Fig. 5

Figure 5. Prechordal plate migration triggers interstitial fluid relocalization by pushing against the overlying epiblast

(A) Schematic of deep cell (DC) radial velocity (V_r) analysis relative to the prechordal plate (ppl) position; ppl leading edge (LE); axial mesendoderm trailing edge (AM TE); animal-vegetal (AV) axis.

(B–E) Representative cross-sections (right panel, close-up on the DC region marked by the red box in the left panel) in gsc::caax-EGFP wild-type (WT) (B and C) and MZoep (D and E) embryos at 7 (B and D) and 7.5 hpf (C and E); dashed white line outlines the yolk syncytial layer (YSL)-DC boundary; dashed red line outlines the deformation of the DC tissue relative to the ppl position; scale bars, 100 μm.

(F and G) Representative heatmaps of DC radial displacement along the AV axis as a function of developmental time in gsc::caax-EGFP WT (F) and MZoep (G) embryos; dotted black lines mark the position of the ppl LE (top) and the position of AM TE (bottom); dashed magenta line indicates the position of the interstitial fluid (IF) ahead of the ppl LE; in (G), the position of ppl LE was superimposed from an average value of ppl position in WT embryos; DC radial velocities are color-coded.

(H) Average DC radial velocities relative to the ppl LE position in gsc::caax-EGFP WT (n = 4) and MZoep (n = 3) embryos; dashed line at ∼50 μm indicates the maximum position along the AV axis at which ppl-mediated radial DC movements are still detectable of the ppl LE; mean ± SEM.

(I) Schematic of IF distribution visualized by a uniformly distributed control dye (top panel) and local ultraviolet (UV) laser photoactivation/uncaging of a caged dye within the IF at the enveloping layer (EVL)-DC boundary, ahead and above of the internalizing ppl (yellow square), and detection of the uncaged dye at the YSL-DC boundary (pink dashed square), ahead of the advancing ppl (bottom panel).

(J–K′) Representative cross-sections of gsc::caax-EGFP WT (J and J′) and MZoep (K and K′) embryos before (50% epiboly; J and K) and after UV laser activation (0′ and 40′, J′ and K′); panels on the right in (J′) and (K′) are close-up views of the indicated regions in the left panels (40′) with dashed pink lines outlining the growing IF accumulation at the YSL-DC boundary, where the accumulation of uncaged-FITC was quantified; top row, control dye (Alexa647) uniformly marking the IF; bottom row, uncaged-FITC distribution; yellow squares mark the UV laser activation site; dashed white lines outline the YSL-DC boundary; scale bars, 100 μm.

(L–M′) IF accumulation area at the YSL-DC boundary ahead of the ppl (L), and corresponding accumulation of uncaged-FITC within that region (M and M′) in gsc::caax-EGFP WT (green area; green line; n = 4) and MZoep (purple area; purple line; n = 4) embryos as a function of time after the onset of internalization (6 hpf) at 0′; values represent the mean. n, number of independent embryo replicates. See also Figure S5 and Videos S4 and S5.