Figure 4

Figure 4

Ectopic Expression of Different Amounts of smad2-CA and smad5-CA mRNA Generates Distinct Embryonic Structures

(A) Ectopic structures were generated by injecting three adjacent blastomeres in 64- to 128-cell-stage embryos.

(B) The floorplate marker shha is expressed throughout the axis (gray arrowhead) of wild-type embryos 24 h post-fertilization (hpf). Injection of smad2-CA mRNA into animal pole blastomeres results in the formation of an ectopic axial structure (black arrowhead) that expresses shha, similar to the results with a squint-mVenus-expressing clone.

(C) krox20 is expressed as a pair in rhombomeres 3 and 5 in the hindbrain (gray arrowhead) of wild-type embryos at 24 hpf. Injection of smad2-CA and smad5-CA mRNA in animal pole blastomeres results in the formation of anterior trunk structures with paired krox20 expression (black arrowhead), similar to the outcome with a squint-mVenus and bmp2b/7-sfGFP-expressing double clone.

(D) hoxc13b is expressed in the tail tip (gray arrowhead) in wild-type embryos at 24 hpf. Injection of smad2-CA and four times more smad5-CA mRNA into animal pole blastomeres results in the formation of a tail structure expressing hoxc13b (black arrowhead), similar to the outcome with a double clone expressing low squint-mVenus and high bmp2b/7-sfGFP (Figure S5).

(E) shha is not expressed in embryos exposed to the Nodal receptor inhibitor SB-505124. Injection of smad2-CA mRNA into animal pole blastomeres results in ectopic shha-positive axial structures despite Nodal receptor inhibition.

(F) krox20 remains expressed (gray arrowheads) in embryos exposed to SB-505124. Injection of smad2-CA and smad5-CA mRNA into animal pole blastomeres stage results in the formation of anterior trunk structures with paired krox20 expression (black arrowheads). Nodal and BMP receptor inhibition by combined exposure to SB-505124 and Dorsomorphin generates embryos with reduced tails compared to the treatment with SB-505124 alone, but krox20 expression persists (gray arrowhead). Injection of smad2-CA and smad5-CA mRNA into animal pole blastomeres results in the formation of anterior trunk structures with paired krox20 expression (black arrowhead) despite Nodal and BMP receptor inhibition. Scale bar in all images, 150 μm.