Input-output relationship of Fgf8a-mediated patterning during gastrulation. (A) smFISH against tbxta and etv4 in early-gastrula stage embryos, after threshold adjustment for background subtraction. (B) smFISH against etv4 in DMSO- (left) and SU5402- (right) treated embryos at early gastrula stage. In both A and B, optical sections of lateral views are shown. Scale bars: 20 μm. (C) Maximum-intensity z-projected images for smFISH against tbxta (top) and etv4 (bottom) at early gastrula (left), and plot of normalized transcript number versus distance from the margin (right). N=13 embryos for tbxta, N=7 for etv4. For A-C, DAPI (blue) was used as a nuclear marker. Overlay with the Fgf8a distribution profile (orange curve) determines its relative extracellular levels corresponding to maximal expression domains of both the transcripts (see blue lines). Peak of Fgf8a-EGFP profile corresponds to an absolute concentration of ∼8 nM. Scale bars: 50 μm. (D) Venus fluorescence in DMSO- (left) and SU5402- (right) treated Tg(etv5b:etv5b-Venus) embryos at early gastrulation. Optical sections of laterally mounted embryos are shown. Scale bars: 50 μm. (E) Sum-intensity z-projected image of Etv5b-Venus from early gastrula-staged transgenic embryos (left) and analysis of nuclear fluorescence intensity as a function of distance from the margin (right). Overlay with the Fgf8a gradient curve (orange curve) shows its minimum relative abundance in the extracellular spaces that correspond to the peak Etv5b output (see blue lines). N=11 embryos. Scale bars: 50 μm. For C and E, exact orientations are shown schematically in insets. Data are mean±s.d.
|
