PMC

Confocal slices of immunostained wild-type (A–C) and Pten mutant embryos (D–F), using anti-GFP (green) to mark the location of the Hex-GFP transgene. Wild-type (A) and Pten^−/− (D) e5.75 embryos stained for anti-phospho-histoneH3 (magenta) to highlight cells in mitosis; phalloidin (red) and DAPI (blue) mark F-actin and nuclei. Wild-type (B) and Pten epiblast deleted (D) e7.5 embryos stained for anti-phospho-histoneH3 (magenta); phalloidin (red) and DAPI (blue). Wild-type (B) and Pten^−/− (E) e6.0 embryos stained for active-caspase3 antibody (red) to label cells undergoing apoptosis. Scale bars = 20 μm.

(A–C) In situ hybridization of e8.5 embryos to show the pattern of expression of Brachyury (T), which marks the primitive streak and midline, and (D–F) Meox1, which is expressed in the paraxial mesoderm. The majority of Pten null embryos show ruffled, accordion-like neural folds (B) and abnormal paraxial mesoderm (E). (C, F). At this stage, ~20% of Pten null embryos display an ectopic anterior-posterior axis; arrows in F indicate ectopic allantoides, suggesting a triplication of the posterior body axis. Anterior is up. Scale bar = 275μm.

(AC) Bright field views of e7.5 embryos. Cells appear to accumulate in the amniotic cavity in some Pten^−/− (B) and Pten-epiblast deleted (C) embryos. Scale bar in A–C = 240μm. (D–F) Transverse sections of e7.5 wild-type (D), Pten^−/− (E) and Pten epiblast-deleted (F) embryos, stained for laminin (green), E- cadherin (red), and DAPI (blue). Arrowheads indicate the location of the primitive streak. E-cadherin down-regulation is normal, indicating that the EMT is normal. Arrows point to mesodermal cells; in contrast to the thin mesodermal wings that extend around the circumference of the embryo in wild-type, mesodermal cells accumulate near the primitive streak in the mutants, indicating that a defect in mesoderm migration is responsible for the accumulation of cells seen in (B and C). Scale bar in D–F = 40μm.

(A–B) In situ hybridization for Brachyury (T), which marks the primitive streak; viewed from the posterior. 50% of e7.5 Pten^−/− embryos (B) show ectopic expression of T. (C, D) In situ hybridization for the expression of Wnt3 at e6.5, lateral view, indicates that Wnt3 is expressed ectopically in Pten^−/− embryos. Arrow indicates position of a cluster of mis-positioned anterior visceral endoderm (AVE) cells near the distal tip of this Pten^−/− embryo at e6.5. (E, F) Expression of the BAT-gal reporter of canonical Wnt signaling at e7.5, lateral view, is mislocalized in Pten^−/− embryos (4/8). In situ hybridization for Foxa2 (G. H) and Lefty2 (I, J), posterior views, show that Lefty2 and Foxa2 are also expressed ectopically in Pten^−/− embryos (3/6 and 3/6 respectively). Bmp4 expression is unchanged in e6.5 Pten^−/− embryos (7/7) (K, L). Scale bar = 250 μm.

e6.5 embryos stained with rhodamine-conjugated phalloidin (red) to reveal the distribution of F-actin. (A) Anterior view of a 3D reconstruction of a Pten^−/− embryo, with and without the green channel for Hex-GFP. Dense F-actin is present at the apical surface of some VE cells, including both Hex⁺ and Hex⁻ cells. Scale bar = 50μm. (B) An optical section through another embryo shows the columnar morphology of cells in the distal region of the visceral endoderm and strong apical F-actin staining. Scale bar = 50μm. (C–E) High magnification images from 3D reconstructions, showing the difference in the F-actin distribution in wild-type (C) and two representative mutant embryos (D, E). Many mutant VE cells show a high level of apical F-actin, including strong foci of F-actin. Scale bar in C–E = 100μm.

