Total Smad2 levels are shown as loading control.