| |
Figure 1.
Graded DV Patterning by the Organizer Depends on Chordin (A) Schematic of DV pattern in the control (Cont.) stage 12.5 embryo. D, dorsal; L, lateral; V, ventral. (B) Whole-mount in situ hybridization with shh (dorsal axial) and sizzled (Szl) (ventral) probes (Mix probes, hereinafter). (C) β-catenin-MO-injected embryo with radial expression of sizzled (100%, n = 20). (D) Radial shh expression in the embryo injected with β-catenin-MO and MOs against four major bmp genes: bmp4, admp, bmp2, and bmp7 (100%, n = 11). (E) Single-blastomere injection of dnBR (shown by the LacZ tracer in red; 200 pg) rescued the DV pattern in the β-catenin-MO-injected embryo (100%, n = 13). (F) Single-blastomere injection of dnBR in the embryo injected with β-catenin- and chordin-MOs. Dorsal expansion of sizzled expression was seen. Compare with (E); 100%, n = 15. (G) Single-blastomere injection of dnBR in the embryo injected with β-catenin- and noggin-MOs. No sizzled expansion (87.5%, n = 16). (H) Summary of (E)–(G). (I–T) Reconstitution experiments using the triple-knockdown embryo with MOs for β-catenin, chordin (Chd), and noggin. (I) through (L) show marker expression in the triple-knockdown embryo at stage 12 (J) sizzled and (L) myoD; (I) and (K) show controls. (M–O) Single-blastomere injection of chordin (the LacZ tracer in red) rescued the DV pattern in the triple-knockdown embryo; 100%, n = 12, for (N) and 85.7%, n = 14, for (O), judged by mixed probes. In (M), at a smaller dosage (6.25 pg), no dorsal-lateral region was observed. (P) Schematic of results in (M) through (O). (Q) through (T) show the effects of overexpression (S) and depletion (T) of sizzled on DV markers in the triple-knockdown embryo rescued by chordin (R) (25 pg); control is shown in (Q). (S) Substantial expansion of shh and myoD (86.7%, n = 15 and 100%, n = 16) by sizzled mRNA injection at the two-cell stage (400 pg). (T) Substantial reduction of shh and myoD (100%, n = 17 and n = 16) by sizzled-MO injection at the two-cell stage. (U and V) Effects of loss (U) and gain (V) of Sizzled’s function on the DV pattern. (U) shows substantial expansion of sizzled (100%, n = 20), and (V) shows substantial expansion of shh (87%, n = 23). See also Figures S1 and S2. |
