Fig. 1

Figure 1. Interstitial fluid accumulation requires Na⁺/K⁺-ATPase activity and relocalizes during axial mesendoderm internalization and migration

(A) Schematic of interstitial fluid (IF) distribution during axial mesendoderm (ME) internalization at 6 h post fertilization (hpf); D, dorsal and S, sagittal views; EVL, enveloping layer; DC, deep cell; YSL, yolk syncytial layer. (A′) Schematic illustrating the principle of IF relocalization analysis at the dorsal side; AV, animal-vegetal axis; pink and blue rectangles indicate the regions of IF accumulation analysis at the YSL-DC and EVL-DC boundaries, respectively, during axial ME internalization and migration (black rectangle).

(B, C, and E–J) Maximum intensity projections (D, dorsal view) and single cross-sections (S, sagittal view) of gsc::caax-EGFP embryos expressing EGFP within the axial ME and injected with fluorescent dextran to mark IF in control (B and C), ouabain-treated (E and F), ouabain-treated and mannitol-injected (G and H), and mannitol-injected (I and J) embryos at 6 hpf (B, E, G, and I), and 8 hpf (C, F, H, and J); dashed white lines outline the anterior axial mesendoderm/prechordal plate (ppl) and, due to the leaky nature of this transgenic reporter line, also the posterior axial ME (pam); dashed blue lines outline the IF at the EVL-DC boundary; dashed pink lines outline the IF at the YSL-DC boundary (S, sagittal view); scale bars, 100 μm. (B′, C′, and E′–J′) IF distribution profiles along the AV axis relative to the position of ppl/pam, marked by gsc::caax-EGFP expression (black lines), in control (green, B′ and C′), ouabain-treated (blue, E′ and F′), ouabain-treated and mannitol-injected (yellow, G′ and H′), and mannitol-injected (red, I′ and J′) embryos at 6 hpf (B′, E′, G′, and I′) and 8 hpf (C′, F′, H′, and J′); multi-color curves represent average values of n = 3 independent embryo replicates of the position of ppl/pam (black lines) and IF distribution at the EVL-DC (blue lines) and YSL-DC (pink lines) boundaries; arrowheads (pink) indicate the IF accumulation ahead of the ppl.

(D) Schematic illustrating the experimental design. Control and ouabain-treated (1 mM, 1–3 hpf) embryos were injected with fluorescent dextran (to mark the IF) either alone or in combination with mannitol (400 mM).

See also Figures S1 and S2 and Videos S1 and S2.