Modelling AVE migration.

<p>(A) Two-dimensional representation of force directions in the vertex model. At each vertex, tension forces act along the edges connecting neighbouring vertices, with unit direction vectors Tc (clockwise) and Ta (anti-clockwise). Pressure forces act normally at the vertex, bisecting the internal angle Φ, with unit direction vector P. (B) On the ellipsoid surface, forces act tangentially. To calculate the forces on a given vertex, its neighbours are projected onto the tangential plane. Unit direction vectors are then determined on this plane. (C) Each cell in the vertex model is 3-D, with associated height and volume. Forces act on the apical surface and depend on quantities such as surface area, edge lengths, height, and perimeter. (D) An initial cell configuration on the ellipsoid surface. Cells highlighted in green are the AVE. The polygon mesh represents the apical surfaces of cells of the VE. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001256#pbio.1001256.s005" target="_blank">Text S1</a> for further details. (E) Comparison of mean polygon number in the ExE-VE and Epi-VE early and late in simulation (roughly equivalent to “distal” and “anterior” embryos). As in wild-type embryos, there is a significant reduction in mean polygon number in the Epi-VE late in simulation as compared to early in simulation (Students <i>t</i> test, <i>p</i><0.001). (F) Frequencies of polygon numbers early and late in simulations. Late in simulations, there is a significant difference in the distribution in the Epi-VE as compared to the ExE-VE, with an increase in four-sided cells and a decrease in six-sided cells (Kolmogorov-Smirnov test, <i>p</i><0.001). There is no significant difference between the distribution in the ExE-VE and Epi-VE early in simulations. Early in simulations: <i>n</i> = 458 Epi-VE and 507 ExE-VE cells from five simulations. Late in simulations: <i>n</i> = 656 Epi-VE and 744 ExE-VE cells from five simulations.</p

Abnormal AVE migration and cellular geometry in mutants with disrupted PCP signalling.

<p>(A) Rosette density (number of rosettes divided by total VE cell number) at different wild-type stages (“pre-AVE”: before AVE induction, <i>n</i> = 9; “distal”: AVE at distal tip before migration, <i>n</i> = 5; “migrating”: AVE migrating, <i>n</i> = 5; and “anterior”: AVE finished proximal migration and moving laterally, <i>n</i> = 4) and in <i>ROSA26^Lyn-Celsr1</i> mutants (<i>n</i> = 7) with disrupted PCP signalling. There is a significant reduction in rosette density in <i>ROSA26^Lyn-Celsr1</i> mutants compared with “migrating” and “anterior” embryos. (A′) The same data as in (A), but depicted as mean number of rosettes per embryo (blue line), and mean number of VE cells per embryo (green bars) at the various stages. <i>ROSA26^Lyn-Celsr1</i> mutants have a comparable number of VE cells to stage matched “anterior” embryos, but show significantly fewer rosettes, leading to the reduced rosette density. (B, B′) En face and profile view of a representative “anterior” embryo, illustrating stereotypical ordered migration of AVE cells. The AVE is marked with a dotted line in (B′) and shows a single group of cells that does not extend more than half-way around the side of the embryo. (C, C′) En face and profile views of an equivalent stage <i>ROSA26^Lyn-Celsr1</i> mutant, showing abnormal AVE migration. AVE cells appear to have broken into several groups (outlined with dotted lines in (C′)) and spread much more broadly within the Epi-VE and even into the ExE-VE. Cell outlines in the embryos in (B) and (C) were visualised by staining for ZO-1 (magenta), and AVE cells by the expression of Hex-GFP (green). Nuclei are visualised with DAPI (dim grey). (D) Comparison of mean polygon number in the Epi-VE and ExE-VE of “anterior” embryos (<i>n</i> = 480 Epi-VE and 409 ExE-VE cells from three embryos) and equivalent stage <i>ROSA26^Lyn-Celsr1</i> mutants (<i>n</i> = 563 Epi-VE and 546 ExE-VE cells from four embryos). As in wild-type “anterior” embryos, the mean polygon number in the Epi-VE of <i>ROSA26^Lyn-Celsr1</i> mutants is significantly lower than that in the ExE-VE. (D′) The same polygon number data grouped according to the VE region. Though the mean polygon number in the ExE-VE is comparable for “anterior” and <i>ROSA26^Lyn-Celsr1</i> embryos, in the Epi-VE it is significantly lower in <i>ROSA26^Lyn-Celsr1</i> embryos, suggestive of increased disequilibrium in cell packing. The scale bar represents 50 µm. <i>p</i> values shown on the graphs were determined using Student's <i>t</i> test.</p

The VE contains multi-cellular rosettes.

<p>(A) A ZO-1 stained embryo in which cells are coloured in to illustrate the presence of junctions where three, four, or five cells meet at a point. (B) Rosettes are formed by five or more cells meeting at a point. A variety of rosettes are shown, including two that share some cells (last panel).</p

Quantitative characterisation of rosettes.

<p>(A) Rosette density (number of rosettes divided by total VE cell number) at different wild-type stages (“pre-AVE”: before AVE induction, <i>n</i> = 9; “distal”: AVE at distal tip before migration, <i>n</i> = 5; “migrating”: AVE migrating, <i>n</i> = 5; and “anterior”: AVE finished proximal migration and moving laterally, <i>n</i> = 4) and in the AVE arrest mutants <i>Nodal^Δ600/lacZ</i> (<i>n</i> = 5) and <i>Cripto</i>^−/− (<i>n</i> = 9). There is a significant increase in rosettes' density in “migrating” embryos as compared to “distal” embryos. The AVE arrest mutants <i>Nodal^Δ600/lacZ</i> and <i>Cripto</i>^−/− show significantly reduced rosette density compared to “migrating” and “anterior” embryos, suggestive of a direct link between rosettes and AVE migration. (A′) The same data as in (A), but depicted as mean number of rosettes per embryo (blue line), and mean number of VE cells per embryo (green bars) at the various stages. “Migrating” embryos have a comparable number of VE cells to “distal” embryos, but have significantly more rosettes, leading to an increase in rosette density. AVE arrest mutants have similar average VE cell numbers to stage matched “anterior” embryos, but show significantly fewer rosettes, leading to the reduced rosette density. (B) Polar plot showing distribution of rosettes in the VE of embryos. Migrating AVE cells were used to determine the anterior of embryos. Rosettes are localised predominantly to the Epi-VE. Within the Epi-VE, rosettes appear to be uniformly distributed with respect to the anterior-posterior axis (<i>n</i> = 39 rosettes from 7 embryos). <i>p</i> values shown on the graphs were determined using Student's <i>t</i> test.</p