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. 2021 Jul 6;10:e64819. doi: 10.7554/eLife.64819

Figure 1. Characterization of the SOX2/T-positive territory of the epiblast.

(A–C) Whole-mount embryos and (E, F) transverse cryosections showing the immunolocalization of SOX2 (A), T (B), T and SOX2 (C, E), and MSGN1 (F) in chicken embryos at stage 5HH. (D) Schematic representation of the expression of T (blue: high; green: low), SOX2 (red), and SOX2/T (gold) in a stage 5HH chicken embryo. The level of the tissue sections in (E, F) is shown with dashed double arrows labeled from 1 to 5 from anterior to posterior. (E’, F’) Higher magnification of the NMP region (level 3, E, F). (E’’, F’’) Higher magnification of the PMP region (level 4, E, F). (n = 7 embryos for whole mount; n = 3 for cryosections). PS regions are defined based on distance from Hensen’s node as described in Psychoyos and Stern, 1996. (G) Maximum intensity projections from confocal images of chicken embryos immunostained for T (green) and SOX2 (red) proteins. Double-positive cells are shown in yellow. White hatched line in the 25-somite embryo marks the end of the neural tube (red) and the NMP region (orange) (n = 23 embryos analyzed in total). (H) Diagram summarizing the experimental procedure to label NMP cells in stage 5HH embryos using electroporation of a fluorescent reporter in the epiblast of the anterior PS region (in green) followed by analysis at the tail bud stage. (I, J) Fate of descendants of cells of the NMP region electroporated at stage 5HH with an H2B-RFP plasmid and imaged in time lapse at the 25-somite stage in the tail bud region. Z-projection from confocal images (I) and tracks (J) of a time-lapse movie showing the movements of the cells in the NMP territory for 10 hr (Video 1). Tracks were color-coded a posteriori. Neural, mesodermal, and NMP cell trajectories are shown in red, green, and gold, respectively. NP: neural plate; NMP: neuromesodermal progenitors; PMP: presomitic mesoderm progenitors; LPP: lateral plate progenitors; EMP: extraembryonic mesoderm progenitors; n: node; epi: epiblast; Meso: mesoderm; PS: primitive streak; NT: neural tube. Arrowheads: Hensen’s node. Asterisk: primitive streak. (A–D, G–J) Dorsal views. Anterior to the top. Scale bar: 100 µm.

Figure 1.

Figure 1—figure supplement 1. Onset of SOX2/T expression cells in chicken.

Figure 1—figure supplement 1.

(A–C) Representative maximum z-projections of confocal images of T and SOX2 whole-mount immunohistochemistry from stage 4 to stage 5HH. (D–F) Representative line profile intensities showing the quantification of the fluorescence intensity of the SOX2 (red) and T (green) staining along the anteroposterior axis of the embryos (y axis). 0 marks the anterior neural plate. Orange arrowheads: position of Hensen’s node representative of the anterior border of the primitive streak (PS). (G) Maximum z-projection of embryos stained in whole mount with SOX2/T antibodies showing the localization and the number of the SOX2/T double-positive cells (in yellow) in the anterior PS region. Within each color channel, we threshold the cells expressing T and SOX2 in the nucleus. The double-positive cells are identified by creating a new image with the thresholded image such as only cells expressing T and SOX2 are visible and quantified. Single positive cells are not shown. (H) Quantification of the number of SOX2/T double-positive cells between stage 4 and 5HH. Dorsal views, anterior to the top. Asterisk marks the tip of PS. White arrowheads: Hensen’s node (n = 18; seven embryos at stage 4HH, four at stage 4+HH, seven at stage 5HH). Scale bar: 100 µm.
Figure 1—figure supplement 1—source data 1. Number of T/SOX2 double-positive cells during early chicken stages.
Figure 1—figure supplement 2. Fate of cells of the anterior PS region at the tail bud stage.

Figure 1—figure supplement 2.

(A) Dorsal z-section of a 25-somite embryo showing the superficial localization of the descendants from the cells co-electroporated at stage 5 HH in the NMP region of the anterior PS with a GAP43-Venus and an H2B-RFP plasmid (marking the membrane and the nucleus, respectively). (B) Diagram showing the dorsal region of the tail bud region where cells were tracked in (C–E). (C–E) Images of tracked cells of the anterior PS that become neural (C), remain NMP, (D) or become PSM (E) cells. NMP cells are circled in gold, neural cells are circled in red, and PSM cells are circled in green (52 cells tracked in three embryos). NT: neural tube; NMP: neuromesodermal progenitors; PSM: presomitic mesoderm; PS: primitive streak. Anterior to the top, dorsal views. Scale bar: 100 µm.