(A) The primitive streak marker T was expressed correctly in a single primitive streak in all Pten-epiblast deleted (Δepi) embryos examined (13/13), as was the anterior streak marker FoxA2. Scale bars = 250 μm. (B) The morphology of e8.5 Pten-epiblast deleted embryos, viewed from the dorsal side. All embryos of this genotype form a single anterior-posterior body axis (n>50). Like the wild-type embryo on the left, the three mutants (on the right) form somites and close the neural tube in the trunk, but have headfolds that fold in irregular patterns. Scale bar =250 μm. (C) SEM views of the ventral side of wild-type and Pten-epiblast deleted e8.5 embryos. In contrast to the single, looping heart tube in wild type, the mutant heart is composed of two tubes that have not fused on the midline (cardia bifida). Scale bar = 100 μm. (D) Pten protein localization in e6.5 wild-type and Pten-epiblast deleted embryos. Arrows indicate the epiblast; arrowheads indicate extraembryonic ectoderm. Pten is detectable in the epiblast, visceral endoderm and extraembryonic ectoderm of wild-type embryos (8/8). Pten protein is not detectable in the epiblast of Pten-epiblast deleted embryos (3/3), although it is still detectable in the extraembryonic ectoderm and visceral endoderm. Scale bar = 50μm.

(A–D) e6.5 embryos, anterior to the left. Cer1, a marker of the AVE, is expressed on the anterior of the wild-type embryo (A). In 37% of Pten^−/−mutant embryos (11/30) (B), most Cer1-expressing cells remain near the distal tip of the embryo. In an additional 13% of Pten^−/−mutant embryos (4/30) Cer1 expression appears to cover most of the embryonic region (C). Cer1 is expressed in the anterior of Pten-epiblast deleted embryos (D; 4/4 embryos). (E–G) Hex-GFP expression (stained with anti-GFP antibody (green); DAPI is blue) in e6.5 embryos, anterior to the left. In wild-type (E), Hex-expressing cells form a contiguous group on the anterior, proximal side of the embryo, while Hex-expressing cells remain distal in Pten^−/− mutants (F) or are scattered over the embryonic region (G), similar to the Cer1 expression pattern. (H–M) Staining for phospho-Akt (S473) (magenta) in wild-type (H–J) and Pten^−/− (K–M) e6.0 embryos. Phospho-Akt was detected at a high level at the membrane of visceral endoderm cells in Pten^−/− embryos, but was not detected above background in the membrane of visceral endoderm cells in the embryonic region of wild-type embryos. The Hex-GFP+ cells are near the distal tip of the mutant embryo, but have migrated toward the anterior in wild type at this stage. (I–J, L–M) p-Akt signal only; high magnification views in J and M. Scale bars in A–G, H–I and K–L = 100μm.

(A–B) 3D reconstructions of e5.75 embryos during AVE migration, showing expression of Hex-GFP (green, staining with anti-GFP antibody) and F-actin (phalloidin, red). White line marks the embryonic/extraembryonic boundary. (A) Wild type, a left/anterior view. Migration is in progress and Hex+ cells have not yet reached the boundary of the embryonic region. (B) The left and right sides a Pten^−/− embryo. AVE cells are more dispersed in the mutant embryo. The leading cells have arrived at the embryonic/extraembryonic border, but many cells remain distally located. Scale bar for A and B = 40μm. (C, D) 3D reconstructions of e6.5 embryos after AVE migration, showing expression of Hex-GFP (green, staining with anti-GFP antibody) and E-cadherin (magenta). (C) Anterior view of wild-type; there are no AVE cells in the back (not shown) or at the distal tip of the embryo. (D) Two views of a Pten^−/−embryo; posterior view to the left and anterior view on the right. AVE cells are present all sides of the embryo, and numerous cells remain distally located. (C) Scale bar for C and D = 50μm. (E–H) Polar plots representing the distribution of AVE cells in embryos like those in (A–D). Each dot represents the position of one Hex⁺ cell. Plots are oriented so the most populated quadrant (the presumptive anterior) is oriented to the left. The position of each cell is indicated both with respect to its position around the circumference of the embryo (angle) and distance migrated along the proximal-distal axis (proximity to center of the plot). Data from 4 wild-type embryos (+/+) at e5.75 (E) and 4 wild-type embryos (+/+) at e6.5 (G) reveal that most Hex+ cells are in the 3 anterior quadrants and have moved away from the distal tip. Data from 3 Pten^−/− mutant embryos at e5.75 (F) and 5 Pten^−/− mutant embryos at e6.5 (H) indicate that cells are more evenly distributed among the quadrants and many more remain near the distal tip. Heterozygous embryos were excluded from this analysis, as they appear to have an intermediate behavior between +/+ and Pten^−/− embryos ().