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Fig. 4. preMBT Nodal signaling is required for mesendoderm induction. (A) Embryos were treated with control medium (nt) or SB5 (200 μM) from the 4-cell stage to stage 10 and harvested for Western blot for P-Smad2/3 or Smad2/3. (B) Embryos were treated with SB5 from the indicated stages until the onset of gastrulation and cultured until stage 40. (C) Embryos were treated with SB5 beginning at the indicated stages, cultured until stage 10, and analyzed by in situ hybridization (ISH) for mesendodermal gene expression (left). control indicates DMSO (vehicle) only. Embryos were also harvested at early gastrula stage (stage 10) for qRT-PCR for the indicated mesendodermal markers and for the ectodermal marker foxi1e (right panel). All samples were normalized to expression levels in untreated embryos at stage 10 (control). control indicates DMSO; B5indicates treatment from 4-cell until gastrula (stage 10). (D) SB5 was added at the 4-cell stage; embryos were washed at the indicated stages, transferred to control medium, and analyzed by ISH for mesendodermal gene expression at stage 10, as in (C). Histogram on right shows qRT-PCR for mesendodermal and ectodermal markers, as in (C). Note that the control and B5(4-cell until stage 10) qRT-PCR data in panel D are identical to those shown in panel C, as qRT-PCR for both groups was performed at the same time. |
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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. |
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Fig. 6. Smad2 rescues mesendodermal gene expression when activated before but not after the MBT. Embryos were injected with a hormone inducible, activated form of Smad2 (GRtSmad2) at the 2-cell stage (along with FLDx as a lineage tracer) and then treated with SB5 beginning at the 4-cell stage. GR-tSmad2 was activated by addition of dexamethasone (dex) before the MBT (stage 7) or after the MBT (stage 9) and embryos were cultured until the onset of gastrulation and then collected for analysis of mesendodermal gene expression by ISH (A) or qRT-PCR (B), as described in Fig. 4. (In panel A, the insets show FLDx immunostaining to reveal the progeny of injected blastoemeres and the numbers in the upper left indicate the frequency of the displayed phenotype/expression pattern). Error bars represent standard deviation for a representative experiment. |
