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. 2023 Oct 30;12:RP89141. doi: 10.7554/eLife.89141

Figure 3. Induction of secondary axes by the GSK3/β-catenin pathway requires membrane trafficking.

Figure 3.

(A) Control embryo at early tailbud. (B) Embryo injected with DN-GSK3 (dominant-negative GSK3-β, 150 pg 1x ventral) mRNA, showing double axes (arrowheads). (C) Injection of 5-(N-Ethyl-N-isopropyl) Amiloride (EIPA) 1x ventral alone showed no phenotypic effect (4 nl,1 mM). (D) Co-injection of DN-GSK3 and EIPA blocked double axis formation. (E) Uninjected control embryo. (F) Activation of Wnt signaling via injection of β-catenin mRNA (80 pg) induced complete twinned axes (arrowheads). (G) Embryo co-injected with β-catenin mRNA and DN-Rab7 (500 pg) showing that membrane trafficking is required for secondary axis formation. (H) Co-injection of β-catenin mRNA and the macropinocytosis inhibitor EIPA blocks axial duplication. The numbers of embryos analyzed were as follows: A = 62, 100%; B = 75, 94% with double axes; C = 76, 100%; D = 70, 98%; E = 70, 100%; F = 73, 98%; G = 69, 75%; H = 82, 97%; four independent experiments (scale bars, 500 μm).