Fig. 5. Charon inhibits Nodal signaling. (A-F) Overexpression of Charon inhibited the effects of Nodal overexpression. The injection of 10 pg of cyclops (cyc) or squint (sqt) RNA, or 100 pg of southpaw (spaw) RNA elicited expansion and/or ectopic expression of goosecoid (gsc) at 6 hpf (A,C,E). Co-injection of 25 pg charon RNA inhibited the effects of cyc, sqt and spaw overexpression (B,D,F). (A-F) Animal pole views, with dorsal to the right. (G) Interaction between Charon and Southpaw. COS7 cells were transfected with expression vectors for Myc-tagged Charon (Charon-Myc), Myc-tagged PRDC (PRDC-Myc), or HA-tagged Spaw (HA-Spaw). The supernatants containing Charon-Myc, PRDC-Myc and HA-Spaw were mixed as indicated, and immunoprecipitated with anti HA- or anti-Myc epitope antibodies. The immunoprecipitates were immunoblotted with anti-HA or Myc antibodies. Arrowheads indicate the position of Charon-Myc (two bands, correspond to approximately 35 and 40 kD). A black asterisk indicates light chains of the antibodies, and white asterisks indicate the position of PRDC-Myc. (H-M) southpaw (spaw) expression (H,I) and charon expression (K,L) at the 12-somite stage (15 hpf). Cross-sections of 12-somite stage embryos showing charon (J) and spaw (M) expression. (N-Q) Two-color staining of charon (purplish) and spaw (red) at the 12 somite (N,O) and 18-somite (18 hpf; P,Q) stages. (H,K,N,P) Lateral views. (I,L,O,Q) Dorsal views of the tailbud.