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Fig. 8. xnr5 and xnr6 can activate Smad2 and regulate their own expression before the MBT (A) 8-cell embryos were injected in one animal cell with fluorescent dextran alone (control) or with xnr1 (50 pg), xnr5 (20 pg), or xnr6 (20 pg) mRNAs. Whole mount ISH for xnr5, xnr6, and chd was performed on embryos fixed at the indicated stages. (B) Embryos from A were analyzed by qRT-PCR at stage 8 (preMBT). (C) 8-cell embryos were injected in one animal cell with GFP-Smad2 (200 pg) alone or with xnr5 mRNA (50 pg). Embryos were fixed at the 512-cell stage, processed for anti-GFP immunostaining, and sectioned for fluorescence imaging. DAPI was used to reveal nuclei. The graph shows the percent of DAPI positive nuclei that also contain GFP-Smad2. (D) Embryos injected as in C were fixed at various stages as indicated and stained for xnr6 expression. ui, uninjected. |
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Fig. 7. Nodal signaling contributes to xnr5/6 expression. (A) SB5 (200 μM) was added at the 4-cell stage and embryos were fixed for ISH at MBT (stage 8.5). Whole-embryos were photographed in vegetal views, dorsal to the top (vegetal view). Embryos bisected along the dorsal/ventral axis prior to ISH are shown with dorsal side to the right (lateral view). The frequency of the expression pattern for each group is indicated in the lower right corner. DMSO was used as a control. (B) To inhibit Nodal signaling, the dominant-negative Nodal receptor (DN-ALK4) was injected (1 ng), along with fluorescent dextran (insets, rust-colored staining), into the 2 right-hand blastomeres of 4-cell embryos. Embryos were fixed at the indicated times and analyzed by ISH. Fluorescent dextran injection alone is shown as control. The number of cells expressing xnr5 or xnr6 in injected clones was counted and significance was determined using the Kruskalallis test. (C) 8-cell embryos were injected in one animal cell with GR-tSmad2 (400 pg) and fluorescent dextran. GR-tSmad2 was activated by addition of dex for a two-hour window beginning at the indicated stages. Embryos were fixed at the end of the window and analyzed by ISH. |
