Figure 6. Limited convergence and ingression of the NMP territory in the epiblast.

(A) Representative snapshots from a time-lapse movie of a stage 5HH chicken embryo in which the epiblast was labeled with nuclear red (Video 4). Tracks of single-labeled nuclei are shown at three different PS levels at three different timepoints (0, 1, and 2 hr) to illustrate differences in longevity of the cells (n = 3 embryos) (NMP: gold; PMP/LPP: green; EMP: blue). (B) Quantification of tracks longevity measured as the ratio of tracks number after 1 and 2 hr divided by the number of tracks at t₀ in each of the colored regions shown in Figure 3A. t₀ marks the start of time-lapse movies of stage 5HH chicken embryos labeled with nuclear red. Gold, green, and blue show tracks in the NMP, PMP/LPP, and EMP domains, respectively, corresponding to the tracks shown in Figure 5A (n = 3; n = 1502 tracks). Two-way ANOVA NMP-PMP/LPP; NMP-EMP. *p<0.05. (C) Representative color-coded time projection showing tracks of epiblast cells at the anterior and posterior PS level after nuclear red labeling in a stage 5HH chicken embryo. The tracks color code represents early timepoints in cyan and later timepoints in yellow (n = 3 embryos). (D) Representative quantification of the angle with the midline of tracks shown in (C). Top: anterior PS region; bottom: posterior PS region (n = 3 embryos). (E) Representative mean lateral to medial speed (V_LM) and anterior to posterior speed (V_AP) over time of epiblast cells labeled with nuclear red in stage 5HH chicken embryos. Y axis represents AP position along the embryo. Color code indicates time of measurement since beginning of the movie (n = 3 embryos). (F) Diagram showing the main direction of epiblast cell movements as a function of their AP position in the epiblast. The length of arrows is proportional to convergence speed. (G) Representative snapshots from a confocal movie of the PS region of a chicken embryo labeled dorsally at stage 5HH with nuclear red and imaged from the ventral side to show epiblast cells ingression (n = 3 embryos). (H) Representative intensity measurement of the nuclear red signal from the ventral side along the PS of the movie shown in (G). Y axis, distance to Hensen’s node (n = 3 embryos). (I) Representative whole-mount immunohistochemistry with anti-laminin (white) in a stage 5HH chicken embryo. Ventral view (n = 3 embryos). Dorsal views, anterior to the top. EMP: extraembryonic progenitors; NMP: neuromesodermal progenitors; PMP: presomitic mesoderm progenitor; LPP: lateral plate progenitor; PS: primitive streak. Arrowhead shows Hensen’s node position. Scale bar: 100 µm.

Figure 6—figure supplement 1. Quantification of longevity, cell speed, and trajectories in time-lapse movies of nuclear red-labeled chicken embryos.

(A) Maximum time color-coded projection showing the tracks of cells labeled with nuclear red in a stage 5HH chicken embryo analyzed at the 6-somite stage. Early to later timepoints are indicated by cyan to magenta to yellow color code. (B, C) Mean medial to lateral speed (V_ML, C) and anterior to posterior speed (V_AP, D) over time of epiblast cells labeled with nuclear red in stage 5HH chicken embryos imaged by confocal imaging in vitro at the 5-somite stage. Y axis represents AP position along the embryo. The color code indicates the time of analysis from purple to yellow where blue is t = 0 min (stage 7HH) and yellow is t = 184 min. (D, E) Color-coded time projection of the epiblast cells labeled in ovo with nuclear red in stage 5HH chicken embryos, imaged by confocal imaging in vitro (D) showing different track angles with the midline quantified in (E). Early timepoints are in cyan, later timepoints are in yellow. Dorsal views, anterior to the top. EMP: extraembryonic progenitors; NP: neural plate; NMP: neuromesodermal progenitors; PMP: presomitic mesoderm progenitor; LPP: lateral plate progenitor; PS: primitive streak (n = 3 embryos). Scale bar: 100 µm.