Fig. 2

Differential behaviours of mesendoderm cells initiate neuroectoderm internalisation.

a Sagittal confocal images of the anterior neural plate (ANP) in wild type (WT) embryos during gastrulation. Anterior (A), posterior (P), dorsal (D), ventral (V) indicated. Cell membranes (mRFP, white) labelled. Yellow dashes: neuroectoderm (NE) ventral and anterior mesendoderm (ME) border. Blue dashes: ME/yolk interface. White line: non-neuroectodermal tissue. Cell membranes (mRFP) in grey LUT. Representative internalising cell (red) illustrates shape change in 2D and 3D over time. b Internalising NE cell depth (red) and lateral cell length (blue) during gastrulation (42 cells, 3 embryos). c) Average ANP strain rates along the AP (upper) and mediolateral (ML, lower) axes of WT embryos. Minimum, green (0); maximum stretching, yellow, maximum compression, blue. Black arrows, average tissue flows. Red dot: AP axis/ANP leading edge intersection. Red dashed circle: initial position of internalisation. d Maximum absolute ANP strain rates along the AP (left) and ML (right) axes of WT embryos during gastrulation. Negative values indicate compression. e Contact time between ME and NE cells [locations marked in (f)] along the AP axis (9 cells, 3 embryos). Kruskal–Wallis test (**, p < 0.01). Central line indicates the median, bottom and top edges of the box indicate the 25^th and 75^th percentiles, respectively. Whiskers extend to the most extreme data points not considered outliers. f Sagittal confocal images showing evolution of ME (circles) and NE (squares) cells at the ME front (blue), mid (red), and rear (orange) during gastrulation. Orientation and labels as in (a). g Sagittal confocal images showing cell-cell contacts between front and rear ME and NE stained for endogenous cdh1 (Fire LUT; white maximum, black minimum), nuclei (cyan) and native gsc:GFP-CAAX signal. Boxed areas are magnified to the right highlighting cdh1 localisation at the front and rear. White arrowheads indicate contacts between ME cells and overlying NE cells. Yellow line demarks interface (slight offset). h Quantification of endogenous cdh1 localisation at cell-cell contacts between NE and ME cells in the front (blue circles) and rear (purple squares) of the collective in a WT embryo. Data is presented as individual normalised intensity values (30 cells, 3 embryos). Two-tailed unpaired t-test (****, p = 2e⁻⁶). i Sagittal (xz) multiphoton image of Tg(actb1:lifeact-EGFP) embryo showing F-actin localisation (Fire-LUT; white maximum, black minimum). White arrowheads indicate F-actin rich protrusions at the ME front. Below, magnified images (xy) and schematics of cells at the ME front with polarised F-actin rich protrusions and ME rear with cortical F-actin localisation. j Number of total cell protrusions observed over time in the front (blue) and rear (purple) of Tg(actb1:lifeact-EGFP) embryos (20 cells each, 3 embryos). Two-sided Mann Whitney test (****, p < 0.0001). k Normalised intensity of F-actin (lifeact-EGFP) measured along the cell periphery (0.5 aligned with direction of collective motion) within single front (blue) and rear (purple) ME cells adjacent to the NE interface (10 cells each, 3 embryos). l Schematic (lateral view) illustrating forces (arrows) exerted by the migrating ME (pink/orange) leading to opposing movements (pink arrow posterior, orange arrow anterior) in the ANP (green), generating mechanical instabilities resulting in NE internalisation and multilayer folding and subsequent tissue extension along the DV and AP axes. Orange dashes: E-cadherin. Purple dashes: F-actin. Cell shapes depicted. All data are presented as the mean ± SEM of 3 embryos, unless otherwise stated. Scale bars: 100 µm. Source data are provided as a Source Data file.