(A) Schemes of mid-sagittal sections of catshark embryos at the stages indicated. The cellular organization of the territories boxed in red is shown in panels B–N. (B–N) Semi-thin (0.5 µm) sections of the territories boxed in panel A, following toluidine Blue staining. (B) Stage 9: cellular organization of the deep mesenchyme. (C–F) Stage 10: (C) cuboidal epithelial-like structure of the superficial cell layer and (D) sub-jacent deep mesenchyme in the anterior region of the blastoderm; (E) columnar epithelial-like structure of the superficial cell layer and (F) sub-jacent deep mesenchyme in the posterior region of the blastoderm. (G–I′) Stage 10+: (G) cuboidal epithelial-like structure of the superficial cell layer and sub-jacent deep mesenchyme in the anterior region of the blastoderm; (I) general structure of the posterior blastoderm margin and (I′) higher magnification of the deep mesenchyme in the territory boxed in panel I, adjacent to the posterior margin; (H) columnar epithelial-like structure of the superficial cell layer. (J–N) Stage 11: (J,K) cuboidal epithelial-like structure of the superficial cell layer and sub-jacent deep mesenchyme in the anterior margin (J) and a more central region of the blastoderm (K); (L) general structure of the posterior involuting blastoderm margin and adjacent deep mesenchyme; (M,N) higher magnifications of panel L showing elongated cells in the deep mesenchyme adjacent to the involuting AME (M) and the cellular organization of the involuting cell layer in its anterior-most aspect (N). Red arrowheads in panels B, G, I′, K and M point to cell protrusions in the deep mesenchyme. Black arrows in panel J show flattened deep cells appearing at this stage close to the anterior margin. Yellow arrows in panel E point to cells showing apical constrictions suggestive of internalizations from the superficial layer.

(A) Scheme showing the experimental procedure used, and the plane and location of the sections shown in panels B–H (red dotted lines). (B–H) DAPI staining (blue) and DiI fluorescence detection (red) on sections of embryos labeled as in panel A. (B) Example of a control stage 10 labeled embryo, cultured for one hour following DiI application. (C,D) Mid-sagittal sections of two embryos labeled in the midline at stage 9 and cultured for 24 hours after DiI application. (E,F) Respectively para-sagittal and mid-sagittal sections of an embryo labeled at lateral and medial levels at stage 10 and cultured for 24 hours after DiI application. Same in panels G,H, with DiI application at stage 10+. White and yellow arrowheads point to DiI labeled cells in the deep mesenchyme or the involuting mesendoderm respectively. Panel I schematizes the types of movements observed for cells derived from the posterior margin depending on both stage and location along the margin (see Results). Whether internalizations by an ingression-like process take place in early gastrulae at the transition zone between the involuting mesendoderm and deep mesenchyme (i.e. the anterior-most aspect of the involuting layer) could not be addressed, due to the inaccessibility of this territory and limitations in embryo culture times (orange question mark). Same abbreviations as in Fig. 1.

(A) Scheme showing the experimental procedure and plane and location of the sections shown in panels C–E (red dotted lines). (B–E) DAPI staining (blue) and DiI fluorescence detection (red) on sections of embryos labeled as in panel A. (B) Example of a control stage 10 labeled embryo, cultured for one hour following DiI application. (C) Mid-sagittal section of an embryo labeled in the center of the blastoderm at stage 9 and cultured for 24 hours after DiI application. (C′) Higher magnification of the territory boxed in panel C showing labeled internalized cells. (D,E) Respectively para-sagittal and mid-sagittal sections of an embryo labeled in the center of the blastoderm at stage 10 and cultured for 24 hours after DiI application. (D′,E′) Higher magnification of the territories boxed in panels D and E showing labeled internalized cells. White arrowheads point to DiI labeled cells in the deep mesenchyme.

Dorsal views of embryos following whole-mount in situ hybridization with ScLeftyB, ScFgf17 and ScDkk1 probes respectively. Views in panels D, E, I, J, N and O are restricted to the posterior part of the blastoderm where elongation of the embryonic axis takes place. Stages are as follows: (A,F,K) stage 9 embryos; (B,G,L) stage 10/10+; (C,H,M) stage 11; (D,I,N) stage 12; (E,J,O) stage 14. Sections of the embryos photographed in panels B, C, F, G, H, K and N are shown as indicated in panels B1, C1, F1, G1, H1, K1, N1 and N2, with the plane and level of section indicated by a red dotted line. White arrowheads point to labeled cells in the deep mesenchyme, black arrowheads point to the labeled part of the involuting mesendoderm. Same abbreviations as in Fig. 1. Scale bars: 500 µm.

(A–C) Dorsal views of embryos following whole-mount in situ hybridization with an ScChd probe. Views in panels B and C are restricted to the posterior part of the blastoderm where elongation of the embryonic axis takes place. Stages are as follows: (A) stage 11+; (B) stage 12; (C) stage 14. Sections of the embryos photographed in panels A and B are shown in panels A1, B1 and B2, with the plane and level of section indicated by a red dotted line. White arrowheads point to the ScChd negative deep mesenchyme, black arrowheads point to the labeled part of the involuting mesendoderm. (D,E) Dorsal views of the posterior margin following whole-mount in situ hybridizations with ScShh. (F) Scheme summarizing the expression patterns observed in endodermal tissues in the catshark. Dotted areas indicate signals in the deep mesenchyme, uniformly colored areas indicate signals in the involuting mesendoderm and at the margin. A dotted black line delimits the extent of the involuting mesendoderm. The combination of genes expressed in each territory is indicated by the color code shown on the left of the scheme. Abbreviations used: ar, archenteron; nt, notochordal triangle; not, notochord; pcm, prechordal mesendoderm. Scale bars: 500 µm.

Dorsal views of catshark embryos following whole-mount in situ hybridizations with the following probes: ScLeftyB (A,B), ScT (C,D), ScChd (E,F), ScOtx5 (G,H), ScLim1 (I,J), ScSox17 (K,L), ScGata6 (M,N), ScHex (O,P). (K1,L1,M1,N1) Higher magnifications of panels K, L, M, N at the level of boxed territories. (A,C,E,G,I,K,M,O) Control embryos. (B,D,F,H,J,L,N,P) SB-505124 treated embryos. All treated embryos shown are class 1 embryos, except those shown in panels L and N, which show examples of class 2 embryos. Control embryos are stage 11 embryos, except those shown in panels K and M, which are stage 12 embryos. (M1′,N1′) Mid-sagittal sections of the posterior margin of control and SB-505124 treated class 2 embryos respectively, following hybridization with a ScGata6 probe. Red arrowheads point to ScLeftyB, ScChd, ScOtx5 and ScLim1 signals at the posterior margin and adjacent inner cell populations (involuting mesendoderm and/or deep mesenchyme), visible in the controls but absent in treated embryos. Yellow arrowheads point to the second phase of ScSox17, ScGata6, and ScHex, observed in control embryos but undetectable following SB-505124 treatment. Thin arrows point to signals in the deep mesenchyme and syncytial nuclei, which are maintained in treated embryos. Red dotted lines in panels M1 and N1 indicate the section planes shown in panels M1′ and N1′. Scale bars: 500 µm.