KRas^V12 cell nuclei deform after they invade

(A and B) Orthogonal and projection views of an invading cell expressing EGFP-KRas^V12 (A, B) and H2B-RFP and mCherry-Utrophin (B′) in XZ (A) or XY (B), with time before and after BCE indicated. Scale bar, 50 μm C, nuclear circularity over time for the cell shown in A, B. D, E, example cells expressing EGFP-KRas^V12 (D and E) and H2B-RFP and mCherry-Utrophin (D′, E′) in the outer epithelium (D) or invaded inside the embryo (E), quantified in F & G. Scale bars, 50 μm F, nuclear circularity in surface (n = 25) and invaded (n = 24) KRas^V12 cells.

(G) Nuclear circularity of surface and invaded KRas^V12 cells located in the yolk (n_surface = 11 and n_invaded= 7), the tail (n_surface = 7 and n_invaded= 10) and the head (n_surface = 7 and n_invaded= 7) of the embryo, where the diagram denotes the different regions classified.

(H and I) EGFP-KRas^V12 cells in an embryo stained for GFP, phospho-histone H2A.X, and DAPI located on the surface (H) or inside the embryo (I). Scale bars, 20 μm.

(J) Percentage of KRas^V12-invaded cells expressing phospho-histone H2A.X in the embryonic surface (n = 6) and inside the embryo (n = 6). In A, B, D, E, H, I, dotted lines outline the cell nucleus. In F, G, J, the error bars are SD(SD), the box the SEM(SEM), and the gray lines, the mean. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Video S1.

Loss of laminin reduces KRas^V12 cell trans-differentiation into mesenchymal phenotypes

(A and B) Sample images of neural tube cells expressing HRas-EGFP in control (A) and lamc1 MO-injected (B) embryos, stained with DRAQ5 and GFP to quantify cell density in C, from 9 control embryos and 6 lamc1 morphants.

(D–G) Images of EGFP-KRas^V12-expressing invaded cells in controls (D, E) and lamc1 mutants (F, G) stained with GFP and N-cadherin, where the dotted lines outline cells of interest, quantified in H and I.

(H) Number of invaded cells per embryo in control (n = 11) and lamc1 mutant (n = 11) embryos.

(I) Percentage of KRas^V12-invaded cells expressing N-cadherin in controls (n = 11) and lamc1 mutants (n = 11).

(J and K) Examples of neuron-like morphology cells expressing EGFP-KRas^V12 in control (J) and lamc1 mutants (I), where arrowheads denote cell bodies, quantified in l.

(L) Percentage of KRas^V12-invaded cells with neuron-like morphologies in control (n = 11) and lamc1 mutants (n = 11). In A, B, D–G, J, K, scale bars represent 20 μm. In C, H, I, L, the error bars indicate the SD, the box the SEM, and the lines, the mean. n.s., not significant, ∗p < 0.05, ∗∗p < 0.01. See also Figure S1.

Embryo confinement in agarose promotes KRas^V12 cell trans-differentiation into mesenchymal phenotypes

(A and B) Example 2 dpf embryos stained for p63 that were grown from 1 to 2 dpf in 0% (A) or 2% agarose (B), with dotted lines outlining embryos. Scale bars, 300 μm.

(C) Area of 2 dpf embryos embedded in 0% (n = 8) or 2% agarose (n = 8) from 1 to 2 dpf.

(D and E) Somite cells expressing HRas-EGFP in embryos grown in 0% (D) and 2% (E) agarose, with dotted lines demarking half somites. Scale bars, 50 μm.

(F) Somite cell density in control (0% agarose) embryos (n = 9) and embryos grown in 2% agarose (n = 5).

(G) Number of cells per somite (in xy slice) in embryos grown in 0% agarose (n = 9) or 2% agarose (n = 5).

(H–K) Examples of invaded EGFP-KRas^V12-expressing cells (highlighted in dotted line) in embryos embedded in 0% (H, I) and 2% (J, K) agarose stained with GFP and N-cadherin. Scale bars, 20 μm.

(L) Number of invaded cells per embryo in controls (n = 25) and 2% agarose embryos (n = 24).

(M) Percentage of KRas^V12-invaded cells expressing N-cadherin from embryos grown in 0% (n = 25) and 2% (n = 24) agarose.

(N and O) Examples of EGFP-KRas^V12 cells with a neuron-like shape in embryos grown in 0% (N) and 2% (O) agarose, where arrowheads denote cell body. Scale bars, 50 μm.

(P) Percentage of KRas^V12-invaded cells adopting a neuron-like morphology in 0% (n = 25) and 2% (n = 24) agarose. In C, F, G, L, M, P, the error bars are SD, the box, SEM, and the lines denote mean. n.s., not significant, ∗p < 0.05, ∗∗∗p < 0.001.

Experimental confinement promotes internal cell masses

(A and B) Representative orthogonal views of 4.5 dpf embryos quantified in C & D expressing EGFP-KRas^V12, stained for e-cadherin, and grown from 1 to 2 dpf in 0% (A) or 2% agarose (B), in XY (A, B), XZ (A′, B′) and YZ (A″, B″), with dotted lines outlining embryos and arrowheads the coordinates of XZ orthogonal sections. Scale bars, 300 μm.

(C) Number of cell masses per embryo in 0% (n = 37) or 2% agarose (n = 22) embryos.

(D) Cell mass areas from embryos grown in 0% (n = 22) or 2% agarose (n = 30). 4.5 dpf control (E) and lamc1 MO-injected (F) embryos expressing EGFP-KRas^V12, as projections (E, F), XZ (E′, F′) and YZ sections (E″, F″). Yellow dotted lines outline embryos and arrowheads mark where orthogonal sections were taken. Scale bars, 300 μm.

(G) Number of cell masses per embryo in controls (n = 16) or lamc1 morphants (n = 8).

(H) Area of cell masses in control (n = 22) or lamc1 MO-injected embryos (n = 2). In C, D, G and H, error bars are the SD, box the SEM and gray lines show the mean. n.s., not significant, ∗p < 0.05.