Figure 1.

Molecular markers reveal subpopulations of definitive endoderm. Whole mount embryos and sections of Sox17 (A-H), Gata5 (I-P) and Gata6 (Q-X) and Gata4 (Y, Z, a-f), analyzed by in situ hybridization (ISH). Stages in bottom right hand corner, anterior to the top, 50 μm thick sagittal sections, lines on whole mount image indicates level of section. Sox17: (A-H) Sox17 in a prestreak blastoderm stage embryo has dynamic expression within Koller's sickle (KS, arrow) with the rostrally extending sickle horns clearly defined by its expression (A). (B) The posterior marginal zone (PMZ, arrowheads) has transcripts in the middle layer, whereas the caudal germ wall (CGW, arrowheads) is negative through all layers. (C) Primitive streak has formed, extending rostrally with Sox17 expressed in a subset of rostral streak cells and in Koller's sickle. (D) Koller's sickle expression down regulates at stage 3c, leaving only the rostral streak positive for transcripts. Note that the central area of anterior tip cells is negative for Sox17 transcripts (arrow). (E) By stage 3d, Sox17-positive endoderm is detected in the early rostral ingressing definitive endoderm (de), anterior and lateral to Hensen's node. (F) In section (arrowheads) definitive endoderm in the lower layer. (G) Maximal streak extension is reached at stage 4+, with definitive endoderm having displaced the hypoblast to the extraembryonic region. Only rostral definitive endoderm now expresses Sox17, with all the definitive endoderm having exited the streak. Axial mesoderm has begun to ingress at stage 4+ (arrowhead in H). Gata5: (I-P) Gata5 expression is strongest in Koller's sickle (KS), with diffuse expression in the posterior hypoblast, PMZ and CGW (I). (J, K) At streak formation (stage 2) restricted expression is detected the caudal two-thirds of the primitive streak, KS (arrow), epiblast PMZ (arrowhead), but not in the CGW (J). (L-N) By stage 3c/d, rostrally displaced hypoblast is positive for transcripts in an initially wide rostral band (arrowheads in section M). For all Gata genes the germinal crescent cells (GCC) are positive (O, W, e), with Gata5 having the best-defined crescent (O, P). Note the broad streak expression (L), spreading laterally as mesoderm and endoderm migrate away (M). (O, P) By stage 4, the central area pellucida is negative for transcripts, with the germ cell crescent (GCC) well defined and junctional mesoderm and endoderm at the lateral edge of the area pellucida strongly positive for transcripts, forming a sharp line at the level of the heart field. Gata6: (Q-X) For Gata6, restricted expression in KS and the lateral sickle horns are well defined (Q). From stage 3b (R), rostrally situated hypoblast has mosaic expression as do all layers of the streak (S). By stage 3c/d (R-V), caudally ingressing endoderm and mesoderm have expression (arrowheads in V). Expression of Gata5 and Gata6 diverges by stage 4, with Gata6 (W, X) marking the a more caudal definitive endoderm subpopulation in addition to a less well-defined junctional population, whereas Gata5 (O) is strongly defined in the lateral endoderm and mesoderm (junctional) population and the center of the embryo is negative for transcripts (compare O, W). Gata4: (Y) Central hypoblast of prestreak embryos expresses Gata4 (see lower layer cells arrowed in Z), with weaker PMZ staining than CGW around the border of the blastoderm (arrowheads). (a) The primitive streak (PS) expresses strongly at stage 3b, except for the anterior tip, as well as diffuse transcripts throughout the area pellucida, becoming restricted to the middle layer of the streak by stage 3c and excluded from the (b, c). (d) The lateral (junctional) mesoderm and primitive streak, exclusive of anterior tip, has strong Gata4 expression by stage 3d. (e, f) By stage 4, with maximal streak extension, expression is lost in the streak, with a fine line of germ cell crescent having expression, and a narrow band of positive junctional mesoderm (JM) cells, similar to Gata5, extending caudally from the heart field. Endoderm cells are negative for transcripts (c, f).